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927 publications mentioning hsa-mir-34a (showing top 100)

Open access articles that are associated with the species Homo sapiens and mention the gene name mir-34a. Click the [+] symbols to view sentences that include the gene name, or the word cloud on the right for a summary.

1
[+] score: 662
Here, in these Daoy-TR-miR-34a cells, we observed that single-pulse tetracycline stimulation promoted an enhancement of miR-34a expression, as a pulse of expression at 1 h after stimulation, followed by a rapid down-regulation at 2 h, and then again an enhancement of miR-34a expression at 4 h, followed by a decrease of expression to 12 h (see Fig. 3A). [score:12]
Therefore, the oncosuppressor activities of miR-34a are likely to correlate with the down-regulation of more than one target at the same time during tumorigenesis (additional possibilities are: c-Met, cyclin D1, cyclin D6, N-Myc, Sirt1, CREB), and future studies are needed to inter-relate these data with those previously reported for targets of miR-34a. [score:10]
While both the miR-34a wild-type precursor and the SNALPs carrying the mature miR-34a down-regulated Dll1 expression, miR-34aMut and SNALP-scrambled (SNALPs containing an unrelated oligonucleotide) did not function, further indicating that there is direct functional regulation by miR-34a on Dll1 protein expression (Fig. 1D). [score:10]
24 h later tetracycline stimulation, when the miR-34a upregulation was exhausted, p21 and p27 expressions were found downregulated. [score:9]
Altogether, these results demonstrate that in MB, miR-34a overexpression controls both autonomous and non autonomous Notch signaling through direct down-regulation of the Dll1 target. [score:9]
In these clones, miR-34a induction lead to early down-regulation of Dll1 at 4 h, followed by an massive down-regulation of the Dll1 protein levels at 12 h. Within this assay, we found that Cyclin D1 was down-regulated at 48 h after induction. [score:9]
MiR-34a overexpression also resulted in transient inhibition of Notch2 signaling 12 h post-transfection, as seen by down-regulation of NICD2 and of its known target: the Hairy and enhancer of split 1 (Hes1) proteins (Fig. 1B). [score:9]
Ji et al. (2009) showed that in MiaPaCa2 pancreatic cancer cells, functional restoration of miR-34a down-regulates CD44 [+]/CD133 [+] cells by inhibiting its downstream target genes Notch and Bcl-2, and impairs tumor-sphere growth in vitro and tumor formation in vivo [20]. [score:8]
Transfection of the miR-34a -expressing vector significantly down-regulated Dll1 reporter activity in Daoy MB cells, while no significant inhibition was seen for the Jag1, Notch1 and Notch2 3’-UTR reporters (Fig. 1A). [score:8]
Using miR-34 direct up-regulation by doxorubicin, we show here that p53 induction results in the down-regulation of Dll1 via miR-34 transcriptional control. [score:8]
Comparative expression analyses have shown that miR-34a is highest within the cerebellar cortex [15] and brain tissues [12], and that it acts as a tumor suppressor in gliomas, by targeting both E2F3 and MYCN, and by regulating cell-cycle and apoptosis genes. [score:8]
In mammals Dll1 has n. 3 compared to other putative targets analised, that have at most n. 2 potential target sites, predicted several miRNA target prediction tools, and this make of Dll1 the most potential and early targetable mRNA by miR34a. [score:8]
Figure 3C gives a graphic representation of the levels of down-regulation of targets by expression of miR-34a upon tetracycline induction. [score:8]
Here, we observed that the two miR-34a -expressing clones showed sustained reductions in Dll1 protein levels and marked down-regulation of NICD2 and Hes1 protein expression (Fig. 1F). [score:8]
If miR-34a attenuated the accumulation of Dll1 induced by the MG132 proteasome inhibitor, we could gain additional indirect evidence that miR-34a down-regulates Dll1. [score:7]
For this reason we think that Dll1 is the only early target of miR-34a in MB, while Notch1 and Notch 2 expression is then controlled by an unknown secondary level mechainism of regulation upon Dll1 negative regulation. [score:7]
As observed in other cell lines, our data show that endogenous miR-34a up-regulation by doxorubicin negatively influences the expression of Dll1, and this confirms our previous data using transient miR-34a regulation. [score:7]
To further validate the direct down-regulation of the expression of the Dll1 protein by miR-34a, we used a miR-34aMut and Stable Nucleic-Acid-Lipid Particles (SNALPs) carrying mature miR-34a. [score:7]
Down-regulation of Dll1 expression by miR-34a negatively regulates cell proliferation, and induces apoptosis and neural differentiation in MB cells. [score:7]
Using an inducible tetracycline on-off mo del of miR-34a expression, we show that in Daoy MB cells, Dll1 is the first target that is regulated in MB, as compared to the other targets analyzed here: Cyclin D1, cMyc and CDK4. [score:7]
We also investigated whether miR-34a was able to influences similarly both Notch 1 and Notch2 receptor signaling in MB cells through its down-regulation of Dll1, considering both cell autonomous (ligand and receptor expressed within the same cell) and non-autonomous [27], [28] (ligand and receptor expressed by two distinct, but neighboring, cells). [score:6]
In gliomas, transfection of miR-34a down-regulates c-Met and CDK6, as also for Notch1 and Notch2, which suggests that miR-34a provides a therapeutic agent for brain tumors, through its targeting of multiple oncogenes [16]. [score:6]
MiR-34a overexpression also results in inhibition of Notch2 signaling and activation of Notch1 in both Daoy and D283-MED MB cells, confirming the inhibitory role of Dll1 on Notch1 activation. [score:6]
Indeed, endogenous expression of miR-34a correlates with down-regulation of Dll1 in other, different, tumor types and cell lines, such as for example, breast cancer cells. [score:6]
In a screening of potential targets within Notch signaling, miR-34a was seen to be a regulator of the Notch pathway through its targeting of Notch ligand Delta-like 1 (Dll1). [score:6]
Real time experiment showed that in Daoy-TR-miR34a both p21 and p27 mRNA were found upregulated from 2 h to 12 h following miR34a expression. [score:6]
Finally, the phosphorylation of S727 of STAT-3 was down-regulated in these miR-34a overexpressing clones (Fig. 5E, S4B). [score:6]
B. Reverse phase proteomic array showing proteins that were down-regulated (top) and up-regulated (bottom) in miR-34a stable clones 1 and 2, compared to an empty vector stable clone. [score:6]
Altogether, these data indicate that the ectopic expression of miR-34a in MB cells can transiently down-regulate Dll1 protein levels, and also influence Notch1 and Notch2 signaling. [score:6]
For this reason, we sought to verify if some of reported targets in the literature (e. g., Cyclin D1, cMyc, CDK4) are down-regulated together with Dll1 by miR-34a in a time -dependent manner in our cell mo del. [score:6]
At present, the target regulation and involvement of miR-34a expression in a range of additional pathways of MB tumorigenesis have been postulated (such as: Bcl2, E2F3 and N-Myc). [score:6]
Down-regulation of Akt phosphorylation on S473 was here observed with miR-34a overexpression in MB cells. [score:6]
We present here a mo del (see Fig. S8) that takes into account the Notch autonomous and non-autonomous cell pathways of activation that can be controlled by p53 activation of miR-34a and inhibition of Dll1 expression. [score:5]
This provides further evidence of direct regulation of endogenous miR-34a on Dll1 expression once it is activated by p53. [score:5]
E. Real-time PCR showing miR-34a expression and representative Western blots showing Dll1 expression of MB (Daoy, UW228 and ONS76) and breast (MCF7 and MDA231T) cells lines, upon 24 h of doxorubicin stimulation. [score:5]
Thus, transient overexpression of MiR-34a, using both transfection and adenovirus infection, inhibits Notch 2 activation, which reduces the levels of the NICD2 protein in the high-cell-density context, but which does not occur at the low cell density (Fig. 2C) further suggesting the existence of additional mechanisms controlled by miR34a that might be due an activation of Notch2 “cell autonomous signaling”. [score:5]
One debatable question raised at this time relates to miR-34a target recognition, following the identification of several gene targets for miR-34a. [score:5]
This thus indicates that in Daoy MB cells, Dll1 act as a repressor on Notch1, and also that through its direct down-regulation effect, miR-34a can then activate Notch1 signaling. [score:5]
Decrease in CD15 [+] and CD133 [+] expression in Daoy cells under hypoxia condition, upon miR-34a overexpression. [score:5]
The histochemistry analyses of the extracted ex-vivo tumors, which included miR-34a adenovirus infections, showed inhibition of NESTIN expression and enhanced proportions of the glial-astrocyte neuronal marker GFAP in the tumors (Fig. 7B). [score:5]
For this reason, we performed additional Dll1 3’-UTR reporter activity assays using miR-34b- and miR-34c-containing expression constructs, and showed that both miR-34b and miR-34c down-regulate Dll1 3’-UTR to the same levels as those seen with miR-34a (Fig. S5D). [score:5]
0024584.g005 Figure 5Decrease in CD15 [+] and CD133 [+] expression in Daoy cells under hypoxia condition, upon miR-34a overexpression. [score:5]
To further validate the previous findings, expression of miR-34a was also determined at each time used for the protein expression analyses (Fig. S1A). [score:5]
C-Myc was not down-regulated by miR-34a induction, neither at the early or the late time points. [score:4]
Figure S8 Mo del of the action of miR-34a upon p53 expression and regulation in MB. [score:4]
This clone was further characterize on its capabilities to activate NICD1, by observing that overexpression of mir34a by tetracycline, result on an up-regulation of NICD1 at 8 hours of induction (see Fig. S2B). [score:4]
In this experiment, we shown that in Daoy cells, transfected with p53 wt expressing vector, doxorubic treatment enhance further mir34a expression compared to Daoy cells transfected with empty vector. [score:4]
In MB, miR-34a Daoy stable clones in which we found Dll1 constitutively down-regulated showed a differentiated phenotype, with an increased level of the glial fibrillary acidic protein (GFAP), as assessed by real-time PCR (Fig. S4C) and by morphological and immunofluorescence analyses (Fig. S4D). [score:4]
MiR-34a affects the typical p53 oncosuppressor activity, by inhibiting cell growth, inducing apoptosis and causing a senescence-like phenotype [13]. [score:4]
We postulated here an additional function of Dll1 once is repressed by miR34a, translating this effect with a further functional regulation of Notch1 and Notch2 receptors. [score:4]
Of note, mutation of the three seed sequences within the 3’-UTR of Dll1 completely abrogated this suppression effect of miR-34a. [score:4]
This confirmed the transient specific down-regulation operated by miR-34a on Dll1. [score:4]
Direct recognition and validation of miR-34a target genes using a luciferase assay and time-course overexpression assays of miR-34a in MB Daoy and D283-MED cell lines. [score:4]
The Notch signaling pathway is known to be relevant in MB development, so we used target-prediction analyses to determine whether miR-34a has any role within Notch signaling. [score:4]
An additional question was raised whether other miR-34 family members can have synergistic actions on Dll1 down-regulation. [score:4]
These results suggest that miR-34a regulates Dll1 expression through three binding sites in the 3’-UTR of the gene that encodes Dll1 (Fig. 1A). [score:4]
Our mo del positions miR-34a as the regulator of the Notch–Delta interactions, further supporting the data presented by Sprinzak et al. (2010) [43], where they found that Notch ligand-Delta has two activities: it transactivates Notch function in neighboring cells, and it cis-inhibits Notch signaling in its own cell. [score:4]
Conversely, within the non-autonomous context, miR-34a function down-regulates Notch2 and significantly increases Notch1 signaling, which enhances differentiation of the adjacent, receiving, cells. [score:4]
In Figure 6D, it can be seen that AdV-GFP-miR-34a infection is mainly driven by adenoviruses reaching those cells that are positioned externally in these spheres (see Movie S1) within the tumor-sphere aggregates (Fig. 6D, see arrows on z3-z4 axes), thus showing the potency for miR-34a up-regulation only in these external cells, with the driving of the differentiation processes into the inner neighboring cells. [score:4]
We additionally explored whether or not the protein half-life during its degradation by the proteasome regulates this observed phenomena of miR-34a controlling Dll1 expression. [score:4]
Those result can be explained by the presence of a functional allele of p53 that can, in turn, induce miR-34a and down-regulate Dll1; this was not seen in cells from the null p53 (p53 [-/-]) mice. [score:4]
Altogether, these data indicate that the endogenous levels of miR-34a can regulate Dll1 protein expression. [score:4]
Within the cell-autonomous context in which miR-34a is up-regulated, an important function arises from the enhancement of both Notch1 and Notch2 signaling, which induces proliferation only in ‘committed’ cells and enhancement of apoptosis derived from the increased number of cells in cycling. [score:4]
The present study started with the hypothesis of additional miR-34a targets as key genes in Notch and Shh signaling. [score:3]
To exclude that the maintained presence of the Dll1 protein was due to the presence of miR-34a and not to the tetracycline, this was repeated with Daoy-TR-EV control cells, which do not overexpress miR-34a in response to tetracycline. [score:3]
A. Real-time PCR showed the time -dependent expression of miR-34a following tetracycline single-pulse stimulation. [score:3]
Hence, miR-34a inhibits cell proliferation, enhances apoptosis, induces cell differentiation and further impairs TPC preservation in vitro. [score:3]
Altogether, our data show a therapeutic benefit on overexpression of miR-34a, as it impairs Akt signaling. [score:3]
MiR-34a endogenous expression and regulation by p53 activation. [score:3]
We observed that in these miR-34a overexpressing clones, the proportion of the active Akt kinase protein (Akt S473) was decreased, while the Akt protein levels did not vary, as validated by Western blotting (Fig. 5E, S4B). [score:3]
F. Real-time PCR time courses showing p27 expression in Daoy-TR-EV and Daoy-TR-miR-34a cells, treated with tetracycline. [score:3]
In vivo, miR-34a overexpression carried by adenoviruses reduces tumor burden in cerebellum xenografts of athymic mice, thus demonstrating an anti-tumorigenic role of miR-34a in vivo. [score:3]
Here, we have demonstrated that miR-34a led to an inhibition of Notch2 activity and a reduction in Hes1 protein levels in MB cells. [score:3]
Here, we have shown that miR-34a targets Notch ligand Dll1 in MB cell lines. [score:3]
Thus, in these Daoy cells, miR-34a overexpression reduced the proportion of TPCs from 7.0% to 2.5% and from 5.0% to 2.0%, respectively (Fig. 5C). [score:3]
We also found PTEN phosphorylated on T380 (Fig. S4B), a sign that pro-apoptosis signaling was occurring in these miR-34a overexpressing clones. [score:3]
Understanding the gene-target network of miR-34a will be of importance for future therapeutic applications. [score:3]
AdV-miR34a infected tumor spheres overexpress both miR34a and the neural differentiating markers at GFAP and Tubb3, respect to AdV-GFP-Mock infected tumor spheres. [score:3]
To dissect out these functions, we generated two different miR-34a -expressing stable clones in Daoy MB cells (Fig. 1F), and then we analyzed this pathway, using Western blotting. [score:3]
We then asked whether miR-34a can affect the endogenous expression of Dll1. [score:3]
Figure S2 A. Real-time PCR showing miR-34a expression in Daoy–miR-34a tetracycline inducible clones (Daoy-TR-miR-34a) at 4 h from tetracycline stimulation, as normalized to sn-U6. [score:3]
Then, using miR-34a expression, neural differentiation is observed only when the tumor spheres are plated at high density (Fig. 6A, S6A), thus underlining that the p53/miR-34a/Dll1 specific axis influences the differentiation processes in a non-autonomous Notch-signaling manner in MB. [score:3]
In doing so, we noted that several predicted targets of miR-34a are key genes of the Notch pathway: Dll1, Jagged1 (Jag1), Notch1 and Notch2, which represent two ligands and two receptors of the Notch pathway, respectively (Table S1). [score:3]
C. Real-time PCR analysis for miR-34a expression in Daoy miR-34a stable clones. [score:3]
Opposite effects of MiR-34a on Notch1 and Notch2 receptors throw the direct targeting of Dll1 in the MB Daoy cell line. [score:3]
The MiR-34 family is directly regulated by the transcription factor p53 [9], [10], [11], and all of the members of this family (miR-34a, mi-R34b and miR-34c) share high sequence similarities [12]. [score:3]
A, B. Real-time PCR showing CD15 (A) and CD133 (B) expression in Daoy cells grown under normoxia and hypoxia conditions (as indicated) for 12 h, after 12 h of infection with AdV-miR-34a or AdV-GFP-mock viruses. [score:3]
B. Left: Real-time PCR analysis showing expression levels of the neural markers Nestin, MAP2, TUJ1, and GFAP in Patch [+/-] P53 [+/-] tumor spheres at 96 h from infection with AdV-miR-34a or AdV-mock viruses. [score:3]
Furthermore, real-time PCR analysis showed some 5.5-fold enhancement of TUj1 and GFAP protein expression, as neural and glial cell markers, respectively, in these MB spheres infected with AdV-GFP-miR-34a from both the Patch1 [+/-] p53 [+/-] mice and the Patch1 [+/-] p53 [-/-] mice (Figs. 6B, S6A, S6B and Movie S1). [score:3]
We thus asked whether by targeting Dll1, miR-34 can impair the proliferation rate of MB cells. [score:3]
Ectopic expression of Dll1 rescued miR-34a -mediated apoptosis in Daoy MB cells. [score:3]
‘Rescue’ experiments using Daoy cells that were stable for the Dll1 cDNA that lacked the 3’-UTR that contained the miR-34a binding sites, attenuated the miR-34a pro-apoptotic effects (Fig. 4C) (measured by caspases 3/7 activity), thus suggesting that in the Daoy cells, direct down-regulation of Dll1 miR-34a is involved in caspase -driven apoptosis. [score:3]
D. Real-time PCR time courses showing p21 expression in Daoy-TR-EV and Daoy-TR-miR-34a cells, treated with tetracycline. [score:3]
At this time, no decrease in Dll1 mRNA levels was detected (data not shown), suggesting an initial effect of miR-34a on Dll1 translation, and then later on Dll1 mRNA cleavage. [score:3]
C. Real-time PCR showing the expression profiles of the neural markers MAP2, MATH3, TUJ1 and GFAP in miR-34a Daoy stable clones 1 and 2 and in an empty vector stable clone. [score:3]
On the other hand in MDA-MB-231 cells, which were unresponsive to doxorubicine treatment, wt-p53 transfection led to an increase of miR34a and p21 expression, both in untreated and doxorubicin treated cells (see Fig. 4G, and Figure S3F). [score:3]
The activation of Notch1 downstream signaling was confirmed by HEY1 protein expression (Fig. 1B) and also by induction of CSL1 transcription factor reporter activity, which was detected at 14 h from miR-34a transfection (Fig. 1C). [score:3]
To generate the wild-type miR-34a and the mutant miR-34Mut adenoviruses, the expression cassettes of each construct were cloned into the shuttle vector Ad5 pVQ-K-NpA. [score:3]
Here, we show that stable nucleic-acid-lipid particles carrying mature miR-34a can target Dll1 in vitro and show equal effects to those of adenovirus miR-34a cell infection. [score:3]
Moreover we had performed additional treatments to verify wheter restoration of p53 wild tipe (wt) isoform in both Daoy and MDA-231-T cell lines led to an enhancement of miR-34a expression. [score:3]
G. Real-time PCR showing miR-34a expression in Daoy, and MDA-231T cells lines transfected with p53 wt, and treated for 12 h with doxorubicin, 18 h later transfection. [score:3]
0024584.g007 Figure 7Orthotopic xenografts of MB Daoy cells overexpressing miR-34a by adenovirus infection: functional effects of miR-34a in vivo. [score:3]
To investigate whether miR-34a enhancement in these Daoy-TR-miR34a influences also the expression of Cdk inhibitors (p21 and p27 proteins), we performed time course experiment upon single-pulse tetracycline stimulation. [score:3]
Tumorigenic cells were isolated from Patch 1 [+/-] p53 [-/-] mice and were infected with AdV-GFP-miR-34a and AdV-GFP-mock 48 h later, Western blotting was carried out, which demonstrated that human miR-34a impaired mDll1 protein expression in the Patch mouse MB cells. [score:3]
Those cells were transfected with p53 wt and 18 h later were stimulates with doxorubicin for 12 h. Real Time experiment was performed, to evaluate miR34a expression, using p21 expression as control. [score:3]
0024584.g003 Figure 3 A. Real-time PCR showed the time -dependent expression of miR-34a following tetracycline single-pulse stimulation. [score:3]
In vivo, we show miR-34a inhibition of tumor growth in orthotopic xenografts of athymic nude mice. [score:3]
*Among the experimentally validated miR-34a targets, the Met and Bcl2 genes were chosen as references for the score values. [score:3]
Following ectopic expression of miR-34a, there was substantial activation of apoptosis in the MB ONS-76, D283-MED and Daoy cell lines (Fig. 4A-C), which resulted from massive caspase activation (as activation of caspases 3/7; p≤0.002; p≤0.02 and p≤0.02, according to cell types, respectively). [score:3]
Altogether, these data support our previous findings and correlate miR-34a function with inhibition of Akt/phosphoinositide 3-kinase (PI3K)/PTEN signaling, which is responsible for maintenance and propagation of TPCs. [score:3]
Orthotopic xenografts of MB Daoy cells overexpressing miR-34a by adenovirus infection: functional effects of miR-34a in vivo. [score:3]
Conversely, within the non-autonomous context, miR-34a enhances the pathway of Notch1, but blocks that of Notch2, which inhibits cell proliferation. [score:3]
Conversely, while expression of miR34a inhibited Notch2 at high cell density impairing this signaling, this phenomena did not have similar effects on Notch1 (analyzed by measuring the amounts of activated NICD1 protein). [score:3]
Apoptosis analysis of MB cells upon miR-34a expression, and doxorubicin stimulation of MB and breast cell lines. [score:3]
Table S1 MiR-34a targets were selected by examining the output of the indicated miRNA databases. [score:3]
Taken altogether, these findings suggest that miR-34a expression has a pro-apoptotic effect and impairs soft-agar colony formation in MB cells. [score:3]
Figure S1 A. Real-time PCR analysis for miR-34a expression in the Daoy cell line following transfection of miR-34a at each time point from 0 h to 16 h. Real-time PCR reactions were normalized to mU6. [score:3]
C. Top: Representative Western blot time courses using 2.5 µM MG132 proteasome inhibitor, performed on Daoy-TR-EV and Daoy-TR-miR-34a cells, as indicated, without and with tetracycline stimulation, using an antibody panel against: Dll1and β-actin. [score:3]
C. Caspase 3/7 assay performed in Daoy cells 24 h from co-transfection with miR-34a and the empty vector or with miR-34a and the mouse Dll1 -expressing vector; and in a Daoy Dll1 stable clone, at 48 h after infection with AdV-GFP-miR-34a or AdV-GFP-mock viruses. [score:2]
Then, in a state of ‘no communication’ between cells (low density of cells (L)), the balance of miR-34a regulation induces preferential Notch1 intracellular signaling activation. [score:2]
Figure 7A illustrates the negative in-vivo regulation of tumorigenesis achieved at 50 days using the AdV-GFP-miR-34a-infected cells. [score:2]
As shown in Figure 4D, miR-34a -expressing clones had a higher fraction of apoptotic cells, compared to the empty vector control clone [30]. [score:2]
As expected, miR-34a expression was not induced in the treated MDA-231T breast cancer cells, which have R280K p53 mutation, that led to an p53 transcriptional activity measured as 0,8%, making these cells unresponsive to doxorubicine treatment [33]. [score:2]
In our assay miR34a did not target Notch1 and Notch2 as previously presented by Li et al., 2009 in glioblastoma. [score:2]
MiR-34a expression resulted in a transient reduction in Dll1 protein levels by 10 h (Fig. 1B). [score:2]
Photon emission shows that within 25 days there is development and engraftment of the tumor burden with the AdV-GFP-mock that is greater than that with AdV-miR-34a. [score:2]
As Dll1 is a known ligand of the Notch1 and Notch2 receptors [21], we investigated whether miR-34a expression can influence the regulation of both of these genes and their pathways. [score:2]
MiR-34a action within a gene-target network. [score:2]
These data provide further supporting evidence that the whole miR-34 family (miR-34a, miR-34b and miR-34c) can regulate Notch signaling through Dll1 in MB. [score:2]
MiR-34a influences inhibition of MB tumor-propagating cells, inducing neural differentiation. [score:2]
We also wanted to understand which other intracellular signaling pathways are affected by miR-34a deregulation. [score:2]
Our study shows that miR-34a is a key negative regulator of Notch ligand Delta-like 1 (Dll1) and influences Notch1 and Notch2 signaling in the cell in both an autonomous and non autonomous manner. [score:2]
MiR-34a overexpression can enhance Notch1 signaling in both autonomous and non-autonomous manners. [score:2]
As revealed using quantitative real-time-PCR, there were significant reductions in both CD15 [+] and CD133 [+] expression in the Daoy AdV-GFP-miR-34a infected cells (p<0.05, p<0.01, respectively) (Fig. 5A, B), as compared to the AdV-GFP-mock-infected cells, and this effect was enhanced in the cells subjected to hypoxia. [score:2]
Bottom right: The same experimental procedures were repeated on Daoy cells using a Dll1 3’-UTR construct with mutations within the miR-34a binding site as the reporter. [score:2]
Figure S3 A. MiR-34a overexpression impairs soft-agar colony formation of D283-MED and ONS76 cells. [score:2]
MiR-34a tetracycline inducible on-off mo del: gene-target network. [score:2]
Thus, we have established here a strong rationale for the development of miR-34a as a novel therapeutic agent against MB TPCs. [score:2]
Since miR-34a precursor sequence is evolutionarily conserved, as is the Dll1 3’-UTR sequence, we determine whether human miR-34a can also regulate murine Dll1 in Patch [+/-] p53 [-/-] MB mouse mo del (Fig 5F). [score:2]
These data further demonstrate that Dll1 is one of the first targets regulated in MB (in comparison with CyclinD1, cMyc, CDK4), and also that miR-34a affects the Notch pathway, driving additional signals that will be further investigated. [score:2]
Mutation of the miR-34a seed-region from the 2 [nd] to the 4 [th] base (miR-34aMut) also resulted in a lack of binding of this miR-34aMut to the Dll1 3’-UTR region. [score:2]
Indeed, as an extension of miR-34a target regulation, this aspect should also be investigated in other Notch activated solid tumors. [score:2]
Negative targeting of Dll1 by MiR-34a influences apoptosis. [score:2]
This activation can be explained by the relatively high expression of miR-34 in this clone, as compared to clone #2 (Fig. S1C). [score:2]
D. Left: Representative confocal GFP immunofluorescence staining of Patch [+/-] P53 [+/-] tumor spheres at 24 h from AdV miR-34a virus infection. [score:1]
To investigate further the functional effects of endogenous miR-34a expression in MB cells, we stimulated UW228, ONS76 and Daoy cells with the genotoxic agent doxorubicin [11], a known p53 inducer. [score:1]
Arroweds denotes that the AdV-miR-34a virus efficiently infects only the cells located in the most external regions of the tumor spheres. [score:1]
D. Representative phase-contrast microscopy images (Leika DMIL, 40×0.22 magnification), showing morphological differences between an empty vector Daoy stable clone (left) and miR-34a Daoy stable clones 1 (middle) and 2 (right). [score:1]
Daoy-Luc cells previously infected with AdV-miR-34a or AdV-GFP-mock viruses were injected into the flanks of the nu/nu mice. [score:1]
Thus, this technology forms the basis for their therapeutic use for the delivery of miR-34a in brain-tumor treatment, with no signs of toxicity described to date in non-human primate trials. [score:1]
This evidence led to a mo del for the potential therapeutic use of miR-34 as a radio-sensitizing agent in p53-mutant breast cancer [14]. [score:1]
Figure S7 A. BLI analysis of 3 etherotopic xenografts performed with Daoy cells previously infected with AdV-miR-34a or AdV-GFP-mock viruses. [score:1]
*:p<0.05 D. Representative Western blot for Daoy cells 10 h from transfection with wild-type or seed-mutated-miR-34a, or at 72 h from treatment with SNALPs carrying miR-34a or SNALP-scrambled, using an anti-Dll1 antibody. [score:1]
Figure S5 A. FACS analyses showing cell counts for CD15 [+] and CD133 [+] subpopulations in Daoy cells grown under normoxia or hypoxia conditions for 12 h, after 24 h of infection with AdV-miR-34a or AdV-mock viruses. [score:1]
Movie S1Tumor spheres isolated from Patch1 [+/-] P53 [-/-] mice infected with AdV-miR-34a show sign of induction of differentiation only when they are not dissociated before the infection. [score:1]
C. Representative FACS analysis for CD15 [+] and CD133 [+] subpopulations in Daoy cells grown under normoxia or hypoxia conditions for 12 h, after 24 h of infection with AdV-GFP-miR-34a or AdV-GFP-mock. [score:1]
fr/”), because of these reported data we reason doxorubicin stimulation may be due in Daoy cells to an enhancement of miR34a transcription. [score:1]
Within the cell autonomous context (right), miR-34a enhances Notch 2 signaling, which induces cell proliferation. [score:1]
D. Representative Western blot for Daoy stable Dll1 clone #1 cells plated at different densities and transfected with wild-type or seed-mutated miR-34a, using, anti-NICD2 and anti-β-Actin antibodies. [score:1]
E. Representative Western blot on Daoy-TR-miR-34a cells 6h later tetracycline stimulation, using an antibody panel against: p21 and β-actin. [score:1]
C. BLI analysis of MB orthotopic xenografts of Daoy cells previously infected with AdV-miR-34a or AdV-GFP-mock viruses. [score:1]
D. Representative Western blot for Daoy cells and two primary human MB cell lines (SaV-MB1 and ViV-dMB) at 72 h of treatment with a SNALP carrying miR-34a or with a SNALP-scrambled, performed using anti-CD133, anti-CD15 and anti-β-Actin antibodies. [score:1]
This hypothesis was further confirmed by fluorescence-activated cell sorting (FACS) analyses, using annexin V and propidium iodide staining in the above-described miR-34a stable clones. [score:1]
Through this technology, we evaluated (at different time points) the levels of the miR-34a protein targets following tetracycline induction, comparing both the non-stimulated and the control Daoy-empty vector tetracycline-inducible cell line (Daoy-TR-EV). [score:1]
The mo del takes into account the control of the p53/miR-34a/Dll1 axis with the Notch cell autonomous and cell non-autonomous pathways. [score:1]
For this purpose, we monitored the levels of the Dll1 protein following time-course Western blotting using Daoy-TR-miR-34a cells. [score:1]
For the FACS analysis, 500,000 viable cells of the empty vector clone and the miR-34a Daoy stable clones were harvested and stained with propidium iodide and an anti-annexin-V antibody. [score:1]
Additional immunofluorescence analyses of these tumor spheres confirmed our previous findings, showing that miR-34a overcomes the loss of p53 and induces mainly neuronal and glial differentiation (Fig. S6A-C). [score:1]
The miR-34a clones show extensive neurite out-growth processes and a more differentiated phenotype. [score:1]
We hypothesize that miR-34a increases the asymmetric division of TPCs at the expense of the symmetric self-renewing division. [score:1]
Here the results presented in vitro by the use of SNALP technology set the basis for their therapeutic uses for the delivering of miR-34a into the cerebellum of affected patients, with this resulting in no signs of toxicity according to the literature data in non-human-primate trials [42]. [score:1]
For the 3’UTRs of Dll1, Notch1 and Jag1, more than one miR-34a -binding site was predicted. [score:1]
Doxorubicin stimulation caused miR-34a induction in ONS-76, UW-228, Daoy and MCF-7 cells (Fig. 4E). [score:1]
Figure S4 A. Representative immunofluorescence analysis of Daoy cells 48 h from infection with AdV-miR-34a or AdV-GFP-mock viruses, stained for Nestin or GFAP. [score:1]
For this reason, we isolated tumor spheres [19] from both Patch 1 [+/-] p53 [-/-] and Patch 1 [+/-] p53 [+/-] mice, and used miR-34a to look for any effects on cell differentiation. [score:1]
As expected, in the presence of both MG132 and tetracycline, the Dll1 protein was not degraded, although due to the miR-34a induction, it was not accumulated either (see Fig. S2B). [score:1]
Adenoviruses carrying the precursor miR-34a induce neurogenesis of tumor spheres derived from a genetic animal mo del of MB (Patch1 [+/-] p53 [-/-]), thus providing further evidence that the miR-34a/Dll1 axis controls both autonomous and non autonomous signaling of Notch. [score:1]
F. Representative Western blot of normal mouse cerebellum, and Patch [+/-] P53 [-/-] and primary Patch [+/-] P53 [-/-] mouse MB cell lines, at 48 h from infection with AdV-GFP-miR-34a or AdV-GFP-mock viruses, carried out using anti-Dll1 and anti-β-Actin antibodies. [score:1]
E. Representative Western blot with a Daoy miR-34a stable clone and a Daoy empty-vector stable clone, using an antibodies panel against: Ak, Akt-S473, STAT3-S727, MEK1/2 S217-221, MARCK S152-156 and β-Actin. [score:1]
Altogether, these data indicate that in MB cells, miR-34a impairs proliferation in vitro, which induces apoptosis. [score:1]
Here, through its extensive effects, miR-34a can negatively influence both the CD133 [+] and CD15 [+] populations of both primary MB cell lines and Daoy cells. [score:1]
B. Representative Western blot time-courses for Daoy (B) and D283-MED (D) cells transfected with miR-34a, using a panel of antibodies against: Dll1, NICD1, Hey1, NICD2, Hes1 and β-Actin. [score:1]
F. Confocal GFP staining on Patch +/- P53 -/- mouse tumor spheres previously infected with AdV-GFP-Mock and then treated with doxorubicin for 12 h. AdV- miR-34a does not exert any prodifferentiating effect at either 24 h or 96 h from infection. [score:1]
However, these effects are cell-type dependent, as miR-34a also supports cell proliferation in HeLa and MCF-7 cells [15]. [score:1]
MiR-34a expression negatively affects CD133 [+]/CD15 [+] tumor-propagating cells, then we assay through reverse-phase proteomic arrays, Akt and Stat3 signaling hypo-phosphorylation. [score:1]
B. Top: Representative Western blot for time-course of tetracycline-stimulated Daoy-TR-EV and Daoy-TR-miR-34a cells, using an antibody panel against: Dll1, CyclinD1, cMYC, CDK4 and β-Actin. [score:1]
Additional data show that doxorubicin treatment of these MB spheres from Patch1 [+/-] p53 [+/-] mice induced neural differentiation, while enhancing miR-34a through p53 activation (Fig. 6D). [score:1]
B. Representative Western blot time course performed on UW228 cells transfected with miR-34a, using an antibodies panel against: Dll1, NICD1, NICD2, Hes1 and β-actin. [score:1]
C. BLI of five mice injected in the fourth cerebellar ventricle with Daoy-Luc cells previously infected with AdV-miR-34a or AdV-GFP-mock viruses. [score:1]
These results were further validated by Western blotting in Daoy cells and in two primary cell cultures extracted from human MBs (classic and desmoplastic) using SNALP-containing oligonucleotides for both miR-34a and an unrelated scrambled oligonucleotide sequence (Fig. 5D). [score:1]
Figure S6 A. Confocal GFP staining on Patch +/- P53 -/- mouse tumor spheres at both 24 h and 96h from AdV-miR34a or AdV-GFP-Mock viruses infection, showing differentiating effect of AdV- miR34a. [score:1]
Right: Representative immunofluorescence staining of Patch [+/-] P53 [+/-] tumor spheres, differentiated following viral delivery of miR-34a, performed using an anti-GFAP antibody. [score:1]
At 12 h from transfection with miR-34a antago-mir or with an unrelated antago-mir, the cells were treated with doxorubicin for 12 h. Untreated cells, or treated and non -transfected Daoy cells, were used as controls. [score:1]
B. Real Time PCR performed on Patch +/- P53 -/- mouse tumor spheres at 48 h from infection with AdV-miR34a or AdV-GFP-Mock viruses. [score:1]
The mature and scrambled control miRNAs had the following sequences: miR-34a (mirbase#MIMAT0000255): 5’-U gG cA gU gU cU uA gC uG gU uG u-3’, Scramble: G uA aU gU uU gG cU cG uG uG cU g (small capitals letters: 2'-O-CH [3] substitutions). [score:1]
We also show here that miR-34a delivery through carrier adenovirus particles can impair tumor growth of Daoy cells, and these data are particularly encouraging, as no signs of toxicity or morbidity were observed in these animals. [score:1]
Representative microscopy images and confocal GFP immunofluorescence staining of Patch [+/-] P53 [+/-] mouse MB spheres at 24 h and 96 h from AdV-GFP-mock (left) or AdV-GFP-miR-34a (right) virus infections. [score:1]
Several studies have confirmed that the miR-34 family is required for normal cell responses to DNA damage following irradiation in vivo. [score:1]
*:p<0.05, **:p<0.005 D. FACS analysis for basal apoptosis of Daoy miR-34a stable clones (clones 1 and 2) and of a Daoy empty-vector stable clone, grown under the same selection conditions. [score:1]
0024584.g001 Figure 1 A. Top: Representative 3’-UTR diagram showing the predicted miR-34a binding sites in individual 3’-UTRs. [score:1]
After 24 h, the medium was replaced with medium containing 50 µg/mL SNALP miR-34a and its control SNALP-scramble, without fetal bovine serum. [score:1]
B. BLI from three mice injected in the fourth cerebellar ventricle with DaoyY-Dll1 #1 Luc cells after infection with AdV-miR-34a virus. [score:1]
Representative three fields of each plate are reported on Figure S3B (cell untrasfected and empty vector or miR34a transfected) which were then counted and plotted to produce histograms represented in Figure S3B. [score:1]
Neural differentiation of tumor spheres by miR-34a. [score:1]
Moreover, in this Daoy-Dll1#1 clone, we observed that miR-34a does not negatively influence Notch2 activation (Fig. 2D), both for the high and low cell density context; these results are further supported by no variations in the Hes1 protein levels (see Fig. 2E). [score:1]
Data are mean BLI values of AdV-miR-34a and AdV-GFP-mock xenografts (n = 5 for each). [score:1]
0024584.g006 Figure 6 A. Differentiating effects of AdV-GFP-miR-34a on tumor spheres from Patch [+/-] P53 [+/-] mice. [score:1]
On the other hand, cerebellum implantation of these Daoy Dll1#1 cell pre-infected with AdV-GFP-miR-34a did not shown impairment of tumorigenesis (Fig. S7B), thus indicating that in vivo Dll1 replacement can rescue miR-34a anti-engraftment effects. [score:1]
These tumor spheres changed their morphology 96 h post-infection with AdV-GFP-miR-34a: they differentiated, inducing neurite sprouting (Fig. 6A). [score:1]
C. Representative Western blot for Daoy cells plated at different densities and transfected with wild-type or seed-mutated miR-34a, and infected with adenovirus carring AdV-GFP-miR-34a and AdV-GFP-mock using anti-NICD1, anti-NICD2 and anti-β-Actin antibodies. [score:1]
D. Immunofluorescence analysis of Patch 1 [+]/ [-] P53 [+]/ [-] tumor spheres at 48h from infection with AdV-miR34a or AdV-GFP-Mock viruses, stained with anti-TUj1 or anti-GFAP antibodies. [score:1]
B. Representative Western blot time courses performed on Daoy-TR-miR-34a cells with tetracycline stimulation, using an antibody panel against: NICD1 and β-actin. [score:1]
A. Differentiating effects of AdV-GFP-miR-34a on tumor spheres from Patch [+/-] P53 [+/-] mice. [score:1]
E. Representative Western blot analysis for Daoy Dll1 clone #1 cells plated at different densities, under basal conditions or at 14 h from transfection with miR-34a or with an empty vector, using anti-Hes1 and anti-β-Actin antibodies. [score:1]
E. Representative Western blot as for (B) on D283-MED cells transfected with miR-34a F. Representative Western blot as for (B) for stable miR-34a clones 1 and 2, a stable empty vector clone, and wild-type Daoy cells. [score:1]
A. Top: Representative 3’-UTR diagram showing the predicted miR-34a binding sites in individual 3’-UTRs. [score:1]
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[+] score: 616
About half the mRNAs down-regulated by miR-34b or miR-34c were also down-regulated by miR-34a, but less than a fifth (91 of 482) of the genes down-regulated after miR-34a overexpression were down-regulated by miR-34b or miR-34c (Fig 2A), suggesting that individual miR-34 miRNAs regulate unique targets. [score:18]
Activation of p53 by cellular stress leads to transcription of miR-34 miRNAs, which in turn can enhance p53 function by: (1) miR-34a -mediated inhibition of multiple negative regulators of p53 to further increase p53 transcriptional activity; and (2) miR-34a -mediated increase of p53 protein stability (miR-34a feed-forward loops); or inhibit p53 function by: (3) direct miR-34a -mediated inhibition of TP53; and (4) direct miR-34 inhibition of many p53-activated genes (negative feedback loops). [score:12]
Although mature miR-34b and miR-34c have sequences almost identical to miR-34a even outside the seed, over -expression of miR-34b or miR-34c, unlike over -expression of miR-34a, had little effect on p53 promoter activity and only weakly up-regulated the mRNA levels of p53 transcriptional targets. [score:10]
Consistent with the inhibitory effect of miR-34a on p53 protein in HepG2 cells, miR-34a overexpression in these cells only modestly induced expression of the p53 transcriptional target CDKN1A, and neither TP53INP1 nor PUMA were induced (Fig 5C). [score:9]
MDM4 levels are likely tightly regulated by miR-34a; this target was one of the most highly enriched mRNAs in the Bi-miR-34a PDs and its mRNA and protein were strongly downregulated after miR-34a over -expression. [score:9]
Consistent with this result, induction of 6 p53 transcriptional targets in HCT116 cells was significantly less after miR-34b or miR-34c overexpression than after miR-34a overexpression (Fig 1D), despite highly elevated miRNA overexpression (S1A Fig). [score:9]
Thus, miR-34a overexpression modulates p53 expression post-transcriptionally, both negatively and positively, via direct targeting of TP53 and indirect enhancement of p53 stability. [score:9]
The net null effect of miR-34a on the p53 response may result from opposing effects of (1) enhancing p53 transcriptional activity by inhibiting its regulators, (2) suppressing the expression of p53 itself and many p53-activated genes, and (3) enhancing p53 stability. [score:8]
We also show that although miR-34a can have a positive effect in p53 transcriptional activity and protein stability, by targeting multiple p53 inhibitor genes (MDM4, SIRT1, MTA2, HDAC1, YY1), as has been previously described [11, 13– 15], it can also have a negative effect on TP53, by directly targeting TP53 mRNA. [score:8]
miR-34a down-regulation of HA-tagged MDM4, expressed without its 3’UTR, was abrogated by silent mutations in the CDS MREs, demonstrating their functionality. [score:7]
As expected, miR-34a overexpression reduced expression of CDK6, a known miR-34a gene target, similarly in p53-sufficient and -deficient cells. [score:7]
However, these positive feedback effects are counterbalanced by two important negative feedback mechanisms uncovered in this study – TP53 is a direct target of miR-34a, recognized by noncanonical 5’UTR and CDS MREs, and about a quarter of p53-transactivated genes are also direct targets of miR-34a. [score:7]
Although all three family members regulated cell cycle progression, miR-34b/c over -expression down-regulated mRNAs that mostly function in different biological processes than miR-34a. [score:7]
Our data confirm a feed forward loop in which p53 and miR-34a activate each other by p53 inducing miR-34a expression and miR-34a suppressing multiple p53 inhibitor genes. [score:7]
482, 163 and 29 mRNAs were significantly down-regulated (fold decrease ≥ 1.5 fold relative to miRNA control) after miR-34a, miR-34b or miR-34c overexpression, respectively (Fig 2A and S1 Table). [score:6]
Highlighted in red are genes that were also significantly down-regulated in the gene microarray analysis of miR-34a over -expressing HCT116 cells. [score:6]
Functional Annotation Analysis of downregulated genes in HCT116 cells overexpressing miR-34 using DAVID Bioinformatics tool. [score:6]
Although TP53 is a direct miR-34a target gene, miR-34a over -expression in p53-proficient cells had variable effects on p53 levels in 7 cancer cell lines, even though miR-34a pulled down TP53 in all lines. [score:6]
miR-34a overexpression in HCT116 cells down-regulated mRNAs of all these genes, except HDAC1 (S2A Fig). [score:6]
Genes down-regulated by miR-34 over -expression in HCT116 cells. [score:6]
When these MREs were cloned downstream of the Renilla luciferase reporter, miR-34a overexpression strongly inhibited luciferase activity and seed-pairing mutations completely restored it (Fig 4D). [score:6]
However, expression of miR-34a-resistant versions of these genes individually, including MDM4, did not reduce the miR-34a -mediated increase of p53 transcriptional activity, suggesting that the coordinated knockdown of multiple inhibitor genes is needed to enhance p53 function. [score:6]
Moreover, mRNAs for 42 of the 221 network genes (19%) were down-regulated at least 20% by miR-34a overexpression in HCT116 cells by mRNA microarray. [score:6]
Multiple p53 inhibitors are direct targets of miR-34a. [score:6]
TP53 is a direct miR-34a target TP53 mRNA was enriched 2.6-fold in the Bi-miR-34a PD in HCT116, suggesting it might be a miR-34a target (S2A Fig and S3 Table). [score:6]
0132767.g003 Fig 3Overexpression of miR-34a-resistant SIRT1 or MDM4 does not inhibit miR-34a -mediated p53 transcriptional activation. [score:5]
Overexpression of miR-34a-resistant SIRT1 or MDM4 does not inhibit miR-34a -mediated p53 transcriptional activation. [score:5]
Although multiple miR-34a targets have been identified, it is not clear which targets determine miR-34a’s contribution to the p53 response. [score:5]
As expected, miR-34 overexpression decreased the miR-34a target gene CDK6 and increased the p53-activated gene CDKN1A, assessed as controls. [score:5]
Although TP53 is a miR-34a target, miR-34a overexpression in p53-proficient HCT116 cells unexpectedly increased endogenous p53 protein in HCT116 cells (Fig 4G). [score:5]
We previously found that the extent of enrichment of a gene’s mRNA in the Bi-miR-34a PD correlates closely with how strongly its expression is suppressed [19]. [score:5]
Four other p53 inhibitors, previously identified as miR-34a targets [11, 13– 15], the deacetylases SIRT1 and HDAC1 [23– 26], the NuRD protein MTA2 [25], and the transcription factor YY1, which represses p53 transcriptional activity and enhances p53 degradation [27, 28], were also enriched. [score:5]
To determine whether suppression of any p53 inhibitor has a predominant role in mediating miR-34a enhancement of p53 transcription, we evaluated the effect of co-transfecting miR-34a and individual miR-34a-resistant p53 inhibitor genes, lacking their 3’UTRs. [score:5]
MDM4 mRNA and protein were strongly suppressed by miR-34a overexpression (S3B Fig). [score:5]
Enforced expression of individual miR-34a resistant p53 -inhibitors does not abrogate miR-34a -mediated p53 transcriptional activation. [score:5]
miR-34a- KO cells showed no significant difference in p53 expression after DOX (Fig 6E) and only a moderate increase in abundance of the p53 inhibitors MDM4, MTA2 and YY1, but no change in SIRT1 or HDAC1 (Fig 6G). [score:5]
Previous reports have shown that miR-34a contributes to p53 function by targeting multiple p53 inhibitors, which include: proteins that deacetylate p53 (SIRT1, MTA2/HDAC1) [23– 26], the negative transcriptional modulator MDM4 [20– 22] and the transcription factor YY1 [27, 28]. [score:5]
S3 FigMDM4, an important inhibitor of p53, is the top enriched p53 network gene (A) Enrichment of mRNAs for 5 p53 inhibitor genes and the housekeeping gene SDHA in the Bi-miR-34a PD relative to control-miRNA (Bi-ctl-miRNA) PD in HCT116 cells, assessed by qRT-PCR. [score:5]
0132767.g001 Fig 1 (A) qRT-PCR analysis of mRNA levels of p53 transcriptional targets, normalized to GAPDH, in miR-34a or cel-miR-67 (control) overexpressing WT and TP53 [-/-] HCT116 cells. [score:5]
MDM4, an important inhibitor of p53, is the top enriched p53 network gene (A) Enrichment of mRNAs for 5 p53 inhibitor genes and the housekeeping gene SDHA in the Bi-miR-34a PD relative to control-miRNA (Bi-ctl-miRNA) PD in HCT116 cells, assessed by qRT-PCR. [score:5]
Thus, miR-34a suppression of multiple p53 inhibitors may be needed to increase p53 activity. [score:5]
miR-34a overexpression increased all but one of the targets tested (BAX, MDM2, PUMA, PIG3, FAS, CDKN1A, GADD45A and TP53INP1, but not PMAIP1 (NOXA)), but only in WT cells (Fig 1A). [score:5]
In mice, miR-34a is expressed in most tissues, while miR-34b/c are predominantly expressed in lung and testis [4, 5]. [score:5]
miR-34a knockout (KO) HCT116 cells had deleted most of the seed region of miR-34a in one chromosome and most of the miR-34a sequence in the other, which abrogated miR-34a expression, by Northern blot and qRT-PCR (Fig 6A and 6B). [score:4]
More importantly, mutation of theses MREs in a full length p53 cDNA expression vector abrogated the decrease in p53 protein levels observed upon miR-34a co-transfection in p53 null HCT116 cells, indicating that these MREs are functional in situ despite their locations. [score:4]
miR-34a knockout does not inhibit the p53 response to genotoxic stress. [score:4]
Two miR-34a MREs, located in the 5’UTR and coding region, mediated miR-34a inhibition of TP53 expression in luciferase reporter assays. [score:4]
TP53 is a direct miR-34a target. [score:4]
In addition, the lack of a strong effect of genetic deletion of miR-34a could also be secondary to functional redundancy provided by the other miR-34 members or other p53-regulated tumor suppressor miRNAs [45– 49] or by the p53-independent miR-449 family, which shares a seed sequence with miR-34 [50]. [score:4]
To determine whether TP53 is a direct miR-34a target, we sought to identify MREs. [score:4]
Since MREs outside the 3’UTR often only weakly regulate gene expression, we next evaluated whether these MREs could mediate miR-34a inhibition in the full length mRNA. [score:4]
Expression of miR-34a-resistant MDM4 containing synonymous CDS-MRE mutations and lacking its 3’UTR significantly reduced miR-34a-enhanced pG13-luc reporter activity, but had no significant effect on CDKN1A, PUMA and TP53INP1 mRNA levels (Fig 3D–3F). [score:4]
0132767.g004 Fig 4 TP53 is a direct miR-34a target. [score:4]
We set out to study how the different miR-34 miRNAs contribute to p53 function, analyze whether they regulate overlapping sets of targets and determine if miR-34 is essential for p53 -mediated function in human cells. [score:4]
S4 Fig MDM4 is a direct miR-34a target that contains multiple 3’UTR and CDS MREs. [score:4]
MicroRNA-34a modulates MDM4 expression via a target site in the open reading frame. [score:4]
MDM4 is a direct miR-34a target that contains multiple 3’UTR and CDS MREs. [score:4]
40% of these down-regulated mRNAs were also pulled down with Bi-miR-34a (S3 Table). [score:4]
To determine whether the miR-34 family might regulate non-overlapping mRNAs, we performed gene microarray analysis of HCT116 cells overexpressing each family member (S1B Fig). [score:4]
Under basal conditions, p53 is highly unstable and its stability is regulated by the ubiquitin ligase MDM2, which is not a miR-34a target. [score:4]
MDM4, which together with MDM2 has the strongest effect on p53 function in knockout mice [40], is the strongest miR-34a target, containing at least 5 miR-34a MREs. [score:4]
An important finding from our study is that TP53 is a direct miR-34a target. [score:4]
This positive effect is enhanced by an unexpected strong increase in p53 protein stability after over -expressing miR-34a. [score:3]
miR-34a function might be more critical in other p53 -dependent processes, such as somatic cell reprogramming or inhibition of EMT (epithelial-mesenchymal transition) and metastasis [51– 56]. [score:3]
miR-34a is not expressed in many cancers because of chromosomal deletion or promoter methylation [8]. [score:3]
miR-34a overexpression increased p53 half-life from ~30 to ~120 min (Fig 4I). [score:3]
To assess the effect of miR-34a on the p53 response, we used qRT-PCR to analyze the effect of miR-34a overexpression on 9 p53-activated gene mRNAs in wild type (WT) and TP53 [-/-] HCT116 cells. [score:3]
Thus, the effect of miR-34a overexpression on p53 levels and function depends on cellular context. [score:3]
TP53 mRNA was enriched 2.6-fold in the Bi-miR-34a PD in HCT116, suggesting it might be a miR-34a target (S2A Fig and S3 Table). [score:3]
Similar results were obtained after expressing miR-34a-resistant versions of HDAC1, MTA2 and YY1 (data not shown). [score:3]
Although the reason for this discrepancy is not clear, it is important to note that the reported difference in miR-34a -induced apoptosis between control and SIRT1 over -expressing HCT116 cells in that study was marginal (no more than 3%) [11]. [score:3]
miR-34a- KO cells proliferated at a faster rate than WT HCT116 cells, consistent with the known roles of miR-34a in inhibiting cell cycle progression and growth factor signaling [19, 35– 38] (Fig 6D). [score:3]
miR-34a- KO cells were generated using TALENs (Transcription activator-like effector nucleases) targeting the miRNA seed (S5 Fig). [score:3]
A better knowledge of the mutual functional dependence between miR-34 and p53 will help to understand miR-34 tumor suppressor function. [score:3]
Our results are in conflict with previous experiments, which showed that enforced SIRT1 expression in HCT116 cells abrogated p53 -dependent apoptosis induced by miR-34a [11]. [score:3]
The number indicates the % of remaining protein, normalized to β-actin, in 3 independent miR-34a overexpressing samples. [score:3]
We next used luciferase reporter promoter assays, in p53-sufficient HCT116 cells, to assess whether miR-34 overexpression enhanced promoter activities of a sequence of 13 tandem repeats of the p53 binding site (pG13-luc) [16] or the promoters of p53-regulated genes, PUMA, CDKN1A (the gene encoding p21/WAF1) and BAX. [score:3]
In contrast to our data, two previous reports described inhibition of p53 -mediated apoptosis using antisense oligonucleotides against miR-34a in HCT116 and U2OS cells treated with 5FU or etoposide respectively [10, 11]. [score:3]
Because miR-34a can affect p53 expression by multiple mechanisms, we investigated the effect of miR-34a overexpression on p53 levels in a panel of tumor lines of different origin (MCF7 and MDA-MB-231 breast carcinomas, R KO and SW480 colorectal carcinomas, HepG2 hepatocellular carcinoma and LN229 glioblastoma). [score:3]
Our observation that only miR-34a overexpression enhances p53 -mediated transcription was surprising since the miR-34 family active strands are highly homologous—the seed (residues 2–9) and residues 11–17 and 19–21 are identical (Fig 1C). [score:3]
Ectopic expression of miR-34a leads to cell cycle arrest, apoptosis or senescence, mimicking p53 activation [9]. [score:3]
mRNA levels of the miR-34a targets CDK4 and CDK6 are shown as controls. [score:3]
In fact miR-34a overexpression increased p53 levels in p53-proficient WT HCT116 cells, which was due to a 4-fold longer protein half-life. [score:3]
The net effect of miR-34a on the p53 response will depend on the relative importance of these pathways, which will be determined by differences in gene expression in each cell. [score:3]
Analysis of miR-34 levels in miR-34 over -expressing samples. [score:3]
Although miR-34a is well known for being a “p53 helper” miRNA [39], miR-34a expression is also induced independently of p53 [57– 59]. [score:3]
miR-34a overexpression increased by 4-fold the luciferase activity of the pG13-luc, CDKN1A and PUMA promoters and increased by 2-fold BAX promoter activity (Fig 1C). [score:3]
Co-transfection of miR-34a-resistant SIRT1, which increased SIRT1 protein, did not significantly reduce miR-34a -induced pG13-luc promoter activity or increase p53 target (CDKN1A, PUMA and TP53INP) mRNAs (Fig 3A and 3C). [score:3]
Inhibition of miR34a using various antisense constructs did not affect the p53 response to DOX (data not shown). [score:3]
When the TP53 3’UTR was cloned downstream of Firefly luciferase, miR-34a overexpression did not change luciferase activity (Fig 4B). [score:3]
Thus MDM4 is directly regulated by miR-34a by binding to at least 2 3’UTR and 3 CDS MREs. [score:3]
0132767.g002 Fig 2 (A) Overlap of genes down-regulated ≥ 1.5 fold in miR-34 OE HCT116 cells compared to control -transfected cells. [score:3]
miR-34a overexpression strongly enhanced the half-life of p53 (4-fold), at least in HCT116 cells. [score:3]
Our previous global transcriptome analysis of miR-34a targets using Bi-miR-34a pull-downs suggested that miR-34a acts as a cellular brake in the proliferative and pro-survival response to growth factor stimulation [19]. [score:3]
MDM4, an important inhibitor of p53 transactivation [20– 22], was the top enriched p53 network gene (68-fold, S3 Table) and the fifth most enriched gene of 2416 Bi-miR-34a -binding mRNAs in HCT116 [19]. [score:3]
Single colonies were tested for miR-34 expression by qRT-PCR and negative colonies were verified by sequencing. [score:3]
However, although miR-34a overexpression significantly reduced CDK4 protein in all lines, it had a variable effect on endogenous p53 protein (Fig 5B). [score:3]
Not unexpectedly, miR-34a-regulated genes were over-represented in genes that regulate the cell cycle, mitosis and cell division, DNA metabolism/replication/repair and the response to stress and DNA damage (Fig 2B). [score:3]
Ectopic expression of miR-34a, but not miR-34b/c, increases p53 transcriptional activity. [score:3]
This discrepancy in HCT116 cells might be explained by potential off-target effects of the miR-34a antagonists. [score:3]
Overexpression of miR-34a, but not miR-34b/c, enhances p53 transcription in HCT116 cells. [score:3]
The miR-34a target CDK4 was also enriched by 3–80 fold. [score:3]
S5 FigThe figure shows the binding sites for each pair of TALENs, left (L) and right (R), targeting miR-34a miRNA (underlined). [score:3]
These data are consistent with a previous report showing differing proteomics profiles in HeLa cells over -expressing miR-34a or miR-34c [41]. [score:3]
More importantly, we show that the net effect of miR-34a over -expression on TP53 levels is cellular context dependent. [score:3]
The mechanism by which miR-34a increases p53 half-life is not known, but its suppression of YY1, whose gene product is known to enhance p53-MDM2 interactions may contribute. [score:3]
In comparison, CDK4, a well-known miR-34a target, was enriched 23-fold and UBC and SDHA housekeeping genes were not enriched. [score:3]
Thus TP53 is a miR-34a target gene that binds to miR-34a through noncanonical 5’UTR and CDS MREs. [score:3]
TALEN designs for targeted deletion of miR-34a miRNA. [score:3]
These secondary gene regulatory networks presumably overlap with the gene network regulated by miR-34a. [score:3]
miR-34a targets many p53 network genes. [score:3]
miR-34a targeting TALENs were generated using the TALE Toolbox kit (Addgene cat#1000000019) [66]. [score:3]
miR-34a overexpression differentially affects p53 levels in p53-sufficient cancer cell lines. [score:3]
Since their initial identification as p53 transcriptional targets, the three members of the miR-34 family have been considered crucial mediators of the p53 response [39]. [score:2]
To examine miR-34a’s role in regulating p53 function, we analyzed how many annotated p53 network genes (S3 Table) were enriched in the Bi-miR-34a PD in HCT116 cells. [score:2]
The miR-34a seed region is in blue, while mutations introduced in the MREs are highlighted in red. [score:2]
We previously used streptavidin pull-downs (PD) of Bi-miR-34a -transfected HCT116 and K562 cells to identify miR-34a-regulated genes [19]. [score:2]
Last, using TALEN-generated (Transcription Activator-Like Effector Nucleases) HCT116 and MCF7 miR-34a knock-out cells, we show that the p53 -mediated response to genotoxic stress is unimpaired in these cells. [score:2]
It is important to note that the effect of knocking out miR-34a on the response to genotoxic stress might differ in different cancer cell lines. [score:2]
As a consequence, knockout of miR-34a had no effect on the p53 response to genotoxic stress in two human cancer cell lines. [score:2]
miR-34a might be a critical regulator of other non-p53 related biological processes, which might differ depending on the cellular context. [score:2]
Because knockdown was incomplete, the lack of an effect could be due to residual miR-34a. [score:2]
After DOX treatment, induction of p53-regulated mRNAs and proteins and apoptosis, assessed by annexin V and propidium iodide (annexin-PI) staining, was comparable in miR-34a- KO and WT cells (Fig 6E and 6F). [score:2]
The miR-34a seed region is highlighted in blue, while mutations (mt) introduced in the MREs are highlighted in red. [score:2]
miR-34a repression of SIRT1 regulates apoptosis. [score:2]
miR-34a and miR-34b/c regulate different biological processes. [score:2]
In WT HCT116 cells, miR-34a is present at ~300 copies/cell under basal conditions and increases to ~1200 copies/cell after DOX. [score:1]
Of note, miR-34a effect on p53 abundance was not related to p53 status since opposing effects were observed in MCF7/HepG2 vs. [score:1]
In all, our data suggest that the role of miR-34a in the p53 response in human cells, integrating the positive and negative effects on p53 network genes, is to stabilize and reinforce the p53 response, rather than promote it. [score:1]
However, WT and miR-34a- KO MCF7 cells did not differ in p53 -mediated apoptosis or cell cycle arrest after DOX (Fig 7D and 7E). [score:1]
Multiple miRNAs, including the miR-34 family, are transcriptionally activated by p53. [score:1]
miR-34a PD mRNA levels were determined by qRT-PCR and plotted as fold change relative to mRNAs pulled down with the control Bi-miRNA (Bi-ctl-miRNA). [score:1]
WT HCT116 cells were transfected with control or miR-34a mimics and protein levels were analyzed by immunoblot 48 hr post-transfection. [score:1]
Of 221 genes, 52 (24%) were enriched at least 2-fold in the Bi-miR-34a PD in HCT116 cells (S2A Fig and S3 Table). [score:1]
The miR-34 family consists of 3 miRNAs—miR-34a on human chromosome 1p36 and miR-34b/c, co-transcribed on human chromosome 11q23. [score:1]
Our results showing that miR-34a is not essential for the p53 mediated response to stress are in agreement with data published by Concepcion et al reporting intact p53 function in miR-34 deficient mice [12]. [score:1]
TP53 mRNA was highly enriched in the Bi-miR-34a PD. [score:1]
A representative blot is shown in (E) and densitometry of p53 relative to β-actin signal in 3 independent experiments in cells transfected with miR-34a relative to cells transfected with control miRNA is shown in (F). [score:1]
Unimpaired p53 response in miR-34a- KO MCF7 cells. [score:1]
Competing negative and positive feedback loops determine the net effect of miR-34a on p53 function. [score:1]
Our study suggests that the role of miR-34a in cancer is complex. [score:1]
miR-34a- KO HCT116 cells have a normal p53 response to genotoxic stress. [score:1]
The enrichment of p53 network genes in the Bi-miR-34a PD was validated by qRT-PCR in independent Bi-miR-34a PDs for 12 randomly selected p53 network genes (S2B Fig). [score:1]
Housekeeping genes UBC and SDHA were not enriched and unbiotinylated miR-34a did not PD any genes (Fig 5A). [score:1]
Pulse-chase analysis of p53 protein in HCT116 cells transfected with control or miR-34a mimics. [score:1]
It may be difficult to predict the antitumor effect of miR-34a, which may depend on the p53 status of the tumor and other tumor-specific genetic and epigenetic changes. [score:1]
Rather than simply promoting p53 function, miR-34a might act at a systems level to affect multiple genes in the p53 network, both positively and negatively. [score:1]
Co-transfection of TP53 cDNA with miR-34a reduced p53 protein by 50% in p53 -null HCT116 cells (Fig 4E and 4F). [score:1]
Thus miR-34 -mediated increased p53 transcription is largely limited to miR-34a. [score:1]
These data together suggest that miR-34a and miR-34b/c serve different biological functions. [score:1]
In addition, a recent study suggests that miR-34a deficiency promotes tumorigenesis only when p53 is haploinsufficient [29]. [score:1]
The bottom panel shows the Northern blot for miR-34a in WT and 34a- KO MCF7 cells. [score:1]
Thus, our results suggest that miR-34a is dispensable for the p53 -mediated response to stress in human cells, as it is in mice [12]. [score:1]
AS-34a indicates a psiCHECK2 reporter containing a perfect match for miR-34a, used as positive control. [score:1]
The representative immunoblot (left) shows HA-tagged MDM4 in 293T cells co -transfected with a plasmid encoding for WT or mutated (mt) HA-MDM4 and with control miRNA or miR-34a mimics. [score:1]
Consistent with these reports all these genes were identified as hits in our Bi-miR-34a PD. [score:1]
To investigate why p53 protein increased, we analyzed p53 protein stability by pulse-chase analysis in control or miR-34a overexpressing HCT116 cells. [score:1]
Thus miR-34a binds to a large proportion of p53 network mRNAs. [score:1]
In summary, although our data confirm the strong interplay of p53 and miR-34a, they suggest a complex functional relationship. [score:1]
Genome-wide transcriptome analysis of miR-34 OE HCT116 cells. [score:1]
Thus sequence determinants outside the seed might profoundly affect miR-34 family function by an unknown mechanism that is worth exploring. [score:1]
Here, we investigated in detail how the different miR-34 family members contribute to p53 function, the miR-34a targets that are relevant for its contribution and how much p53 relies on miR-34a. [score:1]
Future experiments with miR-34 -deficient human cells should address the contribution of miR-34 in these other scenarios. [score:1]
Mean +/- SD of three independent experiments is shown in cells transfected with miR-34 family or cel-miR-67 (M-control) mimics. [score:1]
The mature miR-34a sequence is in red, with the seed sequence underlined. [score:1]
The p53-independent functions of miR-34a may turn out to be more important in vivo than its p53 effects. [score:1]
p53 response gene mRNAs were significantly enriched by miR-34a PD in both cell lines. [score:1]
More importantly, it is unclear how much miR-34a contributes to p53 function. [score:1]
S2 Fig (A) Interactome (Ingenuity) of p53 network genes whose mRNAs were enriched at least 2-fold in the streptavidin PD of Bi-miR-34a relative to Bi-cel-miR-67 control PD in HCT116 cells. [score:1]
Although antagonizing miR-34a in human cells impairs p53 function in a few studies [4, 10, 11], mice genetically deficient in all miR-34 family genes have unimpaired stress responses [12]. [score:1]
miR-34a effect on p53 depends on cellular context. [score:1]
p53- KO HCT116 cells proliferated more slowly than WT cells, suggesting that the increased proliferation of miR-34a- KO cells is p53-independent. [score:1]
miR-34—a microRNA replacement therapy is headed to the clinic. [score:1]
Normalized Firefly luciferase activity, relative to Renilla luciferase activity, after miR-34 transfection is plotted as fold change relative to control miRNA -transfected sample. [score:1]
The bottom panel shows the Northern blot for miR-34a in WT and 34a- KO HCT116 cells. [score:1]
Mo del of miR-34a and p53 interactions. [score:1]
HDAC1, YY1 and MDM4 were more abundant in miR-34a- KO than WT MCF7 cells (Fig 7C), which might have contributed. [score:1]
mRNA in the Bi-miR-34a or Bi-cel-miR-67 control PD and input samples were quantified by microarray and qRT-PCR. [score:1]
Alignment of the miR-34 family with the seed sequence highlighted in red is shown at top. [score:1]
miR-34a KO did not lead to a significant compensatory increase in other family members under basal conditions or during stress. [score:1]
Taken together, our data suggest that miR-34a is dispensable for the p53 -mediated response in human cells. [score:1]
Analysis of p53 network genes, compiled from p53 Knowledgebase, in Biot-miR-34a pull-downs. [score:1]
Our analysis of miR-34a deficient cells focused on the p53 -mediated response to stress. [score:1]
The enrichment ratio is the ratio of mRNA in the Bi-miR-34a PD relative to the control PD, normalized to input levels. [score:1]
0132767.g008 Fig 8Competing negative and positive feedback loops determine the net effect of miR-34a on p53 function. [score:1]
miR-34 levels in transfected samples from Fig 1D (A) and Fig 2 (B), analyzed by qRT-PCR. [score:1]
Because of its role in the p53 pathway and as a tumor suppressor, miR-34a mimics incorporated into liposomes are currently being evaluated for treatment of primary and metastatic liver cancers [61]. [score:1]
In this regard, it has been shown recently that somatic cells from miR-34 deficient mice can be reprogrammed more efficiently [51]. [score:1]
miR-34a KO cells. [score:1]
These 5 mRNAs were enriched in the Bi-miR-34a PD in HCT116 cells at least 10-fold relative to Bi-miRNA-control by qRT-PCR (S3A Fig), confirming the microarray results. [score:1]
PD after transfection of unbiotinylated miR-34a was another control. [score:1]
Thus the TP53 3’UTR does not contain functional miR-34a MREs. [score:1]
Three chromosome 5q11.2 miRNAs (miR-449a/b/c) share a seed sequence with miR-34, and have a tissue distribution similar to that of miR-34b/c [6, 7]. [score:1]
An additional control was PD of unbiotinylated miR-34a. [score:1]
However, additional analysis of the p53 -mediated response to stress in 34a- KO MCF7 cells were consistent with those performed in HCT116 cells, suggesting that, although miR-34a can contribute to p53 function, its contribution is not essential for the p53 -mediated response to stress. [score:1]
miR-34a KO HCT116 cells showed a normal p53 response to stress. [score:1]
Thus the p53 response to genotoxic stress was unimpaired in HCT116 cells deficient in miR-34a. [score:1]
Cells were also transfected with unbiotinylated miR-34a as a negative control. [score:1]
In particular, the effect on p53 is predominantly mediated by miR-34a. [score:1]
We therefore deleted miR-34a in HCT116 cells using TALENs [32– 34] (S5 Fig). [score:1]
We used TALENs to generate cell lines in which the seed region of miR-34a was deleted in both chromosomes to completely eliminate production of the mature miRNA. [score:1]
In support of this idea, miR-34a KO mice exhibit elevated bone resorption and reduced bone mass [60]. [score:1]
In all cells, Bi-miR-34a PD significantly enriched for TP53 mRNA by 3-20-fold relative to Bi-miR-control PD (Fig 5A). [score:1]
0132767.g005 Fig 5 (A) qRT-PCR analysis of the enrichment of TP53 (top) and CDK4 (bottom) mRNAs in Bi-miR-34a PDs in tumor cell lines of different origin. [score:1]
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[+] score: 565
Other miRNAs from this paper: hsa-mir-34b, hsa-mir-34c
Lastly, we validated changes in miR-34a putative target gene expression, including decreased expression of KLF4, SEM3A, and VEGFA transcripts in canine OSA cells overexpressing miR-34a and identified KLF4 and VEGFA as direct target genes of miR-34a. [score:12]
We identified 150 genes containing consensus binding sites for miR-34a that were significantly down-regulated (fold change ≤ -1.4, p-value < 0.05) following enforced expression of miR-34a in OSA8 cells, demonstrating that the miR-34a -downregulated genes are enriched for direct targets of this miRNA (S3 Table). [score:12]
To determine whether putative miR-34a target genes identified by in silico computer -based predictive algorithms are directly regulated by miR-34a, the 3’-UTR of canine KLF4 and VEGFA containing wild-type (WT) or mutant (MUT) miR-34a target sequences were cloned downstream of a luciferase reporter gene in the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Fig 6A). [score:11]
Overexpression of miR-34a significantly altered the gene expression profile of OSA8 cells, including substantial up-regulation (fold change ≥ 1.5) of 12 genes and downregulation (fold change ≤ -1.5) of 99 gene transcripts when compared to control OSA8 cells (Fig 4, p ≤ 0.0001; S2 Table). [score:10]
We validated the expression profiles of select putative miR-34a target genes including Krüppel-like factor 4 (KLF4), Semaphorin 3E (SEMA3E), and Vascular endothelial growth factor receptor A (VEGFA) and confirmed significant down-regulation of these genes in OSA8 and Abrams cells overexpressing miR-34a. [score:10]
Consistent with our results, transcripts for KLF4, SEMA3E, and VEGFA were down-regulated following overexpression of miR-34a in OSA8 cells, suggesting that miR-34a may alter the invasive capacity and migratory behavior of OSA8 cells, in part, by regulating the expression of these genes (Fig 5). [score:9]
Concordant with the observation that miR-34a decreased the expression of SEM3A, KLF4, and VEGFA in canine OSA cell lines and that miR-34a negatively regulates KLF4 and VEGFA transcripts, we found that in primary canine OSA tumors expressing low basal levels of miR-34a, expression of these putative target genes was significantly increased compared to normal osteoblasts possessing high miR-34a levels. [score:9]
Notably, enforced expression of miR-34a significantly altered the transcriptome of canine OSA8 cells and 150 of 773 down-regulated genes (19%) contained consensus binding sites for miR-34a, demonstrating enrichment for direct targets of this miRNA. [score:9]
Transcriptional profiling of canine OSA8 cells possessing enforced miR-34a expression demonstrated dysregulation of numerous genes, including significant down-regulation of multiple putative targets of miR-34a. [score:9]
Predicted miR-34a target genes down-regulated with miR-34a expression in canine OSA8 cells. [score:8]
Similarly, differences in the capacity of miR-34a to inhibit cell viability or suppress cellular migration and invasion have been observed following miR-34a overexpression in different human OSA tumor cell lines [42, 43]. [score:7]
0190086.g005 Fig 5Transcriptional profiling of canine OSA8 cells expressing pre-miR-34a (miR-34a) or empty vector (EV) control was performed by next-generation sequencing to identify genes showing differential expression with miR-34a overexpression. [score:7]
Given the prevalence of miR-34a dysregulation in cancer and its ability to down-regulate the expression of numerous oncogenes across multiple oncogenic pathways, there is ongoing interest in developing miR-34a mimic therapy as a novel therapeutic strategy for cancer. [score:7]
Transcriptional profiling of canine OSA8 cells expressing pre-miR-34a (miR-34a) or empty vector (EV) control was performed by next-generation sequencing to identify genes showing differential expression with miR-34a overexpression. [score:7]
RT-qPCR was performed on OSA8 cells expressing empty vector or miR-34a to validate changes in mRNA expression following miR-34a overexpression. [score:7]
RT-qPCR was performed to independently validate changes in gene expression for putative miR-34a targets (A) KLF4, (B) SEM3AE, and (C) VEGFA altered by miR-34a overexpression in both canine OSA8 and Abrams cell lines. [score:7]
Identification of putative miR-34a target genes dysregulated by miR-34a overexpression in canine OSA cell lines. [score:6]
Lastly, we confirmed direct targeting of KLF4 and VEGFA transcript 3’UTR by miR-34a, suggesting a potential mechanism by which miR-34a inhibits invasion and migration in canine OSA cells. [score:6]
We previously reported a miRNA expression signature that was associated with canine OSA using the nanoString nCounter platform, including down-regulation of miR-34a in primary canine OSA tumors. [score:6]
Prediction of miR-34a binding to the 3’-UTR of genes down-regulated by miR-34a was performed with computer-aided algorithms obtained from TargetScan (http://www. [score:6]
150 genes that were significantly down-regulated (fold change ≤ -1.4, p-value < 0.05) following enforced expression of miR-34a in OSA8 cells were found to contain consensus binding sites for miR-34a using in silico miRNA prediction tools. [score:6]
Similarly, overexpression of miR-34a significantly inhibited cell invasion through Matrigel in the OSA8 and Abrams cells as compared to cells expressing empty vector (Fig 3A). [score:6]
Significantly, several members of this miRNA signature are known to be aberrantly expressed in human OSA tumors, including miR-34a which is also downregulated in canine OSA. [score:6]
This work serves as the foundation for future work to dissect the molecular mechanisms by which miR-34a regulates the invasive capacity of canine OSA cells, with the ultimate goal of identifying new targets for therapeutic intervention in this disease. [score:6]
Enforced expression of miR-34a in canine OSA cell lines reduced cellular invasion and migration and significantly altered the transcriptional profile of OSA cells, including down-regulation of several genes implicated in promoting the metastatic phenotype. [score:6]
Moreover, gene ontology analysis of down-regulated miR-34a target genes showed enrichment of several biological processes related to cell invasion and motility. [score:6]
Enforced expression of miR-34a decreases the invasive capacity of human OSA cell lines in vitro and modulates the progression of OSA lung metastasis in vivo, in part, through direct targeting of oncogenes such as c-Met, sirtuin 1 (SIRT1), and CD44 [42, 43, 54]. [score:6]
Concordant with these data, primary canine OSA tumor tissues demonstrated increased expression levels of putative miR-34a target genes. [score:5]
OSA8 and Abrams cell lines were transduced with empty or pre-miR-34a expressing lentivirus vector and 72 hours post transduction, cells were sorted based on GFP-positivity and overexpression of miR-34a was confirmed by RT-qPCR (Fig 2A). [score:5]
However, this may be explained by differences in the expression of mRNA targets in distinct cell types/tissues that influence the effect of miR-34a on cell behavior. [score:5]
Total RNA was isolated from canine OSA8 cells expressing control or pre-miR-34a lentiviral constructs from three separate transduction experiments and next-generation sequencing was performed to identify differences in gene transcript expression. [score:5]
Concordant with these data, stable overexpression of miR-34a in canine OSA cell lines reduced VEGFA expression with a concomitant decrease in cell invasion and migration. [score:5]
In the canine OSA cell lines, stable overexpression of miR-34a resulted in significant inhibition of cellular invasion and migratory capacity. [score:5]
Putative miR-34a target gene expression is increased in primary canine OSA tumors. [score:5]
Furthermore, the introduction of miR-34a gene constructs into cell lines derived from solid tumors (lung, liver, colon, pancreatic, brain, prostate, bone, ovary) and hematopoietic malignancies (lymphoma, leukemias) induces apoptosis and cell cycle arrest, and inhibits migration and invasion, providing support for a tumor suppressor function of miR-34a [25, 26, 40]. [score:5]
The canine KLF4 and VEGFA 3’UTR clones, which include wild-type (WT) or mutated (MUT) miR-34a seed binding sites were cloned into the PmeI and Xbal sites of pmirGLO dual-luciferase miRNA target expression vector (Promega, Madison, WI, USA). [score:5]
Importantly, miR-34a loss in OSA is associated with enhanced expression of a several targets known to contribute to tumorigenesis including MET, SIRT1, and CDK6 [42, 53, 54] although its contribution to the biology of OSA has not yet been fully elucidated. [score:5]
S2 TableGene transcripts differentially expression in canine OSA8 cells expressing empty vector (EV) or miR-34a as determined by one-way ANOVA comparison test (p < 0.0001). [score:5]
Expression of miR-34a in canine OSA cell lines suppresses cell invasion and migration. [score:5]
0190086.g004 Fig 4Total RNA was isolated from canine OSA8 cells expressing control or pre-miR-34a lentiviral constructs from three separate transduction experiments and next-generation sequencing was performed to identify differences in gene transcript expression. [score:5]
To investigate whether similar alterations in transcript expression occurred following overexpression of miR-34a in other canine OSA cell lines, RT-qPCR was performed for KLF4, SEMA3E, and VEGFA in the canine Abrams OSA cell line expressing either empty control or miR-34a lentivectors (Fig 5). [score:5]
Supervised hierarchical cluster analysis of 111 genes differentially expressed in OSA8 cells expressing either empty vector (EV) or miR-34a (miR34a) as determined by one-way ANOVA comparison test (p ≤ 0.0001). [score:5]
In concordance with the potential role of miR-34a in malignant osteoblast behavior, decreased expression of miR-34a in spontaneous human OSA tumors and low levels of circulating miR-34a in OSA patient plasma are associated with shorter disease-free survival and poor prognosis [44, 45]. [score:5]
Overexpression of miR-34a in canine OSA8 cells significantly alters gene expression. [score:5]
RT-qPCR was performed to validate changes in mRNA expression for selected genes affected by miR-34a over expression. [score:5]
Reporter constructs harboring wild-type (WT) or seed sequence-mutated (MUT) miR-34a binding sites for canine KLF4 and VEGFA were cloned into the pmirGLO Dual-Luciferase miRNA Target Expression Vector. [score:5]
Indeed, the miR-34 family members function as tumor suppressors, inducing apoptosis, cell cycle arrest and senescence, in part, through their interaction with the p53 tumor suppressor network [33, 37– 39]. [score:5]
With respect to OSA, miR-34a expression is decreased in human primary OSA tumor samples when compared to adjacent normal tissues, suggesting a role for miR-34a dysregulation in disease pathogenesis [30]. [score:5]
Ectopic expression of miR-34a in human head and neck squamous cell carcinoma (HNSCC) cells was found to inhibit tumor growth and angiogenesis that was partially reversed by blocking VEGFA production by tumor cells [65]. [score:5]
org) and found that putative miR-34a target genes are highly enriched for biological processes related to cell migration, including GO categories “regulation of cell projection organization” (p-value 7.55 x 10 [−5]), “regulation of locomotion” (p-value 1.96 x 10 [−4]), and “cell projection organization” (p-value 3.90 x 10 [−4]), consistent with the role of miR-34a in mediating cellular invasion and motility in OSA cells (S4 Table). [score:5]
In canine OSA cell lines stably transduced with empty vector or pre-miR-34a lentiviral constructs, overexpression of miR-34a inhibited cellular invasion and migration but had no effect on cell proliferation or cell cycle distribution. [score:5]
As shown in Fig 7, RT-qPCR demonstrated that primary canine OSA tumor tissues express high levels of putative miR-34a target genes, KLF4 and SEMA3E. [score:5]
Given that canine OSA is a well validated spontaneous large animal mo del of the human disease, the purpose of this study was to characterize the impact of miR-34a expression in canine OSA tumor lines to better understand its potential role in the biology of this disease. [score:5]
To determine whether expression levels of putative miR-34a target genes are increased in spontaneous canine OSA tumors, we performed RT-qPCR to evaluate gene transcript expression (KLF4, SEMA3E, and VEGFA) in primary canine OSA tumors and normal canine osteoblast cells. [score:5]
Down-regulated genes (fold change ≤ -1.4) that contained consensus binding sites for miR-34a in their 3’UTR were identified and gene ontology enrichment analysis was performed using the ToppGene database (https://toppgene. [score:4]
With respect to the potential role of miR-34a dysregulation in osteosarcoma (OSA), several studies have demonstrated decreased expression in human OSA. [score:4]
While 2/8 samples showed increased miR-34a expression in metastatic tissues compared to primary tumor biopsies, 4/8 (50%) had decreased miR-34a expression; miR-34a levels were unchanged in 2 patients. [score:4]
KLF4 and VEGFA are direct targets of miR-34a. [score:4]
Our finding that miR-34a expression is decreased in canine OSA is consistent with previous studies demonstrating that miR-34a expression is reduced or absent in human OSA tumor samples compared to paired non-cancerous bone and suggests that loss of miR-34a is a common event in both species [30]. [score:4]
The miR-34 family, most notably miR-34a, is frequently lost or down-regulated in human malignancies including neuroblastoma, breast, lung, and colorectal carcinomas, and osteosarcoma. [score:4]
No effects of miR-34a expression on proliferation of the OSA8 or Abrams cell line were observed at multiple time points when compared to cells expressing empty vector (Fig 2B). [score:4]
Therefore, down-regulation of miR-34a may be associated with the metastatic phenotype in canine OSA. [score:4]
Among the miRNAs implicated in cancer development and progression, the miR-34 family has been intensively studied and data indicate family members function as tumor suppressors in a variety of human cancers [25, 26]. [score:4]
Indeed, a variety of miRNA formulations and target-specific delivery strategies have accelerated the clinical development of miR-34 mimics, Miravirsen (Santaris Pharma) and MRX34 (Mirna Therapeutics) which recently entered first-in-human phase I clinical trials (NCT01829971) in patients with advanced solid tumors [50– 52]. [score:4]
Studies have shown that ectopic expression of miR-34a through various methods such as chemically modified oligonucleotide miR-34a mimics and bioengineered miR-34a prodrugs results in decreased viability, migration and invasion of human OSA cell lines [43, 53, 54]. [score:3]
Gene ontology classification of predicted miR-34a target genes. [score:3]
Transcript levels of putative miR-34a target genes (B) KLF4, (C) SEM3AE, and (D) VEGFA were assessed in primary canine OSA tumors and normal canine osteoblasts using RT-qPCR. [score:3]
Our findings are concordant with data generated in human OSA tumors, suggesting that loss of miR-34a may be common in this disease. [score:3]
To understand the molecular mechanisms underlying the miR-34a associated decrease in cell motility and invasion in canine OSA cell lines, was performed on the OSA8 cells expressing either control or pre-miR-34a lentiviral constructs. [score:3]
RT-qPCR results showed significant down-regulation of miR-34a in canine OSA tumors (p < 0.01) and OSA cells lines (p < 0.05) as compared to normal canine osteoblasts. [score:3]
org) was used to analyze Gene Ontology (GO) classifications of predicted miR-34a target genes. [score:3]
Cells were expanded in culture for 5–7 days and miR-34a expression was determined by RT-qPCR (Applied Biosystems). [score:3]
Linear mixed effects mo dels were applied to OSA8 and Abrams cell line miR-34a expression, proliferation and cell cycling data to take account of the correlation among observations from the same replicates. [score:3]
Although levels of miR-34a expression varied among OSA tumor samples, this may be attributed, in part, to heterogeneity in tumor stroma and/or inflammatory cells present in the tumor microenvironment. [score:3]
Although variability in miR-34a expression levels was observed among primary OSA tumor samples, this may be attributed, in part, to heterogeneity in tumor stroma and/or non-neoplastic cell infiltrates present in the tumor microenvironment. [score:3]
0190086.g002 Fig 2(A) Canine OSA8 and Abrams cells were transduced with pre-miR-34a lentiviral constructs or empty control vector and sorted to greater than 95% purity based on GFP expression 72 hours following infection. [score:3]
MiR-34a directly targets KLF4 and VEGFA in canine OSA cells. [score:3]
Furthermore, gene ontology analysis of these miR-34a target genes demonstrated associations with biological processes related to cell migration. [score:3]
To begin to dissect the underlying mechanisms mediating the observed miR-34a -dependent effects in canine OSA cells, was performed on canine OSA8 cells expressing control or miR-34a lentiviral constructs. [score:3]
These findings are consistent with published data demonstrating that miR-34a expression levels are significantly decreased in human OSA tumor tissues and OSA cell lines [30, 42]. [score:3]
In agreement with our findings in the OSA8 cell line, transcript levels for KLF4, SEMA3E, and VEGFA were substantially decreased following enforced miR-34a expression in the Abrams cell line. [score:3]
No statistically significant difference in cell proliferation or cell cycle distribution was detected in OSA8 or Abrams cells expressing empty vector or pre-miR-34a vector for any of the tested time points (Student’s t-test). [score:3]
Genetically engineered pre-microRNA-34a prodrug suppresses orthotopic osteosarcoma xenograft tumor growth via the induction of apoptosis and cell cycle arrest. [score:3]
Taken together, these findings demonstrate that miR-34a inhibits the invasive capacity and migratory behavior of canine OSA cell lines. [score:3]
We identified putative miR-34a target genes that were common to all three GO categories related to cell migration including Krüppel-like factor 4 (KLF4), Semaphorin 3E (SEMA3E), and Vascular endothelial growth factor A (VEGFA). [score:3]
Concordant with this finding, miR-34a expression did not alter cell cycle status in either the OSA8 or Abrams cells as assessed by propidium iodide (PI) staining (Fig 2C). [score:3]
Gene transcripts altered by miR-34a overexpression in canine OSA8 cells. [score:3]
For example, recent studies demonstrated that miR-34a contributes to cell cycle arrest and apoptosis through its repression of several p53 target genes, including CDK4, CDK6, and Bcl-2 [32, 40, 41]. [score:3]
To investigate the functional consequences of miR-34a expression on OSA cell behavior, we stably expressed miR-34a in the OSA8 and Abrams cells lines. [score:3]
0190086.g003 Fig 3(A) Canine OSA8 and Abrams cells expressing control or pre-miR-34a lentiviral constructs were plated in serum free media in the upper wells of plates fors. [score:3]
Evaluation of putative miR-34a target expression in primary canine OSA tumors. [score:3]
In contrast, this was almost completely abrogated upon transfection of reporter plasmids containing mutant 3’UTRs (pmirGLO-KLF4-3’UTR-MUT or pmirGLO-VEGFA-3’UTR-MUT), indicating that miR-34a negatively regulates KLF4 and VEGFA in OSA8 cells via direct binding to the 3’-UTR of these transcripts. [score:3]
Ectopic expression of miR-34a in canine OSA cells does not affect cell proliferation or cell cycle distribution. [score:3]
Taken together, these findings support the assertion that loss of miR-34a may promote a pattern of gene expression contributing to the metastatic phenotype in canine OSA. [score:3]
identifies miR-34a -induced gene alterations in canine OSA8 cellsTo understand the molecular mechanisms underlying the miR-34a associated decrease in cell motility and invasion in canine OSA cell lines, was performed on the OSA8 cells expressing either control or pre-miR-34a lentiviral constructs. [score:3]
MiRWalk, miRDB) were used to identify predicted miR-34a target genes containing putative miR-34a binding sites within their 3’-UTR. [score:3]
Total RNA was isolated and RT-qPCR was performed as described above immediately prior to plating cells to confirm transduction efficiency miR-34a levels in wild-type (WT), empty vector (EV), and miR-34a expressing cells (* p < 0.05). [score:3]
Furthermore, restoration of miR-34a in human OSA cell lines reduced cell proliferation and migration in vitro and attenuated OSA tumor xenograft growth and metastasis in vivo, in part through targeting of the receptor tyrosine kinase Met [42]. [score:3]
Ectopic expression of miR-34a does not influence cell proliferation or cell cycle distribution in canine OSA cell lines. [score:3]
These data demonstrate that miR-34a contributes to invasion and migration in canine OSA cells and suggest that loss of miR-34a may promote a pattern of gene expression contributing to the metastatic phenotype in canine OSA. [score:3]
Prior studies indicated that miR-34a expression alters the invasive capacity of tumor cells [42]. [score:3]
In our prior work we evaluated miRNA expression signatures in canine OSA and found that canine OSA tumors similarly express low levels of miR-34a. [score:3]
The results of the dual-luciferase assay showed that miR-34a significantly suppressed the luciferase reporter activity of pmirGLO-KLF4-3’UTR-WT and pmirGLO-VEGFA-3’UTR-WT (Fig 6B and 6C). [score:2]
0190086.g007 Fig 7(A) RT-qPCR evaluating mature miR-34a expression in primary canine OSA tumors (N = 9) and normal canine osteoblasts demonstrated that the mean expression of miR-34a was significantly reduced in OSA tumors compared to osteoblasts (Bars: SD. [score:2]
These data provide a further association between miR-34a and regulation of cellular pathways that promote a metastatic phenotype. [score:2]
Concordant with these data, RT-qPCR confirmed that miR-34a expression is substantially decreased in canine OSA tumors as compared to normal canine osteoblasts (Fig 1). [score:2]
MiR-34a expression decreases cell invasion and migration in canine osteosarcoma cell lines. [score:2]
Total RNA was reverse-transcribed and human Taqman miRNA assays were used to detect miR-34a expression. [score:2]
These cross-species data support the notion that miR-34a plays a role in regulating the invasive capacity of OSA cells. [score:2]
Furthermore, p53 -mediated transcriptional regulation of the miR-34 family is conserved across different cell types [33– 37]. [score:2]
Human Taqman miRNA assays (Applied Biosystems) were used according to manufacturer’s instructions to quantify mature miR-34a expression in canine cell lines and tissues (mature miR-34a shares 100% sequence homology between dogs and humans) [46]. [score:2]
We analyzed miR-34a expression levels in primary canine OSA tumors and paired metastases and found that half of metastatic lesions had reduced levels of miR-34a compared with their primary tumor. [score:2]
Furthermore, in a panel of canine OSA cell lines, miR-34a expression was found to be significantly decreased in all of the malignant OSA cell lines compared to that observed in normal canine osteoblasts. [score:2]
However, given the broad capacity for miRNAs to regulate multiple genes and it is likely that miR-34a affects a range of genes that participate in metastasis [14]. [score:2]
Comparison between primary and metastatic tissues with a linear mixed mo dels showed a 1.5-fold (0.9–2.3, 95% confidence interval) decreased expression of miR-34a in metastatic tissues compared to primary OSA tissues (p-value = 0.12). [score:2]
MiR-34a expression is decreased in primary canine OSA tumors and canine OSA cell lines. [score:2]
After confirming overexpression of miR-34a lentiviral constructs, cells were plated immediately for functional assays (cell proliferation, cell cycle analysis, invasion/migration). [score:2]
Our data demonstrate that miR-34a expression is significantly decreased in primary canine OSA tumor tissues and canine OSA cell lines compared to normal canine osteoblasts. [score:2]
RT-qPCR demonstrated that miR-34a expression levels were significantly reduced in primary canine OSA tumors and canine OSA cell lines as compared to normal canine osteoblasts. [score:2]
The impact of miR-34a overexpression on the proliferative capacity of OSA cells was assessed using a bromodeoxyuridine (BrdU) incorporation assay. [score:2]
MiR-34 genes exhibited minimal deletions, loss of heterozygosity (LOH), and epigenetic inactivation in human OSA tumor tissues, demonstrating that other genetic and epigenetic mechanisms may account for the observed decrease expression [30]. [score:2]
Our data demonstrate that expression of miR-34a is significantly decreased in primary canine OSA tumor tissues and cell lines compared to normal canine osteoblasts. [score:2]
MiR-34a expression is reduced in primary canine OSA tumors and OSA cell lines. [score:2]
In contrast, normal canine osteoblasts possessing high basal levels of miR-34a expressed significantly lower levels of KLF4 and SEMA3E compared to OSA tumor samples. [score:2]
RNA sequencing identifies miR-34a -induced gene alterations in canine OSA8 cells. [score:1]
Total RNA was extracted from OSA8 cells transduced with either empty lentivirus (n = 3) or pre-miR-34a lentivirus (n = 3) using the RNeasy Mini Kit (QIAGEN) and was performed at the OSU Comprehensive Cancer Center Genomics Shared Resource as previously described [46]. [score:1]
The purpose of this study was to investigate the potential contribution of miR-34a loss to the biology of canine OSA, a well-established spontaneous mo del of the human disease, and evaluate the functional consequences of altered miR-34a expression in canine OSA cell lines. [score:1]
Canine OSA8 (5 x 10 [4]) or Abrams cells (1 × 10 [5]) transduced with empty vector or pre-miR-34a lentivirus were prepared in serum-free medium and seeded into each insert (upper chamber) and media containing 10% fetal bovine serum was placed in the lower chamber. [score:1]
Deletions of the gene regions harboring these transcripts or CpG promoter methylation with miR-34 gene silencing are frequently observed in human malignancies including neuroblastoma, glioma, breast cancer, non-small cell lung cancer, colorectal cancer, and osteosarcoma [27– 32]. [score:1]
MiR-34a levels were unchanged in two of the paired samples (two already had reduced miR-34a levels in the primary tumor compared to normal osteoblasts), whereas two metastatic tissues exhibited higher miR-34a expression as compared to the primary OSA tumor biopsy. [score:1]
miR-34a is essential for p19(Arf) -driven cell cycle arrest. [score:1]
The propidium iodide staining method was used to assess the effects of miR-34a on cell cycle distribution [49]. [score:1]
Interestingly, enforced expression of miR-34a had no effect on proliferation or cell cycle distribution in the cell lines evaluated. [score:1]
In brief, OSA8 or Abrams cells (5.0 x 10 [6] cells/well) transduced with pre-miR-34a or empty vector control lentivirus were seeded in 6-well plates in 3 mL of RPMI-1640 (OSA8) or DMEM (Abrams) with 10% fetal bovine serum and incubated overnight at 37°C and 5% CO2. [score:1]
Briefly, 2 x 10 [3] Abrams or OSA8 cells transduced with either pre-miR-34a or negative control empty virus were plated in complete medium for 24, 48, or 72 hours in 96-well plates. [score:1]
miR-34a lentivirus infection. [score:1]
The following day, the medium was changed to Stemline (Gibco) with transfection agent TransDux (Systems Biosciences) and either pre-miR-34a or control empty virus was added to cells overnight according to manufacturer’s protocol. [score:1]
MiR-34a expression is decreased in paired canine OSA metastatic lesions compared to primary OSA tumors. [score:1]
The purpose of this study was to investigate the potential contribution of miR-34a loss to the biology of canine OSA, a well-established spontaneous mo del of the human disease. [score:1]
Cells were co -transfected at 80% confluency with 10 pmol control or pre-miR-34a mimic (Systems Biosciences) and 100 ng of WT or MUT KLF4 or VEGFA pmirGLO constructs using Lipofectamine (Invitrogen). [score:1]
0190086.g001 Fig 1(A) RT-qPCR was used to assess mature miR-34a levels in primary canine OSA tumors (N = 20) and canine OSA cell lines (N = 8) relative to normal canine osteoblasts. [score:1]
Effects of miR-34 on OSA cell line migration and invasiveness appear to be at least partially mediated through repression of CD44, the receptor for hyaluronic acid and a well-established marker of cancer cell stemness [43]. [score:1]
The mature miR-34a sequence is located within the second exon of its non-coding host gene whereas miR-34b and miR-34c are co-transcribed and located within a single non-coding precursor (miR-34b/c) [25]. [score:1]
The miR-34 family consists of three evolutionarily conserved miRNAs: MiR-34a, MiR-34b and MiR-34c. [score:1]
miR-34: from bench to bedside. [score:1]
CD511B-1) or pre-miR-34a lentivirus (catalog no. [score:1]
Given the highly metastatic behavior of OSA and data demonstrating that miR-34a influences OSA metastasis in vivo [42], miR-34a expression levels were evaluated in paired primary OSA tumors and metastatic lung lesions from eight canine patients. [score:1]
The limited number of paired tumor samples evaluated in the current study precludes us from drawing conclusions about the expression of miR-34a in OSA metastases. [score:1]
0190086.g006 Fig 6(A) Predicted miR-34a binding sites in the 3’UTR of canine KLF4 and VEGFA. [score:1]
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[+] score: 512
Other miRNAs from this paper: mmu-mir-34c, mmu-mir-34b, mmu-mir-34a, hsa-mir-34b, hsa-mir-34c
Ectopic expression of miR-34a in head and neck cell lines significantly inhibited tumor cell proliferation, migration and colony formation by downregulating the expression of E2F3 and survivin. [score:10]
Since ectopic expression of miR34a inhibits E2F3 expression, we further examined if survivin expression is regulated by miR-34a via E2F3. [score:10]
Overexpression of E2F3a completely rescued survivin expression in miR-34a expressing cells, thereby suggesting that miR-34a may be regulating survivin expression via E2F3a. [score:10]
In addition, ectopic expression of miR-34a in endothelial cells significantly inhibited the expression of the target proteins E2F3a/b, SIRT1, survivin and CDK4 (Figure 7E). [score:9]
Another important observation we made in this study is that ectopic expression of miR-34a in head and neck cancer cells also significantly downregulated survivin expression. [score:8]
In addition, ectopic expression of miR-34a significantly inhibited cell migration even after taking into account the effect of miR-34a on cell proliferation at the same time point (60% cell migration inhibition as compared to 20% cell proliferation inhibition at 24 hrs). [score:8]
Furthermore, our results demonstrate that miR-34a can also regulate tumor angiogenesis by directly inhibiting angiogenic functions of endothelial cells by downregulating a number of key proteins including E2F3, SIRT1, survivin and CDK4. [score:8]
In this study, we looked at changes in protein expression of some of the known targets of mir-34a (e. g. SIRT1, CDK4, E2F3) and found them to be altered upon ectopic miR-34a expression (Figure S1). [score:7]
Interestingly, E2F3a overexpression in UM-SCC-74A cells significantly rescued tumor cells from miR-34a -mediated inhibition of cell proliferation (56%, Figure 4C) and colony formation (86%, Figure 4D), whereas E2F3b was only partially effective in reversing miR-34a -mediated inhibition of cell proliferation (19%, Figure 4C) and colony formation (26%, Figure 4D). [score:7]
Representative photographs of SCID mice bearing tumors at day 18 and dissected tumors are shown in Figure 5C-F. Ectopic expression of miR-34a expression in both the tumor cell lines significantly inhibited tumor growth in SCID mice (Figure 5). [score:7]
miR-34a Expression is Significantly Downregulated in Primary Tumor Samples and Tumor Cell Lines from the Head and Neck Cancer Patients. [score:6]
miR-34a Inhibits Tumor Angiogenesis by Downregulating VEGF Secretion from Tumor Cells. [score:6]
miR-34a is a tumor-suppressor that is frequently downregulated in a number of tumor types. [score:6]
Interestingly, miR-34a inhibited tumor angiogenesis by blocking VEGF production by tumor cells as well as directly inhibiting endothelial cell functions. [score:6]
Recently, HPV infection was shown to down-regulate miR-34a levels by destabilizing tumor suppressor p53 protein in cervical cancer [47], [48]. [score:6]
miR-34a inhibits tumor angiogenesis by downregulating VEGF secretion from tumor cells. [score:6]
Interestingly, miR-34a inhibited tumor angiogenesis by blocking VEGF secretion by tumor cells as well as directly inhibiting endothelial cell functions. [score:6]
Expression of miR-34a is downregulated in HNSCC cell lines and patient’s tumor samples. [score:6]
In our previous study, we have shown Bcl-2 expression is significantly elevated in tumor -associated endothelial cells (EC-Bcl-2) [39], [40] and our results from this study suggest that miR-34a expression is significantly decreased in Bcl-2 expressing endothelial cells as compared to endothelial cells containing empty vector alone (EC-VC) (Figure 7A). [score:6]
In our real-time PCR analysis of the head and neck cell lines, miR-34a expression was markedly downregulated in all the HNSCC cell lines that we tested. [score:6]
VEGF is a key angiogenic protein and recent studies have shown that a number of miR-34a target proteins including E2F3, Myc and c-met can regulate VEGF expression [36], [37], [38]. [score:6]
Our results demonstrate that miR-34a expression is significantly downregulated in primary tumors from head and neck cancer patients as well as in head and neck cancer cell lines. [score:6]
Similar to HNSCC cell lines, miR-34a expression was also significantly downregulated in tumor samples from head and neck cancer patients. [score:6]
miR-34a was originally discovered as a potential tumor suppressor that is downregulated and induces apoptosis in neuroblastoma cells [20]. [score:6]
Figure S1 miR-34a significantly downregulates SIRT1, CDK4, E2F3a/b and survivin protein expression. [score:6]
As both E2F3 isoforms were downregulated in miR-34a transfected cells, we performed isoform-specific rescue experiments to understand the specific contribution of E2F3a and E2F3b in miR-34a mediated tumor suppressor function. [score:6]
miR-34a Inhibits Tumor and Colony Formation by Downregulating E2F3 and Survivin. [score:6]
In addition, miR-34a also significantly inhibited tumor angiogenesis by downregulating a key angiogenic factor VEGF. [score:6]
miR-34a overexpression also markedly downregulated E2F3 and survivin levels. [score:6]
Indeed, miR-34a expression was markedly downregulated in all the head and neck cancer cell lines that were examined (Figure 1B). [score:6]
Ectopic expression of miR-34a inhibits tumor cell proliferation and colony formation. [score:5]
0037601.g007 Figure 7 A: miR-34a expression in endothelial cells expressing Bcl-2 (EC-Bcl-2) or vector alone (EC-VC) was analyzed by real time RT-PCR. [score:5]
miR-34a Inhibits Tumor Growth, in vivo To confirm the anti-tumor effects on miR-34a in vivo, we implanted two head and neck cell lines either expressing miR-34a or scrambled control in the right or left flanks of SCID mice, respectively. [score:5]
Our results demonstrate that ectopic expression of miR-34a strongly suppresses multiple tumorigenic functions (e. g. proliferation, and colony formation) of head and neck cancer cells. [score:5]
We next examined if head and neck tumor cell lines also exhibit similar downregulated expression of miR-34a as compared to normal keratinocyes. [score:5]
These tumor suppressive effects of miR-34a are mediated by changes in a number of target mRNAs including MYCN, CDK4, cyclin D1, SIRT1 and Bcl-2 [18], [51], [52], [53]. [score:5]
We next examined if overexpression of miR resistant E2F3a or E2F3b could rescue survivin expression in miR-34a treated cells. [score:5]
Ectopic expression of miR-34a also significantly inhibited tumor growth and tumor angiogenesis in a SCID mouse xenograft mo del. [score:5]
A: miR-34a expression in endothelial cells expressing Bcl-2 (EC-Bcl-2) or vector alone (EC-VC) was analyzed by real time RT-PCR. [score:5]
In this study we show that miR-34a mediates its tumor suppressor effects predominantly through E2F3a isoform, as overexpression of miR resistant E2F3a significantly rescued the effects of miR-34a on cell proliferation and colony formation. [score:5]
Overexpression of E2F3a was able to completely rescue survivin expression in miR-34a transfected UM-SCC-74A cells, whereas E2F3b was only partially effective (Figure 4E). [score:5]
Ectopic expression of miR-34a in HNSCC cell lines significantly inhibited tumor cell proliferation, colony formation and migration. [score:5]
Our rescue experiments with miR resistant E2F3a suggest that miR-34a may be inhibiting survivin expression via E2F3a [35]. [score:5]
Expression of both E2F3a and E2F3b proteins was markedly downregulated in miR-34a transfected cells as compared to cells transfected with scrambled control RNA (Figure 4A). [score:5]
*, represent a significant inhibition (p<0.05) of cell proliferation in miR-34a expressing tumor cells at 48 hrs as compared to SC. [score:4]
We next sought to identify the target mRNAs of miR-34a that regulate head and neck tumor cell function. [score:4]
To confirm that miR-34a regulates the endogenous expression of E2F3a and E2F3b, we ectopically introduced miR-34a in UM-SCC-74A cells and examined E2F3a/b levels in these cells 72 hrs post transfection. [score:4]
*, represent a significant inhibition (p<0.05) of tumor cell colony formation in miR-34a expressing tumor cells as compared to SC. [score:4]
miR-34a mediates its biological function by downregulating E2F3 and survivin levels. [score:4]
UM-SCC-74A tumors expressing miR-34a showed a significant decrease in blood vessel density (83%) as compared to tumors expressing scrambled control RNA (Figure 6A-B). [score:4]
*, represent a significant inhibition (p<0.05) of cell migration in miR-34a expressing tumor cells as compared to SC. [score:4]
Taken together, these findings suggest that dysregulation of miR-34a expression is common in HNSCC and modulation of miR34a activity might represent a novel therapeutic strategy for the treatment of HNSCC. [score:4]
Similarly, Scapoli et al, have shown in their recent study that miR-34a levels are significantly downregulated in head and neck squamous cell carcinoma [44]. [score:4]
*, represent a significant inhibition (p<0.05) of cell proliferation in miR-34a expressing tumor cells as compared to SC. [score:4]
It has been shown that VEGF levels can be regulated by a number of miR-34a target proteins including E2F3, Myc and c-met [36], [37], [38]. [score:4]
The comparatively higher miR-34a levels in these cell lines could most likely be due to the presence of wild-type p53 in these cells as miR-34a has been shown to be a direct transcriptional target of p53 [23], [24]. [score:4]
In addition, survivin promoter activity is also regulated by E2F3, a key miR-34a target protein [35]. [score:4]
Our results demonstrate that miR-34a is significantly downregulated in HNSCC tumors and cell lines. [score:4]
To further validate the cell line findings of inverse relationship between miR-34a and its target proteins survivin and E2F3, we examined miR-34a, survivin and E2F3 mRNA levels in the primary head and neck tumor samples. [score:3]
miR-34a was ectopically expressed in tumor cells and VEGF (VEGF A) levels in the culture supernatants was quantified by. [score:3]
miR-34a inhibits endothelial cell proliferation, migration and tube formation. [score:3]
F: miR-34a, survivin and E2F3 mRNA expression from primary tumors of 15 head and neck cancer patients was analyzed by real time RT-PCR. [score:3]
0037601.g002 Figure 2 A: Tumor cells (UM-SCC-74A) were transfected with miR-34a or scrambled control microRNAs (SC) and miR-34a expression was analyzed by quantitative real time PCR (RT-PCR). [score:3]
A: Representative photographs of UM-SCC-74A tumors expressing scrambled control microRNAs (SC) or miR-34a. [score:3]
A: Tumor cells (UM-SCC-74A) were transfected with miR-34a or scrambled control microRNAs (SC) and miR-34a expression was analyzed by quantitative real time PCR (RT-PCR). [score:3]
miR-34a Inhibits Head and Neck Tumor, Colony Formation and Migration. [score:3]
In addition, miR-34a significantly inhibited tumor growth and tumor angiogenesis in a SCID mouse xenograft mo del. [score:3]
However, it is not known if miR-34a -mediated regulation of survivin promoter activity is due to its direct effect or mediated via another protein. [score:3]
We next examined if miR-34a inhibits tumor angiogenesis by blocking VEGF production by the tumor cells. [score:3]
In order to confirm that ectopic expression of miR-34a stayed throughout the in vivo experiments, we isolated RNA from UM-SCC-74A tumor samples at the end of study and performed real time RT-PCR. [score:3]
Therefore, we next examined if ectopic expression of miR-34a affected tumor angiogenesis. [score:3]
0037601.g001 Figure 1 A: miR-34a expression was examined in primary tumor samples (n = 15) or in adjacent normal mucosa (n = 15) of head and neck cancer patients by quantitative real time PCR (RT-PCR) and statistical significance was analyzed by Mann-Whitney test (p value 0.022). [score:3]
Whereas only 20% cell proliferation inhibition (Figure 2) was observed at the same time point (24 hrs), thereby suggesting that miR-34a predominantly affected cell migration at 24 hrs. [score:3]
Indeed, ectopic expression of miR-34a was maintained in the tumor cells throughout the study (Figure 5G). [score:3]
Ectopic expression of miR-34a in transfected cells (UM-SCC-74A) was confirmed by RT-PCR (Figure 2A). [score:3]
These results provide a novel mechanistic role for the miR-34a-E2F3a-survivin axis in mediating miR-34a tumor suppressor function. [score:3]
miR-34a Inhibits Tumor Growth, in vivo. [score:3]
We also observed an inverse correlation between miR-34a and survivin expression (low miR-34a and high survivin) in most of the tumor samples from HNSCC patients. [score:3]
Ectopic Expression of miR-34a in HNSCC Cells and Endothelial Cells. [score:3]
Similar ectopic expression of miR-34a was observed in UM-SCC-74B cells (data not shown). [score:3]
miR-34a Inhibits Endothelial, Migration and Tube Formation. [score:3]
However, very little is known about the role and expression status of miR-34a in head and neck cancers. [score:3]
Recent studies have highlighted the role of miR-34a as a tumor suppressor in a number of tumor types including prostate cancer, hepatocellular carcinoma, neuroblastoma and colon cancer [17], [18], [19], [20], [21], [22]. [score:3]
D: Representative photographs of UM-SCC-74B tumors expressing scrambled control microRNAs (SC) or miR-34a. [score:3]
We therefore selected two HNSCC cell lines (UM-SCC-74A and UM-SCC-74B) that are HPV negative and contain functional wild-type p53 for the in vitro and in vivo experiments, although they were not among the cell lines with the lowest miR-34a expression. [score:3]
C: UM-SCC-74A cells overexpressing E2F3a, E2F3b or vector alone (VC) were ectopically transfected with miR-34a or scrambled control (SC). [score:3]
To confirm the anti-tumor effects on miR-34a in vivo, we implanted two head and neck cell lines either expressing miR-34a or scrambled control in the right or left flanks of SCID mice, respectively. [score:3]
Figure S2 miR-34a significantly inhibits tumor cell proliferation. [score:3]
The miR-34a expression in patient samples and head and neck cancer cell lines (Figure 1) was analyzed by Mann-Whitney test. [score:3]
Ectopic expression of miR-34a significantly inhibited cell proliferation in UM-SCC-74A cells (MTT assay; Figure 2B & Figure S2 and Xcelligence assay; Figure 2C) and UM-SCC-74B cells (Figure 2D). [score:3]
miR-34a inhibits tumor growth in vivo. [score:3]
Rescue experiments using microRNA resistant E2F3 isoforms suggest that miR-34a -mediated inhibition of cell proliferation and colony formation is predominantly mediated by E2F3a isoform. [score:3]
miR-34a expression in tumor samples, HNSCC cell lines and endothelial cells was examined by real time PCR. [score:3]
Taken together, our results demonstrate an important tumor suppressor and anti-angiogenic function for miR-34a in head and neck cancers. [score:3]
Transfection of miR-34a in endothelial cells significantly inhibited cell proliferation (85%) and migration (84%) (Figure 7B-C). [score:3]
miR-34a significantly inhibits tumor cell migration. [score:3]
Similarly, miR-34a significantly inhibited the ability of endothelial cells to form tubular structure on Matrigel (Figure 7Da-b). [score:3]
E: UM-SCC-74A cells overexpressing E2F3a, E2F3b or vector alone (VC) were ectopically transfected with miR-34a or SC. [score:3]
A: miR-34a expression was examined in primary tumor samples (n = 15) or in adjacent normal mucosa (n = 15) of head and neck cancer patients by quantitative real time PCR (RT-PCR) and statistical significance was analyzed by Mann-Whitney test (p value 0.022). [score:3]
org) search revealed several growth regulatory mRNAs that contains conserved miR-34a recognition sites in their 3′-UTR. [score:2]
The strong tumor suppressor effect of miR-34a on HNSCC cell lines observed during in vitro assays was further supported by its effects on in vivo xenograft tumor growth. [score:2]
This remarkable decrease in miR-34a in head and neck tumors suggested to us that its deregulation in HNSCC may be playing a role in the progression of head and neck cancer. [score:2]
We next examined if miR-34a could directly affect angiogenic function of endothelial cells in vitro. [score:2]
*, represent a significant inhibition (p<0.05) of tumor growth in miR-34a group cells as compared to SC. [score:2]
B: miR-34a expression was analyzed in 9 head and neck cancer cell lines by RT-PCR and compared with 3 normal human keratinocyes [human oral keratinocytes (HOK); human epidermal keratinocytes, adult (HEKa) and human epidermal keratinocytes, neonatal (HEKn)]. [score:2]
Our results show that miR-34a expression is significantly decreased (Mann-Whitney test; p value 0.0226) in head and neck tumors as compared to adjacent normal tissue (Figure 1A). [score:2]
*, represent a significant inhibition (p<0.05) of tumor angiogenesis in miR-34a group as compared to SC. [score:2]
Similarly, miR-34a levels were inversely correlated with E2F3 levels (Pearson r value 0.81; p value 0.0002). [score:1]
G: miR-34a levels in UM-SCC-74A tumors at the end of the in vivo experiments. [score:1]
We did not observe any significant differences in miR-34a levels in tumor samples from the different head and neck sub-sites. [score:1]
Lipofectamine-2000 was used to transfect miR-34a in HNSCC cell lines and human endothelial cells. [score:1]
In addition, tumor samples from HNSCC patients showed an inverse relationship between miR-34a and survivin as well as miR-34a and E2F3 levels. [score:1]
To examine the inverse relationship between miR-34a and survivin expression in patient samples (Figure 4F), miR-34a levels for each tumor sample was plotted against the inverse of survivin levels and Pearson correlation coefficient was calculated. [score:1]
At the end of incubation, miR-34a and lipofectamine solution was carefully added to 6-well plates containing cells. [score:1]
E: Microvessel density in tumor samples from scrambled control microRNAs (SC) or miR-34a groups. [score:1]
0037601.g004 Figure 4 A: UM-SCC-74A cells were transfected with miR-34a or scrambled control (SC). [score:1]
C: VEGF levels (pg/ml per 10 [6] tumor cells) in culture supernatants from UM-SCC-74A cells transfected with miR-34a or scrambled control microRNA (SC). [score:1]
Recently, miR-34a was shown to decrease survivin promoter activity [34]. [score:1]
UM-SCC-74A cells were transfected with miR-34a or SC. [score:1]
miR-34a effect on tumor growth and tumor angiogenesis was examined by in vivo SCID mouse xenograft mo del. [score:1]
14/15 patient samples showed low levels of miR-34a and high levels of survivin mRNA. [score:1]
One sample (Patient # 10) showed high miR-34a and low survivin levels (Figure 4F). [score:1]
Recently, miR-34a was shown to modulate survivin promoter activity [34]. [score:1]
F: VEGF levels (pg/ml per 10 [6] tumor cells) in culture supernatants from UM-SCC-74B cells transfected with miR-34a or scrambled control microRNA (SC). [score:1]
Untreated UM-SCC-74A cells produced very high levels of VEGF (1247 pg/ml/10 [6]cells) and miR-34a transduction significantly reduced VEGF production (56%) (Figure 6C). [score:1]
Next, we examined the role of miR-34a on head and neck tumor cell proliferation, colony formation and migration. [score:1]
50 nM of pre-miR-34a or scrambled control RNA was used to transfect head and neck cancer cell lines. [score:1]
Similarly, miR-34a transfection in UM-SCC-74B cells significantly reduced VEGF production (45%) (Figure 6F). [score:1]
In another study, Wald et al, also did not observe any significant difference in miR-34a levels in HPV -positive verses HPV -negative head and neck cancer cell lines [49]. [score:1]
In separate tubes, miR-34a (50 nM) and lipofectamine 2000 were diluted in OPTI-MEM medium and incubated for 5 minutes. [score:1]
In our in vivo tumor growth study, in addition to smaller tumor size, we also observed that tumors were visually less vascular in miR-34a transduced group. [score:1]
We decided to conduct an in-depth analysis of the effect of miR-34a and E2F3 in HNSCC. [score:1]
Indeed, miR-34a transfection in UM-SCC-74A cells significant decreased survivin protein levels (Figure 4E). [score:1]
A piece of each of the primary tumor was used to extract RNA to confirm the presence of miR-34a by real-time PCR. [score:1]
Tumor cells transfected with miR-34a or SC were mixed with 100 µl of Matrigel and injected subcutaneously in the left and right flanks of SCID mice respectively (n = 5). [score:1]
Precursor human miR-34a or scrambled control miRNA (Applied Biosystems) transfection in HNSCC tumor cells and EC was performed using Lipofectamine 2000 (Invitrogen) as per manufacturer’s instructions. [score:1]
All 15 tumor samples showed an inverse relationship between miR-34a levels and survivin (Pearson r value 0.89; p value 0.0001). [score:1]
E-H: Effect of miR-34a on tumor cell colony formation (E-F; UM-SCC-74A and G-H; UM-SCC-74B) was examined by culturing tumor cells (5,000) in 60 mm Petri dishes for 14 days. [score:1]
Percentage cell migration for cells ectopically expressing miR-34 was calculated by adjusting migration index for tumor cells transduced with SC to 100%. [score:1]
We measured miR-34a expression levels in 30 frozen samples from head and neck cancer patients (15 tumors and 15 adjacent normal controls) by TaqMan real time RT-PCR. [score:1]
After incubation, miR-34a and lipofectamine were mixed together and further incubated for 30 minutes. [score:1]
B: Microvessel density in tumor samples from scrambled control microRNAs (SC) or miR-34a groups. [score:1]
However, little is known about the role of miR-34a in head and neck squamous cell carcinoma (HNSCC). [score:1]
In this study, we examined if loss of miR-34a in head and neck cancers promotes tumor growth and tumor angiogenesis. [score:1]
Partial rescue of miR-34a function by E2F3b in our studies could be due to some functional overlap often observed in E2F3a and E2F3b [28], [30]. [score:1]
We examined miR-34a levels in 3 different normal keratinocyte cell types [human oral keratinocytes (HOK); human epidermal keratinocytes, adult (HEKa) and human epidermal keratinocytes, neonatal (HEKn)] and 9 head and neck cancer cell lines. [score:1]
Functionally miR-34a was found to affect tumor cell proliferation, apoptosis, senescence, invasion, metastasis and drug resistance [21], [22], [23], [25], [26], [27]. [score:1]
0037601.g005 Figure 5Tumor cells transfected with miR-34a or SC were mixed with 100 µl of Matrigel and injected subcutaneously in the left and right flanks of SCID mice respectively (n = 5). [score:1]
Green and red circles are used to highlights tumor cells transfected with scrambled control (SC) or miR-34a, respectively. [score:1]
A: UM-SCC-74A cells were transfected with miR-34a or scrambled control (SC). [score:1]
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Kumamoto K Nutlin-3a activates p53 to both down-regulate inhibitor of growth 2 and up-regulate mir-34a, mir-34b, and mir-34c expression, and induce senescenceCancer Res. [score:11]
In support, using complementary gain-of-function and loss-of-function approaches, we demonstrated that miR-34a inhibits downstream cell survival signaling by directly targeting Ang1/Tie2 in vitro and in vivo, an effect that inhibits apoptosis, neutrophil accumulation, and vascular injury in vivo. [score:8]
Furthermore, miR-34a inhibitor increased the expression of the downstream targets of miR-34a (Supplementary Fig.   3K). [score:7]
These findings coupled with the improved survival observed in miR-34a “knockout” studies in neonatal BPD mice (Figs.   5, 6, 8) suggest that therapies directed at inhibiting miR-34a expression may ameliorate BPD. [score:7]
To identify the molecular targets of miR-34a, we examined the predicted miR-34a targets using bioinformatics tools, focusing our attention on the regulators of lung inflammation and injury. [score:6]
In addition, miR-34a -inhibitor treatment increased miR-34a targets Ang1 and Tie2 expression in hyperoxia-exposed MLE12 cells as compared to scrambled control (Fig.   4c). [score:6]
While this observation that miR-34a modulated Ang1/Tie2 signaling in lung epithelial cells suggested an association, it was important to assess whether miR-34a can directly target Ang1/Tie2 according to the in silico target-prediction analysis. [score:6]
Next we transfected MLE12 cells with different concentrations of miR-34a mimic and noted that at 50 nm concentration the expression of Ang1 and Tie2 proteins were markedly decreased (Fig.   3a), as well as other downstream targets of miR-34a (Supplementary Fig.   3J). [score:5]
Furthermore, cleaved caspase3 expression was also decreased in miR-34a -inhibitor treated group (Fig.   4c, d). [score:5]
As in the neonatal lungs, the expression of miR-34a downstream targets were also decreased in MLE12 cells (Supplementary Fig.   3F, G). [score:5]
Administration of miR-34a inhibitor in BPD mice also reduced the TUNEL -positive score (Fig.   8d) and reduced cleaved-caspase3 expression (Fig.   8e). [score:5]
Shan W Activation of the SIRT1/p66shc antiapoptosis pathway via carnosic acid -induced inhibition of miR-34a protects rats against nonalcoholic fatty liver diseaseCell Death Dis. [score:5]
Pharmacologic miR-34a inhibition has clinical translational potential as a viable therapeutic option in the treatment of neonatal patients to prevent/ameliorate BPD. [score:5]
Using three available prediction algorithms (Targetscan, miRANDA, and Pictar), we then produced a comprehensive list of all possible miR-34a targets. [score:5]
To address whether miR-34 expression was required and sufficient for the hyperoxia -induced lung injury and inflammation leading to the BPD pulmonary phenotype, we next asked whether only miR-34a overexpression itself was sufficient, in the absence of hyperoxia i. e., in RA. [score:5]
Mechanistically, we were able to show that miR-34a mimic in RA was able to decrease the expression of the downstream targets (Ang1, Tie2, SCF, c-kit, Notch2, and Sirt1) in MLE12 cells as well as in vivo (Fig.   7d, e). [score:5]
Interestingly, Trp53 siRNA increased the expression of miR-34a downstream targets Ang1 and Tie2 in MLE12 cells (Supplementary Fig.   3H). [score:5]
First, hyperoxia induces miR-34a expression in lung T2AECs of newborn mice and human infants with the clinically relevant diagnoses of RDS, evolving and established BPD suggesting translational significance. [score:5]
Recent reports demonstrate that inhibition of the miR-34 family does not promote tumorigenesis, supporting the potential for therapeutic suppression of this family as a treatment for BPD [56]. [score:5]
As shown in Fig.   1c, in T2AECs, hyperoxia (95% O [2]) gradually induced the expression of mature miR-34a at 4 h. We did not find any significant changes in miR-34a expression in hyperoxia-exposed lung endothelial cells or macrophages (Supplementary Fig.   1C, D). [score:5]
Downstream targets of the miR34a signaling pathway include Ang1 and its receptor Tie2, and the anti-apoptotic protein Bcl2; decreased expression of both are known to increase cell death in hyperoxia -induced lung injury mo dels and BPD. [score:5]
To determine whether the effects of miR-34a are limited to the hyperoxia -induced BPD mo del or could be dependent on other injury mediators, we tested the expression of miR-34a in lungs of our TGF-β1 doxycycline-inducible overexpressing transgenic mouse mo del. [score:5]
Overexpression of miR-34a inhibited the activity of a luciferase reporter construct containing Ang1 and Tie2 3′ UTRs (Fig.   3b, c). [score:5]
Finally, in comparison to controls, miR-34a inhibitor treated mice had significantly increased Ang1 and Tie2 as well as Sirt1 and Bcl2 protein expression in hyperoxia-exposed lungs (Fig.   8k). [score:5]
Silencing of miR-34a ameliorates the apoptotic response in vitro and in vivo, leading to suppressed epithelial apoptosis; this, in turn, is associated with restoration of alveolarization, enhanced angiogenesis and improvement in pulmonary vascular development. [score:4]
In addition, miR-34a expression is also regulated by Trp53 in both our in vitro and in vivo hyperoxia-exposed/BPD mo dels. [score:4]
miR-34a downregulates Ang1-Tie2 signaling in developing lungs. [score:4]
Given that the miR-34 family has been implicated in the p53 tumor suppressor network, and that p53 pathway defects are common features of human cancer [25], miR-34 inhibition therapy is considered a promising therapeutic approach [26]. [score:4]
Taken together, our data suggest that miR-34a inhibitor treatment improves the alveolar and vascular development in the hyperoxia-exposed BPD mouse mo del, at least in part, via the Ang1/Tie signaling pathway. [score:4]
In addition, miR34a, by suppressing the Ang1/Tie2 signaling pathway and enhancing cell death, results in dysregulated vascularization in the lung. [score:4]
To identify the downstream mechanism of miR-34a-regulated protection, we used mRNA databases to identify targets and revealed many apoptosis/inflammation associated genes. [score:4]
Sirt1, Notch2, CDKs, and Bcl2 are predicted targets of miR-34a and these have previously been described as important factors in lung development and injury 63– 66. [score:4]
In vivo, Ang1/Tie2 was significantly upregulated by miR-34a antagonism, and this signaling was able to ameliorate the neonatal BPD phenotype. [score:4]
Hyperoxia upregulates miR-34a in T2AECs in developing lungs. [score:4]
Furthermore, downregulated miR-34a had the opposite effect suggesting that miR-34a can significantly affect cellular biological function. [score:4]
These effects are associated with differential regulation of downstream targets of miR-34a, impacting on inflammatory and angiogenic pathways. [score:4]
These data are shown in Supplementary Fig.   2D, E, where miR34a expression is significantly increased in RA and BPD, compared to WT controls, in p53 absence/inhibition. [score:4]
miR-34a expression was significantly increased with hyperoxia exposure and reached their maximum levels at PN7 (almost 10-fold); even the BPD mo del showed a significant increase in miR-34a expression as compared to RA control (Fig.   1a; Supplementary Fig.   1B). [score:4]
Collectively, our findings support miR-34a as a novel therapeutic target in regulating hyperoxia -induced acute lung injury (HALI) and BPD. [score:4]
We obtained lung tissues and noted that miR34a expression was decreased with hypoxia and decreased TGFβ signaling (using inducible dominant -negative mutation of the TGF-beta type II receptor (DNTGFbetaRII) mice) or a combination of the two exposures (Supplementary Fig.   9C). [score:4]
Interestingly, reconstitution of rAng1 in miR-34a overexpressing epithelial cells underlined their critical importance in miR-34a -mediated effects on cell survival regulation. [score:4]
Importantly, it should be pointed out that miR-34a deletion/inhibition enhanced pulmonary vascular development and indices of PAH in hyperoxia-exposed neonatal mice. [score:4]
Fig. 9Improved effects of miR-34a inhibition occurs via Ang1-Tie2 signaling. [score:3]
d The wild-type Ang1 3′ UTR reporter vector was co -transfected into the MLE12 cells with either the scrambled or miR-34a inhibitor. [score:3]
h, i Bar graphs showing lung IL-1β and IL-6 in RA and BPD mice treated with miR-34a inhibitor or scrambled control. [score:3]
We honed onto Ang1 and its receptor, Tie2 (Tek) as potential targets of miR-34a, as they have conserved miR-34a seed sequence in its 3′ UTR (Supplementary Fig.   3A). [score:3]
Since Akt and Erk pathways are important for epithelial cell survival and growth in response to hyperoxia [34] and Ang1/Tie2 signaling activates Akt and Erk by phosphorylating them, we examined the effect of miR-34a inhibitor in vitro. [score:3]
Of note, miR-34 family members also have been recognized as tumor suppressor miRNAs. [score:3]
While previous studies have reported the expression of miR-34a in neonatal and adult lung injury 11, 51, none, to the best of our knowledge, has comprehensively mechanistically defined the role of miR-34a in HALI and BPD in developing lungs. [score:3]
d Representative graph shows TUNEL -positive cells (%) in NB WT mice lungs treated with miR-34a inhibitor or control. [score:3]
miR-34a inhibitor treatment improves hyperoxia -induced BPD. [score:3]
Phosphorylation of Akt and Erk was increased in miR-34a inhibitor transfected cells treated with recombinant Ang1 (Fig.   9e). [score:3]
Such an approach can be done by utilizing surfactant (which is used routinely in preterm neonates with RDS) as a delivery vehicle for a miR-34a inhibitor. [score:3]
Deletion/inhibition of miR-34a globally and locally in type 2 alveolar epithelial cells (T2AECs) limits cell death and inflammation with injury and improves the pulmonary and pulmonary arterial hypertension (PAH) phenotypes in BPD mouse mo dels. [score:3]
Chang TC Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosisMol. [score:3]
Similarly miR-34a inhibitor transfection increased 3′ UTR activity of Ang1 only (Fig.   3D). [score:3]
In contrast, hyperoxia-exposure to neonatal T2AECs led to decreased Ang1/Tie2 protein levels (Fig.   2g, h) as well as other downstream targets of miR-34a, Sirt1, and Notch2 (Supplementary Fig.   3I). [score:3]
To address the role of miR-34a being specifically expressed in T2AECs in mediating alveolarization, we used SPC-CreER/miR-34a [fl/fl] mouse line to disrupt miR34a specifically in T2AECs. [score:3]
In addition, there was significantly increased Ang1, Tie2, SCF, and Notch2 expression in the miR34a (−/−) BPD mice lungs (Fig.   5i). [score:3]
g Morphometric analysis of lung histology sections of NB WT and miR-34a KO expressed as chord length and analyzed using Image J software. [score:3]
To utilize an in vitro mo del, we used MLE12 cells, and noted that the expression of miR-34a was highest with 95% O [2] exposure at 24 h (Fig.   1d) and with 60% O [2] exposure at 48 h (Fig.   1e). [score:3]
In an effort to localize the specific lung compartment, we checked miR-34a expression in freshly isolated neonatal lung T2AECs, endothelial and macrophage cells. [score:3]
Fig. 10Lungs of infants with RDS and BPD have increased miR-34a expression. [score:3]
To examine lung regenerative capacity in PN4 mouse lungs, these were stained with PCNA, which revealed increased cell proliferation in the lungs treated with miR-34a inhibitor (Fig.   8j). [score:3]
Fig. 1Expression of miR-34a in hyperoxia exposed NB lungs and type 2 cells. [score:3]
miR-34 overexpression in RA restores the BPD phenotype. [score:3]
b, c Morphometric analyses of lung histology sections of NB WT mice miR-34a inhibitor at PN14. [score:3]
In response to hyperoxia, pri-miR-34a was rapidly induced, with the highest expression reaching 15-fold at PN2, after which it began to decline (Fig.   1b), most likely due to the processing of pri-miR-34a into the pre-forms and mature forms. [score:3]
a miR-34a expression in cell pellets obtained from tracheal aspirates of neonates in the first PN week, who subsequently did or did not develop BPD. [score:3]
Bernardo BC Therapeutic inhibition of the miR-34 family attenuates pathological cardiac remo deling and improves heart functionProc. [score:3]
Conversely, overexpression of miR-34a in room air (RA) worsened the BPD pulmonary and PAH phenotypes, while the addition of miR-34a in the miR-34a deletion mice mo del exposed to hyperoxia led to reiteration of the BPD pulmonary phenotype. [score:3]
Third, we experimentally validate the mechanistic angiogenic, inflammatory, cell death and cell proliferation pathways of miR-34a, focusing on the role of vascular downstream targets, Ang1 and Tie2, and show that Ang1 treatment is protective of the BPD pulmonary and associated PAH phenotypes. [score:3]
Importantly, lung delivery of miR-34a inhibitor repressed activation of the cell death pathway and reduced the lung BPD phenotype in hyperoxia-exposed mice. [score:3]
The class III histone deacetylator, Sirt1 is also a downstream target of miR-34a, and a decrease in Sirt1 has been associated with enhanced transcription of pro-inflammatory mediators and BPD. [score:3]
Next, we studied the kinetics of miR-34a expression in hyperoxia-exposed lungs at PN2, PN4, PN7, and PN14 (using lung samples from the mouse mo del of BPD). [score:3]
Similarly, in situ hybridization showed higher expression of miR-34a in epithelial linings of lungs of neonates with RDS especially with RDS 3-7 and RDS >7 days of PN age, mostly localized to T2AECs (Fig.   10b). [score:3]
a MLE12 cells were transfected with different concentrations of miR-34a mimic in RA and western blots for Ang1 and Tie2 expression were performed. [score:3]
k shows increased Ang1 and Tie2 in miR-34a inhibitor treated PN4 lung samples. [score:3]
Taken together, our data show that T2AEC-specific deletion of miR-34a is sufficient to rescue the BPD phenotype in hyperoxia; conversely, increased expression miR-34a in RA is sufficient to re-create the BPD pulmonary phenotype. [score:3]
e analysis of cleaved and total Caspase 3 was performed on MLE12 cells transfected with miR-34a inhibitor or scrambled control. [score:3]
Furthermore, we used antenatal LPS administration (mimicking chorioamnionitis) with/without additional PN hyperoxia exposure in a neonatal rat mo del, and noted increased expression of miR34a in the lungs, but only when PN hyperoxia exposure was present (Supplemental Fig.   9D). [score:3]
Furthermore, we also found miR-34a targets Ang1 and Sirt1 were decreased in TGF-β1 TG mouse lung samples (Supplementary Fig.   9B). [score:3]
f, g Bar graphs showing BAL neutrophils count and myeloperoxidase activity in RA and BPD mice treated with miR-34a inhibitor or scrambled control. [score:3]
Most importantly, we demonstrate the feasibility and efficacy of in vivo miR-34a inhibition as a protective therapeutic option to ameliorate BPD and associated PAH. [score:3]
Moreover, miR-34a overexpression in room air alone was sufficient to produce the BPD phenotype in neonatal mice (Fig.   7a, b). [score:3]
Tamoxifen -induced Cre recombinase activity markedly decreased miR-34a expression in PN4 lung (Fig.   6a). [score:3]
Second, using genetic gain-of-function and loss-of-function strategies (including deletion of miR-34a specifically in T2AECs), we comprehensively prove a causal detrimental role of increased miR-34a; conversely, inhibition of miR-34a was protective of the BPD pulmonary and associated PAH phenotypes. [score:3]
miR-34a inhibition improved PAH in the mouse BPD mo del. [score:3]
a Representative images of lung histology (H&E stain) of NB WT mice mo dels of RA or BPD were treated with miR-34a inhibitor (20 µM; PN2 and PN4) intranasal. [score:3]
a Schematic of tamoxifen -induced deletion of miR-34a in Spc CRE -expressing miR-34a [fl/fl] mice. [score:3]
Zhang F Roles of microRNA-34a targeting SIRT1 in mesenchymal stem cellsStem Cell Res. [score:3]
We administered 5 µl (20 µM concentration) of miR-34a inhibitor (or scrambled control) at PN2 and PN4 intranasally, during hyperoxia exposure. [score:3]
e Activation of Erk and Akt signalling by rAng1 in miR-34a inhibitor or scrambled control transfected MLE12 cells as analyzed by western blot of phosho-Erk1/2, total Erk1/2 protein, phospho-Akt and total Akt protein, shown at the indicated time points. [score:3]
We also show that administration of recombinant Ang1, one of the downstream targets of miR-34a, ameliorates the BPD pulmonary and PAH phenotypes. [score:3]
a Representative graphs showing miR-34 expression in WT NB mice exposed to hyperoxia for 2, 4, and 7 days after birth and in the BPD mo del. [score:3]
Human BPD infant lungs have increased miR-34a expression. [score:3]
These data led us to hypothesize that Ang1/Tie2 may be functional downstream targets of miR-34a in the inflammatory/apoptotic response to hyperoxia in lung epithelial cells. [score:3]
Additional downstream targets of miR-34a (Notch2, Sirt1, c-kit, p-ckit, and SCF) were also decreased upon hyperoxia exposure in PN4 neonatal lungs (Supplementary Fig.   3C−E). [score:3]
To answer this question, we co -transfected miR-34a mimic/inhibitor with Ang1/Tie2 3′ UTRs in MLE12 cells. [score:3]
To determine whether the increased T2AEC expression of miR-34a seen in the above studies is causally related to impairment of lung BPD phenotype, we used mice in which miR-34a deletion was conditionally induced exclusively in T2AECs. [score:3]
Given that genetic deletion of miR-34a was associated with complete protection from hyperoxia -induced changes in lung morphometry and inflammation, we next sought to block miR-34a as a therapeutic strategy in NB WT mice exposed to hyperoxia, using a miR-34a inhibitor via the intranasal route. [score:3]
Moreover, we identify the underlying molecular mechanisms by analyzing specific inflammatory/vascular/survival -associated targets of miR-34a. [score:3]
miR-34a inhibitor treated animals demonstrated attenuated right ventricular hypertrophy (RVH), as indicated by right ventricle (RV)/left ventricle (LV) ratio and Fulton’s Index (Supplementary Fig.   6E, F). [score:3]
In addition, hyperoxia decreases cell proliferation via CDK4 and cyclin D1, both targets of miR34a. [score:3]
Lung histology showed that, in comparison to the scrambled group, intranasal treatment with miRNA-34a inhibitor in neonatal mice significantly improved the BPD pulmonary phenotype, specifically in terms of chord length and septal thickness (Fig.   8a–c). [score:3]
We transfected these cells with miR-34a inhibitor, miR-34a mimic and scrambled controls and exposed to 48 h hyperoxia. [score:3]
Next, we transfected Trp53 siRNA in MLE12 cells and neonatal PN4 lungs, but only noted a modest (non-significant) decrease in miR-34a expression (Supplementary Fig.   2B, C). [score:3]
miR-34a regulation in other mo dels BPD/lung injury. [score:2]
These data suggest that the mechanism of PAH in BPD mice is, to some degree, regulated by the miR-34a/Ang1/Tie2 axis. [score:2]
b Primary miR-34a expression is shown in hyperoxia exposed and BPD murine lung tissue as compared to controls. [score:2]
miR-34a regulates epithelial-mesenchymal transition in BPD. [score:2]
b Representative graph showing significantly deccreased Annexin V and PI staining positive cells in miR-34a inhibitor transfected group compared to its control in hyperoxia exposed MLE12 cells. [score:2]
Mohan M Kumar V Lackner AA Alvarez X Dysregulated miR-34a-SIRT1-acetyl p65 axis is a potential mediator of immune activation in the colon during chronic simian immunodeficiency virus infection of rhesus macaquesJ. [score:2]
The expression of miR-34a was significantly higher in TA cell pellets from individuals who went on to develop BPD and/or died, compared to controls (Fig.   10a). [score:2]
This hyperoxia -induced cell death response was significantly increased in the presence of miR-34a mimic (mostly Annexin V+Propidium iodide positive) and decreased with miR-34a inhibitor (mostly Annexin V positive) transfection as compared to scrambled controls (Supplementary Fig.   4, Fig.   4a, b). [score:2]
In contrast, we noted decreased expression of Ang2 in miR34a (−/−) mice lungs upon hyperoxia exposure at PN4 as well as being significantly decreased in BPD mice lungs at PN14, compared to respective controls (Fig.   5j, k). [score:2]
s and quantification showing decreased expression of Ang2 in miR-34a KO lungs compared to WT, upon exposure to hyperoxia (n = 2). [score:2]
Since several publications have shown that miR-34a expression is regulated by Trp53 25, 26, we evaluated and noted that Trp53 was acetylated upon hyperoxia exposure to MLE12 cells (Supplementary Fig.   2A). [score:2]
Collectively, these data demonstrate that miR-34a is a critical component of the neonatal mouse response to hyperoxia and regulates inflammation and alveolarization in HALI and BPD. [score:2]
Our data thus reveal a critical role of miR-34a and the downstream Ang1/Tie2 signaling and the transition between the pro-inflammatory and anti-inflammatory phenotypes, which is believed to be important for the molecular regulation of functional shaping of T2AECs apoptosis and proliferation and the related BPD phenotype. [score:2]
In conclusion, we show that miR-34a contributes to neonatal murine BPD by influencing T2AECs apoptosis through regulation of anti-apoptotic Ang1/Tie2 signaling. [score:2]
As previously reported [30], we observed decreased vascular growth in BPD animals compared to RA mice lungs, which was improved in miR-34a inhibitor treated animals, confirmed by quantification (Supplementary Fig.   6C, D). [score:2]
Hence, increased miR-34a results in increased inflammation, impaired alveolarization and dysregulated vascularization in the developing lung—the hallmarks of “new” BPD. [score:2]
s showing increased expression of Tie2, Ang1, SCF, and Notch2 in miR-34a KO lungs as compared to WT. [score:2]
Recent direct evidence suggests that miR-34a is correlated with potential inflamed states, including the staphylococcal enterotoxin B -induced acute inflammatory lung injury [51], hepatic ischemia/reperfusion injury [52], high-fat diet induced hepatic steatosis [53], cardiac aging, and myocardial infarction 54– 56 and acute kidney injury [57]. [score:2]
Overall, our results would suggest that there is a potential role for miR-34a regulating EMT in the BPD mo del, but further experiments (beyond the scope of this manuscript) would be required to be definitive. [score:2]
To firmly establish the mechanistic role of miR-34a, given the impact on Ang1 expression, we evaluated the effect of Ang1 administration in the regulation of lung epithelial cell survival pathway and the BPD pulmonary phenotype. [score:2]
We noted increased expression of miR-34a at PN10 in the neonatal mouse lungs as compared to transgene negative animals (Supplementary Fig.   9A). [score:2]
Liu XJ MicroRNA-34a suppresses autophagy in tubular epithelial cells in acute kidney injuryAm. [score:2]
ks and quantification of the same showing significantly decreased expression of Ang2 in miR-34a KO lungs as compared to WT, in the BPD mo del at PN14 (n = 3). [score:2]
c image showing increased Tie2 and Ang1 and decreased cleaved caspase 3 in miR-34a inhibitor transfected group compared to its control in hyperoxia exposed MLE12 cells. [score:2]
i NB WT and miR-34a KO mice were exposed to hyperoxia from PN day 1−4. [score:1]
Given the potential role of miRs in the pathogenesis of BPD, in this study, we reveal that lung miR-34a levels are significantly increased in neonatal mice lungs exposed to hyperoxia. [score:1]
f A proposed schema for the role of miR-34a in the pathogenesis of BPD. [score:1]
Fig. 6Inducible deletion of miR-34a from type 2 epithelial cells improves BPD. [score:1]
In addition, in the PN7 HALI mo del, Ang1 treatment showed improved Ki67 staining levels similar to that of the miR-34 (−/−) mice lungs (Supplementary Fig.   7). [score:1]
Fig. 5Deletion of miR-34a results in improvement of BPD. [score:1]
Hence, miR-34a deletion in T2AECs is sufficient to protect the newborn lung to develop the BPD pulmonary phenotype, upon hyperoxia-exposure. [score:1]
a Representative images of lung histology (H&E stain) of NB WT mice mo dels of RA or BPD were treated with miR-34a mimic (20 µM; PN2 and PN4) intranasal. [score:1]
miR-34a deletion was induced by maternal tamoxifen administration (2 mg) from PN1−PN5 in the NB pups, via maternal milk. [score:1]
h Bar graph showing the percentage of TUNEL -positive cells indicating the apoptosis quantification in WT and miR-34a KO BPD mo dels. [score:1]
In addition, miR34a−/− mice [71] and conditional miR-34 [fl/fl] [72] (JAX laboratory) and SPC-CreER (gift from Brigid Hogan, PhD, Duke University, USA) were housed in the Yale and Drexel Universities Animal Care Facilities (New Haven, CT and Phila delphia, PA, respectively). [score:1]
b The wild-type Ang1 3′ UTR reporter vector was co -transfected into the MLE12 cells with either the N. C. mimic or miR-34a mimic c The WT Tie2 3′ UTR reporter vector was co -transfected into the MLE12 cells with either the N. C. mimic or miR-34-a mimic. [score:1]
c Morphometric analyses of lung histology sections of NB WT mice RA or BPD treated with miR-34a mimic and control. [score:1]
Takano M Nekomoto C Kawami M Yumoto R Mantell LL Role of miR-34a in TGF-beta1- and drug -induced epithelial-mesenchymal transition in alveolar type II epithelial cellsJ. [score:1]
To determine the contribution of miR-34a to HALI, we examined WT and miR-34a (−/−) mice exposed to hyperoxia and noted that miR-34a (−/−) NB mice in hyperoxia had better survival than WT mice (Fig.   5a). [score:1]
c Bar graph showing the morphometric analysis of lung histology sections of NB miR-34a KO mice exposed to RA or 100% O [2] at PN7. [score:1]
Importantly, the PAH indices worsened upon exposure to miR34a -mimic in the miR34a -mimic treated miR-34a (− /− ) hyperoxia-exposed mice (Supplementary Fig.   6E, F). [score:1]
We also evaluated miR34a expression in p53 null mutant and Trp53 siRNA treated mice in room air and our BPD mo del at PN14. [score:1]
Choi YJ miR-34 miRNAs provide a barrier for somatic cell reprogrammingNat. [score:1]
b Next, we used ISH to detect miR-34a in human neonatal lungs. [score:1]
Representative bar graph showing tamoxifen deletion of miR-34a in Spc CRE positive miR-34 KO lungs (T2-miR34a [−/−]). [score:1]
Similar protective responses were noted in the miR-34a (−/−) mice (Supplementary Fig.   6G, H). [score:1]
Additionally, T2AECs miR-34a deletion decreased TUNEL -positive cells (Fig.   6c) and lung inflammation as demonstrated by a decrease in lung neutrophils in the BALF and a significant decrease in tissue MPO activity in T2-miR34a [−/−] lungs (Fig.   6d, e). [score:1]
These data would suggest that hyperoxia and/or ventilation -induced injuries to the developing lung are accompanied by alterations in the miR-34a-Ang1/Tie2 signaling axis in human neonates. [score:1]
A proposed schema for the role of miR-34a in the pathogenesis of BPD is shown in Fig.   10f. [score:1]
Intriguingly, miR-34a is also increased in RDS, evolving and established BPD patients, indicating its potential role in human neonatal lung injury. [score:1]
f Representative H&E stained images of alveolar regions from lungs from WT and miR-34a KO mice from RA and BPD groups. [score:1]
To evaluate the human disease relevance of these findings, we examined whether miR-34a is increased in the TA and/or lungs of babies with RDS/BPD. [score:1]
Kim HJ Carbon monoxide protects against hepatic ischemia/reperfusion injury by modulating the miR-34a/SIRT1 pathwayBiochim. [score:1]
c Freshly isolated type 2 epithelial cells were used for measuring miR-34a expression in room air (RA) and after 4 h and 16 h HYP (95% O [2]). [score:1]
to the developing lung leads to production and release of the primary (Pri-miR-34a), which is processed into the mature form of miR-34a. [score:1]
We next addressed the question whether hyperoxia could increase transcription of miR-34a. [score:1]
Therefore, miR-34a may be an indicator of inflammation/injury, especially since its role in cell death and cell cycle is well established 58– 60. [score:1]
b Representative images of lung histology (H&E stain) of NB miR-34a KO mice exposed to RA or 100% O [2] at PN7. [score:1]
We crossed miR-34a- floxed mice (miR-34a [fl/fl]) with SPC-Cre-ER mice. [score:1]
Consistent with above studies, we also observed that when miR-34a was augmented in neonatal lung, cell proliferation, and angiogenesis levels were notably attenuated, and apoptosis was significantly increased. [score:1]
Deletion of T2AEC-specific miR-34a reverses the BPD phenotype. [score:1]
b Representative graph shows chord length analysis of lung histology (H&E stain) of NB T2-miR34a − /−mice BPD mo del along with controls. [score:1]
Finally, using three independent cohorts of human samples, we show the significant association of increased miR-34a and localization to T2AECs in neonates with respiratory distress syndrome (RDS) and BPD. [score:1]
In addition, provision of miR-34a to the miR34a (−/−) BPD mo del re-creates the BPD pulmonary phenotype. [score:1]
a NB WT (n = 8) and miR-34a KO (n = 11) mice were exposed to hyperoxia from PN day 1–15 and were monitored for survival. [score:1]
We thus propose Ang1/Tie2 signaling as the major factor in miR-34a -mediated BPD. [score:1]
Other studies have indicated that miR-34a attenuates cell proliferation, invasion and EMT 35, 61, 62. [score:1]
Briefly, lung sections were subjected to deparaffinization, incubation with 0.5% pepsin solution (20 min at 37 °C in humidified chamber), dehydration, and hybridization with either 40 nM Biotin LNA miR-34a probe, at 55 °C for 3 h. Subsequently sections were washed, blocked and incubated with streptavidin-AP reagent for 20 min and applied with alkaline phosphatase solution containing nitro-blue tetrazolium and 5-bromo-4-chloro-3=indolyphosphate (BCIP/NBT) for 1 h. Finally sections were dehydrated, mounted, and examined under microscope. [score:1]
Hyperoxia-exposed WT mice had a maximal increase in neutrophils and MPO activity, which were significantly decreased in the miR-34a (−/−) mice lungs (Fig.   5d, e). [score:1]
miR-34a global deletion renders mice resistant to HALI/BPD. [score:1]
Concepcion CP Intact p53 -dependent responses in miR-34 -deficient micePLoS Genet. [score:1]
We provide evidence of the in vivo relevance of miR-34a in hyperoxia -induced neonatal human and murine lung injury. [score:1]
The same trend was observed in inflammatory bronchoalveolar lavage fluid (BALF) interleukin (IL)-1β and IL-6 levels, both of which were increased in WT, but not as much in miR-34a (−/−) mice, with IL-6 levels being significantly decreased, at PN7, upon hyperoxia-exposure (Supplementary Figs.   5A, 5B). [score:1]
Li X Lian F Liu C Hu KQ Wang XD Isocaloric pair-fed high-carbohydrate diet induced more hepatic steatosis and inflammation than high-fat diet mediated by miR-34a/SIRT1 axis in miceSci. [score:1]
To test this, we intranasally administered miR-34a mimic in WT and miR-34a (−/−) mice, and confirmed the restoration of miR-34a levels (Supplementary Figs.   6A, 6B). [score:1]
d, e Hyperoxia increased the numbers of neutrophils and BAL myeloperoxidase (MPO) in neonatal mouse lungs, and the deletion of miR-34a attenuated the hyperoxia -induced increase in neutrophil numbers in the BPD mouse mo del. [score:1]
j NB WT and miR-34a KO mice were exposed to hyperoxia from PN day 1−4. [score:1]
d analysis of Ang1, Tie2, SCF, c-Kit, and Notch2 was performed on MLE12 cells transfected with miR-34a mimic or scrambled control eting of Tie2, Ang1, Notch2, and Sirt1 was performed on miR-34a mimic treated RA and BPD mice lungs. [score:1]
miR-34a was detected in lungs from WT mice breathing RA and increased markedly after exposure to 100% O [2] (Supplementary Fig.   1A). [score:1]
miR-34a increases apoptosis in lung epithelial cells. [score:1]
We confirmed that tamoxifen injection alone had effect on lung morphometry (Supplementary Fig.   5C) in WT, Cre, or miR-34a [fl/fl] mice. [score:1]
Taken together, our data would suggest that hyperoxia as well as increased TGFβ signaling is a major contributor to the increased levels of miR34a. [score:1]
Importantly, as was the case with alveolarization (Fig.   7a–c), there was decreased vascular density (equivalent to control and scrambled miR -treated BPD lungs) in the miR34a -mimic treated miR-34a (−/− ) mice hyperoxia-exposed BPD lungs (Supplementary Fig.   6C, D). [score:1]
Figures  7a, b show that administration of miR-34a mimic is sufficient to elicit the BPD phenotype in RA. [score:1]
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[+] score: 455
Other miRNAs from this paper: hsa-mir-34b, hsa-mir-34c
Western blot analysis (Figure 2 A ) showed that transfection of miR-34 mimics downregulated expression of target genes, Bcl-2, Notch1 and Notch2 at the protein level, but had no effect on Bcl-xL and Mcl-1 expression, indicating the target gene knock-down by miR-34 mimics affects transcripts harbouring miR-34 target sites. [score:15]
Interestingly, the Notch2 expression inhibition at the protein level by miR-34 was not accompanied by inhibition at the mRNA level, in agreement with previous reports that miRNA inhibits target gene expression post-transcriptionally, with or without mRNA degradation [11], [20]. [score:13]
Our results show that miR-34 restoration in human pancreatic cancer MiaPaCa2 and BxPC3 cells inhibited the expression of target genes, Bcl-2, Notch1 and Notch2; significantly inhibited clonogenic cell growth and invasion; induced apoptosis and G1 and G2/M arrest; and sensitized the cells to chemotherapy and radiation. [score:9]
Restoration of miR-34 down-regulates target genes' expression. [score:8]
This strategy was explored in the current study, where p53 downstream target miR-34 was restored in p53-mutant pancreatic cancer MiaPaCa2 cells with a high level of Bcl-2 and low levels of miR-34s, resulting in downregulation of Bcl-2 and Notch1-2, together with the inhibited clonogenic cell growth and invasion; increased apoptosis and G1 and G2/M arrest in cell cycle; and sensitization to chemotherapy and radiation. [score:8]
Our results are consistent with the notion that Bcl-2 is a direct target of miR-34 and miR-34 potently inhibits Bcl-2 expression. [score:8]
This multi-mode action of miR-34 provides a therapeutic advantage over the siRNA -based therapies in that miR-34 has multiple targets, can work on multiple cell signalling pathways at the same time, leading to synergistic effects which may translate into improved clinical efficacy for pancreatic cancer patients with p53 deficiency and advanced disease. [score:7]
Figure 2 B shows the qRT-PCR analysis of the potential target genes; miR-34 mimics potently inhibited BCL2 and Notch1 gene expression, consistent with the Western blot data. [score:7]
Our results demonstrate that miR-34 is involved in MiaPaCa2 cell growth; miR-34 restoration inhibits the clonogenic growth, and inhibition of endogenous miR-34 by miR-34 inhibitors promotes the growth. [score:7]
We have recently shown that expression of miR-34s is dramatically reduced in p53-mutant gastric cancer cells and that the restoration of miR-34 expression inhibited the cancer cell growth [6]. [score:7]
These data indicate that the CD44+/CD133+ cells were the target cells of miR-34, i. e., miR-34 exerts its tumor-suppressing activity via inhibiting the CD44+/CD133+ cells. [score:7]
However, our study is the first report showing that miRNA miR-34 inhibits pancreatic CD44+/CD133+ CSC, potentially via inhibiting downstream target “stem cell genes” such as Notch and Bcl-2. Interestingly, miR-34a and miR-34b are among the short-list of the stem cell-specific miRNAs discovered by Dr. [score:7]
A, miR-34 restoration down-regulates target proteins Bcl-2, Notch1 and Notch2, no effects on Mcl-1. MiaPaCa2 cells were transfected with miR-34 mimics or non-specific control miRNA mimic (NC mimic) (100 pmol per well in 6-well plates by Lipofectamine 2000) for 48 hours, then collected for Western blot analysis. [score:6]
0006816.g002 Figure 2 A, miR-34 restoration down-regulates target proteins Bcl-2, Notch1 and Notch2, no effects on Mcl-1. MiaPaCa2 cells were transfected with miR-34 mimics or non-specific control miRNA mimic (NC mimic) (100 pmol per well in 6-well plates by Lipofectamine 2000) for 48 hours, then collected for Western blot analysis. [score:6]
Restoration of miR-34 expression in the pancreatic cancer cells by either transfection of miR-34 mimics or infection with lentiviral miR-34-MIF downregulated Bcl-2 and Notch1/2. [score:6]
The report from He et al. indicated that ectopic expression of miR-34 induces cell cycle arrest in both primary and tumor-derived cell lines, which is consistent with the ability of miR-34 to downregulate a program of genes promoting cell cycle progression [11]. [score:6]
B, qRT-PCR analysis shows that the target gene Bcl-2 is downregulated in miR-34-MIF clone. [score:6]
Recently, the three members of the miR-34 family were found to be directly regulated by p53 and the functional activity of miR-34 indicated a potential role as a tumor suppressor [6], [7], [8], [9], [10], [11], [12]. [score:5]
MiaPaCa2 and BxPC3 cells have very low expression levels of both primary and mature miR-34a,b,c but high levels of the miR-34 target genes BCL2 and Notch1, and different levels of Notch2–4 (Figure 1 ). [score:5]
We have also shown that the CD44+/CD133+ tumorsphere-forming and tumor-initiating cells have high Bcl-2 and loss of miR-34 expression, indicating that miR-34 and its target Bcl-2 might be involved in cancer stem cells. [score:5]
qRT-PCR was performed to determine the expression levels of potential miR-34 target genes [6]. [score:5]
Restoration of miR-34 inhibits the clonogenic growth of MiaPaCa2 cells, whereas inhibition of miR-34 promotes cell growth. [score:5]
We also assessed in parallel the expression of presumptive miR-34-regulated target genes and proteins, using the primers and methods as we described recently [6]. [score:5]
The miR-34 -mediated reduction of the CD44+/CD133+ CSC is associated with the potent and simultaneous inhibition of its downstream target genes Notch and Bcl-2, genes involved in stem cells self-renewal and survival, so-called “stem cell genes” or “stemness genes” [6], [41], [42], [43]. [score:5]
C, qRT-PCR analysis of the expression levels of miR-34 target genes in human pancreatic cancer cell lines as well as normal human fibroblast WI-38 cells. [score:5]
More significantly, miR-34 restoration inhibits the CD44+/CD133+ tumor-initiating cells or CSC, accompanied by significant inhibition of tumorsphere growth in vitro and tumor formation in vivo. [score:5]
The transfected miR-34 mimics inhibited the luciferase reporter gene expression, which is controlled by Bcl-2 3′UTR in the promoter region (Figure 2 C ). [score:5]
Our above results have shown that miR-34 potently inhibits Bcl-2 expression and cell growth and increases cell death and response to chemo-/radiotherapy in the overall population of MiaPaCa-2 cells. [score:5]
Here again, miR-34 shows the advantage of its multi-target potential, as both stem cell genes Notch and Bcl-2 are inhibited by miR-34 at the same time, a potent synergy may be achieved in blocking both Notch signalling pathway and the anti-apoptotic function of Bcl-2 in tumor-initiating cells or cancer stem cells. [score:5]
We also examined the effect of inhibition of endogenous miR-34 on cell growth by mi RIDIAN miR-34 inhibitors. [score:5]
Next, we carried out qRT-PCR analysis of the sorted MiaPaCa2 cells to assess whether there is any difference in these populations as to the expression levels of miR-34 and its target genes. [score:5]
Interestingly, although miR-34a inhibited the Bcl-2 expression in the total population as well as in both the sorted CD44+/CD133+ cells and CD44−/CD133− cells, the effect of miR-34a on the CD44+/CD133+ population was most dramatic (miR-34a 1.65±1.15 vs. [score:5]
As shown in Figure 3 A–B, miR-34 restoration significantly inhibited the clonogenic cell growth, with miR-34a mimic inducing >80% inhibition of colony formation compared to NC mimic (18.3±3.8 colonies/well vs. [score:4]
Chang et al. reported that 15 pancreatic cancer cell lines have at least a 2-fold reduction in miR-34a expression as compared to expression in normal pancreatic ductal epithelial cell lines [8]. [score:4]
Our data support the view that miR-34 may be involved in pancreatic cancer stem cell self-renewal, potentially via the direct modulation of downstream targets Bcl-2 and Notch, implying that miR-34 may play an important role in pancreatic cancer stem cell self-renewal and/or cell fate determination. [score:4]
Our data support the view that miR-34 may be involved in pancreatic cancer stem cell self-renewal, potentially via the direct modulation of downstream targets Bcl-2 and Notch, implying that miR-34 may play an important role in pancreatic cancer stem cell self-renewal and/or cell fate determination, at least in the p53-mutant MiaPaCa2 mo del. [score:4]
Among the target proteins regulated by miR-34 are Notch pathway proteins and Bcl-2, suggesting the possibility of a role for miR-34 in the maintenance and survival of cancer stem cells. [score:4]
A, Western blot shows Bcl-2 protein is downregulated in miR-34a-MIF clone. [score:4]
Our data provide the first evidence that miR-34 is involved in pancreatic CSC self-renewal, potentially via the direct modulation of downstream targets Notch and Bcl-2. Our results provide novel insight into how miR-34 works in pancreatic cancer cells with p53 loss of function. [score:4]
The results demonstrate that the transfected miR-34s are functional and confirm that Bcl-2 is a direct target of miR-34, consistent with earlier reports [8], [10], [21]. [score:4]
In addition, because more than 50% of primary human cancers have mutations inactivating p53 function, the findings provided impetus to explore the functional restoration of miR-34 as a novel approach to inhibit cancers with p53 loss-of-function. [score:4]
CD44+/CD133+ cells are tumorsphere-forming and tumor-initiating cells with high Bcl-2 and loss of miR-34 expression. [score:3]
There is an inverse correlation in the expression levels of miR-34 and Bcl-2 in Q2 versus Q3, e. g., Q2 cells (with enriched cancer stem cells) have high Bcl-2 and low miR-34, Q3 cells (non-tumorigenic cells) have low Bcl-2 and high miR-34 levels. [score:3]
MiaPaCa2 and BxPC3 cells were infected with the FIV lentiviral system expressing miR-34a (miR-34a-MIF) or vector control (MIF), and the infected cells were selected via antibiotic resistance (Zeocin 50 µg/mL, Invitrogen), as we recently described [6]. [score:3]
miR-34 restoration inhibits the MiaPaCa2 tumor formation in nude mice. [score:3]
In conclusion, our study demonstrates that miR-34 may restore, at least in part, the tumor suppressing function of p53 in p53 -deficient human pancreatic cancer cells. [score:3]
Similar results were also observed in BxPC3 cells, where lentiviral miR-34a restoration significantly inhibited the clonogenic growth (Figure 7 A ) and tumorspheres (Figure 7 B ). [score:3]
As shown in Figure 8 A, miR-34a restoration significantly inhibited MiaPaCa2 tumor formation in vivo (miR-34a mimic 2/10 versus NC mimic 10/10, P<0.0001, n = 10). [score:3]
The miR-34 expression data were normalized to that of Actin and the relative levels are shown (set unsorted cells = 1). [score:3]
miR-34 restoration leads to a significant reduction of CD44+/CD133+ cells and inhibition of tumorsphere growth. [score:3]
The feline immunodeficiency virus lentiviral system expressing miR-34a (miR-34a-MIF), or vector control (MIF), was used to infect MiaPaCa2 and BxPC3 cells and the infected cell population was selected via Zeocin resistance [6]. [score:3]
Our results show that miR-34 restoration caused an 87% reduction of the CD44+/CD133+ tumorsphere-forming and tumor-initiating CSC in MiaPaCa2 cells with p53 loss of function, accompanied by a significant inhibition of tumorsphere growth in vitro and tumor formation in vivo. [score:3]
By modulating CSC, the restoration of tumor suppressor miR-34 may provide a novel therapeutic approach for p53 -deficient pancreatic cancer. [score:3]
We are currently carrying out more detailed mechanism studies to delineate the involvement of Notch signaling pathway in miR-34 -induced inhibition of pancreatic CSC and its role in chemo/radiosensitization of pancreatic cancer with p53 loss of function. [score:3]
miR-34a restoration resulted in an 87% reduction of the CD44+/CD133+ cells, accompanied by significant inhibition of tumorsphere growth in vitro as well as tumor formation in vivo. [score:3]
More significantly, we show that miR-34 restoration led to an 87% reduction of the CD44+/CD133+ CSC, accompanied by significant inhibition of tumorsphere growth in vitro as well as tumor formation in vivo. [score:3]
mi RIDIAN miRNA miR34a,b,c mimics and negative control miRNA mimic (NC mimic), mi RIDIAN miR-34 inhibitors and negative controls were obtained from Dharmacon (Chicago, IL) [6]. [score:3]
Our data are consistent with the reported tumor suppressor function of miR-34 [6], [8], [9], [11], [22]. [score:3]
miR-34 restoration inhibits the MiaPaCa2 tumor initiation in nude mice. [score:3]
We previously reported that the Bcl-2 protein is regulated directly by miR-34 [10]. [score:3]
Our data provide the first evidence that miR-34 is able to inhibit CD44+/CD133+ tumorsphere-forming and tumor-initiating cancer stem cells in p53-mutant pancreatic cancer, implying that miR-34 might play a role in the self-renewal of pancreatic cancer stem cells. [score:3]
The feline immunodeficiency virus (FIV) lentiviral system expressing miR-34a (miR-34a-MIF) or vector control (MIF), as well as a lentiviral packaging system, were purchased from System Biosciences (SBI, Mountain View, CA). [score:3]
miR-34 restoration inhibits MiaPaCa2 cell clonogenic growth and leads to caspase-3 activation and apoptosis. [score:3]
Twenty-four hr after miR-34 mimic transfection of MiaPaCa2 cells with miR-34 mimics (100 pmol per well in 6-well plates), the expression of potential target genes was measured by qRT-PCR with SYBR Green PCR System (TaqMan). [score:3]
Transcription of the three miRNA miR-34 family members was recently found to be directly regulated by p53. [score:3]
Our results demonstrate that miR-34 may restore, at least in part, the tumor suppressing function of the p53 in p53 -deficient human pancreatic cancer cells. [score:3]
Our study demonstrates that miR-34 may restore, at least in part, the tumor suppressing function of p53 in p53 -deficient cancer cells. [score:3]
Next, we examined whether miR-34 restoration could sensitize the pancreatic cancer cells with a high level of endogenous Bcl-2 expression to chemo- and radiotherapy. [score:3]
Figure S3Restoration of miR-34 by MIF lentiviral system inhibited MiaPaCa2 tumorspheres. [score:3]
More significantly, miR-34 restoration led to an 87% reduction of the tumor-initiating cell population, accompanied by significant inhibition of tumorsphere growth in vitro and tumor formation in vivo. [score:3]
This effect on cell cycle is similar to that of p53 restoration as we previously reported [23], [24], [27], [28], indicating that miR-34 restoration can exert effects akin to restoration of p53 tumor suppressor function, at least in part, in the cells with p53 loss of function. [score:3]
miR-34 significantly inhibited the invasion potential of MiaPaCa2 cells (Figure S2). [score:3]
Restoration of miR-34 by MIF lentiviral system decreases the CD44+/CD133+ MiaPaCa2 cells and inhibits tumorspheres from the sorted CD44+/CD133+ cells. [score:3]
At present, the linkages between p53, the downstream target miR-34 and presumptive pancreatic cancer stem cells are unknown. [score:3]
Another important finding from the current study is that our data provide a potential link between the tumor suppressor miR-34 and the tumor-initiating cells or cancer stem cells. [score:3]
This miR-34a -induced reduction of the CD44+/CD133+ cells was accompanied by reduced tumorsphere formation and smaller size of the tumorspheres (Figure 6 C ), and associated with a significant reduction of Bcl-2 expression in the CD44+/CD133+ tumorsphere-forming cells (Figure 6 D ). [score:3]
0006816.g003 Figure 3MiaPaCa2 cells were transfected with miR-34 mimics or inhibitors, 24 hr later the cells were seeded in 6-well plates (200 cells/well, in triplicates). [score:3]
Taken together, the published studies suggest miR-34 family members may have tumor suppressor function downstream of p53. [score:3]
More importantly, our results demonstrate for the first time that the CD44+/CD133+ tumor-initiating cells, or pancreatic cancer stem cells, have a low level of miR-34 accompanied by a high level of Bcl-2, suggesting a potential link of miR-34 and its target Bcl-2 to pancreatic cancer stem cells. [score:3]
Restoration of miR34a inhibits the clonogenic growth and tumorspheres of BxPC-3 cells. [score:3]
MiaPaCa2 cells were transfected with miR-34 mimics or inhibitors, 24 hr later the cells were seeded in 6-well plates (200 cells/well, in triplicates). [score:3]
miR-34 restoration significantly inhibited clonogenic cell growth and invasion, induced apoptosis and G1 and G2/M arrest in cell cycle, and sensitized the cells to chemotherapy and radiation. [score:3]
Western blot analysis revealed that Bcl-2 protein was down-regulated in miR-34a-MIF cells as compared with the MIF vector control cells (Figure S1 A), consistent with qRT-PCR analysis of the Bcl-2 mRNA level (Figure S1 B). [score:3]
MiaPaCa2 cells were infected with feline immunodeficiency virus (FIV) lentiviral system expressing miR-34a (miR-34a-MIF) or control (MIF), and selected for stable cells by Zeocin-resistance. [score:3]
They are single-stranded chemically enhanced oligonucleotides that can effectively inhibit the endogenous mature miR-34. [score:3]
0006816.g007 Figure 7BxPC-3 cells were infected with lentiviral miR-34a expression system (miR-34a) or vector control (MIF), and the infected cells were sorted for GFP positive cells by FACS. [score:3]
Figure S2 Restoration of miR-34 inhibits the invasion of MiaPaCa2 cells. [score:3]
e. m, n = 2. C, miR-34a restoration inhibits tumorspheres from the sorted CD44+/CD133+ cells. [score:3]
We examined a series of human pancreatic cancer cell lines, MiaPaCa2, BxPC3, Capan1, Capan2, Panc-1, and the normal human lung fibroblast cell line WI-38, for miR-34a,b,c expression. [score:3]
Restoration of miR-34 may hold significant promise as a novel molecular therapy for human pancreatic cancer with loss of p53–miR34, potentially via inhibiting pancreatic cancer stem cells. [score:3]
It has been reported that miR-34 targets Notch, c-Met and Bcl-2, genes involved in the self-renewal and survival of cancer stem cells [10], [11], [14]. [score:3]
B, Quantitative real-time PCR analysis of the potential target genes' mRNA levels after miR-34 mimic transfection in MiaPaCa2 cells. [score:3]
Since p53 tumor suppressor function is mediated in part via induction of apoptosis [23], [24], we examined the effect of miR-34 restoration on apoptosis-induction in MiaPaCa2 cells transfected with miR-34 mimics. [score:3]
miR-34 restoration could thus re-build, at least in part, the p53 tumor suppressing signalling network in pancreatic cancer cells lacking functional p53. [score:3]
As shown in Figure 5 D, the CD44+/CD133+ (Q2) cells have a high level of Bcl-2 expression but loss of miR-34a/b/c as compared with CD44−/CD133− (Q3) cells or the unsorted (total) cells. [score:2]
Delineating the role of miR-34 in regulation of cell growth and tumor progression, and its potential link to tumor-initiating cells or cancer stem cells may provide a basis for exploring its potential as a novel treatment strategy. [score:2]
miR-34 inhibitors induced an almost 20% increase in clonogenic growth as compared with control (120.3±2.9 colonies/well vs. [score:2]
E, Colony formation assay shows the miR-34a inhibits clonogenic growth of the miR-34-MIF. [score:2]
However, mutation in the Bcl-2 3′UTR complementary to the miR-34 seed sequence abolished this effect, indicating that the observed activity is sequence-specific. [score:2]
0006816.g006 Figure 6 A–B, CD44 and CD133 FACS analysis of the MiaPaCa2-miR-34a-MIF and MiaPaCa2-MIF cells. [score:1]
Lentiviral miR-34a infection. [score:1]
We identified that CD44+/CD133+ MiaPaCa2 cells are enriched with tumorsphere-forming and tumor-initiating cells or cancer stem/progenitor cells with high levels of Notch/Bcl-2 and loss of miR-34. [score:1]
Similar results on tumorspheres were observed with the total cells from the miR-34a-MIF cells (Figure S3). [score:1]
A, miR-34 restoration sensitizes the cells to chemotherapeutic agents. [score:1]
Sharp's group in their pioneer miRNA study [47], supporting the link of miR-34 to CSC. [score:1]
C, Restoration of miR-34 leads to caspase-3 activation. [score:1]
miR-34a restoration significantly reduced the CD44+/CD133+ cells. [score:1]
MiaPaCa2 cells were transfected with miR-34a mimic or NC mimic for 24 hr. [score:1]
Restoration of miR-34 sensitizes MiaPaCa2 cells to chemotherapy and radiation. [score:1]
MiaPaCa2 CD44+/CD133+ cells are tumorsphere-forming cells that have high Bcl-2 and loss of miR-34. [score:1]
MiaPaCa2 cells were transfected with miR-34a mimic or NC mimic and placed in the Transwell inserts, cultured for two days, observed under microscope (A) and quantified (B). [score:1]
Another potential role for miR-34 in cancer initiation and progression may be a link to tumor-initiating cells or cancer stem cells (CSC). [score:1]
For cell cycle and apoptosis analysis by flow cytometry, MiaPaCa2 cells were transfected with miR-34 mimics or NC mimic in 6-well plates, trypsinized 24 hr later and washed with phosphate-buffered saline, and fixed in 70% ethanol on ice. [score:1]
To evaluate the long-term effects of the miR-34 restoration, we also employed a lentiviral system to express miR-34a. [score:1]
We examined the roles of miR-34 in p53-mutant human pancreatic cancer cell lines MiaPaCa2 and BxPC3, and the potential link to pancreatic cancer stem cells. [score:1]
To investigate the potential role of miR-34 in pancreatic cancer stem cells, we examined whether miR-34 restoration could inhibit the CD44+/CD133+ cells and their self-renewal potential. [score:1]
Cells were also co -transfected with 100 pmol of each miR-34 mimic or NC mimic, as indicated, using Lipofectamine 2000. [score:1]
As shown in Figure 3 C, transient transfection of miR-34 mimics resulted in significantly increased activation of caspase-3, a key indication of the cells undergoing apoptosis [25]. [score:1]
Figure 6 A shows that miR-34a restoration significantly decreased the CD44+/CD133+ cells (0.58±0.08% versus MIF control 1.93±0.19%, P = 0.022, n = 2), an 87% reduction (Figure 6 B ). [score:1]
miR-34a, miR-34b and miR-34c mimics all had similar activities. [score:1]
MiaPaCa2 cells were transfected with miR-34a mimic or NC mimic for 24 hours. [score:1]
miR-34 restoration by transfection of MiaPaCa2 cells with miR-34 mimics. [score:1]
These findings suggest that miR-34 mimics may hold significant promise as a novel molecular therapy for human pancreatic cancer with loss of p53–miR34, potentially via modulating pancreatic cancer stem cells. [score:1]
The stable MiaPaCa2-miR-34a-MIF or MiaPaCa2-MIF cells were plated for tumorsphere formation as described in. [score:1]
miR-34 restoration sensitizes MiaPaCa2 cells to chemo- and radiotherapy. [score:1]
Our results show that the CD44+/CD133+ MiaPaCa2 cells, even though only comprising 1–2% of total cell population, have much higher levels of Bcl-2 and Notch1, and lower levels of miR-34a,b,c, while CD44−/CD133− cells have levels comparable to that of total population. [score:1]
Our data demonstrate that miR-34 restoration can overcome chemo-/radioresistance of the pancreatic cancer cells that have high levels of Bcl-2 and low basal levels of miR-34s, and are dependent on Bcl-2 for survival and resistance to therapy. [score:1]
MiaPaCa2 cells were transfected with miR-34 mimic or NC mimic for 24 hr, plated in 96-well plates (5,000 cells/well), and treated with serially diluted chemotherapeutic agents, in triplicates. [score:1]
miR-34a has been reported to be involved in p53 -mediated apoptosis in colon cancer and pancreatic cancer [8], [9]. [score:1]
B, miR-34 restoration increases caspase-3 activation induced by gemcitabine or X-ray radiation in MiaPaCa2 cells. [score:1]
Error bar indicates S. D. D, miR-34a-MIF cells grows slower than MIF control cells. [score:1]
0006816.g004 Figure 4 A, miR-34 restoration sensitizes the cells to chemotherapeutic agents. [score:1]
Figure S1 shows the characterization of expression changes in the miR-34a-MIF cells. [score:1]
Loss of miR-34 has been linked to chemoresistance of cancer [13]. [score:1]
The stable MiaPaCa2-miR-34a-MIF or MiaPaCa2-MIF cells were placed in the Transwell inserts, cultured for two days, observed under microscope (C) and quantified (D). [score:1]
In the current study, we have examined the effects of functional restoration of miR-34 by miR-34 mimics and lentiviral miR-34a on human p53-mutant pancreatic cancer MiaPaCa2 cells, as well as the potential link to the pancreatic cancer stem cell self-renewal. [score:1]
Cells were transfected with miR-34a mimic or NC mimic. [score:1]
MiaPaCa2 cells were co -transfected with the Bcl-2 3′UTR Luciferase Reporter or its mutant, b-gal vector, together with either miR-34 mimics or NC mimic. [score:1]
miR-34 mimics resulted in significant G1 and G2/M arrest and a reduction of cells in S phase (Figure 3 D ), consistent with other reports on miR-34 restoration in various cancer mo dels [6], [7], [8], [10], [11], [21], [22], [26]. [score:1]
0006816.g008 Figure 8MiaPaCa2 cells were transfected with miR-34a mimic or NC mimic for 24 hours. [score:1]
Transfection of miR-34 mimics. [score:1]
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7
[+] score: 428
Other miRNAs from this paper: hsa-mir-19a, hsa-mir-320c-1, hsa-mir-320c-2
In PCT, miR-34a promotes proliferation and suppresses apoptosis by targeting Growth arrest specific1 (GAS1), which can suppressor that inhibits cancer cell proliferation and induces apoptosis through inhibition of RET receptor tyrosine kinase, via PI3K/Akt/Bad pathway [26]. [score:11]
Combining with our previous studies and bioinformatics analysis, we expected that CD44 gene was a direct target of mir-34a, siRNA -mediated knockdown of CD44 partially phenocopied mir-34a overexpression suggesting that the pro-apoptotic role of mir-34a may be mediated primarily through CD44 regulation, whereas restoring the expression of CD44 attenuated the function of mir-34a in bladder cancer cells. [score:10]
Here, we identified mir-34a as down-regulated in bladder cancer, and verified that microRNA-34a functions as an anti-metastatic microRNA and suppresses angiogenesis in bladder cancer by directly targeting CD44. [score:9]
Our study proved that mir-34a functions as an anti-metastatic microRNA and suppresses angiogenesis by directly targeting CD44 in bladder cancer and subsequently suppress CD44 that regulates transcription of a variety of genes in bladder cancer cells. [score:9]
And previous studies reported that upregulated the expression of miR-34a could exchange the expression of beta-catenin [41, 50]. [score:8]
Besides that, we evaluated the expression of CD44 in bladder cancer cell lines and found the expression of CD44 was up-regulated in these cell lines (Figure  1D, p < 0.01), suggesting a negative correlation between the expression of CD44 and miR-34a in bladder cancer cell lines, which was shown in Figure  1E (R [2] = 0.5988 p = 0.0412). [score:8]
Our study defines a major metastasis and angiogenesis suppressive role for mir-34a, a microRNA functions as a tumor suppressor in bladder cancer by directly targeting CD44, which would be helpful as a therapeutic approach to block bladder cancer metastasis. [score:8]
Here, on the basis of our functional tests, findings that miR-34a was frequently downregulated in bladder cancer tissues and that miR-34a could suppress cell migration and invasion, and suppress tubefomation. [score:8]
To determine whether miR-34a could repress CD44 expression or not by targeting its binding sites in the CD44 3’-UTR, the PCR products containing intact target sites or a mutation of miR-34a seed recognition sequence (Figure  6F) were inserted into the luciferase reporter vector. [score:8]
MiR-34a has been described as a “star” miRNA in cancer research, which commonly functions as a tumor suppressor and is down-regulated in many human cancers [19], Furthermore, the aberrant miR-34a expression has been linked to chemotherapy resistance in a variety of cancer [20- 25]. [score:8]
Accordingly, identification of CD44 as a miR-34a target gene may explain, at least in part, why overexpression of miR-34a suppressed the migration, invasion, and angiogenesis of bladder cancer cells. [score:7]
shown that miR-34a overexpression increased E-cadherin expression but decreased β-catenin and Vimentin expression (Figure  3C). [score:7]
These results were consistent with the findings that overexpression of mir-34a suppressed the angiogenesis, migration and invasion of bladder cancer cells in vitro, providing further evidence that CD44 was involved in mir-34ameiated suppression of bladder cancer cells. [score:7]
Immunoblot showed that miR-34a consistently promoted E-cadherin protein expression and inhibited N-cadherin, Beta-catenin and Vimentin protein expression in all these cell lines. [score:7]
In this study, we focus on it that microRNA-34a functions as an anti-metastatic microRNA and suppress angiogenesis in bladder cancer by directly targeting CD44. [score:6]
Our studies focus on it that microRNA-34a functions as an anti-metastatic microRNA and suppresses angiogenesis in bladder cancer by directly targeting CD44 could further enrich the theory of biology. [score:6]
Although there are many related research, that microRNA-34a functions as an anti-metastatic microRNA and suppresses angiogenesis in bladder cancer by directly targeting CD44 still need further illuminated. [score:6]
ting indicated that transfection of CD44 restored the downregulation of CD44 induced by stable miR-34a overexpression. [score:6]
Knockdown of CD44 by siRNA inhibits tube-formation and increased CD44 expression could efficiently reverse the effect of anti-angiogenesis of miR-34a in bladder cancer cells A, EMT related factors and VEGF was detected by qPCR. [score:6]
All data presented here strongly support our hypothesis that the tumor-suppressive effect of miR-34a is mediated by directly targeting CD44. [score:6]
Six synthetic, chemically modified short single or double stranded RNA oligonucleotides (miR-34a mimics, mimics NC, miR-34a inhibitor, inhibitor NC, agomir-miR-34a and agomir-NC) were purchased from Ribo Biotech (Guangzhou, China). [score:5]
Mir-34a overexpression inhibits bladder cancer cell invasion and metastasis by modulating EMT related proteins in vivo and vitroTo assess changes in cell migration, 5637 and T24 cells transfected with hsa-miR-34a mimics or FAM-labeled pre-miR-NC (negative control) were allowed to migrate through a transwell membrane into complete media. [score:5]
CD44 protein expression was inhibited in miR-34a transfected bladder carcinoma cells. [score:5]
C, The expression of mir-34a in blood stream positively correlates with the expression in bladder tissues (R2 = 0.3036 P = 0.0411, Pearson’s correlation). [score:5]
E, CD44 expression was detected by western blot after overexpression of miR-34a and corresponding negative control i 5637 and T24. [score:5]
Mir-34a has been reported taken great part in the occurrence and development of some human cancer contains bladder cancer [25], prostate cancer [30], cervical carcinoma [48], renal cancer [49] by targeting certain targets. [score:5]
To confirm the synergistic effect of CD44 and miR-34a in tumorigenesis, we pulled down the expression of CD44 by specific siRNA resulting in decreased capability of cell angiogenesis, invasion and migration (Figure  4C and Figure  5A), which is in parallel with miR-34a overexpression in all the bladder cancer cell lines. [score:5]
A better understanding of the role of miR-34a as a potential therapeutic target in bladder genetic analyses suggested that bladder is a genetic disease involving multiple types of gene changes, including miRNAs by the recent decades of research [27, 28]. [score:5]
Because above results indicated the negative regulation of CD44 expression by miR-34a, we hypothesized that knockdown of CD44 should have a similar effect on cultured bladder cancer cells. [score:5]
Moreover, knockdown of miR-34a with anti-miR-34a inhibitor increased the luciferase activity in 5637 and T24 cells (Figure  6G), whereas mutation of miR-34a recognition site abolished these effects (Figure  6G). [score:5]
C and D, mir-34a expression was detected by qPCR after infection of miR-34a mimics or inhibitor and corresponding negative control in 5637 and T24 for 48 hours. [score:5]
Figure 2 Overexpression of mir-34a inhibits bladder cancer cell invasion and migration and tube formation. [score:5]
Additionally, we found that significantly reduced expression of circulating mir-34a in bladder cancer (Figure  1B, p < 0.01), and next, we compared the expression of mir-34a in bladder cancer tissues and bloodstream, showing a positive correlation between them (Figure  1C, R2 = 0.3036 p = 0.0411). [score:4]
In summary, our research has demonstrated that miR-34a is significantly downregulated in bladder cancer tissues and bloodstream. [score:4]
On the other hand, miR-34a is up-regulated in some specific type of cancer, such as papillary thyroid carcinoma (PTC) [26]. [score:4]
Compared with SV-HUC-1 cells, these six bladder carcinoma cell lines had a significantly lower level of miR-34a expression, meanwhile, we evaluated the expression of miR-34a in bladder tissues, and we found that mir-34a was downregulated in bladder tissues, compared with the corresponding adjacent control, (Figure  1A, P < 0.01). [score:4]
In the subsequent mechanism research, mir-34a has many different certain targets in regulating different kinds of human cancer. [score:4]
These results indicated that miR-34a directly and specifically interacted with the target site in the CD44’-UTR. [score:4]
ting indicated that transfection of CD44 rescued the miR-34a–induced downregulation of CD44 (Figure  4B). [score:4]
MiR-34a overexpression can inhibit bladder cell migration, invasion, tubefomation in vitro and metastasis and angiogenesis in vivo. [score:4]
Next, we investigated the direct effects of miR-34a on CD44 expression in bladder cancer cell lines; we conducted the miRNA overexpression experiments. [score:4]
Interestingly, as predicated by TargetScan and previous study, miR-34a could negatively regulate different kinds of mRNAs including CD44 in some human cancers. [score:4]
We not only proved that mir-34a was significantly downregulated in bladder cancer tissues and cell lines but also that circulating miR-34a levels are reduced in bladder cancer, and their levels were positively relevance. [score:4]
Furthermore, CD44 is a direct and functional target of miR-34a, and the functions that CD44 -mediated can be reversed by miR-34a in bladder cells. [score:4]
Mir-34a overexpression inhibits bladder cancer cell invasion and metastasis by modulating EMT related proteins in vivo and vitro. [score:4]
CD44 has been identified to be the direct target of miR-34a in prostate cancer, bladder cancer and renal cancer [25, 28, 30]. [score:4]
In vivo metastasis, assays also demonstrated that overexpression of mir34a markedly inhibited bladder cancer metastasis. [score:4]
Figure 1 mir-34a was downregulated in bladder cancer and circulatingmiR-34a levels are reduced in bladder cancer. [score:4]
Combination with other pales of new specific biomarkers and restoration of miR-34a expression may be a potential diagnostic and therapeutic strategy to assess the prognosis and treatment of bladder cancer in the future. [score:3]
There have few reports on the expression of circulating mir-34a in bladder cancer, and the level of miR-34a was correlated with the level of mir-34a in bladder cancer tissues. [score:3]
The data indicated that miR-34a functions as a tumor suppressor in bladder cancer. [score:3]
Oligonucleotide and lentivirus were used to overexpress miR-34a. [score:3]
The tube formation of endothelial cells was suppressed by treatment with the medium preconditioned by stable transfection of bladder cancer cells with miR-34a precursor (Figure  2E). [score:3]
B, Transfection of CD44 rescued the angiogenic capabilities of miR-34a -overexpressing cells. [score:3]
indicated that miR-34a could promote E-cadherin and inhibited N-cadherin, Beta-catenin and Vimentin mRNA in all these cell lines. [score:3]
Our data in vivo suggested that altered expression of miR-34a played a role in bladder cancer cellular metastasis and angiogenesis. [score:3]
Mir-34a expression’s loss in human bladder cancer and circulating mir-34a level was reduced in human bladder cancer. [score:3]
The expression of mir-34a was detected by quantitative real-time PCR. [score:3]
The CD44/mir-34a expression differences between cancer and control were analyzed using Student’s t test within SPSS (Version 17.0 SPSS Inc. [score:3]
The miR-34a expression was analyzed by Mann–Whitney test, Wilcoxon matched pairs test for paired data. [score:3]
CD44 overexpression eliminated the pro-apoptotic effects of miR-34a which is similar to what’s observed in medulloblastoma [51]. [score:3]
CD44 was a candidate target of mir-34a and mir-34a interacted with a putative binding site in the CD44 3’UTR. [score:3]
A, relative expression of mir-34a expression levels were evaluated by qPCR in bladder cancer. [score:3]
In our study, we identified a crucial tumor-suppressive miRNA, mir-34a, which plays an important role in the progression in bladder cancer. [score:3]
These data indicated that the circulating level of mir-34a is proportionately associated with bladder cancer mir34a expression and circulating miRNAs may be able to be used as biomarker for bladder cancer. [score:3]
The mean vessel density within tumors decreased after stable overexpression of miR-34a. [score:3]
B, relative expression of circulating mir-34a expression levels were evaluated by qPCR in bladder cancer. [score:3]
Aberrant miR-34a expression has been linked to chemotherapy resistance in a variety of cancer. [score:3]
MiR-34a function as an anti-metastatic microRNA via directly targeting CD44 in bladder cancer. [score:3]
Figure 3 Overexpression of miR-34a attenuated the metastasis and angiogenesis of bladder cancer in vivo. [score:3]
These results suggest that the role of miR-34a is possibly tumor-specific and highly dependent on its targets in different cancer cells. [score:3]
The lentivirus mediating the overexpression of miR-34a and the control vector were supplied by JIKAI Company Shanghai China. [score:3]
These results suggested that miR-34a could inhibit EMT in bladder cancer cell (Figure  2I, Figure  2J and Figure  4A). [score:3]
Figure 4 mir-34a targets CD44 in bladder cancer cells and the validation of transfection/infection. [score:3]
As shown in Figure  2A, mir-34a overexpression significantly reduced 5637 and T24 cell invasion by approximately 32% and 56%. [score:3]
Increased CD44 expression could efficiently reverse the effect of anti-metastatic of miR-34a in bladder cancer cells. [score:3]
C, miR-34a expression after mimics transcription was detected by qPCR. [score:3]
A and B, mir-34a expression was detected by qPCR after infection of miR-34a lentivirus and corresponding negative control in 5637 and T24 for 4 days. [score:3]
50 nmoles of miR-34a mimics/inhibitor or adjacent negative control and 50 ng pmirGLO-30-UTR vector were co -transfected into cells using X-treme GENE transfection reagent (Roche Applied Science). [score:3]
Overexpression of miR-34a precursor into bladder cells resulted in increase of miR-34a levels (Figure  6A-D). [score:3]
E and F, increased CD44 expression could efficiently reverse the effect of anti-angiogenesis of miR-34a in bladder cancer cells. [score:3]
Gain-of-function experiments investigated that increased mir-34a expression suppressed tube formation and reduced cell migration and invasion. [score:3]
A, hematoxylin/eosin (HE) and immunohistochemical staining revealed that stable transfection of miR-34a precursor resulted in decreased expression of VEGF, CD44 and CD31 within tumors. [score:3]
It was reported that p53 had a miR-34a -dependent integrated mechanism to regulate glucose metabolism [39]. [score:2]
However, despite the fact that more and more evidences indicated mir-34a plays a crucial role in the biology processes of bladder cancer, the role of mir-34a in the development and progression remains elucidated in bladder cancer. [score:2]
Compared with the negative control, mir-34a overexpression led to 55% and 65% reduction of migratory cells in 5637 and T24, respectively (Figure  2A). [score:2]
E, MiR-34a inversely correlates with CD44 expression (R2 = 0.5988 P = 0.0412, Pearson’s correlation). [score:2]
Some important prior studies including ours have reported on the dysregulation of various miRNAs including mir-34a in bladder cancer [22, 25, 27]. [score:2]
In intravenous injection assay, bioluminescence imaging revealed that fluorescence signal in lenti-mir-34a group were significantly weaker than lenti-NC group, which mean that less metastasis is formed in lung after mir-34a overexpression (Figure  2C). [score:2]
Additionally, we identified that EMT (epithelial-mesenchymal transition) related proteins could be regulated by mir-34a which indicated that mir-34a could partially reserve EMT. [score:2]
Luciferase assay was carried out to verify the precise target of miR-34a. [score:2]
According to the previous study in prostate cancer [30] dual luciferase reporter assay was used and determined CD44 was also exactly the target of miR-34a in bladder cancer cells (Figure  6G). [score:2]
F and G, The seed regions of the miR-34a target sites in CD44 and the luciferase activity assay. [score:2]
Mir-34a suppressed the angiogenesis of bladder cancer cells in vivo and vitro. [score:2]
This novel miR-34a/CD44/EMT related factor provides new insight into the mechanisms underlying tumor metastasis in bladder cancer. [score:1]
Figure 5 The anti-angiogenesis functions of miR-34a were mediated by reducing the production of CD44. [score:1]
Some previous studies indicate that CD44 promotes the invasion and angiogenesis of tumor cells [25, 28]; we further investigated the effects of miR-34a overexpression and CD44 restoration on cultured bladder cancer cells. [score:1]
Lenti-miR-34a and Lenti-NC were purchased from Genechem Biotech (Shanghai, China). [score:1]
Restoration of miR-34a could markedly decrease CD44 protein (Figure  6E). [score:1]
The correlation between miR-34a level and CD44 protein expression was calculated by Spearman’s correlation. [score:1]
In the CD44 3’-UTR, there was one potential binding site of miR-34a with high complementarity (Figure  6F), the miR-34a- binding site was located at bases 1855–1878 and 3399–3422 of the CD44 3’-UTR, respectively (Figure  6F). [score:1]
Bladder cancer cell miR-34a CD44 Metastatic Angiogenesis Bladder cancer is the most common urinary tract malignancy and the fifth most common malignancy in the developed world and is the most common urological tumor in China. [score:1]
The anti-metastatic activity of miR-34a was tested in nude mice T24 lung metastasis mo del as described previously [25]. [score:1]
The plasmids were transfected into bladder cancer cells stably transfected with empty vector or miR-34a precursor. [score:1]
Figure 6 The anti-metastatic functions of miR-34a were mediated by reducing the production of CD44. [score:1]
HUVECs were cultured in the following media: Culture Media of 5637 and T24 cells transfected with mir-34a and NC. [score:1]
To assess changes in cell migration, 5637 and T24 cells transfected with hsa-miR-34a mimics or FAM-labeled pre-miR-NC (negative control) were allowed to migrate through a transwell membrane into complete media. [score:1]
Moreover, stable transfection of miR-34a precursor resulted in a decrease in CD31 -positive mean vessel density within tumors (Figure  3A). [score:1]
To investigate the hypothesis that miR-34a may influence the CD44 expression in bladder cancer, combination with the previous reports, computational prediction was done by miRNA databases. [score:1]
T24 cell was stably infected with Lenti-miR-34a or Lenti-NC containing GFP label. [score:1]
Subsequently, we proved that restoration of CD44 could rescue the antitumor effects of miR-34a by co-transfection of miR-34a mimics and CD44 in bladder cancer cell lines (Figure  4E and Figure  5B). [score:1]
CD31, an endothelial cell–specific marker which stained in T24 tumors to evaluate for blood vessel density, the immunohistochemistry results showed that blood vessel quantification reduced dramatically in the T24 tumors over -expressing mir-34a. [score:1]
Tumor cells stably infected with miR-34a lentivirus established significantly fewer metastatic colonies. [score:1]
These results indicated that miR-34a remarkably decreased the angiogenesis of bladder cancer cells in vivo and vitro. [score:1]
Besides, although several other studies have investigated circulating miRNAs, they demonstrated that mir-34a plays a key role in NAFLD and colorectal cancer pathogenesis is supported by the evidence that serum levels of circulating mir-34a increased in participants with NAFLD and colorectal cancer, and they put forward that mir-34a may present a therapeutic target for NAFLD and colorectal cancer treatments, and a novel biomarker to predict NAFLD and colorectal cancer susceptibility and progression [44, 45]. [score:1]
In fact, the relationship between mir-34a and CD44 may have more connotations. [score:1]
Tumor cells stably infected with miR-34a lentivirus established significantly fewer metastatic colonies (Figure  3B). [score:1]
The luciferase activity normalized to that of firefly was significantly reduced in the tumor cells stably transfected with miR-34a precursor (Figure  6G), and the effect was abolished by mutating the putative miR-34a–binding site within the 3’-UTR of CD44 (Figure  6G). [score:1]
All the results above indicated that mir-34a takes a great role in the recurrence and progression in bladder cancer and this may have future application as part of a biomarker profile in bladder cancer. [score:1]
In the xenograft tumors, the VEGF and CD44 were also reduced by stable transfection of miR-34a precursor (Figure  3A). [score:1]
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[+] score: 425
Recently, microRNA-34a (miR-34a) was identified as a novel class of tumor-suppressive miRNAs that is downregulated in several human cancers, including breast, lung, and liver; re-introduction of miR-34a mimics the inhibition of cancer cell growth and metastasis [19– 21]. [score:8]
MicroRNA-34a inhibits migrations and invasion in BC cells in vitroTo investigate miR-34a's effects on BC cell growth, migration, and invasion, pre-miR-34a for the overexpression of precursor miR-34a (pre-miR-34a), or anti-miR-34a for the overexpression or inhibition of miR-34a, were transiently transfected into the MBC cell lines, BT-549 (Figure 2D) and MDA-MB-435 (Supplementary Figure 2), respectively. [score:7]
The downregulation of miR-34a expression in breast could be associated with the frequent deletion of chromosome 1p36, TP53 mutations and CpG methylation of the miR-34a promoter in these tumors [52, 54]. [score:7]
Prediction potential targets of hsa-miR-34a in the 3′-UTR of EMT-TFs was found by using the bioinformatics analysis based on the target prediction tools TargetScan (http://www. [score:7]
Therefore, we proposed that EMT-TFs over -expression might suppress miR-34a expression of BC. [score:7]
The expression levels T-47D, BT-549, MDA-MB-231, and MDA-MB-435 were expressed as relative to the miR-34a expression level of MCF-7 cells, non-metastatic BC cell lines (mean±SD). [score:7]
We next explored whether miR-34a is able to directly target NOTCH1, TWIST1 and ZEB1 gene expression at the post-transcriptional level by regulating the activity of mRNA (Figure 6). [score:7]
Here, we found that miR-34a potentially inhibits TFs, like TWIST1 and ZEB1, and EMT -associated protein NOTCH1 expression upon the 3′-untranslated region (3′-UTR) in BC cells. [score:7]
However, overexpression of miR-34a did not substantially affect SLUG, ZEB2 expression (P ≥ 0.05; Figures 2B-2C) and SNAIL1 expression (data not shown). [score:7]
The above data showed that miR-34a is significantly downregulated in MBC tissues, suggesting that the downregulation of miR-34a most likely affects the progression of MBC. [score:7]
Up-regulated miR-34a contributed to a significant down-regulation of cell migration and invasion. [score:7]
NOTCH1, SLUG, TWIST1, and ZEB1/2 expression levels, in contrast to miR-34a, were verified in BC cell lines, which confirmed that NOTCH1 and EMT-TFs partially inhibit miR-34a expression in cells (Figures 3A-3E). [score:7]
We have consequently revealed that miR-34a is downregulated in MBC tissues and suppresses cancer metastasis by affecting several malignancy endpoints. [score:6]
Upon overexpression, miR-34a reduced NOTCH1, TWIST1, and ZEB1 levels, although it expressed a less significant reduction in their corresponding protein levels (P > 0.05; Figures 2B-2C), indicating both transcriptional and post-transcriptional regulations. [score:6]
Accordingly, overexpression of miR-34a acts as a mediator of tumor suppression by transcriptionally regulating TP53, NOTCH, and transforming growth factor beta (TGF-β) signaling pathways [14, 20, 22– 25]. [score:6]
Ectopic expression of miR-34a inhibits the cell migration and invasion in BT-549 BC cells. [score:5]
Transient transfection of miR-34a significantly inhibits cell proliferation, cell migrations, cell survival and cell invasion of BC cell lines by targeting NOTCH1, ZEB1, and TWIST1. [score:5]
However, the relative expression of the SLUG and ZEB2 and miR-34a expression was not significantly co-related in BC cell lines (Figures 3B and 3E, respectively). [score:5]
It is possible that miR-34a may interact with 3′-UTR of EMT-TFs by stem-loop structure and change the conformation of the binding and landing site of other EMT-activation proteins, possibly inhibiting the protein expression of EMT and disrupting the post transcriptional modification pattern through methylations and ubiquitin-proteasome pathway [57]. [score:5]
Since different cancer cell types display differences in the expression and relevance of EMT regulators [30– 32], current attention is focused on systematically analyzing EMT-TFs’ regulatory mechanism and EMT-TFs/miR-34a feedback loop, like the role of TWIST1, SLUG, ZEB1/2 and SNAIL1 in promoting BC migration, invasion and metastasis. [score:5]
Previous studies indicated that miR-34a significantly suppressed proliferation, invasion, and metastasis of BC cells, which correlated to the overexpression of some EMT-TFs [19, 58, 59]. [score:5]
Relative expression levels of NOTCH1 A., SLUG B., TWIST1 C., and ZEB1 D. and ZEB2 E. were detected in non-metastatic BC cell lines (MCF7 and T47D), and MBC cell lines (MDA-MB-231, MDA-MB-435 and BT-549), in comparison to relative expression levels of miR-34a using qRT-PCR. [score:5]
Moreover, we found a significant inverse correlation between miR-34a expression and EMT-TFs expression. [score:5]
The forced miR-34a expression in MBC cell line BT-549 powerfully inhibits cell migrations and invasion. [score:5]
EMT-TFs demonstrate high expression levels in MBC cell lines in front of miR-34a expression. [score:5]
To our knowledge, the correlation between downregulation of miR-34a expression and EMT-TFs regulations in BC was investigated systematically in this study for the first time. [score:5]
We also revealed that miR-34a expression is down-regulated in MBC tissues compared to normal tissues. [score:5]
A number of studies have suggested that miR-34a is potentially a regulatory microRNA that is usually up-regulated in breast, pancreatic, renal and gastric cancer [47– 50]. [score:5]
The expressions of miR-34a was normalized against the expression level of U6 snRNA. [score:5]
To investigate miR-34a's effects on BC cell growth, migration, and invasion, pre-miR-34a for the overexpression of precursor miR-34a (pre-miR-34a), or anti-miR-34a for the overexpression or inhibition of miR-34a, were transiently transfected into the MBC cell lines, BT-549 (Figure 2D) and MDA-MB-435 (Supplementary Figure 2), respectively. [score:5]
Despite some limitations in sample size, the current data showed an inverse correlation between miR-34a expression and EMT-TFs expression in MBC tissues, indicating that miR-34a could be a promising and novel noninvasive biomarker in BC diagnosing. [score:5]
Our results clearly showed that miR-34a's expression did not significantly inhibit cell proliferation in BT-549 cells. [score:5]
Our results indicated that miR-34a inhibits TWIST1, ZEB1, and NOTCH1 expression upon their 3′-UTR activity in BC cells and reduces the metastatic and invasive features of MBC effectively. [score:5]
Both proteins and gene analysis showed that TQ+miR-34a significantly inhibited the mRNA level expression of NOTCH1 (P = 0.073; Figure 7A) TWIST1 (P = 0.062; Figure 7B) and ZEB1 (P = 0.005; Figure 7C) in the BT-549 cell line. [score:5]
Correlations between relative expression levels of NOTCH1 A., SLUG B., ZEB1 C., and ZEB2 D. with miR-34a expression level. [score:5]
Figure 3Relative expression levels of NOTCH1 A., SLUG B., TWIST1 C., and ZEB1 D. and ZEB2 E. were detected in non-metastatic BC cell lines (MCF7 and T47D), and MBC cell lines (MDA-MB-231, MDA-MB-435 and BT-549), in comparison to relative expression levels of miR-34a using qRT-PCR. [score:5]
Thus, co- delivery of TQ and miR-34a could produce synergistic effects on tumor growth and migrations, and a nanosystem -based co- delivery of tumor suppressive miRNAs and natural compound TQ may be a promising combination for therapeutic strategy against MBC by down -regulating TWIST1, NOTCH1 and ZEB1 in the near future. [score:4]
A number of studies have concluded that knockdown of some EMT-TFs in cancer cell lines interacting with miR-34a inhibits cancer progression and metastasis [19, 33, 55, 56]. [score:4]
These results suggested that miR-34a directly targets multiple genes of NOTCH1, TWIST1, and ZEB1 in vitro. [score:4]
MicroRNA-34A inhibits BC progression by targeting TWIST1, NOTCH1, and ZEB1. [score:4]
Interestingly, this is the first report demonstrating that the co- delivery of miR-34a and TQ is able to inactivate the downstream of the EMT signaling pathway by directly targeting TWIST1 and ZEB1 in vivo. [score:4]
TWIST1, NOTCH1 and ZEB1 are direct targets of miR-34a. [score:4]
Co- delivery of miR-34A and Thymoquinone inhibits BC metastasis by regulating EMT-TFs. [score:4]
In considering both gene and protein levels, TWIST1, SLUG, and ZEB1 expressions were higher compared to the lower expression of miR-34a. [score:4]
Ahn et al. (2012) demonstrated that ZEB1 targeted miR-34a as a direct tragedian of miR-34a in human lung cancer [60]. [score:4]
The relative luciferase values were measured and normalized by luciferase reporter assay after 48 h. NOTCH1, TWIST1, and ZEB1 expression were directly down-regulated by miR-34a. [score:4]
The qRT-PCR and analysis were conducted to investigate whether miR-34a expression could remarkably affect the EMT-TFs SNAIL1, SLUG, TWIST1, ZEB1 and ZEB2, and NOTCH1 expression levels. [score:3]
MBC metastatic breast cancer BC breast cancer EMT epithelial to mesenchymal transition ZEB zinc-finger E-box -binding bHLH basic helix–loo–helix SLUG zinc finger protein SNAI2 EMT-TFs EMT-inducing transcription factors TWIST1 Twist-related protein 1 miR-34a MicroRNAs-34a TGF-β transforming growth factor beta WHO World Health Organization UICC Union for International Cancer Control RLU relative light unit RFS relapse free survival OS overall survival DMFS distant metastasis free survival TQ Thymoquinone PPS post progression survival SD standard deviation UTR untranslated region LSD least significant difference qRT-PCR quantitative reverse transcriptase-PCR ROS reactive oxygen species. [score:3]
Expression of miR-34a in BC cell lines and BC specimens. [score:3]
In general, a significant correlation between miR-34a expression and clinicopathological features of MBC patients was observed. [score:3]
Previous investigational screenings for miR-34a expressions in different tissues and cancer cell types found that the expression of miR-34a is variable in BC [30– 32]. [score:3]
In addition, some extent of reduction in ZEB2 was also observed, by TQ through either treatment or miR-34a overexpression (Supplementary Figure 3). [score:3]
Relationship between miR-34a expression level and clinicopathological features of MBC patients. [score:3]
In conclusion, we indicate that the lower level of expression of miR-34a could be a promising and novel biomarker for MBC. [score:3]
Strikingly, the inhibited-miR-34a in BT-549 BC cells showed more significant migrations and invasions after 12 h of cell culture. [score:3]
Here, we did not observe significant changes in cell migration in MDA-MB-435 cell lines with anti-miR-34a overexpression (Supplementary Figure 2); thus, we concluded that miR-34a functions are dependent on MBC cell line types. [score:3]
The aforementioned outcomes might help describe a possible aim on why EMT-TFs/TQ/miR-34a contributes to MBC progression, helping in the identification of a novel therapeutic target of MBC progression. [score:3]
Figure 5 Data indicate significantly negative associations in EMT-TFs and miR-34a expressions in MBC patients. [score:3]
The relative expression of miR-34a in MBC tissues was significantly related to certain clinicopathological features, like progesterone receptor and Ki-67 proliferation marker status. [score:3]
Synergic effects of thymoquinone (TQ) and miR-34a on mRNA (top panel) and protein (bottom panel) level expression of EMT-TFs. [score:3]
First, prediction on-line databases, like miR base databases, identified that NOTCH1, TWIST1 and ZEB1 were potential downstream targets of miR-34a through their interactions in the 3′-UTR region (Figure 6A). [score:3]
Now, the details of how miR-34a physically interacts with EMT-TFs and how this interaction inhibits MBC remain unclear. [score:3]
The plasmids for miR-34a of miRNA inhibitors (HmiR-AN0440-SN-5), precursor miRNA clone in non-viral vectors (HmiR0005-MR04) and respective negative controls (CmiR-AN0001-SN, CmiR0005-MR04) were purchased from GeneCopoeia, Inc, USA. [score:3]
Furthermore, miR-34a expression levels were inversely correlated with Ki-67 (r = −0.23, P = 0.002; Figure 1C). [score:3]
The over-expressed miR-34a cells reduced NOTCH1, TWIST1, and ZEB1 levels. [score:3]
Consistently, after the inhibition of miR-34a, TWIST1, NOTCH1 and ZEB1 protein levels were significantly increased. [score:3]
B. The relative expression of EMT-TFs in BT-549 cells transfected with miR-34a mimic were analyzed by qRT-PCR. [score:3]
Therefore, miR-34a suppresses several malignancy parameters in human breast tumor cells, but has no obvious effects on cancer cell growth. [score:3]
Consequently, several miRNAs, including miR-1 [40], miR-34a [30, 41, 42], miR-100 [43], miR-125b [44], and miR-142 [45] have been proposed to inhibit or reduce the EMT process during the advanced stage of BC [46]. [score:3]
As shown in Figure 2A, the miR-34a expression in BT-549 cells transfected with miR-34a mimic was significantly increased. [score:3]
Data indicate significantly negative associations in EMT-TFs and miR-34a expressions in MBC patients. [score:3]
Interestingly, the average level of miR-34a expression was consistently and significantly lower in tissues of MBC (n = 33) than that of normal breast samples (n = 15) (Figure 1A). [score:3]
Pre-miR-34a restoration inhibits cell migration and invasion in comparison to the pre-controls. [score:3]
Our study seeks to address that miR-34a expression is lower in human BC tissues than in normal breast tissues. [score:3]
Inverse relationship between levels of miR-34a in 48 human breast specimens with Ki67 C. PR expression D. The diamond indicates human BC specimens (n = 33), and circles represent paired adjacent normal tissue (n = 15). [score:3]
There was no expression of the miR-34a in the MDA-MB-435 BC cells. [score:3]
Nevertheless, these reports on the interaction between miR-34a expression and the inactivation of EMT signaling pathway are still limited [46]. [score:3]
Figure 1D presents a positive correlation between the miR-34a expression level ratios and PR. [score:3]
Molecular mo del of BC metastasis suppressed via TQ/miR-34a-initiated signaling pathways. [score:3]
Although there is no statistical significance observed currently, further research might exhibit a significant correlation between miR-34a expression level and tumor size (p value is 0.08) as well as histological grade (p value is 0.075) if we increase the sample size. [score:3]
We found that TWIST1 ratios were not significantly correlated with the frequencies of miR-34a expression (r = −0.093, P = 0.532; data not shown). [score:3]
The plasmids for miR-34a of miRNA inhibitors (HmiR-AN0440-SN-5), precursor miRNA clone in non-viral vectors (HmiR0005-MR04) and respective negative controls (CmiR-AN0001-SN, CmiR0005-MR04) (GeneCopoeia, Inc, USA) were transfected into 1×10 [5] cells per well in 24 well plates (Qiagen, Hilden, Germany). [score:3]
In general, it was observed significant correlation between the miR-34a expression levels and some clinicopathological features of MBC patients. [score:3]
The underlying mechanisms governing MBC remained largely undefined, with few studies proposing the inhibitions of EMT-TFs/miR-34a feedback loop as a mo del for the dissemination process. [score:3]
miR-34a resides on the chromosomal locus 1p36.23 and is involved directly and indirectly with many different oncogenic processes, including growth, survival, differentiation, proliferations, migration, invasion, and immune responses [20]. [score:3]
However, all these findings indicated that miR-34a might have a significant impact on the tumorigenesis of human cancer by targeting differential EMT signaling pathways other than those in BC. [score:3]
Studies indicated that frequencies of miR-34a expression in the BC are controversial [51– 53]. [score:3]
However, clinicopathological features, such as human epidermal growth factor receptor-2, estrogens receptor, and TP53, did not exhibit a significant association with miR-34a expression (Supplementary Figures 1A-1C). [score:3]
In considering both tumor and normal samples, miR-34a was inversely correlated with NOTCH1 (r = −0.563, P = 0.001; Figure 5A), SLUG (r = −0.374, P = 0.009; Figure 5B), ZEB1 (r = −0.505, P = 0.001; Figure 5C), and ZEB2 (r = −0.317, P = 0.028; Figure 5D) expressions. [score:3]
In our current study, we found that EMT-TFs/TQ/miR-34a axis plays critical roles in regulating MBC contribution. [score:2]
MicroRNA-34a expression in human BC cell lines and tissues. [score:2]
MicroRNA-34a inhibits migrations and invasion in BC cells in vitro. [score:2]
Figure 8 shows the transcriptional regulators of EMT and a representation of some points of intersection at TQ/miR-34a-inducing signaling pathways. [score:2]
We therefore presented the first comprehensive report of epigenetic inactivation of the EMT-TFs/miR-34a pathway in human BC that can potentially alter the stability of these regulations in EMT and mesenchymal state, consequently contributing to metastasis. [score:2]
Evidence implies that miR-34a is a promising non-invasive biomolecular tool for gene regulation in the metastatic process by coordinating TP53, NOTCH1, and TGF-β-pathways [27– 29]. [score:2]
Mutation was generated in the NOTCH1, and ZEB1 3′UTR sequence in the complementary site for the seed region of miR-34a. [score:2]
These results clearly show that TQ/miR-34a is a potential therapeutic agent for further development in the management of BC/MBC. [score:2]
B. Total RNA was prepared from the cell lysates and expression of miR-34a was quantified by TaqMan MicroRNA Assays. [score:2]
MicroRNA-34a expression correlates to clinicopathological features. [score:2]
The epigenetic inactivation of the EMT-TFs/miR-34a pathway can potentially push the equilibrium of these regulations toward EMT, thereby contributing to metastasis. [score:2]
Information about regulators and mediators of EMT-TFs/miR-34a loops is still murky and unknown. [score:2]
TQ, Thymoquinone; miR-34a, microRNAs-34a; PI3K/AKT, phosphatidylinositol 3-kinase (PI3K)/Akt; ROS, reactive oxygen species; MAPK, Mitogen-Activated Protein Kinase; ZEB, zinc-finger E-box -binding; EMT, epithelial to mesenchymal transitions, MET, mesenchymal to epithelial transitions; EMT-TFs, EMT-inducing transcription factors. [score:1]
Based on these results, we created a molecular mo del demonstrating the synergistic effect of miR-34a and TQ in MBC cell line (Figure 8). [score:1]
Figure 2 A. The BT-549 cells were transfected with plasmid pre-miR-34a and pre-miR-34a-controls as indicated. [score:1]
Co- delivery of TQ/miR-34a leads to repression of metastatic, cell differentiation, and EMT factures in front of inducing of apoptosis and TP53 activations and MET features of BC cell. [score:1]
Figure 6 A. miR-34a binding sequences at the 3′-UTR of NOTCH1, TWIST1 and ZEB1. [score:1]
All these results suggested that miR-34a is able to inactivate the EMT signaling pathway with mediation of NOTCH1, TWIST1 and ZEB1 in MBC cell lines. [score:1]
A. miR-34a binding sequences at the 3′-UTR of NOTCH1, TWIST1 and ZEB1. [score:1]
Despite this, it appears that the reduction of EMT-features and their modifications through the interaction between miR-34a and EMT-TFs may be a major functional mechanism of miR-34a in our present study. [score:1]
Testing for hsa-miR-34a (Cat# 4427975) was purchased from ABI. [score:1]
It is likely the initiation binding feedback loop of EMT-TFs /miR-34a is different depending on the types of cancer cell and physiological features. [score:1]
However, co-transfection of miR-34a, along with the NOTCH1, TWIST1, and ZEB1-mut 3′-UTR or control reporter genes, did not show significant effects on the luciferase activity. [score:1]
With this background, we investigated the synergistic effects of TQ and miR-34a on the expression of EMT -associated proteins. [score:1]
BT-549 cells were transfected with either the wild-type or mutant NOTCH1 B., TWIST1 C., ZEB1 D. -3′UTR luciferase reporter genes, together with different concentrations of miR-34a mimic or controls (0, 10, 30, and 90 ng). [score:1]
So far, it is unclear whether miR-34a epigenetically silences EMT by binding to EMT-TFs in BC. [score:1]
Although previous studies have demonstrated that EMT-TFs played a fundamental role in the initiation, aggregation, progression, and metastasis of BC by inducing the activation of NOTCH1, TP53, and TGF-β cascade [10, 13, 33], EMT-TFs’ expression patterns, in comparison to miR-34a, have not been well characterized in human BC. [score:1]
D. miR-34a dependently inhibited, migration and invasive characteristics of BT-549 cells. [score:1]
Importantly, miR-34a does not have any inhibitory effects on SLUG and ZEB2 in BC cells we investigated. [score:1]
There is some evidence supporting this finding, showing the negative correlations of miR-34a with the biomarkers of most cancers. [score:1]
A. The BT-549 cells were transfected with plasmid pre-miR-34a and pre-miR-34a-controls as indicated. [score:1]
Thus, the obtained data proposes that co- delivery of TQ and miR-34a could produce synergistic effects on tumor growth and migrations. [score:1]
Altogether, these data suggested that NOTCH1 (Figure 3A), TWIST1 (Figure 3C), and ZEB1 (Figure 3D) are reverse correlated with miR-34a in the both MBC and BC cell lines (especially BT-549 and MDA-MB-435 cell lines) (P≤0.001). [score:1]
In addition, miR-34a levels were significantly lower in some MBC cell lines in comparison to non-metastatic BC cell line MCF-7 (Figure 1B). [score:1]
4) 0.541  No (18) 3 (9.4) 15 (46.9) PR  Negative (22) 6 (12.5) 16 (33.3) 0.02  Positive (26) 11 (22.9) 15 (31.2) ER  Negative (18) 4 (8.3) 14 (29.2) 0.121  Positive (30) 13 (27.1) 17 (35.4) P53  Negative (34) 13 (32.5) 21 (52.5) 0.60  Positive (6) 2 (5.0%) 4 (10.0) Ki67  ≤ 30 (27) 14 (29.2) 13 (27.1) 0.03  > 30 (21) 3 (6.2) 18 (37.5) Her2/neu  Low/weak (27) 10 (20.8) 17 (35.4) 0.517  Moderate/strong (21) 7 (14.6) 14 (29.2): miR-34a, MicroRNAs-34a; TNM, TNM classification of malignant tumors; ER, estrogens receptor; PR, progesterone receptor; HER2, human epidermal growth factor receptor-2. Data are presented as a number for all others. [score:1]
The miR-34a expression which measured by stem-loop qRT-PCR in transfected cells was significantly increased. [score:1]
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Besides, over -expression of miR-34a also inhibited the expression of TGF-β/Smad4 downstream targets N-cadherin and induced the expression level of E-cadherin. [score:11]
Our results showed forced up-regulation of miR-34a significantly inhibited the protein expression of Smad4, and inhibited CC cells invasion and migration. [score:10]
Since miRNAs are generally involved in the pathogenesis of cancer by directly regulating the expression of their targets at a post-transcriptional level, we applied bioinformatic methods to predict the potential targets of miR-34a. [score:9]
Based on the contrasting expression patterns of miR-34a and Smad4 in most of the EHCC tissues and our in vitro data, we proposed that miR-34a is involved in the pathogenesis of EHCC by directly inhibiting the protein expression of Smad4. [score:8]
Snail, which is a downstream target of TGF-β/Smad4 signaling pathway, was also decreased by increasing miR-34a expression but increased by using miR-34a inhibitor in EHCC cells. [score:7]
As a direct transcriptional target of p53, decreased expression of miR-34a is partly due to the mutations of p53 in tumors [8]. [score:7]
Taken together, our results suggest that miR-34a inhibits invasion and migration by targeting Smad4 to suppress EMT through TGF- beta/Smad signaling pathway in human EHCC. [score:7]
The protein expression levels of Smad4 and Snail, which are downstream targets of TGF-β/Smad4 pathway, were found decreased by transfection with the miR-34a mimics but increased by transfection with a miR-34a inhibitor in both QBC939 and HuCCT1 cells (Fig.   3d). [score:7]
Fig. 1miR-34a expression in human EHCC tissues and CC cell lines, and the relationship between miR-34a expression and disease-free survival in EHCC patients. [score:7]
The Kaplan-Meier curve of disease-free survival in patients with high miR-34a expression (n = 13) and low miR-34a expression (n = 14) (***P < 0.01). [score:7]
These data suggest that Smad4 expression was primarily inhibited by miR-34a at the translational level. [score:7]
Recent research has found that down-regulation of miR-34a leads to a switch from Mnt (MAX network transcriptional repressor) to c-Myc expression during cholestatic cholangiocarcinogenesis in a mouse mo del [9]. [score:6]
Our data revealed that miR-34a directly targets the 3′-UTR of Smad4, and that ectopic expression of miR-34a represses Smad4 protein level in CC cell lines. [score:6]
Kaplan-Meier analysis showed that down-regulation of miR-34a was correlated with decreased disease-free survival (Fig.   1c, P = 0.004). [score:6]
Even a down-regulation of miR-34a with negative staining for Smad4 proteins were found in 7 patients, low expression of miR-34a is associated with positive staining for Smad4 protein in 18 EHCC samples. [score:6]
In the present study, we showed that the expression of miR-34a was down-regulated in EHCC tissues. [score:6]
miR-34a functions as a tumor-suppressor miRNA, and can target many downstream genes in the development and progression of carcinogenesis. [score:6]
The expression pattern of individual miRs with strict tissues, the clinical-feature-specificity or the different target genes involved in the unique regulation network of EHCC may all involved in the effect of miR-34a on Smad4 [35, 45]. [score:6]
Ltd, China) were used to inhibit and increase miR-34a expression respectively. [score:5]
The median disease-free survival time was 13.07 months and 23.54 months in low- and high- miR-34a group, respectively (*P < 0.05) Table 2 Multivariate logistic regression analysis for screening the influencial factors of miR-34a expression Variable b OR (95 % CI) P value Intercept − − 0.0266 Clinical stages −3.4965 0.030 (0.004 ~ 0.253) 0.0013(I + II versus III + IV) Note: All clinicopathological features were employed for variable selection in the logistic regression analysis using a stepwise backward method. [score:5]
Moreover, Snail protein expression level was decreased by transfection with miR-34a mimics but increased by transfection with miR-34a inhibitor in both QBC939 and HuCCT1 cells (Fig.   3d). [score:5]
Together, these results confirmed that Smad4 is a direct target of miR-34a and is regulated by miR-34a in CC cell lines. [score:5]
Taken together, our data provide new insights into the potential contribution of miR-34a in inhibition the progression of EHCC, and suggest miR-34a is a useful molecule target for developing new therapeutic method against EHCC. [score:5]
miR-34a expression in human EHCC tissues and CC cell lines, and the clinicopathological significance of miR-34a expression in EHCC patients. [score:5]
c qRT-PCR analysis of expression of miR-34a treated with miR-34a mimics or miR-34a inhibitor in QBC939 and HuCCT1 cells (***P < 0.01). [score:5]
Smad4 was over-expressed in most of the EHCC patients and was further demonstrated as one of the downstream targets of miR-34a, which was involved in the progression of EHCC. [score:5]
qRT-PCR analysis revealed that the expression level of miR-34a was markedly decreased in all of the CC cell lines in comparison with the expression levels in HiBECs (P < 0.01, Fig.   1b). [score:5]
qRT-PCR analysis of Smad4 mRNA expression levels treated with miR-34a inhibitor or mimic in QBC939 and HuCCT1 cells. [score:5]
Smad4 mRNA levels were not significantly influenced by the over -expression or inhibition of miR-34a in vitro. [score:5]
d analysis of Smad4 and Snail expression treated with miR-34a inhibitor or mimic in QBC939 and HuCCT1 cells. [score:5]
These results indicate that miR-34a participates in the regulation of cell migration and invasion in EHCC cells through down-regulation of TGF-β/Smad4 signaling pathway. [score:5]
a miRNA target prediction screened one computative miR-34a binding site at Smad4-three prime untranslated region (3′-UTR). [score:5]
However, Smad4 mRNA levels were not significantly influenced by the over -expression or inhibition of miR-34a in vitro (Additional file 3: Figure S1). [score:5]
Moreover, lower expression of miR-34a correlated with the decreased disease-free survival in these EHCC patients. [score:5]
Therefore, our studies demonstrated that miR-34a functions as a tumor-suppressor miRNA by inhibiting TGF-β/Smad -induced EMT in CC cells. [score:5]
Moreover, partial mutation of the perfectly complementary sites in the 3′-UTR of Smad4 abolished the suppressive effect due to the disruption of the interaction between miR-34a and Smad4 (Fig.   3b). [score:4]
Thus, miR-34a could also regulate some downstream targets in the progress of metastasis or invasion of EHCC. [score:4]
These data suggest that miR-34a expression is inversely correlated with Smad4 in EHCC patients, and miR-34a might play a critical role on Smad4 regulation in over a half but not all of the EHCC patients. [score:4]
Thus, our data showed that activation of miR-34a could antagonize Smad4 -mediated TGF-β induction of EMT process through regulation of E-cadherin and N-cadherin expression. [score:4]
miR-34a directly targets Smad4 in CC cells. [score:4]
Recent studies have found that miR-34a represses RhoA, a regulator of cell migration and invasion, by suppressing c-Myc–Skp2–Miz1 transcriptional complex that activates RhoA in human prostate cancer cells [37]. [score:4]
Our clinical analysis showed that down-regulation of miR-34a is correlated with lymphatic metastasis and advanced clinical stages. [score:4]
Some reports have demonstrated that miR-34a to be an important anti-oncogene by regulation of different downstream targets in various types of cancers [7, 8, 10– 14, 26– 29]. [score:4]
Down-regulation of miR-34a has been found in left and median bile duct ligation (LMBDL) mouse livers [9]. [score:4]
Moreover, Smad4 was demonstrated as a direct transcriptional target of miR-34a in CC. [score:4]
Up-regulation of miR-34a represses the EMT via TGF-β/Smad signaling pathway in CC cell lines. [score:4]
Fig. 3Smad4 is a direct miR-34a target. [score:4]
These data are consistent with the decreased expression levels of miR-34a in the digestive system cancers which has been demonstrated by several groups [9, 26– 29]. [score:3]
Decreased expression of miR-34a in EHCC patients is correlated with lymphatic metastasis, advanced clinical stages and overall survival rate. [score:3]
In the present study, miR-34a expression levels were detected in EHCC tissues, and CC cell lines. [score:3]
miR-34a was also found to reduce cell proliferation and invasiveness partially through its inhibitory effect on Delta-like 1 (DLL1) in choriocarcinoma [11]. [score:3]
miR-34a showed lower expression levels in specimens with lymphatic metastasis (P = 0.004, Table  1) and in the advanced clinical stages (stage III, IV vs I, II) (P < 0.001, Table  1). [score:3]
Among these, miR-34a is one of the earliest known tumor suppressors and is commonly deleted in various types of cancers. [score:3]
Smad4 is further demonstrated as one of the targets of miR-34a, which was involved in the migration and invasion in EHCC cells. [score:3]
Although there are a few samples with both miR-34a down-regulation and negative staining for Smad4 proteins by IHC, the protein level of Smad4 was increased in most of our EHCC tissues compared with NBD tissues. [score:3]
Loss of the miR-34a expression leads to an induction of Smad4 and activation of TGF-β/Smad4 signaling pathway, which accelerate CC cells invasion and migration via EMT. [score:3]
miR-34a suppresses the activation of TGF-β/Smad4 signal -induced invasion and migration in CC cell lines. [score:3]
Moreover, miR-34a can suppress tumor metastasis and invasion through a variety of signaling pathways in several cancers [10– 14]. [score:3]
These controversial results may due to the different experimental mo dels and/or the various functions of miR-34a in different diseases. [score:3]
miR-34a expression levels were detected in EHCC tissues, adjacent non-tumor tissues, normal bile duct (NBD) specimens of patients and cholangiocarcinoma (CC) cell lines by quantitative real-time polymerase chain reaction (qRT-PCR). [score:3]
In summary, our results have identified miR-34a as a tumor suppressive miRNA in human EHCC, which acts at least in part through the repression of Smad4. [score:3]
Original magnification, 100× and 400× respectively for each slide Based on the Sanger miRNA database and TargetScan software, one potential binding site of miR-34a in the 3′-UTR of Smad4 (from 2995 to 3002) was predicted (Fig.   3a). [score:3]
With the significance level for removal set at 0.05, only clinical stages was screened into the final mo del In order to determine the clinical significance of miR-34a target genes in EHCC, Sanger miRNA database (http://www. [score:3]
Immunohistochemistry was carried on to identify the downstream target gene of miR-34a in EHCC patients. [score:3]
The contrast results were observed when the CC cells were treated with the miR-34a inhibitor. [score:3]
The results of multivariate logistic regression analysis also showed that miR-34a expression related with clinical stages (P = 0.0013, Table  2). [score:3]
For those EHCC specimens, which did not have inverse correlation of miR-34a and Smad4 expression, we speculate that other factors might antagonize or interfere with the effect of miR-34a on Smad4. [score:3]
Interestingly, recent report suggested that miR-34a plays a critical role in the progression of cardiac fibrosis by increasing Smad4 expression to activate TGF-β1 [38]. [score:3]
Logistic regression analysis was performed to determine a negative correlation between Smad4 protein expression level and miR-34a level (B = −3.035, P = 0.01) among the total 27 EHCC tissues. [score:3]
miR-34a and Smad4 protein levels are inversely expressed in human EHCC tissues. [score:3]
These results identified Smad4 as a novel target of miR-34a in the EMT process of EHCC. [score:3]
We also found activation of miR-34a suppresses the invasion and migration through TGF-β/Smad4 signaling pathway in vitro. [score:3]
However, these inductions were inhibited by the treatment with miR-34a mimics in both QBC939 and HuCCT1 cells (Fig.   5b). [score:3]
c miR-34a predicts disease-free survival in EHCC patients. [score:3]
Correlation analysis between relative expressions of Smad4 and miR-34a was examined by logistic regression analysis. [score:3]
It was found that transfection with miR-34a significantly suppressed cell migration in both QBC939 and HuCCT1 cells. [score:3]
Moreover, the expression levels of miR-34a and Smad4 are inversely correlated in human clinical specimens of EHCC. [score:3]
Moreover, activation of miR-34a suppressed invasion and migration through TGF-beta/Smad4 signaling pathway by epithelial-mesenchymal transition (EMT) in vitro. [score:3]
Data are plotted as the average number of cells per field of view from three different experiments (original magnification: ×100) The relative expression level of miR-34a in EHCC tissues was significantly lower than the NBD and the adjacent non-tumor tissues (P < 0.01, Fig.   1a). [score:3]
b The mRNA expression level of miR-34a in 4 CC cell lines (QBC939, HuCCT1, RBE and HCCC9810) compared to HiBECs determined by qRT-PCR (***P < 0.01). [score:2]
analysis showed that compared with TGF-β treatment alone, transfection of miR-34a mimics increased E-cadherin expression levels while decreasing Smad4 and N-cadherin protein levels (Fig.   4a). [score:2]
Computational search, functional luciferase assay and western blot were further used to demonstrate the downstream target of miR-34a in CC cells. [score:2]
a The mRNA expression profile of miR-34a in 27 primary EHCC tissues compared to the adjacent non-tumor tissues and 7 normal bile duct tissues (NBD) determined by qRT-PCR (***P < 0.01). [score:2]
a The migration and invasive properties of EHCC cells treated with the empty vector, miR-34a mimic or miR-34a inhibitor were analyzed using a cell invasion assay in transwell chambers. [score:2]
Data are plotted as the average number of cells per field of view from three different experiments (original magnification: ×100) miR-34a is one of the most prominent miRNAs implicated in the development and progression of human cancers [7]. [score:2]
Cell morphology, invasion and migration assays were further applied to confirm the anti-carcinogenic effects of miR-34a through the downstream target. [score:2]
In the present study, we found that miR-34a expression was significantly decreased in human EHCC tissues and CC cell lines when compared with the adjacent non-tumor tissues, NBD tissues and the HiBECs. [score:2]
miR-34a expression was significantly decreased in human EHCC tissues and CC cell lines when compared with the adjacent non-tumor tissues and normal bile duct tissues. [score:2]
Moreover, both miR-34a and TGF-β/Smad4 pathway have been shown to involve in mediating metastasis and invasion in various types of cancers including cholangiocarcinoma [23, 24]. [score:1]
Cholangiocarcinoma miR-34a Smad4 Epithelial-mesenchymal transition Transforming growth factor-beta Cholangiocarcinoma (CC) is a bile duct cancer, and is classified anatomically as intrahepatic CC (IHCC) or extrahepatic CC (EHCC). [score:1]
As Smad4 is the common-smad protein for the transduction of TGF-β signaling pathway, which plays important roles through EMT in carcinogenesis [16], the repression of Smad4 by miR-34a may impair this signaling pathway in EHCC. [score:1]
miR-34a was found correlated with the migration and invasion in EHCC patients. [score:1]
Further investigation showed that miR-34a suppresses the activity of a luciferase reporter gene fused with the 3′-UTR of Smad4 mRNA, which is dependent on the miR-34a binding sequence. [score:1]
Thus, miR-34a was an independent prognostic indicator in EHCC. [score:1]
These data suggest that miR-34a is more likely involved in the metastasis or invasion during the progression of EHCC. [score:1]
Cells transfected with miR-34a scramble oligos were used as the controls (original magnification: ×200) EMT is often linked to a gain in the migratory and invasive properties of cells [19]. [score:1]
However, the role of miR-34a in EHCC has yet to be elucidated. [score:1]
The results indicated that only miR-34a was selected into the mo del (P = 0.002). [score:1]
A chemically modified antisense oligonucleotide and a synthetic miR-34a mimic (GenePharm Co. [score:1]
Quantitative analyses of the migrated and invasion cells are also shown (right panel, **P < 0.05 and ***P < 0.01 indicates miR-34a mimic, TGF-β or a mixed miR-34a and TGF-β vs. [score:1]
In this study, we evaluated the expression and effects of miR-34a on EHCC. [score:1]
To further investigate the role of miR-34a in the progression of EHCC by its ability to repress EMT, we examined the effects of miR-34a on the downstream targets of TGF-β/Smad4 pathway in both QBC939 and HuCCT1 cells. [score:1]
The cells were transfected with miR-34a mimics or scramble oligos, and simultaneously treated with TGF-β. [score:1]
β-actin levels were used as internal loading controlFurthermore, to investigate the biological function of miR-34a in CC cells, we transfected miR-34a mimic oligonucleotides or miR-34a inhibitor oligonucleotides into the EHCC cell lines (QBC939 and HuCCT1) to further increase or decrease the endogenous level of miR-34a (Fig.   3b). [score:1]
The fragment containing miR-34a binding sites in the Smad4 3′-UTR was amplified by PCR and inserted downstream of the firefly luciferase gene in a pGL3-promoter vector (Promega, Madison, WI, USA). [score:1]
a Both of the QBC939 and HuCCT1 cells were transfected with miR-34a mimics or scramble oligos, and treated with or without TGF-β simultaneously. [score:1]
Several reports have also shown that the levels of transcription factors driving EMT are controlled by miRNAs including miR-34a [26, 41– 44]. [score:1]
$$$P < 0.01 means a mixed miR-34a and TGF-β vs. [score:1]
The miR-34 family members share high sequence homology [7]. [score:1]
However, the anti-tumor function of miR-34a in EHCC is still not clear yet. [score:1]
To further evaluate the clinical value of miR-34a in EHCC patients, we divided the patients into two groups according to the median value (5.113) of the expression level of miR-34a. [score:1]
Representative images of cells in the lower section of a transwell chamber are shown to demonstrate the migration and invasive properties of QBC939 and HuCCT1 cells when transfected with the empty vector, miR-34a mimic, TGF-β or a mixed miR-34a and TGF-β (left panel). [score:1]
However, no association of miR-34a was observed with age, gender, tumor size, different pathological types, cell differentiation and Bismuth classification (P > 0.05, Table  1). [score:1]
org/) were used to predict the candidates of miR-34a. [score:1]
Cells transfected with miR-34a scramble oligos were used as the controls. [score:1]
These data suggest that miR-34a could antagonize Smad4 -mediated TGF-β induction of EMT in vitro. [score:1]
Both QBC939 and HuCCT1 cells were transfected with miR-34a mimics then treated with or without 5 ng/ml of TGF-β for 24 hrs. [score:1]
We identify miR-34a could mediate TGF-β/Smad4 signaling pathway induced EMT in the progression of cholangiocarcinoma. [score:1]
To further investigate whether miR-34a suppresses cell invasion and migration through TGF-β/Smad4 signaling pathway in EHCC, both QBC939 and HuCCT1 cells were transfected with miR-34a mimics then treated with or without TGF-β. [score:1]
β-actin levels were used as internal loading control Furthermore, to investigate the biological function of miR-34a in CC cells, we transfected miR-34a mimic oligonucleotides or miR-34a inhibitor oligonucleotides into the EHCC cell lines (QBC939 and HuCCT1) to further increase or decrease the endogenous level of miR-34a (Fig.   3b). [score:1]
For further characterization of miR-34a expression in CC cell lines, HiBECs, EHCC cell line QBC939, HuCCT1 and the IHCC cell lines RBE and HCCC9810 were examined. [score:1]
The morphological changes of EHCC cells were detected after transfected with miR-34a mimics and/or treated with TGF-β. [score:1]
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[+] score: 398
Studies have shown that miR-34a suppresses brain tumor growth by targeting c-Met and Notch [25], suppresses proliferation and migration through the down-regulation of c-Met in uveal melanoma cells [26], and inhibits migration and invasion through down-regulation of c-Met expression in human hepatocellular carcinoma cells [24]. [score:17]
Studies have reported that miR-34a directly repressed the expression of c-Met in HeLa cells [16], suppressed brain tumor growth by targeting c-Met [25], and acted as a tumor suppressor in uveal melanoma cell proliferation and migration through the down-regulation of c-Met [26]. [score:13]
Over -expression of miR-34a down-regulated the expression of c-Met protein and mRNA simultaneously, suggesting that miR-34a functions as tumor suppressors probably through down -regulating c-Met in osteosarcoma. [score:11]
We focused on miR-34a because not only previous reports demonstrated that the expression of miR-34a was significantly decreased in primary osteosarcoma samples as compared with adjacent normal tissues [23], but also the mutations of p53 tumor suppressor gene, which directly regulates the expression of miR-34a, was also found in 20–60% of sporadic osteosarcomas [16], [37]. [score:9]
In addition, miR-34a could specifically down-regulate the expression of the metastasis related gene c-Met, indicating that miR-34a may function as a tumor gene suppressor through down -regulating c-Met oncogene. [score:9]
Therefore, in SOSP-9607 cells, we further investigated whether over -expression of miR-34a also down-regulated the expression of c-Met and whether c-Met inhibited the migration and invasion as that in other cells. [score:8]
These results together indicate that miR-34a down-regulates c-Met expression in osteosarcoma at the translational level and reduces mRNA stability simultaneously. [score:8]
Studies have demonstrated that miR-34a, which is a direct target of the p53 tumor suppressor gene, functions as a tumor suppressor and is associated with the tumor growth and metastasis of various human malignances. [score:8]
Therefore, up-regulated expression of miR-34a was very effective on inhibiting the tumor growth and metastasis behaviors of osteosarcoma cells both in vitro and in vivo. [score:8]
We predicted putative miR-34a target genes by using online softwares, such as TargetScan 5.1 and PicTar, and finally obtained several putative target genes that are correlated with the tumor growth or metastasis (Table 1, 2). [score:7]
The results presented in this study demonstrated that over -expression of miR-34a could inhibit the tumor growth and metastasis of osteosarcoma probably through down regulating c-Met. [score:6]
miR-34a is a direct transcriptional target of p53 tumor suppressor [16]. [score:6]
miR-34a plays its tumor inhibitory effect by down -regulating its targets such as CDK4, CDK6, E2F3, E2F5 et al [18], [19]. [score:6]
However, considering that the miR-34a-c-Met pathway may show different patterns in different cell backgrounds, we further investigated whether over -expression of miR-34a also down-regulated the expression of c-Met in osteosarcoma cells. [score:6]
miR-34a functions as a tumor suppressor gene by down -regulating its targets such as CDK4, CDK6, E2F3, E2F5 et al [18], [19]. [score:6]
miR-34 family are direct transcriptional targets of p53 tumor suppressor [16]. [score:6]
Therefore, we inferred that miR-34a may play an important role in inhibiting tumor metastasis and proliferation through down -regulating multiple target genes, including genes in Notch and Wnt signaling pathways. [score:6]
These results indicated that pcDNA-miR34a can up-regulate miR-34a expression in both SOSP-9607 cells and SAOS-2 cells, which facilitated the further study of miR-34a functions in osteosarcoma. [score:6]
We supposed that miR-34a may prove to be a promising gene therapeutic agent which functions as a tumor suppressor gene through down -regulating multiple target oncogenes in osteosarcoma. [score:6]
Over -expression of miR-34a partially inhibited proliferation, migration and invasion of osteosarcoma cells in vitro, as well as the tumor growth and pulmonary metastasis of osteosarcoma cells in vivo. [score:5]
By using TargetScan 5.1 and PicTar, we predicted putative genes of miR-34a which have not been experimentally identified yet, and finally obtained several putative targets which are correlated with tumor growth or metastasis, such as BCL6, CCNE2, CD97, CSNK1G3, CTNND1, DLL1, DKK1, GAS1, IGFBP3, LEF1, PGEA1, POFUT1, R-RAS, RUNX2, UHRF2, VCL, VEGFA, etc. [score:5]
Meanwhile, mutations of the two c-Met 3′UTR -binding sites abolished the ability of miR-34a to regulate luciferase expression (Figure 6C). [score:5]
Meanwhile, mutations of the two c-Met 3′UTR -binding sites abolished the ability of miR-34a to regulate luciferase expression. [score:5]
As expected, c-Met also regulated the migration and invasion of SOSP-9607 cells, and c-Met is indeed a direct target of miR-34a in SOSP-9607 cells. [score:5]
These results indicate that miR-34a functions as a tumor suppressor gene and can be used as a potential target in the gene therapy of osteosarcoma. [score:5]
For the first time, we reported that over -expression of miR-34a inhibited growth and metastasis of osteosarcoma cells both in vitro and in vivo. [score:5]
we demonstrated that over -expression of miR-34a significantly suppresses proliferation,migration and invasion of SOSP-9607 cells in vitro. [score:5]
The result showed that the miR-34a expression level of G418 sellected cells, in the orthotopic tumors after 6 weeks of inoculation, was indeed over-expressed as that in vitro. [score:5]
Osteosarcoma cells over -expressing miR-34a exhibited a significant decrease in the expression levels of c-Met mRNA and protein simultaneously. [score:5]
Therefore, to thoroughly understand the regulatory mechanism of miR-34a in osteosarcoma,it is critical to identify more target genes that mediate the miR-34a induced regulation of tumor growth and metastasis. [score:5]
It has been reported that hepatocellular carcinoma cells transfected with miR-34a mimics are inhibited of both migration and invasion [24], and the expression of miR-34a is associated with the tumorigenesis of osteosacoma [23]. [score:5]
SOSP-9607 cells were transiently transfected with 50 nM of miR-34a mimics, mimics NC, miR-34a inhibitor and inhibitor NC (Genepharma, China), respectively. [score:5]
The inactivating mutations of p53 often cause a decreased expression of miR-34a in tumors [17]. [score:4]
In the present studies, we performed in vitro and in vivo experiments to evaluate the effects of miR-34a on tumor growth and metastasis of SOSP-9607 cells, as well as the expression of c-Met, because c-Met is a direct target of miR-34a and correlated to the metastasis potential of tumors. [score:4]
We also repeated the luciferase assay to validate c-met as a target of miR-34a in osteosarcoma cells SOSP-9607, and tested whether c-Met inhibits the migration and invasion of SOSP-9607 cells as that in other cells. [score:4]
Meanwhile, the miR-34a expression levels in the orthotopic tumors were also tested, and the result showed that the orthotopic tumors in the miR-34a group expressed higher miR-34a levels as compared with control group (Figure 4E). [score:4]
These results indicated that miR-34a may suppress tumor growth and metastasis in osteosarcoma cells through down -regulating c-Met oncogene. [score:4]
6. c-Met is a target of miR-34a, and regulates the migration and invasion of osteosarcoma cells. [score:4]
Mol Pharmacol 54 Wei JS Song YK Durinck S Chen QR Cheuk AT 2008 The MYCN oncogene is a direct target of miR-34a. [score:4]
Predicting and identifying the miR-34a -targeting genes offer experimental basis for further research on regulatory mechanism of miR-34a. [score:4]
And there are other putative miR-34a target genes beside c-Met which could potentially be key players in the development of osteosarcoma. [score:4]
0033778.g001 Figure 1(A) Schematic representation of miR-34a expression vector. [score:3]
The procedure of miR-34a eukaryotic expression vector construction was referred to the method described previously [65]. [score:3]
The prediction of miR-34a putative target genes was performed using bioinformatics methods based on sequence similarity between miRNAs and mRNAs. [score:3]
Finally, there are other putative miR-34a target genes beside c-Met which may mediate the miR-34a induced inhabitation of tumor growth and metastasis in osteosarcoma. [score:3]
So, we further tested the miR-34a expression levels in the orthotopic tumors 6 weeks after inoculation. [score:3]
5. miR-34a inhibits pulmonary metastasis of osteosarcoma in vivo. [score:3]
7. Putative Targets of miR-34a. [score:3]
3. miR-34a inhibits migration and invasion of osteosarcoma in vitro. [score:3]
Furthermore, there are other putative miR-34a target genes which could potentially be key players in the growth and metastasis of osteosarcoma cells. [score:3]
Both of the previous reports suggested that miR-34a may function as a tumor suppressor in osteosarcoma. [score:3]
Because we couldn't maintain the G418 selection in vivo, the selected cells may not maintain miR-34a over -expression as that in vitro. [score:3]
Moreover, Li et al. found that miR-34a inhibits migration and invasion of human hepatocellular carcinoma cells [24]. [score:3]
4. miR-34a inhibits tumor growth of osteosarcoma in vivo. [score:3]
Taken together, these results strongly suggest that miR-34a can inhibit osteosarcoma metastasis and might be prevention of metastasis and recurrence in osteosarcoma patients. [score:3]
The results demonstrated that miR-34a also significantly inhibited the capacities of orthotopic tumor growth and lung metastasis in vivo. [score:3]
Bioinformatics methods based on sequence similarity between miRNAs and mRNAs were used to predict the putative target genes of miR-34a. [score:3]
We also tested SAOS-2 cells transiently transfected with either pcDNA3.1 or pcDNA-miR34a (Figure 3E, F, G, H) and SOSP-9607 cells transiently transfected with either miR-34a mimics or inhibitors (Figure 2C, D, E, F), the results were similar to that of stable transfected SOSP-9607 cells. [score:3]
Putative Targets of miR-34a. [score:3]
It will be interesting to verify the putative target genes and further investigate the mechanism by which miR-34a functions as a tumor suppressor gene in osteosarcoma. [score:3]
We also performed the prediction of miR-34a putative target genes which are correlated to tumor growth and metastasis by using bioinformatics analysis. [score:3]
2. miR-34a inhibits proliferation of osteosarcoma in vitro. [score:3]
Then the orthotopic tumors were weighed, the miR-34a expression levels in the orthotopic tumors were tested by real time RT-PCR, and the number of pulmonary matastatic tumor nodules was counted under a low-powered dissecting stereomicroscope. [score:3]
Both of the results indicated that ectogenous miR-34a can significantly inhibit the tumor growth of osteosarcoma in vivo. [score:3]
Interestingly, many genes in Notch and Wnt signaling pathways are putative targets of miR-34a. [score:3]
We also tested SAOS-2 cells transiently transfected with either pcDNA3.1 or pcDNA-miR34a (Figure 2B) and SOSP-9607 cells transiently transfected with either miR-34a mimics or inhibitors (Figure S2A, B). [score:3]
Meanwhile, there are several genes which mediate the miR-34a induced tumor growth inhibition, such as BCL2, E2F3 and MYCN in neuroblastoma [52], [54], mitogen-activated protein kinase kinase 1 (MEK1) in human chronic myelocytic leukemia cell line K562 [53] and SIRT1 in prostate cancer PC3 cells [58]. [score:3]
Finally, the results from bioinformatics analysis demonstrated that there were multiple putative targets of miR-34a that may be associated with the proliferation and metastasis of osteosarcoma, including factors in Wnt and Notch signaling pathways. [score:3]
The results demonstrated that, in SOSP-9607 cells, the luciferase activity of the pmiR-Met UTR-Wt construct was significantly inhibited after the introduction of miR-34a, similar to those reported by others [27]. [score:3]
And then, we reviewed experimentally identified miR-34a target genes, such as BCL2 [52], CCND1 [18],CDK6 [18], E2F3 [38], JAG1 [40], Mek1 [53], MET [25], MYCN [54], NOTCH1 [55], NOTCH2 [55], SIRT1 [56], WNT1 [57] et al (Table 2). [score:3]
Among them, there are several genes, which mediate the inhibition of miR-34a induced metastasis in human malignances, such as Notch1 and JAG1 in cervical carcinoma and choriocarcinoma cells [40] and c-Met in in human hepatocellular carcinoma cells [24]. [score:3]
Experimentally identified Targets of miR-34a. [score:3]
It is supposed that the miR-34a-c-Met pathway might be a general regulator of tumor growth and metastasis in a wide range of human malignances, including brain tumor, uveal melanoma, hepatocellular carcinoma and osteosarcoma. [score:2]
These results indicate that miR-34a plays an important role in the development of osteosarcoma. [score:2]
For validation of c-Met as a target genes of miR-34a in osteosarcoma cells, luciferase assay was performed as described previously [26]. [score:2]
It is likely that miR-34a may also regulate other genes beside c-Met. [score:2]
The results showed that cells in miR-34a group expressed a higher level of miR-34a as compared with control group and blank group,respectively. [score:2]
Pang et al. found that MiR-34a suppresses invasion of cervical carcinoma and choriocarcinoma cells [40]. [score:2]
We examined the c-Met expression level of osteosarcoma cells in three groups SOSP-9607 cells, and observed a significant decrease of c-Met mRNA and protein levels in miR-34a group cells as compared with blank group and control group. [score:2]
For establishing stable transfectants, SOSP-9607 cells were transfected with either pcDNA-miR34a vector or pcDNA3.1 vector. [score:1]
Representative photographs of migrated and invaded SAOS-2 cells (pcDNA3.1, SAOS-2 cells transiently transfected with pcDNA3.1; pcDNA-miR34a, SAOS-2 cells transiently transfected with pcDNA-miR34a) on the membrane at a magnification of 100× (E, F). [score:1]
Therefore, experiments with SOSP-9607 cells were divided into three groups as blank group (SOSP-9607 cells), control group (stable SOSP-9607 cells transfected with pcDNA3.1) and miR-34a group (stable SOSP-9607 cells transfected with pcDNA-miR34a). [score:1]
The miR-34a expression levels in SAOS-2 cells transiently transfected with either pcDNA3.1 or pcDNA-miR34a was also measured, and a similar result was shown (Figure 1C). [score:1]
In conclusion, the results presented here demonstrated that miR-34a has great biological effects on the growth and metastasis of osteosarcoma cells both in vitro and in vivo. [score:1]
However, tumor nodules (arrows) in miR-34a group were smaller and distributed in a lower concentration as comparing with control group. [score:1]
However, the functions of miR-34a in osteosarcoma have not been totally elucidated. [score:1]
Two groups of stable transfected cells (control group, stable SOSP-9607 cells transfected with pcDNA3.1; and miR-34a group, stable SOSP-9607 cells transfected with pcDNA-miR34a) were injected into proximal tibia of young nude mice as described in methods, respectively. [score:1]
The inactivation and absence of miR-34a is related to the pathogenesis of a variety of tumors [17], [21], [22], including osteosarcoma [23]. [score:1]
By contrast, cells in miR-34a group produced much smaller tumors (Figure 4A, C). [score:1]
Figure S1 DNA sequencing of plasmid pcDNA-miR34a. [score:1]
Initial links to tumorigenesis emerged from Welch et al. who found that miR-34a, whose encoding gene is on chromosome 1p36, is correlated with tumor occurrence and frequently missed in human neuroblastoma [38]. [score:1]
However, we can't imprudently assume that miR-34a would also have biological activity in vivo. [score:1]
The mean tumor weight±SD of orthotopic tumors were as follows: miR-34a group 1.132±0.177 g, control group 1.768±0.341 g (Figure 4B, D). [score:1]
There is a highly conserved p53 binding site which is approximately 30 kb above the miR-34a encoding gene [39]. [score:1]
Since pulmonary metastases are responsible for mortality of patient carrying osteosarcoma, miR-34a may prove to be a promising gene therapeutic agent. [score:1]
An average of 26.2±12.4 metastatic tumor nodules were detected per lung in miR-34a group, while mice in control group produced an average of 96.7±20.5 metastatic tumor nodules per lung (Figure 5B), indicating that miR-34a significantly decreased tumor colonization in the lung. [score:1]
Finally, because pulmonary metastases are responsible for mortality of patient carrying osteosarcoma, miR-34a may prove to be a promising gene therapeutic agent. [score:1]
To facilitate the investigation of the effects of miR-34a on osteosarcoma, a has-miR-34a eukaryotic expression vector, named pcDNA-miR34a, was constructed (Figure 1A). [score:1]
miR-34a is a member of an evolutionarily conserved miRNA family, miR-34s. [score:1]
Therefore, it is of great significance to further study the function and mechanism of miR-34a in Osteosarcoma. [score:1]
However, the effects of miR-34a on osteosarcoma have not been totally elucidated. [score:1]
SOSP-9607 cells were transfected with either pcDNA-miR34a or pcDNA3.1 and then G418-slected for 6 weeks to generate two stable SOSP-9607 cells (stable SOSP-9607 cells transfected with pcDNA3.1 and pcDNA-miR34a, respectively). [score:1]
The restriction enzyme cutting sites of BamH I and Hind III were underlined; the pri-miR-34a sequences were highlighted. [score:1]
, strongly indicated that miR-34a was an important participant in the reduction of migratory and invasive potential of osteosarcoma in vitro. [score:1]
Representative photographs of migrated and invaded SOSP-9607 cells (Blank, SOSP-9607 cells; control, stable SOSP-9607 cells transfected with pcDNA3.1; miR-34a, stable SOSP-9607 cells transfected with pcDNA-miR34a) on the membrane at a magnification of 100× (A, B). [score:1]
It will be interesting to further investigate the mechanism by which miR-34a functions as a tumor suppressor gene in osteosarcoma. [score:1]
However, there is no study on the role of miR-34 in osteosarcoma metastasis. [score:1]
The results from RT-PCR demonstrated that the endogenous c-Met mRNA level in miR-34a group cells was also significantly decreased (Figure 6B). [score:1]
The results showed that the growth velocities of orthotopic tumors in miR-34a group (stable SOSP-9607 cells transfected with pcDNA-miR34a. ) [score:1]
The lungs of miR-34a group mice contained less and smaller spontaneous metastases as comparing with control group (Figure 5C). [score:1]
The miR-34a expression levels in three groups were measured using Stem-loop Real-time RT-PCR. [score:1]
miR-34a also plays an important role in the p53 -induced cell cycle arrest, cell senescence, apoptosis and other biological behavior [20]. [score:1]
However, the role of miR-34a in osteosarcoma has not been totally elucidated. [score:1]
In brief, two groups SOSP-9607 cells (control group, stable SOSP-9607 cells transfected with pcDNA3.1; miR-34a group, stable SOSP-9607 cells transfected with pcDNA-miR34a) were harvested by treatment with trypsin-EDTA (Invitrogen), washed twice with PBS, and resuspended in PBS. [score:1]
WNT1 pictar Proliferation; cell migration; Wnt signaling pathway To facilitate the investigation of the effects of miR-34a on osteosarcoma, a has-miR-34a eukaryotic expression vector, named pcDNA-miR34a, was constructed (Figure 1A). [score:1]
The mRNA levels of c-Met in three groups of SOSP-9607 cells (blank, SOSP-9607 cells; control, stable SOSP-9607 cells transfected with pcDNA3.1; miR-34a, stable SOSP-9607 cells transfected with pcDNA-miR34a. ) [score:1]
The stable transfectants were then expanded and the expression of miR-34a was evaluated by real time RT-PCR. [score:1]
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[+] score: 370
Other miRNAs from this paper: hsa-mir-34b, hsa-mir-34c
Consistent with these C. elegans results, of a published GEO dataset for hippocampus of wild-type adult male C57BL/6 mice 44 revealed upregulation of genes related to extracellular matrix, cell adhesion, basement membrane and anti-apoptosis when mir-34 was knocked down by adeno -associated viral (AAV)- delivered mir-34 sponges (180 upregulated and 36 downregulated genes, FDR < 0.01) (Table S3). [score:11]
We observed reduced stress resistance in both mir-34 mutants and overexpressors, supporting the role of this feedback inhibition loop in regulation of mir-34 and DAF-16 levels to reduce the fluctuations in daf-16 and myc network target expression levels under stress conditions. [score:10]
We conclude that mir-34 upregulation is necessary for inducing developmental arrest with correct morphogenesis and enhanced survival of dauers, and that this role of mir-34 relies on a functional insulin signaling receptor, DAF-2. mir-34 is regulated by DAF-16, PQM-1 and DAF-12The insulin signaling pathway regulates dauer-related phenotypes and responses to stress conditions by regulating nuclear localization of its downstream target transcription factor, DAF-16/FOXO 32 33. [score:10]
According to this regulatory loop, if miR-34 becomes upregulated above threshold levels, mir-34 expression is lowered via the feedback inhibition of DAF-16, which results in reduced stress resistance. [score:9]
Supplementary Table 1. Supplementary Table 2. Supplementary Table 3. P mir-34 [2.2kb] ::gfp is expressed in various tissues during development of C. elegans and its expression is upregulated in dauers. [score:9]
We conclude that mir-34 upregulation is necessary for inducing developmental arrest with correct morphogenesis and enhanced survival of dauers, and that this role of mir-34 relies on a functional insulin signaling receptor, DAF-2. The insulin signaling pathway regulates dauer-related phenotypes and responses to stress conditions by regulating nuclear localization of its downstream target transcription factor, DAF-16/FOXO 32 33. [score:9]
A mir-34 rescue strain, which has a single copy insertion of mir-34 and restores expression of miR-34 to 70% of the wild type level (Fig. 2A), as well as a mir-34 overexpression strain (mir-34OE), which has 4 copies of mir-34 and expresses miR-34 3-fold higher than wild type (Fig. 2A), rescue these morphological defects (Fig. S1). [score:7]
Genes that were expressed higher in the absence of mir-34 were significantly enriched for DAF-16 binding elements (DBE) and AGO-CLIP supported miR-34 targets, whereas genes which were expressed higher in the wild-type dauers were significantly depleted for DAF-16 binding but enriched for PQM-1 binding the DAE (Fig. 4B). [score:7]
These data suggest that differential expression of genes between wild-type and mutant dauers is the result of both direct regulation by miR-34 and indirect regulation via DAF-16/PQM-1 binding. [score:7]
We observed a large overlap between class 1 genes, dauer related genes, and genes that were up-regulated in mir-34(gk437) dauers, and between class 2 genes, non-dauer genes, and genes that were down-regulated in mir-34(gk437) dauers (Fig. S4). [score:7]
The same expression patterns were observed in dauers of the transgenic P mir-34 [5kb] ::gfp reporter 26, suggesting that all crucial regulatory elements for dauer related upregulation of mir-34 were located within the 2.2 kb upstream region. [score:7]
If miR-34 expression helps in establishing the stress response program, then gene expression changes when mir-34 is overexpressed under normal conditions should overlap with stress response genes that are observed in WT animals grown under heat stress. [score:7]
Although the observed changes in daf-16 expression upon miR-34 overexpression are not large, combined with the experimental AGO-CLIP data 39 and DAF-16::GFP reporter analysis results, they suggest direct regulation of daf-16 by miR-34. [score:7]
In mir-34OE and mir-34 mutant dauers a large number of genes were differentially expressed compared to WT dauers (1157 and 4652, respectively; Fig. 4B), consistent with the observed phenotypes and upregulated mir-34 expression pattern in dauers. [score:7]
GO term analysis of genes that were upregulated in the mir-34(gk437) dauer background and had ALG-1 binding sites and/or MIRZA scores higher than 100 revealed upregulation of genes encoding glycoproteins, cytoskeletal genes, intermediate filaments, extracellular matrix proteins, transmembrane and transport proteins (Table S2). [score:7]
In mutants that enhance temperature -induced dauer formation, the P mir-34 [2.2kb] ::gfp transgene expression patterns were similar to those seen in the wild-type (WT) background, implying that high mir-34 expression derived from differential gene expression at the dauer stage, and not from starvation conditions (Fig. 1C). [score:7]
mir-34 expression is regulated by the dauer larva gene expression program. [score:6]
Stress -induced upregulation of mir-34 expression was observed in starved worms, dauers and older adults 26 31, which was recapitulated by our P mir-34 [2.2kb] ::gfp transgenic line (Fig. 1A–D). [score:6]
Further evidence for a DAF-16- mir-34 feedback inhibition loopFinally, we sought additional evidence for daf-16 regulation by miR-34 in a daf-16::gfp reporter strain and in gene expression data. [score:6]
In this study, we demonstrated that miR-34 levels are upregulated to sustain a gene expression program that is associated with morphological and metabolic adaptation of stress. [score:6]
mir-34 expression is regulated by the dauer larva gene expression programTo study the relationship between mir-34, cell cycle arrest and stress, we focused on the dauer stage of C. elegans, which is the stress-resistant diapause stage that forms under harsh environmental conditions such as crowding, high temperatures and starvation 28 29. [score:6]
The highest expression levels were observed with insulin signaling pathway mutant dauers (Fig. 1C ii and iii), suggesting the possibility for the involvement of DAF-16/FOXO in the regulation of mir-34 expression. [score:6]
Furthermore, in N2 animals shift from 20 °C to 25 °C does not significantly change daf-16 levels (6% change, P = 0.523) but overexpression of miR-34 at 25 °C leads to a 25% downregulation of daf-16 compared to WT at 20 °C (P = 0.012). [score:5]
However, several other autophagy-related genes (lgg-1, atg-13, atg-16.2, unc-51, bec-1) were downregulated in mir-34OE dauers compared to mir-34 mutants (Table S1), which may suggest autophagy inhibition by mir-34 as was proposed in the aforementioned study 57. [score:5]
Detailed MIRZA alignment of the miR-34 target in daf-16 mRNA shown in panel C. (E) Expression of DAF-16::GFP is elevated with temperature in amphid neurons (indicated by arrows) in mir-34(gk437) mutants but not in wild-type animals. [score:5]
Small internal promoter deletions in the region bound by these TFs revealed that DAF-12 binding elements, insulin response element (IRE) and GA-repeats were required for mir-34 expression in hypodermis and seam cells (Fig. S2), and DAF-16 was necessary for activation of P mir-34 [2.2kb] ::gfp expression in dauers (Fig. 1C and Fig. S3A) and in amphid neurons, especially AWC neurons of adults (Fig. S3D). [score:5]
A good target would have a MIRZA score above 50, and Table S1 also lists how many miR-34 targets were found in a gene, and their cumulative MIRZA score. [score:5]
Effects of mir-34 deletion or overexpression on gene expression under various conditions. [score:5]
miR-34 targets daf-16To understand the molecular programs underlying phenotypic changes observed in the mir-34 mutant and overexpression strains, we identified experimentally supported targets of miR-34 using Argonaute crosslinking and immunoprecipitation (AGO-CLIP) data generated by Grosswendt et al. 39, in combination with calculated by MIRZA software 40. [score:5]
These results suggest that mir-34 upregulation is dependent upon DAF-16 in the dauer stage. [score:4]
We also observed that upregulation of P mir-34 [2.2kb] ::gfp in dauers is abolished by mutating this region, and in the daf-16(mu86) background. [score:4]
While in wild-type animals temperature shift from 20 °C to 25 °C resulted in 1891 and 2425 down- and up- regulated genes respectively (Fig. 4D), the number of differentially expressed genes was smaller in mir-34(gk437) (1192/1709 genes) and mir-34OE (1008/1442) backgrounds (Fig. 4D). [score:4]
Thus, our results suggest that mir-34 is involved in a feedback inhibition loop that includes the daf-16 and myc networks to regulate a stress response program in C. elegans (Fig. S6). [score:4]
Furthermore, analysis of P mir-34 [2.2kb] ::gfp in excretory gland cells in various mutant background showed a direct correlation between DAF-16 levels and reporter expression (Fig. S3B,C). [score:4]
Finally, we sought additional evidence for daf-16 regulation by miR-34 in a daf-16::gfp reporter strain and in gene expression data. [score:4]
Additionally, mdl-1 and mxl-3, from the Myc-like interaction network in C. elegans, showed mir-34 dependent downregulation under high temperature growth, suggesting that the myc network is a part of the stress response pathway that is modulated by daf-16 and mir-34. [score:4]
We showed that mir-34 mutation results in morphogenesis defects of dauers, which correlates with our transcriptome analysis results that shows deregulation of cell adhesion, cytoskeleton, ECM and basement membrane related gene categories both in of mir-34 mutant dauers and mir-34 knockdown mouse hippocampus. [score:4]
Therefore, we think that upregulation of mir-34 is necessary for correct morphogenesis of tissues to ensure long survival of dauers. [score:4]
Additionally, ATG9A did not show a significant change in expression levels in mir-34 knockdown in male mouse hippocampus (Table S3). [score:4]
We identified the minimal promoter region responsible for mir-34 upregulation by generating several P mir-34 [2.2kb] ::gfp strains with shorter upstream regions relative to the initial 2.2 kb promoter (Fig. 3A). [score:4]
mir-34 is regulated by DAF-16 and targets daf-16. [score:4]
Upregulation of mir-34 in hypodermis and seam cells and the morphological defects of mir-34(gk437) mutants correlate with these GO terms. [score:4]
miR-34 targets daf-16. [score:3]
Construction of mir-34 rescue and overexpression strains. [score:3]
The predicted miR-34 target region is located in the last coding exon of daf-16, not far from the stop codon (Fig. 3C). [score:3]
The mdl-1 gene promoter is bound by DAF-16 and PQM-1, according to modENCODE data 35, and the mdl-1 mRNA is targeted by miR-34 according to AGO-CLIP data 39 and MIRZA prediction, although the MIRZA score is modest (Table S1). [score:3]
qPCR validation of miR-34 expression. [score:3]
Furthermore, according to our combined analysis of MIRZA scores and AGO-CLIP data, one of the top predicted miR-34 targets is daf-16/FOXO. [score:3]
We observed higher DAF-16::GFP levels in mir-34(gk437) mutants grown at high temperatures (P = 0.0175, t test), accompanied by higher levels of nuclear localization of the translational fusion protein in amphid neurons, however, there were no significant differences under normal growth conditions (Fig. 3E,F). [score:3]
P mir-34 [2.2kb] ::gfp levels were similar to WT levels in daf-2(e1370);daf-16(mu86) background (Fig. S3D), suggesting that other factors were also involved in mir-34 induction upon inhibition of insulin signaling pathway. [score:3]
miR-34 expression is necessary for inducing stress response programs. [score:3]
The number of differentially expressed genes was highly diminished in mir-34(gk437) mutants when the comparison was done in the daf-2(e1370) background, where nuclear DAF-16 levels are saturated (Fig. 4C). [score:3]
Construction of mir-34 rescue and overexpression strainsP mir-34 [2.2kb]:: mir-34 was cloned into MosSCI plasmid and isolated plasmid was microinjected into N2 worms together with marker plasmids. [score:3]
The levels of daf-16 mRNA decreased by 12% and 8%, respectively, in adults and dauers overexpressing miR-34 at 20 °C, although the statistical significance of these changes is low (adjusted P value 0.41 and 0.58 respectively). [score:3]
We demonstrated DAF-16 dependent changes in the transcriptomes of animals that lack and overexpress mir-34. [score:3]
To understand the molecular programs underlying phenotypic changes observed in the mir-34 mutant and overexpression strains, we identified experimentally supported targets of miR-34 using Argonaute crosslinking and immunoprecipitation (AGO-CLIP) data generated by Grosswendt et al. 39, in combination with calculated by MIRZA software 40. [score:3]
At 25 °C, overexpression of miR-34 in adults results in a 17% decrease of daf-16 levels (adjusted P = 0.161). [score:3]
However, we found that in C. elegans atg-9 mRNA expression was lower in mir-34 and daf-2;mir-34 backgrounds, and did not observe a significant change in atg-9 transcript levels in adult stages (Table S1). [score:3]
mir-34 regulates dauer morphogenesis and survival dependent upon the insulin signaling pathwayWe investigated the role of mir-34 upregulation in dauers by studying dauer morphogenesis and survival in mir-34 mutants. [score:3]
Native levels of mir-34 expression are required for correct morphogenesis of dauers and dauer survival. [score:3]
In predauers, P mir-34 [2.2kb] ::gfp expression was increased in amphid neurons, especially in AWC neurons. [score:3]
In line with these findings, our results suggest that mir-34 has an evolutionarily conserved function in orchestrating responses to stresses, by modulating expression levels of DAF-16/FOXO and the Myc network. [score:3]
Additionally, glucose supplementation reduced P mir-34 [2.2kb] ::gfp levels (Fig. S3C), and prolonged stress conditions resulted in reduction of P mir-34 [2.2kb] ::gfp expression in many tissues of the worms. [score:3]
Further evidence for a DAF-16- mir-34 feedback inhibition loop. [score:3]
There were only 28 such genes for which expression increased with temperature in WT animals but decreased in mir-34(gk437) animals (Fig. 4H), including mdl-1 and mxl-3. mdl-1 is a basic helix-loop-helix (bHLH) transcription factor that acts as a part of the Myc-like interaction network in C. elegans. [score:3]
The analysis of these strains indicated that sequences between 0.5 kb and 1.2 kb upstream of mir-34 gene are essential for its regulation (Fig. 3B). [score:2]
These results suggest that indeed mir-34 plays a direct role in establishing the stress response program. [score:2]
The combination of AGO-CLIP evidence and highly-scoring MIRZA prediction suggests that daf-16 is very likely regulated by miR-34 and suggests existence of a negative feedback-loop between daf-16 and mir-34. [score:2]
mir-34 is regulated by DAF-16, PQM-1 and DAF-12. [score:2]
Other transcription factors, which showed same pattern as mdl-1 in terms of sensitivity to mir-34 levels included nhr-23, egl-13 and zip-7. NHR-23 is a critical co-regulator of functionally linked genes involved in growth and molting. [score:2]
The survival defect of mir-34 mutants required a functional insulin signaling pathway, where DAF-16 nuclear localization levels are not saturated, and probably DAF-16 activity is more prone to regulation by mir-34. [score:2]
Additionally, compared to WT dauers, mir-34(gk437) mutants had a shorter body size, and worms that overexpress mir-34 had a slightly longer body size (Fig. 2B). [score:2]
Additionally, the mir-34(gk437) mutation enhanced dauer formation in daf-7(e1372) mutant background but it did not have an effect on dauer formation in daf-2(e1370) mutants (Fig. 2D). [score:2]
mir-34 regulates dauer morphogenesis and survival dependent upon the insulin signaling pathway. [score:2]
We investigated the role of mir-34 upregulation in dauers by studying dauer morphogenesis and survival in mir-34 mutants. [score:2]
Since daf-16(mu86) null mutants cannot form dauers, this mutation was introduced into the daf-7(e1372);P mir-34 [2.2kb] ::gfp strain, which is dauer constitutive at 25 °C, in order to investigate whether the high expression of mir-34 in daf-7(e1372) mutant dauers was dependent upon DAF-16. [score:2]
For example, mir-34 deletion mutants do not show any abnormal morphological, developmental or biological phenotypes under standard laboratory culture conditions. [score:2]
According to a previous study the autophagy-related mRNA ATG9A was regulated by mir-34 in mammalian cells 57. [score:2]
Which genes might be particularly sensitive to miR-34 levels in this stress response program? [score:1]
To study the relationship between mir-34, cell cycle arrest and stress, we focused on the dauer stage of C. elegans, which is the stress-resistant diapause stage that forms under harsh environmental conditions such as crowding, high temperatures and starvation 28 29. [score:1]
Thus, a strong daf-2/dauer transcriptional signature is present in mir-34(gk437) mutant dauers, as it is also evident from our transcriptome analysis (Fig. S5A (i, ii)). [score:1]
In line with these results, both mir-34(gk437) and mir-34OE adults were more sensitive to hypoxia, heat stress, and starvation. [score:1]
Although mir-34 mutant dauers exhibit more a dauer-related transcriptional signature, changes in gene category representation are accompanied by the body defects and short survival rates of mir-34 mutants. [score:1]
This suggests that precise levels of miR-34 are required to elicit proper response to heat stress, and that deviations from these levels impair the stress response program. [score:1]
qPCR was performed on N2, mir-34(gk437) and N2 and mir-34(gk437) worms carrying P mir-34 [2.2kb]:: mir-34 transgene by using TaqMan kit. [score:1]
This finding was in line with the lower amount of phenotypic changes observed between daf-2(e1370) and daf-2(e1370);mir-34(gk437) mutant worms. [score:1]
These transcription factors may also be responsible for miR-34 dependent transcriptome and phenotypic changes under stress conditions. [score:1]
At the same time, there was no significant difference between daf-2(e1370) and daf-2(e1370);mir-34(gk437) worms in terms of body size and survival (data not shown). [score:1]
Around 80% (250 dauers tested) of mir-34(gk437) dauers that were selected from starved plates had locomotion defects, and were rolling along their body axis. [score:1]
How to cite this article: Isik, M. et al. MicroRNA mir-34 provides robustness to environmental stress response via the DAF-16 network in C. elegans. [score:1]
Transcriptome analysis reveals the crosstalk between DAF-16 and mir-34To investigate the possible crosstalk between mir-34 and DAF-16, we performed microarray gene expression analysis for several genetic backgrounds and stress conditions (Table S1, Fig. 4). [score:1]
It has a MIRZA score of 625, and exhibits perfect complementarity to nucleotides 1–8 of the mature miR-34 sequence (Fig. 3D). [score:1]
Transcriptome analysis reveals the crosstalk between DAF-16 and mir-34. [score:1]
This suggests that the C. elegans myc network may play an important role in modulating a stress response program downstream of mir-34. [score:1]
By engineering promoter truncations we showed that an IRE sequence, which binds DAF-16, is present in mir-34 promoter. [score:1]
P mir-34 [2.2kb]:: mir-34 was cloned into MosSCI plasmid and isolated plasmid was microinjected into N2 worms together with marker plasmids. [score:1]
To investigate the possible crosstalk between mir-34 and DAF-16, we performed microarray gene expression analysis for several genetic backgrounds and stress conditions (Table S1, Fig. 4). [score:1]
MIRZA, miR-34 target predictions calculated by MIRZA. [score:1]
ALG – presence of AGO-CLIP regions from Grosswendt et al. 39, overlapping miR-34 MIRZA predictions. [score:1]
Furthermore, mir-34(gk437) mutant dauers exhibited a lower survival rate (at both 20 °C and 25 °C) than WT dauers (Fig. 2C). [score:1]
However, miR-34 is critical in the DNA damage response in both mammals and C. elegans. [score:1]
To address this question, we looked for genes that responded oppositely to heat stress in WT and mir-34(gk437) animals. [score:1]
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[+] score: 368
Other miRNAs from this paper: hsa-mir-34b, hsa-mir-34c
We demonstrate that re-enforced expression of miR-34a in pancreatic CSCs upon treatment with SAHA, causes an inhibition in the mRNA expression of all the components of Notch pathway, Notch receptor Notch1, Notch 3 and its ligand Jagged1 and downstream Notch target gene Hes1 expression (Fig. 5A). [score:11]
These data suggest that up-regulation of miR-34a by SAHA may have functional significance on EMT regulation by inhibiting Zeb-1. Cadherin switch has been shown to occur during EMT regulation. [score:8]
Since 5-Aza-dC inhibited the expression of VEGF through up-regulation of miR34a, we next examined the effects of 5-Aza-dC on VEGF-B 3′UTR-luciferase reporter activity (Fig. 4C). [score:8]
Since SAHA inhibited the expression of SIRT1 through up-regulation of miR34a, we next examined the effects of SAHA on SIRT1 3′UTR-luciferase reporter activity. [score:8]
Since SAHA inhibited the expression of VEGF through up-regulation of miR34a, we also examined the effects of SAHA on VEGF-B 3′UTR-luciferase reporter activity. [score:8]
These data suggest that up-regulation of miR-34a by SAHA may have functional significance on EMT regulation by inhibiting Zeb-1. Cadherin switch has been shown to occur during EMT regulation. [score:8]
It has also been shown to be hyper-methylated in breast, ovarian, colon, lung and haematological malignancies and downregulate CDK6 translation thereby demonstrating the tumor suppressor role of miR-34a [28], [29], [30]. [score:8]
Since SAHA inhibited the expression of Zeb-1 through up-regulation of miR34a, we also examined the effects of SAHA on Zeb1 3′UTR-luciferase reporter activity. [score:8]
We have demonstrated here that re -expression of miR-34a in pancreatic cancer stem cells on treatment with SAHA, causes an inhibition in the mRNA expression of all the components of Notch pathway. [score:7]
To prove this hypothesis, we examined if the reduced expression of miR-34a in pancreatic cancer could be restored upon treatment with a DNA methylation inhibitor, 5-Aza-dc and/or a histone deacetylase inhibitor, SAHA. [score:7]
Treatment of pancreatic CSCs with SAHA and 5-Aza-dC alone induced the expression of E-Cadherin and inhibited the expression of N-Cadherin, thereby showing that miR-34a is a strong inducer of an epithelial phenotype (Fig. 6D). [score:7]
SAHA down regulates the expression of SIRT1 through upregulation of miR-34a, which can now participate in a positive feedback loop leading to further activation of p53. [score:7]
Modulation of the expression of miR-34a by SAHA inhibited the mRNA expression of various components of the Notch pathway, thus suggesting that miR-34a may be involved in pancreatic CSC self-renewal. [score:7]
Interestingly, SAHA also inhibited the expression of VEGF thus suggesting that by inhibiting the Notch pathway, miR-34a may play an anti-angiogenic and anti-invasive role as well. [score:7]
Furthermore, manipulating the expression of miR-34a by using miR-34a antagomiR altered the protein expression of its target genes (Fig. 4A and B). [score:7]
5-Aza-dC inhibited the expression of cyclin D1 and CDK4 and on the contrary induced the expression of p27 [/KIP1], and these effects were abrogated in the presence of miR34a antagonist. [score:7]
Upregulation of miR-34a inhibits cell proliferation, and induces apoptosis and cell cycle arrest in human pancreatic CSCs. [score:6]
The tumor suppressor TP53 has been identified as a transcriptional regulator of miR-34a and several recent studies have implicated the miR-34 family of miRNAs in the p53 tumor suppressor network [20], [26]. [score:6]
MicroRNA-34a (miR-34a) is a transcriptional target of p53 and is down-regulated in pancreatic cancer. [score:6]
These observations are in concert with published studies of Yamakuchi et al [46], where they have shown that p53 acetylation on lysine 382 is increased after ectopic miR-34a expression and also by Fujita et al who have reported the down-regulation of SIRT1 by miR-34a [47]. [score:6]
Our results indicate that 5-Aza-dC and SAHA modulate the protein expression levels of known direct target genes of miR-34a. [score:6]
Further, to understand the functional role of miR-34a in the regulation of pancreatic cancer progression the effect of 5-Aza-dC and SAHA on the protein expression of putative targets of miR-34a in pancreatic CSCs was examined. [score:6]
These data suggest that MiR-34a inhibits SIRT1 expression through a miR-34a -binding site within the 3′ UTR of SIRT1. [score:5]
Expression of miR-34a in pancreatic CSCs and cell lines was significantly induced on treatment with chromatin modifiers, demethylating agent 5-aza-2′-deoxycytidine (5-Aza-dC) or the histone deacetylase inhibitor, SAHA (vorionostat). [score:5]
Reduced expression of miR-34a in pancreatic cancer could be a resultant of either transcriptional regulation due to p53 mutations as these are very frequent [22] or through epigenetic silencing. [score:5]
We demonstrate in this study that miR-34a expression is down regulated in human pancreatic CSCs and pancreatic tumor derived cell lines, irrespective of their p53 mutational status. [score:5]
Since miR-34a is involved in the self renewing capacity and metastasis of pancreatic CSCs, expression of putative targets of miR-34a in pancreatic CSCs on treatment with 5-Aza-dC and SAHA was examined by (Fig. 3A and B). [score:5]
The expression of miR-34a was normalized against the expression of another small RNA, RNU48 as endogenous normalization control. [score:5]
Recently, miR-34a family members were found to be directly regulated by TP53, and the functional activity of miR-34a indicated a potential role as a tumor suppressor. [score:5]
These data suggest that up-regulation of miR-34a by 5-Aza-dC and SAHA may have functional significance on the regulation of Notch and self-renewal. [score:5]
Treatment of pancreatic CSCs with the chromatin-modulating agents resulted in the inhibition of Bcl-2, CDK6 and SIRT1, which are the putative targets of miR-34a. [score:5]
Furthermore, miR-34a inhibits SIRT1 expression through a miR-34a -binding site within the 3′ UTR of SIRT1. [score:5]
Inhibtion of self-renewal genes that are targets of miR-34a in pancreatic CSCs. [score:5]
These chromatin modifying agents can also regulate the downstream targets of miR-34a, thus suggesting the role of miR-34a restoration in regulating the pancreatic tumorigenesis and providing a functional basis for the epigenetic inactivation of this miRNA in pancreatic cancer. [score:5]
Re -expression of miR-34a in human pancreatic cancer stem cells (CSCs) and in human pancreatic cancer cell lines upon treatment with 5-Aza-dC and SAHA strongly inhibited the cell proliferation, cell cycle progression, self-renewal, epithelial to mesenchymal transition (EMT) and invasion. [score:5]
These data suggest that up-regulation of miR-34a by SAHA may have functional significance on the regulation of VEGF. [score:5]
Expression of putative targets of miR-34a. [score:5]
In conclusion, we demonstrate that miR-34a is a tumor suppressor gene which targets multiple critical oncogenic pathways. [score:5]
Further inhibition of miR-34a by antgomiR abrogates the effects of 5-Aza-dC and SAHA in restoration of the expression of miR-34a in pancreatic CSCs suggesting that 5-Aza-dC and SAHA regulate the stem cell characteristic through miR-34a (Fig. 1C). [score:4]
MiR-34a acts as a suppressor of neuroblastoma tumorigenesis by targeting the mRNA encoding E2F3 and reducing E2F3 protein levels [27]. [score:4]
Enhanced or knockdown expressions of miR-34a were performed by transfection with miR-34a precursor (Ambion) or anti-miR-34a (Ambion), respectively. [score:4]
Consistent with previous studies in pancreatic cancer [20], we observed a global decrease in the expression of miR-34a in pancreatic CSCs and cell lines independent of their p53 mutational status relative to non-neoplastic pancreatic epithelial cells (Fig. 1A). [score:4]
Pancreatic cancer cell lines ASPC-1(p53wt), MiaPACA-2 (p53mutant) and pancreatic cancer stem cells were treated with SAHA (3 µM) or Aza-5dC (4 µM) for 24 h. The expression of miR-34a was quantified using quantitative reverse transcriptase polymerase chain reaction (RT-PCR-Taqman) and Taqman Real Time Assays, and normalized to RNU48 expression. [score:4]
miR-34a is localized on human chromosome 1p36, which is a region associated with a variety of cancers, and has been shown to be down-regulated in pancreatic cancer [20]. [score:4]
Our data reveal that the chromatin modulators, 5Aza-dC and SAHA can epigenetically restore the expression of miR-34a independent of the p53 mutational status in pancreatic CSCs and pancreatic cancer tumor derived cell lines. [score:4]
5Aza-dC and SAHA can epigenetically restore the expression of miR-34a independent of the p53 mutational status in pancreatic cancer. [score:4]
These results demonstrate that miR-34a might play an important role in the regulation of pancreatic tumorigenesis by inhibiting the self-renewal capacity of pancreatic CSCs, metastasis and invasion. [score:4]
miR-34a is itself a transcriptional target of p53 suggesting a positive feedback between p53 and miR-34a. [score:3]
By comparison, anti-miR34a blocked the inhibitory effects of SAHA on luciferase activity. [score:3]
Reduction of miR-34a gene methylation and altered acetylation pattern by the use of chromatin-modifying agents resulted in concomitant reactivation of miR-34a expression. [score:3]
To further understand the effect of up-regulation of miR-34a expression on the invasive potential of pancreatic CSCs, we next investigated the effect of 5-Aza-dC and SAHA on the pancreatic CSCs using scratch migration, soft agar colony formation, and transwell boyden chamber invasion assays. [score:3]
Restoration of miR-34a expression in pancreatic CSCs by the chromatin modifiers significantly reduced in vitro migration, invasion and anchorage-independent growth. [score:3]
By comparison, anti-miR34a blocked the inhibitory effects of SAHA on Zeb-1 3′UTR-Luciferase activity. [score:3]
Taken together, SAHA resulted in inhibition of Snail, Slug and Zeb1 transcription (Fig. 6B), thereby suggesting a role of miR-34a in the reversal of the EMT transition in pancreatic CSCs. [score:3]
Restoration of the expression of miR-34a in pancreatic cancer cells. [score:3]
By comparison, anti-miR34a blocked the inhibitory effects of 5-Aza-dC on luciferase activity. [score:3]
Effect of miR-34a restoration on expression of stem cell renewal genes. [score:3]
Another plausible explanation of this could be that in addition to the epigenetic silencing of miR-34a, SIRT1 a downstream target of miR-34a may be a key player through a SIRT1-p53 pathway. [score:3]
Chromatin modulators inhibit cell viability and promote apoptosis in pancreatic cancer stem cells on restoration of miR-34a. [score:3]
By comparison, anti-miR34a blocked the inhibitory effects of SAHA on RBP-Jκ-luciferase activity. [score:3]
Effect of 5-Aza-dC and SAHA on the putative targets of the miR-34a in pancreatic CSCs. [score:3]
0024099.g001 Figure 1 (A) Relative expression of miR-34a was quantified in human pancreatic cancer cell lines ASPC-1(p53wt), MiaPACA-2 (p53mutant), human pancreatic cancer stem cells (PanCSC) and human pancreatic normal ductal epithelial cells (HPNE). [score:3]
MiR-34a upregulation by these agents also induced acetylated p53, p21 [WAF1], p27 [KIP1] and PUMA in pancreatic CSCs. [score:3]
Expression and restoration miR-34a in pancreatic CSCs and cell lines. [score:3]
miR-34a is highly expressed in normal tissues, like testis, lung, adrenal gland and spleen, although its physiological function is unknown. [score:3]
Our present studies have revealed that expression levels of miR-34a were significantly reduced in pancreatic CSCs and pancreatic cancer tumor cells independent of their p53 mutational status, compared to normal pancreatic ductal epithelial cells. [score:3]
The data also suggest that epigenetic modification of regulatory sequences in CpG islands and deacetylation of histones may contribute to miR-34a silencing in pancreatic cancer. [score:2]
Our results demonstrate that miR-34a might play an important role in the regulation of pancreatic tumorigenesis. [score:2]
Based on this premise, we quantified the expression of miR-34a in human pancreatic CSCs and pancreatic cancer cell lines irrespective of the p53 mutation status, compared to normal pancreatic ductal epithelial cells using TaqMan miRNA assays. [score:2]
The miR-34a responsive genes are highly enriched for those that regulate cell-cycle progression, cellular proliferation, apoptosis, DNA repair, and angiogenesis thereby providing a functional basis for the epigenetic inactivation of this miRNA in pancreatic cancer [31]. [score:2]
This finding substantiates our hypothesis that miR-34a may be thus epigenetically regulated in pancreatic cancer. [score:2]
Therefore, understanding the contributions of these mechanisms in the down regulation of miR-34a in pancreatic cancer, which may contribute to the malignancy of the pancreas, could be of relevance. [score:2]
Role of miR-34a restoration on the EMT regulation in pancreatic cancer cells and CSCs. [score:2]
Inhibition of miR-34a by antagomiR abrogates the effects of 5-Aza-dC and SAHA, suggesting that 5-Aza-dC and SAHA regulate stem cell characteristics through miR-34a. [score:2]
The comparative Ct (ΔΔCt) method was used to determine the expression fold change of miR-34a in pancreatic cancer cells compared to normal pancreatic epithelial cells. [score:2]
By comparison, SAHA had no effect on SIRT1 mutant 3′UTR-luciferase activity (containing no functional miR34a binding site). [score:1]
The restoration of miR-34a by pharmacological intervention thus provide novel prospects for clinical innovation laying the ground work for in vivo experiments in the future. [score:1]
This is thus far, the first demonstration of inhibition of pancreatic CSC characteristics by epigenetic modulation of miR-34a by therapeutic intervention using 5-Aza-dC and SAHA. [score:1]
To better understand the biological significance of the restoration of miR-34a, ASPC-1 cells were treated with or without SAHA and its effects on the cell cycle distribution was examined by PI staining using flow cytometry (data not shown). [score:1]
The present study thus demonstrates the role of miR-34a as a critical regulator of pancreatic cancer progression by the regulating CSC characteristics. [score:1]
Therefore, restoration of miR-34a expression by 5-Aza-dC and SAHA in pancreatic CSCs will provide not only unique tools for the investigation of miRNA function, but also promising reagent to boost patient response to existing chemotherapies or as standalone cancer drug. [score:1]
SAHA and 5-Aza-dC induced miR-34a-luciferase activity, suggesting a functional role of miR34a in pancreatic CSCs. [score:1]
Transfection with antagomiR for miR-34a was alone sufficient to abrogate this effect. [score:1]
This study aimed to investigate the functional significance of miR-34a in pancreatic cancer progression through its epigenetic restoration with chromatin modulators, demethylating agent 5-Aza-2′-deoxycytidine (5-Aza-dC) and HDAC inhibitor Vorinostat (SAHA). [score:1]
Furthermore, SAHA induced G2/M arrest was abrogated in the presence of miR-34a antagonist relative to the negative control oligonucleotides thus suggesting the involvement of miR-34a in the cell cycle arrest by SAHA (data not shown). [score:1]
In pancreatic CSCs, modulation of miR-34a induced apoptosis by activating caspase-3/7. [score:1]
We next examined the transcriptional regulation of miR-34a in CSCs by miR-34a-luciferase reporter assay (Fig. 1D). [score:1]
Thus it explains how epigenetic silencing of miR-34a would interrupt this feedback, resulting in lower p53 activity, thereby providing a selective advantage to the pancreatic cancer cells. [score:1]
These results demonstrate that 5-Aza-dC and SAHA induces the restoration of miR-34a in pancreatic cancer cells. [score:1]
0024099.g004 Figure 4 (A), Pancreatic CSCs were transiently transfected with either negative control (scrambled) or anti-miR34a oligonucleotide and treated with Aza-5dC (4 µM) for 48 h. was performed to measure the expression of cyclin D1, CDK2, p27 and VEGF. [score:1]
This lead us to hypothesize that miR-34a, thus may be epigenetically silenced in pancreatic cancer. [score:1]
Pancreatic CSCs were transiently transfected with either negative control (scrambled) or anti-miR34a oligonucleotide, and treated with SAHA (3 µM) or Aza-5dC (4 µM) for 24 h. RNA was extracted to measure the expression of miR34a by q-RT-PCR as described above. [score:1]
Effect of miR-34a restoration on epithelial-mesenchymal transition of pancreatic CSCs. [score:1]
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Since we found that c-Myc is a target of miR-34a, thus downregulating the expression of c-Myc, we examined whether the downregulation of c-Myc by miR-34a results in reduction of RhoA expression by suppressing assembly of the c-Myc–Skp2–Miz1 complex. [score:15]
c-Myc overexpression partially rescued RhoA expression (Fig. 5A, compare lane 4 with 3) and miR-34 -induced suppression of invasion (Fig. 5B), suggesting that miR-34a inhibits invasion, at least partially, via RhoA reduction by targeting c-Myc. [score:11]
We found that miR-34a expression is downregulated prostate cancer tissue and that miR-34a re -expression strongly inhibits cell proliferation, in vivo xenograft growth and cell invasion. [score:10]
We studied whether miR-34a affected the expression level of CAD and NUC using DRB (5.6-di-chloro-1-b-D-ribofuranosyl-bensimidazole) which specifically suppresses expression of c-Myc-responsive CAD and NUC by inhibiting P-TEFb [40]. [score:9]
miR-34a reduced RhoA expression and overexpression of c-Myc reversed the reduction of RhoA and inhibition of cell invasion. [score:7]
By targeting c-Myc, miR-34a reduced the c-Myc-Miz-Skp2 complex which induces RhoA transcription and inhibited cell invasion, showing that miR-34a indirectly regulates RhoA. [score:7]
c-Met reversed miR-34 -induced suppression of invasion, indicating that miR-34a inhibits invasion, at least partially, by targeting c-Met (Figs. 7B and C). [score:7]
miR-34a expression is downregulated in prostate cancer. [score:6]
Since ectopic expression of miR-34a suppressed PC-3 cell proliferation, we next studied the effects of miR-34a on apoptosis in PC-3 cells using flow cytometry. [score:5]
We also found miR-34a targets c-Met and inhibits PC-3 cell invasion. [score:5]
These data document the fact that miR-34a suppresses RhoA activation at the initiation of transcription via targeting c-Myc. [score:5]
At week 5, tumor sizes of control xenografts were about 5 times larger than those of miR-34a xenografts, indicating that the ectopic expression of miR-34a significantly suppressed tumor growth in vivo (Figs. 1D and E). [score:5]
p53 has been found to target the miR-34 family [4], [5], [6] and the ectopic expression of miR-34 genes has drastic effects on cell proliferation and survival. [score:5]
c-Met overexpression partially rescues suppression of cell invasion by miR-34a. [score:5]
We also showed that miR-34a inhibited PC-3 cell invasion by targeting c-Met. [score:5]
We found that miR-34a decreased CAD and NUC mRNA expression in PC-3 cells while DRB restored the expression (Fig. 6B). [score:5]
We showed that miR-34a inhibited c-Myc by binding to its 3′UTR in PC-3 cells and suppressed assembly of the c-Myc transcriptional complexes. [score:5]
miR-34a inhibits RhoA and suppresses assembly of c-Myc transcriptional complex. [score:5]
c-Myc overexpression partially rescues RhoA suppression by miR-34a. [score:5]
To determine whether inhibition of invasion by miR-34a could be reversed via restoration of c-Myc, we transfected PC-3 cells with a c-Myc expression plasmid together with pre-miR-34a. [score:5]
The effects of miR-34a on these c-Myc functional complexes provide direct evidence of how miR-34a functions to suppress c-Myc. [score:4]
Mutation of the putative miR-34a target sites in these UTRs decreased the response to miR-34a. [score:4]
These results confirm that miR-34a directly targets c-Met and c-Myc via binding to their 3′UTRs in PC-3 cells. [score:4]
We report here that miR-34a was downregulated in prostate cancer tissues. [score:4]
Real-time RT-PCR showed that the expression level of miR-34a was lower in malignant prostate cells compared with the average of the expression level of miR-34a in the normal tissues (data not shown). [score:4]
Our results demonstrate that in prostate cancer PC-3 cells miR-34a suppresses assembly and function of the c-Myc complex that activates or elongates transcription, revealing a novel role of miR-34a in the regulation of transcription by c-Myc. [score:4]
We demonstrated that miR-34a suppressed RhoA transcription by reducing the c-Myc–Skp2–Miz1 transcriptional complex. [score:3]
Thus, inhibition of c-Myc is thought to be a significant function of miR-34a. [score:3]
All cancer tissues showed lower miR-34a levels compared with matched adjacent normal regions, demonstrating that miR-34a is downregulated in cancer (Fig. 1A). [score:3]
miR-34a expression was analyzed by real-time PCR and was normalized to that of the control. [score:3]
miR-34a targets c-Myc and c-Met. [score:3]
miR-34a inhibits invasion and migration of PC-3 cells. [score:3]
miR-34a inhibited cell proliferation in vitro, in vivo tumor growth and promoted apoptosis in prostate cancer cells. [score:3]
An HIV -based lentiviral packaging system including a plasmid expressing miR-34a or vector control was purchased from GeneCopoeia (Rockville, MD). [score:3]
As p53 has been found to target miR-34a and since, cell-cycle arrest and apoptosis are also end points of p53 activation, the miR-34a gene may be a mediator of p53 function. [score:3]
IP also showed that miR-34a decreased the levels of these components in the c-Myc immunoprecipitates [lanes 4 (control) and 6], indicating that miR-34a suppressed assembly of the c-Myc–Skp2–Miz1 transcriptional complex in PC-3 cells. [score:3]
These results suggested that miR-34a reduced the recruitment of the c-Myc–Skp2–Miz1 complex to the RhoA promoter and reduces RhoA expression. [score:3]
0029722.g006 Figure 6 (A) miR-34a suppresses the formation of the endogenous c-Myc-P-TEFb complex. [score:3]
s with miR-34a -expressing PC-3 cells showed that miR-34a repressed luciferase activity. [score:3]
We examined the expression levels of miR-34a in laser capture microdissected (LCM) prostate cancer tissues (n = 10) and matched adjacent normal regions by real-time PCR. [score:3]
miR-34a suppresses the c-Myc transcriptional complex of RhoA. [score:3]
We employed a lentiviral system to express miR-34a. [score:3]
miR-34a inhibits cell invasion and migration. [score:3]
IP also showed that miR-34a decreased the amount of p-TEFb in the c-Myc immunoprecipitates in PC-3 cells [lanes 4 (control) and 6], indicating that miR-34a suppressed assembly of the c-Myc–P-TEFb complex in PC-3 cells. [score:3]
miR-34a inhibits cell proliferation of PC-3 cells. [score:3]
Figure S4 miR-34a target sequences of c-Met and c-Myc. [score:3]
miR-34a reduced colony numbers to about 20% of that of control, showing that miR-34a significantly inhibited the colony forming ability of PC-3 cells (Figs. 1B and C). [score:3]
0029722.g002 Figure 2 (A) miR-34a inhibits invasion of PC-3 cells. [score:3]
PC-3 cells were transfected with a c-Met expression plasmid together with pre-miR-34a. [score:3]
We found that c-Myc is a target of miR-34a in PC-3 cells, and we studied the effect of miR-34a on RhoA activation via the c-Myc–Skp2–Miz1 transcriptional complex. [score:3]
The HIV lentiviral system, expressing miR-34a or vector control, was used to infect PC-3 cells and the infected cells were selected with puromycin. [score:3]
Xenograft tumors from PC-3 cells overexpressing miR-34a were smaller than xenograft tumors from the control cells. [score:3]
miR-34a suppresses the c-Myc-P-TEFb complex. [score:3]
The alteration of the c-Myc complexes by miR-34a also suggests that the suppression of the assembly of the c-Myc complexes may involve unknown mechanisms in addition to the simple reduction in quantity of c-Myc protein by miR-34a since miR-34a has dramatic effects on cellular processes. [score:3]
These results indicate that miR-34a repressed these genes by suppressing the c-Myc-pTEFb complex. [score:3]
We found that ectopic expression of miR-34a increased PC-3 cell apoptosis to about4 times of that controls (Fig. 1F and Fig. S3), demonstrating that miR-34a has apoptotic activity in PC-3 cells. [score:3]
0029722.g001 Figure 1(A) Relative miR-34a expression in laser capture microdissected (LCM) prostate cancer tissues (C) and matched adjacent normal regions (N). [score:3]
PC-3 cells were infected with the HIV -based lentivirus expressing miR-34a or vector control (1.5×10 [6] infectious units of virus (IFU) (in 20 µl), and the infected PC-3 cells were selected with puromycin (0.5 ug/ml). [score:3]
Our study demonstrates here that miR-34a abrogates the c-Myc-P-TEFb complex by targeting c-Myc. [score:3]
Therefore, we studied effects of miR-34a on c-Met and invasion since miR-34a targets c-Met (Fig. 1). [score:3]
Suspensions of the stable miR-34a expressing cells or the control cells (1×10 [7] cells in 100 µl RPMI medium) were subcutaneously injected into female nude mice (strain BALB/c nu/nu; Charles River Laboratories, Inc. [score:3]
It has been suggested that miR-34a inhibits cell invasion in a c-Met -dependent manner [41], [42]. [score:3]
miR-34a expression at 72 h of the transfection was analyzed by real-time PCR and was normalized to that of the control (NC). [score:3]
miR-34a inhibits cell growth. [score:3]
Since miR-34a targets c-Myc, we performed immunoprecipitation (IP) to study assembly of the c-Myc-pTEFB transcription elongation complex in mir-34a -transfected PC-3 cells. [score:3]
miR-34a also suppressed the c-Myc-P-TEFb complex that plays a key role in controlling the elongation phase of transcription by RNA polymerase II (Pol II), indicating one of the mechanisms by which miR-34a has dramatic effects on cellular function. [score:3]
Our results demonstrate that miR-34a suppresses assembly and function of the c-Myc complex that elongates transcription. [score:3]
Figure S2 miR-34a suppresses proliferation of prostate cancer cells. [score:3]
Figure S1 miR-34a expression in PC-3 cells. [score:3]
We cloned the putative miR-34a targets in the 3′UTRs into a luciferase construct. [score:3]
miR-34a targets oncogenes in PC-3 cells. [score:3]
To examine the effects of the ectopic expression of miR-34a on in vivo tumor growth, we subcutaneously injected the stable miR-34a or the control cell line into nude mice. [score:3]
Real-time PCR showed miR-34a decreased RhoA mRNA level (Fig. 4A) and revealed that miR-34a reduced RhoA protein expression (Fig. 4B). [score:3]
miR-34a suppresses assembly of c-Myc-P-TEFb complex. [score:3]
In conclusion we have shown for the first time that miR-34a suppresses assembly and function of the c-Myc–Skp2–Miz1 complex that activates RhoA and the c-Myc-pTEFB complex that elongates transcription of various genes. [score:3]
Our results demonstrate that miR-34a suppresses assembly and function of the c-Myc complex that activates transcription of RhoA. [score:3]
During oncogene -induced senescence, miR-34a was also found to target c-Myc [15]. [score:3]
MTS assay showed that miR-34a inhibited PC-3 cell proliferation by about 40% on day 4 but had no significant effect on LNCaP and DU145 cells (Fig. S2A). [score:2]
We also assayed miR-34a expression levels in malignant (PC-3, LNCaP and DU145 cells) and non-malignant prostate RWPE-1 cells. [score:2]
miR-34a also reduced levels of transcription factor E2F1 which regulates the cell cycle, DNA replication and cell proliferation (Fig. 3B). [score:2]
Real-time RT-PCR revealed that the expression level of miR-34a was markedly lower in PC-3 cells compared with non-malignant epithelial prostate cell RWPE-1 cells (data not shown). [score:2]
Therefore, our study reveals a novel role of miR-34a in the regulation of transcription by c-Myc. [score:2]
We performed soft agar colony formation assay using the infected PC-3 cells expressing miR-34a or control. [score:2]
ChIP assay revealed miR-34a suppresses the recruitment of c-Myc to the RhoA promoter. [score:2]
We also compared the expression levels of miR-34a in malignant prostate cells and the laser capture microdissected (LCM) prostate normal tissues (n = 10). [score:2]
The transient transfection of pre-miR-34a increased miR-34a levels in prostate cancer cells (Fig. S1A). [score:1]
To study the effect of miR-34a on the growth of prostate cancer cells, we transiently transfected several cell lines with pre-miR negative control (NC) or pre-miR-34a. [score:1]
0029722.g005 Figure 5 (A) PC-3 cells were transiently transfected with pCMV6-ENTRY only or pCMV6-ENTRY-c-Myc for 8 h followed by transient transfection with pre-miR negative control (NC) or pre–miR-34a for 72 h. Protein level was analyzed by. [score:1]
Our study partly gives an account of the global and profound effects of miR-34a on the cellular processes demonstrated here and in previous reports [4], [5], [7], [8]. [score:1]
Figure S3 miR-34a induces apoptosis in PC-3 cells. [score:1]
0029722.g007 Figure 7 (A) PC-3 cells were transiently transfected with pCMV6-ENTRY only or pCMV6-ENTRY-c-Met for 8 h followed by transient transfection with pre-miR negative control (NC) or pre–miR-34a for 72 h. Protein level was analyzed by. [score:1]
These results indicate that the complexes were altered by miR-34a, suggesting a new complex assembly. [score:1]
Transient transfection of pre-miR-34a also caused significant morphological changes in PC-3 cells (Fig. S2B). [score:1]
c-Met has 2 predicted binding sites for miR-34a in its 3′-UTRs (Fig. S3). [score:1]
Time course of tumor growth in nude mice after subcutaneous injection of a stably transfected miR-34a PC-3 cell line or control cell line. [score:1]
We examined the effects of miR-34a transfection on the protein levels of these genes. [score:1]
Generation of stable miR-34a cell lines. [score:1]
Transfection of miR-34a reduced the protein levels of c-Met and c-Myc, protein in PC-3 cells (Fig. 3B). [score:1]
We performed immunoprecipitation (IP) to study assembly of the c-Myc–Skp2–Miz1 transcriptional complex in miR-34a transfected cells. [score:1]
PC-3 cells were transfected with pre-miR negative control (NC) or pre-miR-34a for 3 days. [score:1]
These results indicate that miR-34a binds to the 3′-UTRs of c-Myc and c-Met (Fig. 3A). [score:1]
The stable transfection of pre-miR-34a increased miR-34a levels in PC-3 cells (Fig. S1B). [score:1]
RWPE-1 cell line was cultured in keratinocyte growth medium supplemented with 5 ng/mL human recombinant epidermal growth factor and 0.05 mg/mL bovine pituitary extract (Invitrogen, Carlsbad, CA) Cells in 6-well plates were transfected with 30 nM pre-miR negative control (NC) or pre-miR-34a (Applied Biosystems, Foster City, CA) using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. [score:1]
The c-Myc complexes which were induced by miR-34a were heterogeneous since the ratios of the components were different from those in the control immunocomplexes (Figs. 4C and 6A). [score:1]
Here we report the effects of miR-34a on the assembly of c-Myc functional complexes, the c-Myc–Skp2–Miz1 and c-Myc-P-TEFb complexes. [score:1]
Ectopic miR-34a causes cell-cycle arrest in the G1 phase [6], [7] and apoptosis [7], [8]. [score:1]
PC-3 cells were transiently transfected with pre-miR negative control (NC) or pre-miR-34a for 72 h. (A) RhoA mRNA level was analyzed by real-time PCR. [score:1]
PC-3 cells were treated with 20 µM of DRB for 12 h and were transiently transfected with pre-miR negative control (NC) or pre–miR-34a for 48 h. CAD and NUC mRNA level was analyzed by real-time PCR. [score:1]
Cells in 24-well plates were transfected with 30 nM pre-miR negative control (NC) or pre-miR-34a (Applied Biosystems) using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. [score:1]
Accumulating evidence has shown the importance of P-TEFb in malignancy, thus linking miR-34a to P-TEFb is a significant finding in cancer biology. [score:1]
Oncogenes, c-Met and c-Myc, have predicted binding sites for miR-34a in their 3′-UTRs (Fig. S4). [score:1]
The results clearly revealed that miR-34 reduced invasion of PC-3 cells to 20% of that of controls (Figs. 2A and B). [score:1]
0029722.g004 Figure 4 PC-3 cells were transiently transfected with pre-miR negative control (NC) or pre-miR-34a for 72 h. (A) RhoA mRNA level was analyzed by real-time PCR. [score:1]
PC-3 cells were transiently transfected with pre-miR negative control (NC) or pre–miR-34a for 72 h and total cell lysates were immunoprecipitated with c-Myc antibody or IgG (control), followed by analysis. [score:1]
RWPE-1 cell line was cultured in keratinocyte growth medium supplemented with 5 ng/mL human recombinant epidermal growth factor and 0.05 mg/mL bovine pituitary extract (Invitrogen, Carlsbad, CA) Cells in 6-well plates were transfected with 30 nM pre-miR negative control (NC) or pre-miR-34a (Applied Biosystems, Foster City, CA) using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. [score:1]
A recent report indicates that PC-3 cells have characteristics of prostatic small cell carcinoma and not of adenocarcinoma [38], however we used PC-3 cells for further study because the proliferation suppression effect of miR-34a was significant, which was consistent with the previously reported data [36]. [score:1]
In this study we demonstrated for the first time the functional effects of miR-34a on c-Myc transcriptional complexes in PC-3 prostate cancer cells. [score:1]
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[+] score: 351
Figure 9 Note: A. histogram analysis of the expression of miR-34a in each group; B. histogram analysis of the mRNA expression of Notch1 in each group; C. histogram analysis of the mRNA expression of Notch4 in each group; D. histogram analysis of the mRNA expression of Hes1 in each group; *, P < 0.05 compared with the blank group and the NC group; NC, negative control; Hes1, hairy and enhancer of split 1. (Figure 10) showed that at 48 h after transfection with miR-34a mimics, liver cancer Huh7 cells had down-regulated protein expressions of Notch signaling pathway-related Notch1, Notch4 and Hes1 and apoptosis-related Bcl-2 and Bcl-xL, but had up-regulated expressions of cell cycle-related P21 and apoptosis-related Bax when compared with the blank group and the NC group (all P < 0.05). [score:17]
As miR-34a can significantly down-regulate the expressions of Bcl-2, Bcl-w and Bcl-xL [35], and up-regulate the expressions of Bax and P21 [36], it can be concluded that the low expression of miR-34a could results in the inhibition of cell apoptosis, which is in consistent with our findings. [score:15]
Figure 4 Note: A. the mRNA expressions of miR-34a and Notch receptors during LR; B. the protein expressions of Notch receptors during LR; Hes1, hairy and enhancer of split 1. Note: A. the mRNA expressions of miR-34a and Notch receptors during LR; B. the protein expressions of Notch receptors during LR; Hes1, hairy and enhancer of split 1. An online predicting software Target Scan was used to identify the target site on which Notch1 bound to miR-34a. [score:13]
For instance, miR-17~92 facilitated LR in an oestrogen -dependent manner [16], an increased expression of miR-34a led to inhibited hepatocyte proliferation during the late phase of LR [17], and miR-21 was upregulated in the early stage of LR, which targeted Pellino-1 to regulate NF-kappaB signaling [18]. [score:11]
Moreover, the expressions of miR-34a, P21 and Bax were up-regulated, while the expressions of Notch receptors, and Bcl-2 and Bcl-xL were down-regulated in this group. [score:11]
According to the results of qRT-PCR (Figure 9), the liver cancer Huh7 cells in the miR-34a inhibitor group had decreased expression of miR-34a and increased mRNA expressions of Notch1, Notch4 and Hes1 in comparison to the blank group and the NC group (all P < 0.05), while the cells in the miR-34a mimics group had increased expression of miR-34a and decreased mRNA expressions of Notch, Notch4 and Hes1 when compared with the blank group and the NC group (all P < 0.05). [score:10]
According to qRT-PCR and (Figure 4), it was found that from 0 d to 1 d after PH, the protein and mRNA expressions of Notch1, Notch 4 and Hes1 kept increasing, while the expression of miR-34a decreased to its lowest level at 1 d. Since 1 d, the mRNA and protein expressions of Notch4 started to decrease, and they dropped to the lowest level at 5 d, after which the expressions gradually increased to the level before liver resection. [score:9]
Figure 4 Note: A. the mRNA expressions of miR-34a and Notch receptors during LR; B. the protein expressions of Notch receptors during LR; Hes1, hairy and enhancer of split 1. An online predicting software Target Scan was used to identify the target site on which Notch1 bound to miR-34a. [score:9]
Additionally, miR-34a could down-regulate expression of Notch1, hence low expression of miR-34a contributes to the development and progression of human malignancies through promotion of Notch1, including pancreatic cancer and prostate cancer [38– 40]. [score:9]
Besides, it is found that over -expression of miR-34a led to reduced expression of Notch receptors (Notch1 and Notch4) and Notch target gene (Hes1), suggesting that miR-34a regulates Notch signaling pathway in a negative manner. [score:8]
In cervical cancer and choriocarcinoma, forced expression of miR-34a could inhibit Jagged1 and Notch1 expression, thereby causing a reduced invasion capacity of tumor cells [9]. [score:7]
Besides, miR-34a suppressed the proliferation and induced apoptosis of human glioma U87 cells by reducing the expression of target gene Notch1 [29]. [score:7]
In general, the mRNA expression of Notch1 was negatively related to that of miR-34a, and the protein expression of Notch1 decreased from 1 d to 5 d and increased from 5 d to 7 d. As for the expression of Hes1, it presented two ascending tendencies: the first peak appeared 1 d, and the second at 5 d which was, however, a little lower than the first peak. [score:7]
In contrast, liver cancer Huh7 cells transfected with miR-34a inhibitors had increased protein expressions of Notch1, Notch4, Hes1, Bcl-2 and Bcl-xL but decreased expressions of P21 and Bax when compared with the blank group and the NC group (all P < 0.05). [score:6]
Though miR-34a was proved to target Notch signaling pathway in multiple tumors, such as glioblastomas, breast cancer, cervical cancer and choriocarcinoma [9, 12, 13], the role of miR-34a interacting with Notch signaling pathway in liver cancer remains to be discussed, so this study would like to explore whether miR-34a affects LR and cancer development by targeting Notch signaling pathway. [score:6]
In conclusion, the present study revealed that miR-34a could promote LR and suppress the development of liver cancer by inhibiting Notch signaling pathway. [score:6]
To conclude, at the early stage of LR, the expression of miR-34a was significantly in negative correlation with the expressions of Notch1, Notch4 and Hes1. [score:5]
Our study demonstrates that miR-34a regulated LR and the development of liver cancer by inhibiting Notch signaling pathway. [score:5]
At 48 h and 72 h, the growth of cells in the miR-34a mimics group were inhibited obviously, with its OD value being significant lower than those in the blank group and the NC group (both P < 0.05), while the growth of cells in the miR-34a inhibitors group was speeded up, with its OD value being statistically higher than those in the blank and NC groups (both P < 0.05). [score:5]
MicroRNA-34a (miR-34a), belonging to miR family with high conservation, is encoded on human chromosome 1 and is considered as a suppressor in human malignancies through its regulation of target genes [7]. [score:5]
Cells were divided into four groups: blank group (without any transfected sequence), NC group (transfected with negative control sequence 5′-UUCUCCGAACGUGUCACGUTT-3′), miR-34a mimics group (transfected with miR-34a mimics sequence 5′-ACCGUCACAGAAUCGACCAACA-3′), and miR-34a inhibitors group (transfected with miR-34a inhibitor sequence 5′-ACAACCAGCUAAGACACUGCCA-3′). [score:5]
Finally, it's demonstrated that the low expression of miR-34a in liver cancer Huh7 cells resulted in a larger proportion of cells in the S phase, facilitated cell proliferation and lower cell apoptosis rate, implying that miR-34a acts as a suppressor in liver cancer. [score:5]
Our data indicated that miR-34a regulated LR and liver cancer development by targeting Notch signaling pathway. [score:5]
Expressions of Notch signaling pathway-related proteins and cell cycle and apoptosis-related proteins in the blank, NC, miR-34a mimics and miR-34a inhibitors groups. [score:5]
A previous study found that miR-34a mimic might be a novel target molecule for the treatment of liver cancer as it significantly suppressed tumor cell growth [8]. [score:5]
Expressions of miR-34a and Notch receptor mRNA in the blank, NC, miR-34a mimics and miR-34a inhibitors groups. [score:5]
The inactivation of apoptosis is central to cancer development, therefore low expression of miR-34a can results in the occurrence and progress of cancer [37]. [score:4]
In line with these studies, we found that in liver cancer Huh7 cells in the miR-34a mimics group, miR-34a expression significantly increased, while the expressions of Notch1, Notch4 and Hes-1 decreased when compared with the blank and NC groups. [score:4]
Figure 6 Note: transfection of miR-34a mimics inhibited cells growth, while transfection of miR-34a inhibitors speeded up cells growth; *, P < 0.05 compared with the blank group and the NC group; NC, negative control. [score:4]
Note: transfection of miR-34a mimics inhibited cells growth, while transfection of miR-34a inhibitors speeded up cells growth; *, P < 0.05 compared with the blank group and the NC group; NC, negative control. [score:4]
It is why miR-34a was gradually up-regulated to normal level in our study. [score:4]
Deregulation of miR-34a occurs owing to altered p53 expression [22]. [score:4]
In addition, elevated miR-34a in the late phase of LR can greatly repress the proliferation of rat hepatocytes by down -regulating INHBB and Met expression in regenerating livers [23], as the silencing of INHBB could greatly decelerate the growth of rat hepatocytes in LR and an increase in Met could cause impaired LR [23, 24]. [score:4]
Growth of xenograft tumor in nude mice in the blank, NC, miR-34a mimics and miR-34a inhibitors groups. [score:3]
There was no significant change in the expression of miR-34a in the SH group. [score:3]
Cell cycle in the blank, NC, miR-34a mimics and miR-34a inhibitors groups detected by PI staining. [score:3]
Therefore, that variation of p53 during LR is believed to contribute to the changing expression of miR-34a. [score:3]
Hereafter, the expression of miR-34a in the PH group began to increase and reached the highest level at 5 d which was significantly higher than that in the SH group (P < 0.05). [score:3]
In early stage of LR, the expressions of Notch receptors and miR-34a were negatively correlated. [score:3]
was applied detect to the expressions of miR-34a and Notch receptor mRNA. [score:3]
Expression of miR-34a during LR. [score:3]
The results of qRT-PCR were presented in Figure 3. The miR-34a expression at 0.5 d after PH decreased to half of the level at 0 d (P < 0.05), and it reached the lowest level at 1 d which was about a quarter of that in the SH group (P < 0.05). [score:3]
In the study, the expression of miR-34a decreased at the beginning of LR and then gradually increased to normal level, indicating the involvement of miR-34a in LR. [score:3]
According to the results of PI staining (Figure 7), the proportions of cells in G1 phase in the blank, NC, miR-34a mimics and miR-34a inhibitors groups were (56.68 ± 2.23)%, (56.80 ± 2.33)%, (57.65 ± 2.04)% and (55.33 ± 2.19)%, respectively; the proportions of cells in S phase in the four groups were (26.06 ± 1.01)%, (26.97 ± 2.21)%, (15.63 ± 1.68)% and (34.72 ± 2.55)%, respectively; the proportions of cells in G2/M phase in the four groups were (17.26 ± 1.22)%, (16.23 ± 0.12)%, (26.72 ± 0.36)% and (9.95 ± 0.37)%, respectively. [score:3]
Notch 1 is a direct target gene of miR-34a [28], as the dual-luciferase reporter assay verified that miR-34a bound to the 3′ UTR -binding sites of Notch 1 mRNA in the present study. [score:3]
The results suggested that miR-34a could inhibit tumorigenicity of liver cancer cells. [score:3]
The findings provide a tantalizing hint that miR-34a might be a new therapeutic target for liver cancer. [score:3]
U6 was used as the internal reference of miR-34a, and β-actin as the internal reference of rest target genes. [score:3]
According to the results for Annexin V/PI staining (Figure 8), the apoptosis rates of cells in the blank, NC, miR-34a mimics and miR-34a inhibitors groups at 48 h after transfection were (1.89 ± 0.22) %, (1.91 ± 0.25) %, (18.31 ± 1.56) % and (0.91 ± 0.06) %, respectively. [score:3]
Meanwhile, the sequences of Notch1-3′-untranslated region (UTR)-WT (wild-type Notch 3′UTR) and Notch1-3′-UTR-MT (mutant-type Notch1 3′UTR missing the binding site of miR-34a) were designed. [score:3]
However, there was no statistically significant difference in the expressions of miR-34a, and Notch1, Notch4 and Hes1 mRNA between the blank group and the NC group (all P > 0.05). [score:3]
miR-34a targeted Notch1. [score:3]
The expressions of miR-34a and Notch receptors in rats. [score:3]
The growth of xenograft tumor in nude mice the blank, NC, miR-34a mimics and miR-34a inhibitors groups. [score:3]
This study aimed to investigate the role of microRNA-34a (miR-34a) in regulating liver regeneration (LR) and the development of liver cancer in rats by targeting Notch signaling pathway. [score:3]
The target gene of miR-34a was analyzed using biological prediction website (microRNA. [score:3]
In the experiment of tumor formation in nude mice (Figure 11), liver cancer cells in the miR-34a mimics group had attenuated tumorigenicity, with reduced tumor volume and weight (0.430 ± 0.044 g) in nude mice, while those in the miR-34a inhibitors group had obviously heavier tumor (1.125 ± 0.151 g), which showed significant difference in comparison to the blank group (0.759 ± 0.103 g) and the NC group (0.795 ± 0.123 g) (all P < 0.05). [score:3]
Cell cycle in the blank, NC, miR-34a mimics and miR-34a inhibitors groups. [score:3]
Cell apoptosis in the blank, NC, miR-34a mimics and miR-34a inhibitors groups. [score:3]
Cell proliferation in the blank, NC, miR-34a mimics and miR-34a inhibitors groups. [score:3]
The miR-34a inhibitors group showed contrary tendencies. [score:3]
Human liver cancer Huh7 cells were transfected and divided into blank, negative control (NC), miR-34a mimics and miR-34a inhibitors groups. [score:3]
Cell apoptosis in the blank, NC, miR-34a mimics and miR-34a inhibitors groups at 48 h after transfection detected using Annexin V/PI staining method. [score:3]
From these two aspects, it could be well concluded that miR-34a is an inhibitor in liver cancer. [score:3]
Expression of miR-34a in the PH and SH groups during LR. [score:3]
Compared to the blank and NC groups, the cell growth was inhibited, cell cycle was mainly arrested in the G2/M phase and cell apoptosis rate increased in the miR-34a mimics group. [score:2]
Note: A. the sequence of 3′-UTR where Notch1 mRNA bound to miR-34a; B. dual-luciferase reporter gene assay, which indicated that miR-34a mimics could inhibit the luciferases activity of miR-34a/Notch1-WT plasmid, while it had no effect on the luciferases activity of miR-34a/Notch1-MT; *, P < 0.05; WT, wild type ; MT, mutant type. [score:2]
Compared with the blank group and the NC group, the miR-34a mimics group had increased percentage of cells in G2/M phase and decreased percentage of cells in S phase (both P < 0.05), indicating that the proliferation of liver cancer Huh7 cells was significantly inhibited. [score:2]
Compared to the SH group, miR-34a expression in liver tissues in the PH group decreased first and then increased to the normal level during LR. [score:2]
Compared with the blank group and the NC group, the miR-34a inhibitors group had decreased percentage of cells in G2/M phase and increased percentage of cells in S phase (both P < 0.05), suggesting increased number of liver cancer Huh7 cells in mitotic phases. [score:2]
MiR-34a targeted Notch1. [score:2]
Figure 5 Note: A. the sequence of 3′-UTR where Notch1 mRNA bound to miR-34a; B. dual-luciferase reporter gene assay, which indicated that miR-34a mimics could inhibit the luciferases activity of miR-34a/Notch1-WT plasmid, while it had no effect on the luciferases activity of miR-34a/Notch1-MT; *, P < 0.05; WT, wild type ; MT, mutant type. [score:2]
Compared with cells in the blank group and the NC group, the liver cancer Huh7 cells in the miR-34a inhibitors group had significantly lower apoptosis rate, while those in the miR-34a mimics group had significantly higher apoptosis rate (all P < 0.05). [score:2]
Figure 5A shows the sequence of 3′-UTR where Notch1 mRNA bound to miR-34a. [score:1]
Additionally, the tumor growth in the miR-34a mimics group was reduced. [score:1]
It was found that miR-34a mimics exerted no significant influence on the luciferases activity of miR-34a/Notch1-MT plasmid (P > 0.05), but it led to 65% reduction in the luciferases activity of miR-34a/Notch1-WT plasmid (P < 0.05) (Figure 5B). [score:1]
The miR-34a induces cell-cycle arrest, apoptosis or senescence in cancer cells [33]. [score:1]
Association between miR-34a and Notch receptors at the early stage of LR. [score:1]
Inactivation of miR-34a has been found in colorectal, urothelial, mammary, ovarian and renal cell carcinomas [41]. [score:1]
The inactivation of Notch1 signaling pathway by miR-34a was also proved to attenuate the aggressiveness of prostate cancer [30]. [score:1]
The liver cancer cell lines (Huh7) were transfected with miR-34a/Notch1-WT or miR-34a/Notch1-MT recombinant plasmids. [score:1]
The miR-34a is wi dely known for anti-oncogenic activity in liver cancer, and the activation of miR-34a by the transcription factor p53 suggests its potential role in the modulation of hepatic cell behavior [19– 21]. [score:1]
However, further studies are needed to test the effect of miR-34a on more hepatic cell types to further support our finding. [score:1]
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Our results showed that miR-34a inhibited luciferase activity significantly, whereas no effect was observed when their respective target sites were mutated, suggesting that miR-34a directly targets c-Myc and FoxM1 via binding to the 3′ UTRs in liver cancer cells (Figure 4D, 4G). [score:8]
In contrast, in HCC cells with p53 inactivation, the suppressive miR-34a was unable to inhibit FoxM1 or c-Myc expression, leading to the sustained activation of telomerase and the subsequent telomere length maintenance. [score:7]
Further, when endogenous p53 expression was knocked down by a p53 siRNA in the four p53 wild-type liver cancer cells, miR-34a expression was also attenuated (Figure 6F). [score:6]
Since the same chromosome 1p36 region, which is the tumor suppressor candidate, is also frequently deleted in cancer, thus the down-regulation of miR-34a could be partially due to the deletion of 1p36 [27]. [score:6]
Results showed that compared with negative control cells, ectopic expression of miR-34a significantly inhibited telomerase activity and induced telomere shortening, as represented by hTERT mRNA expression and relative T/S ratio (telomere to single copy gene), respectively (Figure 3B–3C). [score:6]
The data showed that among the hTERT activators examined, FoxM1 and c-Myc were the most down-regulated genes by miR-34a overexpression (Figure 4A). [score:6]
After 48 hours, the expression level of miR-34a was significantly upregulated when transfected with synthetic miR-34a duplex (Figure 3A). [score:6]
The expression of miR-34a was firstly reported to be down-regulated in rat during hepatocarcinogenesis induced by a methyl -deficient diet [25]. [score:6]
Meanwhile, results from the qRT-PCR and SA- β-gal staining suggested that the overexpression of miR-34a is able to rescue the siRNA-TP53 -mediated hTERT activation and senescence inhibition, further demonstrating the regulatory roles of p53 in miR-34a -induced senescence (Figure 6H–6I). [score:6]
The transfection of miR-34a duplex oligoribonucleotides (sense, 5′-UGG CAG UGU CUU AGC UGG UUG UU-3′; antisense 5′-CAA CCA GCU AAG ACA CUG CGA AA-3′), mimics control (sense, 5′-UUC UCC GAA CGU GUC ACG UTT-3′; antisense, 5′-ACG UGA CAC GUU CGG AGA ATT-3′), inhibitor anti-miR-34a (5′-ACA ACC AGC UAA GAC ACU GCC A-3′), and inhibitor control (5′-CAG UAC UUU UGU GUA GUA CAA-3′) were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the procedure recommended by the manufacturer. [score:5]
Our above data showed that miR-34a inhibited the expression and activity of telomerase significantly. [score:5]
Thus, by computing the correlation coefficient using the NCI-60 expression profiling data, we quantified the correlation strength between miR-34 familiy and telomerase reverse transcriptase (hTERT) expression profile in different cancer cell lines (except neurologic cancer cells). [score:5]
Figure 8 In normal cells, the miR-34a activated by p53 would target FoxM1 and c-Myc expression, resulting in the inactivation of telomerase and the subsequent telomere length shortening. [score:5]
In normal cells, the miR-34a activated by p53 would target FoxM1 and c-Myc expression, resulting in the inactivation of telomerase and the subsequent telomere length shortening. [score:5]
As altered expression of miR-34a would contribute to the impaired telomerase activity, we discovered similar effects of miR-34a mimics and FoxM1 siRNA on the hTERT expression, with possible synergistic effect when transfected together, as revealed by qRT-PCR and western blot (Figure 5C–5D). [score:5]
Thus, by computing the correlation coefficient using the NCI-60 expression profiling data, we quantified the correlation strength between p53 and miR-34a expression. [score:5]
Our data showed that miR-34a, miR-34b and miR-34c were underexpressed in more than half HCC samples compared with the adjacent tissues, suggesting that down-regulation of miR-34 famlily might be involved in the hepatic carcinogenesis (P < 0.05, Figure 1A–1B). [score:5]
Expression correlation between miR-34 family and hTERT or TP53 expression profile were analyzed using those two database. [score:5]
Kaplan–Meier curves showed that patients with underexpressed miR-34a and miR-34b had poorer overall survival and higher recurrence rates than those with higher expression (P < 0.05), whereas no substantial difference was observed for miR-34c (P > 0.05, Figure 1C–1D). [score:5]
In this study, we clearly demonstrate that miR-34a suppresses the expression of FoxM1 and c-Myc, which activates telomerase activity and extends telomere length, providing a rationale for the accelerated senescence by miR-34a in cancer cells (Figure 8A–8B). [score:5]
The miR-34 family is frequently downregulated in cancer partly due to the inactivation of p53 [19]. [score:4]
Contradictorily, other reports showd that miR-34a was also up-regulated in the HCCs in a chemical -induced HCC mo del [26]. [score:4]
However, in this study, we showed that miR-34a, which was also regulated by p53, induced telomerase activity by targeting FoxM1/c-Myc pathway, for the first time to demonstrate miR-34a as a bridge between p53 and telomere -dependent pathways in senescence. [score:4]
miR-34 family is frequently down-regulated in HCC and associates with poor prognosis. [score:4]
In human HCCs, Li et al. previously reported that miR-34a was down-regulated in 19 of 25 (76%) human HCC tissues. [score:4]
To gain insight into the biological role of miR-34 family in human HCC development, we examined the expression levels of miR-34 family in 75 paired HCC samples by qRT-PCR. [score:4]
Furthermore, in our study, the down-regulated miR-34a level was demonstrated to be correlated with tumor malignant features and poor prognosis. [score:4]
Next we sought to find out the molecular mechanism by which miR-34a regulates telomerase expression. [score:4]
Recently, the miR-34 family (a, b and c) has gained attention as they were identified as p53 targets and regulate p53 -mediated cycle arrest and apoptosis [18]. [score:4]
In the current study, we report for the first time that miR-34a induces telomere -dependent senescence in HCC cells via targeting FoxM1/c-Myc pathway, which is regulated by p53. [score:4]
These evidence suggest that by targeting FoxM1/c-Myc pathway, miR-34a might possess a significant role in the regulation of telomerase activity, telomere length, and the subsequent cellular senescence. [score:4]
However, with respect to the up-regulated miR-34a level, there might be alternative mechanisms that remain to be identified. [score:4]
In the present study, we provided evidence that miR-34a, the novel prognostic marker in cancer, regulated senescence and viability in HCC tissues and cell lines, at least, by targeting the FoxM1 and c-Myc gene, in the telomere pathway. [score:4]
Our data demonstrate that upregulation of miR-34a would induce cellular senescence and growth arrest in human HCC. [score:4]
Although Jin et al. suggested that miR-34a might regulate the telomere length by targeting PNUTS, the specific mechanism still remains elusive [33]. [score:4]
Figure 4 (A) Heat maps of gene expression changes after transfected with miR-34a mimics, as revealed by qRT-PCR. [score:3]
To our expectation, the p53 protein expression significantly increased after treatment with H [2]O [2] or cisplatin in the wild-type p53 cells and the levels of miR-34a also increased accordingly (Figure 6D–6E). [score:3]
Furthermore, to verify the idea that miR-34a represses FoxM1 and c-Myc through these sites, we generated luciferase reporter constructs, in which the miR-34a seed target or the specific 3′-UTR is placed behind the luciferase gene. [score:3]
miR-34a targets c-Myc and FoxM1. [score:3]
Recently, Bai et al. suggested that miR-34a promoted renal senescence by suppressing mitochondrial antioxidative enzymes with a concomitant increase in reactive oxygen species (ROS), providing a new mechanism of senescence pathway by miR-34a [30]. [score:3]
As shown in Figure 6B–6C, the miR-34a expression positively correlated with the p53 level (P < 0.05), which was consistent with the NCI-60 data. [score:3]
In addition, our data also provide in vivo evidences for the HCC treatment by enforced expression of the lentivirus -based miR-34a. [score:3]
miR-34a inhibits telomerase activity and induces telomere shortening. [score:3]
To further verify this hypothesis, we then examined the p53 and miR-34a expression in four liver cancer cells by qRT-PCR and western blot. [score:3]
Figure 2 (A, B) Relationship between miR-34 family levels and hTERT mRNA expression in NCI-60 cell lines. [score:3]
miR-34a targets FoxM1 and c-Myc in liver cancer cells. [score:3]
By target prediction, it turned out that FoxM1 and c-Myc have predicted binding sites for miR-34a in their 3′ UTRs (Figure 4B, 4E). [score:3]
Subcutaneous administration of miR-34a caused significant suppression of tumor growth of both HHCC and 7721 cells (Figure 7). [score:3]
In addition, they identified the c-Myc as target of miR-34a, which was consistent with our result. [score:3]
Of different liver cancer cell lines, HepG2, SMMC-7721, HHCC, and SK-Hep-1 cells were transiently transfected with a synthetic miR-34a duplex (miR-34a mimic), or an oligonucleotide complementary to the miR-34a sequence to block its function (anti-miR-34a), or negative control scrambled siRNA (control mimics and control inhibitor). [score:3]
Figure 3 (A) After 48 h of miR-34a mimics or inhibitor transfection, miR-34a level was analyzed by qRT-PCR. [score:3]
Comparatively low levels of miR-34a expression were demonstrated in several types of cancers [23, 24]. [score:3]
Thus we wondered whether miR-34a could affect any of the transcriptional factor expression. [score:3]
The red, white, and blue right-hand panel indicates log (base 2) of expression ratios after miR-34a mimics transfection. [score:3]
miR-34a inhibits telomerase activity via FoxM1/c-Myc signal pathway. [score:3]
As shown in Figure 2C, the hTERT mRNA expression appears to be inversely correlated with the levels of miR-34a (P < 0.05), which is consistent with the NCI-60 data. [score:3]
Taken together, these results suggested that miR-34a inhibits telomerase activity significantly, probably via the FoxM1/c-Myc signal pathway. [score:3]
When focusing on the genes potentially affect telomerase activity, we identified FoxM1 and c-Myc as targets of miR-34a. [score:3]
As well as transcriptionally regulated by p53, Christoffersen et al. reported that miR-34a was also regulated independently of p53 during oncogene -induced senescence [29]. [score:3]
Figure 7Administration of miR-34a with lentivirus suppresses cancer cell growth in vivo (A) Photographs illustrating representative features and growth curves of HHCC and 7721 tumors in nude mice after injection of LV-miR-34a or control LV-GFP. [score:3]
Figure 6 (A) Relationship between miR-34a and p53 expression levels in NCI-60 cell lines. [score:3]
Administration of miR-34a with lentivirus induces senescence -associated growth arrest in vivoSubcutaneous administration of miR-34a caused significant suppression of tumor growth of both HHCC and 7721 cells (Figure 7). [score:3]
Administration of miR-34a with lentivirus suppresses cancer cell growth in vivo. [score:3]
Taken together, These data suggest that suppression of cell proliferation by miR-34a is mainly associated with the induction of senescence-like phenotypes, probably via telomerase and telomere pathway. [score:3]
Subsequently, western blot also confirmed that the levels of FoxM1, c-Myc and hTERT were decreased after treatment with TP53 siRNA, indicating that the miR-34a induced senescence is probably regulated by p53 (Figure 6G). [score:2]
Figure 1 (A, B) miR-34a, miR-34b and miR-34c expression were significantly decreased in HCC compared with the corresponding adjacent tissues using qRT-PCR analyses. [score:2]
Since previously studies reported that miR-34a is mostly activated in the presence of wild-type p53 function, we wondered whether the miR-34a induced senescence is also regulated by p53. [score:2]
miR-34a induced senescence is regulated by p53. [score:2]
As shown in Figure 6A, we found a positive correlation of p53 and miR-34a in the NIC-60 data (P < 0.05), indicating that p53 might regulate the miR-34a function in cellular senescence. [score:2]
The mRNA expression was assayed in triplicate and normalized to different reference genes, such as the GAPDH, U6 (miR-34a). [score:2]
As shown in Figure 2A–2B, only miR-34a is inversely correlated with the telomerase activity (P < 0.05), indicating the potential regulating roles of miR-34a in telomerase activity. [score:2]
The cellular senescence caused by miR-34a also resulted in remarkable inhibition of cell proliferation, as represented by the MTT assay (Figure 3F). [score:2]
Consistent with this result, we discovered that the expression of miR-34a was reduced in 52 of 75 (69%) human HCC tissues compared with the adjacent tissues. [score:2]
Mir-34a was transfected to several HCC cell lines and expression of the transcriptional factors was examined by qRT-PCR analysis. [score:2]
Our finding that miR-34a regulates telomere length and telomerase expression prompted us to further investigate the pro-senescent effect of miR-34a in HCC. [score:2]
In addition, tumor tissues treated with miR-34a showed a significantly decreased immunostaining of Ki67 and PCNA, suggesting the in vivo anti-proliferation effect of miR-34a (Figure 7B–7C). [score:1]
However, the correlations between miR-34a and other tumor index such as tumor size (r = –0.05, P > 0.05) and TNM staging (r = –0.094, P > 0.05) fail to reach statistical significance. [score:1]
Results showed that miR-34a shortened telomere length significantly, suggesting the miR-34a -induced tumor growth arrest was probably due to the telomere -associated senescence (Figure 7F). [score:1]
We then examined the relationship between miR-34 family and telomerase activity in 75 HCC samples by qRT-PCR. [score:1]
Correlation of miR-34 family levels with telomerase activity. [score:1]
Administration of miR-34a with lentivirus induces senescence -associated growth arrest in vivo. [score:1]
We observed that introduction of miR-34a into liver cancer cells caused senescence-like phenotypes, with positive staining for senescence -associated β-galactosidase (SA β-gal) and enlarged cellular size (Figure 3D–3E). [score:1]
Previous reports showed that miR-34 -induced senescence in cancer cell is all in the form of telomere-independent cell cycle arrest. [score:1]
Once the tumor size reached ~50 mm [3], mice were treated with intratumoral injections (at days 0) of 1 × 10 [7] pfu/ml of the LV-miR-34a or LV-control lentivirus construction on each side. [score:1]
Subsequently, we tested whether miR-34a had an effect on telomerase activity and telomere length in vitro. [score:1]
However, so far all the reported examples of senescence induction by miR-34a are telomere-independent [31, 32]. [score:1]
This novel miR-34a -mediated mechanism offers a new potential strategy for cancer therapy. [score:1]
To further explore the potential roles of miR-34 family in affecting malignant characteristics, the expression levels of miR-34 family in tumor tissues were used to build a signature of prognosis. [score:1]
In this study, we found that miR-34a might induce senescence in HCC via the modulation of telomerase activity, providing new insights into the mechanism underlying HCC senescence. [score:1]
miR-34 family expression is associated with malignant characteristics in patients with HCC. [score:1]
However, in either the p53 mutant Huh-7 cells or the p53 deficient Hep3B cells, there is no change in either p53 or miR-34a after treatment with H [2]O [2] or cisplatin (Figure 6D–6E). [score:1]
miR-34a induces senescence -associated growth arrest in liver cancer cells. [score:1]
Since miR-34a targets FoxM1 and c-Myc, which were demonstrated to be the hTERT transactivators, we further investigated whether. [score:1]
Thus, we speculate that miR-34a might play varied roles during the carcinogenesis of HCCs caused by different mechanisms. [score:1]
However, discrepancies emerged when we focused on the miR-34a level in HCC. [score:1]
With the transfection of miR-34a duplex (miR-34a) in cancer cell lines, we found that miR-34a reduced the protein levels of c-Myc and FoxM1 significantly (Figure 4C, 4F). [score:1]
With no surprise, similar effects were also observed for miR-34a mimics and c-Myc siRNA (Figure 5E–5F). [score:1]
2) Ref Ref Tumor thrombosis  No 62(82.7) 41(80.4) Ref Ref 47(81.0) Ref Ref  Yes 13(17.3) 10(19.6) 1.42(0.71–2.85) 1.34(0.66–2.70) 11(19.0) 1.39(0.72–2.68) 1.17(0.58–2.35) TNM staging  I+II 29(38.7) 22(43.1) Ref Ref 24(41.4) Ref Ref  III+IV 46(61.3) 29(56.9) 0.98(0.56–1.71) 0.66(0.37–1.19) 34(58.6) 1.09(0. 64–1.84) 0.82(0.46–1.44) In the correlation analysis, the miR-34a level was showed to be negatively correlated with AFP level (r = –0.236, P < 0.05), suggesting some connections of these markers in the prognosis of HCC patients. [score:1]
Previous studies demonstrated that miR-34 family could induce cellular senescence by participating in cell cycle arrest. [score:1]
2) Ref Ref Tumor thrombosis  No 62(82.7) 41(80.4) Ref Ref 47(81.0) Ref Ref  Yes 13(17.3) 10(19.6) 1.42(0.71–2.85) 1.34(0.66–2.70) 11(19.0) 1.39(0.72–2.68) 1.17(0.58–2.35) TNM staging  I+II 29(38.7) 22(43.1) Ref Ref 24(41.4) Ref Ref  III+IV 46(61.3) 29(56.9) 0.98(0.56–1.71) 0.66(0.37–1.19) 34(58.6) 1.09(0. 64–1.84) 0.82(0.46–1.44)In the correlation analysis, the miR-34a level was showed to be negatively correlated with AFP level (r = –0.236, P < 0.05), suggesting some connections of these markers in the prognosis of HCC patients. [score:1]
In addition, this relationship was also verified by the multivariate Cox regression analysis, which demonstrates that both miR-34a and miR-34b could be independent prognostic factors for the overall survival and recurrence in HCC patients underwent surgical resection (Table 1). [score:1]
Overview of telomere related pathways for miR-34a -induced senescence. [score:1]
Until recently, it was unknown whether miR-34 family could induce cellular senescence in HCC in a telomere -dependent way. [score:1]
Lentiviral constructs containing pre-miR-34a (LV-miR-34a) was purchased from GeneChem (Shanghai, China). [score:1]
The association between miR-34a and senescence has been wi dely studied. [score:1]
By analyzing nutlin-3a -treated cells, Kumamoto et al. firstly demonstrated that miR-34 family was involved in the p53 -dependent senescence pathway [28]. [score:1]
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In addition, miR-34a regulates phorbol ester -induced megakaryocytic differentiation of K562 cells by inhibiting cell proliferation and inducing cell cycle arrest, through direct regulation of its known targets cyclin -dependent kinase 4 and 6 (CDK4 and CDK6), MYB and mitogen-activated protein kinase kinase 1 (MEK1) [15]– [16]. [score:8]
Importantly, several reports showed that members of the miR-34 family are direct p53 targets, and their upregulation induces apoptosis and cell cycle arrest [8]– [13]. [score:7]
In mice, miR-34a is ubiquitously expressed with the highest expression in the brain [7], whereas miR-34b and c are mainly expressed in lung tissue [8]. [score:7]
miR-34a transcription is directly activated by p53, and in turn miR-34a regulates the expression of some p53 target genes [9], [11], [12]. [score:7]
In this study, we demonstrated a novel function for miR-34a during neuronal differentiation, implicating its expression in the appearance of postmitotic neurons and on the regulation of neurite elongation via SIRT1 downregulation. [score:7]
Rather, miR-34a-targets, such as CDK4 and CDK6, as well as MEK1, which regulate cell cycle progression and inhibit cell proliferation, may constitute potential mediators of miR-34a effect on postmitotic neurons. [score:6]
Notably, SIRT1 expression markedly decreased from the third day of differentiation, which also corresponds with miR-34a upregulation. [score:6]
C. Under resveratrol treatment, overexpression of miR-34a resulted in increased proportion of GFAP [+] cells (right), while miR-34a downregulation had the opposite effect (left). [score:6]
miR-34a may mediate neurite outgrowth by downregulating SIRT1 expression. [score:6]
However, a recent publication reported that miR-34a expression was not involved in SIRT1 downregulation during human embryonic stem cell differentiation [32]. [score:6]
Here, we have identified the role of miR-34a in regulating mouse neural stem (NS) cell differentiation by mechanisms that involve SIRT1 downregulation and increased p53-DNA binding activity. [score:5]
When evaluating the expression of both miR-34a and SIRT1, we found that miR-34a expression was significantly upregulated from the third day on, increasing ∼11 fold (p<0.001) at day 6, when compared with undifferentiated cells (Fig. 2A). [score:5]
This data might be particularly important in the specific case of gliomas, since miR-34a has been extensively studied as a therapeutic target for tumor suppression. [score:5]
SIRT1 expression levels decrease after miR-34a overexpression. [score:5]
Importantly, a similar increase in acetylated p53 was observed after miR-34a overexpression, suggesting that miR-34a may increase p53 acetylation and subsequent p53 transcriptional activity due to decreased SIRT1 expression. [score:5]
Recently, miR-34a was shown to modulate mouse embryonic stem cell differentiation by regulating silent information regulator 1 (SIRT1) expression levels [17]. [score:5]
In this scenario, siRNA transfection would result in increased SIRT1 downregulation when compared with miR-34a overexpression. [score:5]
Based on this, downregulation of SIRT1 mediated by miR-34a would result in increased levels of acetylated p53. [score:4]
In addition, members of the miR-34 family have been identified as direct p53 targets. [score:4]
Moreover, under certain conditions, miR-34a may upregulate astrocytic differentiation through a SIRT1-independent mechanism. [score:4]
miR-34a upregulation increases acetylation of p53 and p53-DNA binding activity. [score:4]
As miR-34a negatively regulates SIRT1 expression, we would have expected to have an opposite effect of miR-34a on GFAP [+] cells. [score:4]
To further explore this hypothesis, we upregulated miR-34a expression levels and evaluated p53-DNA binding activity by EMSA. [score:4]
In addition, miR-34a can also regulate Notch-1 and Notch-2 protein expression [36], which could contribute to the observed final outcome. [score:4]
Therefore, resveratrol treatment further enhanced upregulation of astrocytic differentiation after miR-34a modulation. [score:4]
Given that miR-34a negatively regulates SIRT1 expression and SIRT1 induces the astroglial lineage, we would have expected an opposite effect of miR-34a in GFAP [+] cells. [score:4]
SIRT1 protein was significantly decreased after miR-34a overexpression (p<0.05) (Fig. 2D), suggesting that the effect of miR-34a on the number of postmitotic neurons and neurite elongation might be mediated by SIRT1. [score:3]
These results support the observation that miR-34a may modulate neurite outgrowth by targeting SIRT1. [score:3]
Interestingly, under resveratrol treatment, overexpression of miR-34a resulted in a significant increase in GFAP [+] cells by ∼3 fold (p<0.001) (Fig. 5C), while transfection with anti-miR-34a under the same conditions resulted in a decrease in GFAP [+] cells by ∼17% (p<0.05). [score:3]
A. Expression of miR-34a throughout the differentiation period. [score:3]
Similarly to miR-34a overexpression, transfection with SIRT1 siRNA also resulted in significantly increased total neurite output by ∼3.5-fold (p<0.0001) (Fig. 3D). [score:3]
In fact, the authors observed no increase in miR-34a expression after induction of differentiation into embryonic bodies. [score:3]
Although the number of neuritis was not changed, miR-34a overexpression led to a significant increase by ∼22% (p<0.05) in the length of neurites, suggesting that miR-34a modulates not only the appearance of postmitotic neurons but also plays a potential role in neurite elongation (Fig. 1F). [score:3]
miR-34a may have a wide range of molecular targets during cell differentiation. [score:3]
Moreover, the effect of siRNA -mediated silencing of SIRT1 on neurite number/output was more pronounced than the effect of miR-34a overexpression. [score:3]
A. Representative immunoblot showing increased p53 acetylation at lysine 379 after SIRT1 silencing or overexpression of miR-34a. [score:3]
In addition, acetylation of p53 (Lys 379) and p53-DNA binding activity were increased and cell death unchanged after miR-34a overexpression, thus reinforcing the role of p53 during neural differentiation. [score:3]
This is in accordance with previous studies showing that miR-34a overexpression did not affect human astrocytes [36]. [score:3]
Nuclear extracts from p53- and miR-34a -overexpressing cells were prepared as previously described [61]. [score:3]
These results suggested that miR-34a overexpression increased p53-DNA binding activity. [score:3]
Our results provide new insight into the molecular mechanisms by which miR-34a modulates neural differentiation, suggesting that miR-34a is required for proper neuronal differentiation, in part, by targeting SIRT1 and modulating p53 activity. [score:3]
Flow cytometry analysis revealed substantial alterations in NeuN expression, when cells were transfected with either anti- or pre-miR-34a (Fig. 1A). [score:3]
Therefore, multiple targets could contribute to its effects in astrogliogenesis induced by miR-34a. [score:3]
C. Immunofluorescence detection of NeuN expression in 6 day cells transfected with pre-miR-Control and pre-miR-34a for 72 h. Scale bar, 50 µm. [score:3]
Nevertheless, the precise role of miR-34a during neuronal differentiation and the relevance of SIRT1 targeting have not been reported. [score:3]
miR-34a is a well-known miRNA involved in various cellular functions that acts as a tumor suppressor [9], [11]. [score:3]
miR-34a overexpression increased postmitotic neurons and neurite elongation of mouse NS cells, whereas anti-miR-34a had the opposite effect. [score:3]
Additional studies, possibly using genetic backgrounds with reduced activity of regulatory pathways modulated by resveratrol would be necessary to confirm the capacity and the mechanism of miR-34a to regulate astrogliogenesis. [score:3]
This is in agreement with previous observations that miR-34a can suppress cell-cycle genes and induce a neural phenotype [35]. [score:3]
In addition, we have shown that this differentiation process is associated with the modulation of pro-apoptotic miRNA expression, including miR-16, let-7a and miR-34a [31]. [score:3]
D. Representative immunoblot (top) and corresponding densitometry analysis (bottom) showing decreased SIRT1 expression in pre-miR-34a transfected cells. [score:3]
SIRT1 expression is modulated by miR-34a during neural differentiation. [score:3]
Specifically, overexpression of miR-34a resulted in increased total neurite output. [score:3]
We previously determined that miR-34a expression was concomitant with the appearance of postmitotic neurons and astrocytes during mouse NS cell differentiation [31]. [score:3]
The effect of miR-34a overexpression on the percentage of postmitotic neurons was also confirmed by immunocytochemistry, revealing increased number of NeuN [+] cells after pre-miR-34a transfection (Fig. 1C and D). [score:3]
SIRT1 was identified as a target of miR-34a, which may mediate the effect of miR-34a on neurite elongation. [score:3]
miR-34a overexpression increases acetylation of p53 and p53-DNA binding activity. [score:3]
Nevertheless, transfection with p53 siRNA did not affect miR-34a expression, suggesting that induction of miR-34a during mouse NS cell differentiation is p53 independent (data not shown). [score:3]
SIRT1 has recently been suggested to be a target of miR-34a during embryonic stem cell differentiation [17]. [score:3]
Our results suggested that miR-34a indirectly regulates p53, possibly through a SIRT1 -dependent mechanism. [score:3]
F. Neurite number and total neurite output are given to quantify the effect of miR-34a overexpression on cellular morphology. [score:3]
miR-34a is involved in monocyte-derived dendritic cell differentiation by targeting JAG1 [14]. [score:3]
miR-34a expression was modulated using 100 nM of pre/anti-miR negative control and pre/anti-miR-34a (Applied Biosystems, Foster City, CA). [score:3]
Importantly, the EMSA results show that total nuclear proteins capable of binding to the p53-cons probe markedly increase under miR-34a overexpression (Fig. 4C). [score:3]
miR-34a overexpression also resulted in altered cellular morphology. [score:3]
miR-34a expression was analyzed by quantitative Real Time-PCR using specific Taqman primers and GAPDH for normalization. [score:2]
Curiously, miR-34a was shown to positively regulate the proportion of GFAP [+] cells when cells were pre -treated with resveratrol. [score:2]
miR-34a appears to be a key player parting the p53 regulatory network [37]. [score:2]
D. Immunofluorescence showing increased number of GFAP [+] cells under resveratrol treatment and miR-34a overexpression (bottom) when compared with controls (top). [score:2]
The induction of miR-34a during mouse NS cell differentiation and the concomitant decrease of SIRT1 suggest that p53 may be involved in this regulation. [score:2]
In conclusion, our results support a role for miR-34a in the regulation of neural differentiation. [score:2]
Therefore, it is possible that the particular environment generated by resveratrol treatment would render the cells sensitive to the modulation of miR-34a, revealing an additional miR-34a function, i. e. the capacity to regulate the glial lineage. [score:2]
Modulation of miR-34a had no effect on the number of Nestin [+] and β-III Tubulin [+], while the effect on GFAP [+] cells resulted in a slight tendency for a positive regulation. [score:2]
miR-34a promotes astrocytic differentiation under resveratrol treatment. [score:1]
Cells transfected with either control or pre-miR-34a at 3 days of differentiation were also processed for SIRT1 detection by immunoblotting. [score:1]
miR-34a is a member of the miR-34 family, which in mammals also includes miR-34b, and -34c [6]. [score:1]
Astrogliogenesis can be modulated by miR-34a through a SIRT1-independent mechanism. [score:1]
However, there is growing evidence to suggest a role for miR-34a during cell differentiation. [score:1]
Evaluation of miR-34a expression levels by quantitative Real Time-PCR. [score:1]
In addition, miR-34a -mediated silencing of SIRT1 may be necessary for correct establishment of specific differentiation programs during neural stem cell differentiation. [score:1]
Several signaling pathways can be potentially affected by miR-34a, and implicated in the transition of dividing neuronal precursors to immature postmitotic neurons. [score:1]
Pre- and anti-miR-34a, and respective controls, were transfected into differentiating mouse NS cells at 3 days. [score:1]
Next, we evaluated whether overexpression of miR-34a resulted in decreased SIRT1 at the protein level. [score:1]
A. Representative histograms of Nestin, β-III Tubulin, NeuN and GFAP detection in anti-miR-control (red line) and anti-miR-34a -transfected cultures (blue line) (top), or in pre-miR-control (red line) and pre-miR-34a -transfected cultures (blue line) (bottom). [score:1]
miR-34a may modulate neuronal outgrowth through a SIRT1 -mediated mechanism. [score:1]
Here we show that miR-34a promoted the appearance of postmitotic neurons. [score:1]
To determine the effect of miR-34a on neural differentiation, we explored the effect of miR-34a modulation on the percentage of neural progenitors (Nestin [+]), neuronal precursors (β-III Tubulin [+]), postmitotic neurons (NeuN [+]) and astrocytes (GFAP [+]). [score:1]
Instead, p53 seems to act downstream of miR-34a in this cellular context. [score:1]
These results suggested that the effect of miR-34a on the percentage of postmitotic neurons was not mediated by SIRT1. [score:1]
This suggests that under certain conditions, miR-34a may have the capacity to influence commitment toward astrogliogenesis by a SIRT1-independent mechanism. [score:1]
Neurite number, total neurite output and the length of longest neurite were determined to evaluate the effect of SIRT1 silencing and miR-34a overexpression on neurite outgowth. [score:1]
miR-34a modulates the proportion of NeuN -positive cells and neurite outgrowth. [score:1]
Immunofluorescence analysis of GFAP after pre-miR-34a transfection corroborated this observation (Fig. 5D). [score:1]
Efficiencies of miR-34a modulation and SIRT1 silencing were assessed by quantitative real-time RT-PCR and immunoblotting, respectively. [score:1]
Here we report an additional effect of miR-34a on neurite elongation. [score:1]
Nevertheless, while the present study provides a foundation for the role of miR-34a in neural stem cell differentiation, the role of p53 in this pathway remains unclear. [score:1]
Mouse NS cells were transfected using 100 nM of either anti- or pre-miR-34a at 3 days, and collected after 24 and 72 h, respectively. [score:1]
miR-34a modulates the appearance of postmitotic neurons and neurite outgrowth. [score:1]
Recently, miR-34a has been implicated in the differentiation of monocyte-derived dendritic cells, human erythroleukemia cells, and mouse embryonic stem cells. [score:1]
We found that transient transfection of miR-34a into mouse NS cells had almost no effect on the astrocytic subpopulation. [score:1]
To confirm that SIRT1 participates in miR-34a -mediated neural differentiation, we initially determined whether SIRT1 modulation affects the percentage of postmitotic neurons. [score:1]
For this purpose, cells were fixed at 3 and 6 days, for SIRT1 and miR-34a modulation, respectively, and stained with the marker β-III Tubulin to identify neuronal precursor cells. [score:1]
Further, the percentage of NeuN [+] cells significantly increased by 93% after pre-miR-34a transfection for 72 h (p<0.05) (Fig. 1B). [score:1]
C. EMSA showing increased p53-DNA binding activity in pre-miR-34a transfected cells for 48 and 72 h. D. Representative Annexin V-APC/PI staining showing absence of cell death after miR-34a modulation. [score:1]
However, the function of miR-34a in the control of the differentiation program of specific neural cell types remains largely unknown. [score:1]
These results are in accordance with previous data showing a p53-independent role for miR-34a during megakaryocytic differentiation of K562 cells [16]. [score:1]
Thus, we examined the effect of miR-34a on acetylation of p53. [score:1]
Our results showed that the specific effect of miR-34a on the appearance of postmitotic neurons was not mediated by SIRT1. [score:1]
0021396.g001 Figure 1Mouse NS cells were transfected using 100 nM of either anti- or pre-miR-34a at 3 days, and collected after 24 and 72 h, respectively. [score:1]
miR-34a is encoded by its own transcript, whereas miR-34b and miR-34c share a common primary transcript. [score:1]
D. Quantification data of NeuN -positive cells assessed by immunocytochemistry after miR-34a modulation. [score:1]
B. Quantification data of NeuN -positive cells assessed by flow cytometry after miR-34a modulation. [score:1]
Interestingly, in conditions where SIRT1 was activated by pharmacologic treatment with resveratrol, miR-34a promoted astrocytic differentiation, through a SIRT1-independent mechanism. [score:1]
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It had been confirmed that cisplatin treatment upregulated miR-34a expression regardless of p53 status [9], while our results also indicated there was no significant correlation between miR-34a and P53 expression following cisplatin treatment (Figure  2A and Additional file 1), so the cisplatin induced epigenetic alteration could be a reasonable explanation for the upregulation of miR-34a expression. [score:13]
To explore whether promoter hypermethylation leads to the suppression of expression, we examined the expression of miR-34a in bladder epithelial cell lines treated with the DNA methylation inhibitor, 5-aza-dC. [score:9]
These results indicated that the downregulation of CD44 expression following cisplatin treatment was mainly due to cisplatin -induced endogenous miR-34a upregulation. [score:9]
Firstly, by using a CD44 siRNA, we demonstrated that downregulation of CD44 could efficiently inhibit cell proliferation (Figure  6A), decrease colony and sphere formation ability (Figure  6B and C) in all three MIBC cell lines, which correlated with the effect of overexpressed miR-34a. [score:8]
Increased miR-34a expression significantly sensitizes bladder cancer cells to cisplatin treatment and inhibits the tumorigenicity and proliferation of cancer cells in vitro and in vivo through targeting CD44. [score:7]
miR-34a is the most significantly inducted miRNAs by p53 and ectopic miR-34a expression induces apoptosis, cell-cycle arrest, senescence and alters cancer cell chemosensitivity through direct targeting multiple genes in p53 signaling pathway, such as Sirt-1, CDK6, E2F3 and Bcl-2 [12- 14]. [score:6]
Downregulation of CD44 by siRNA led to similar effect of miR-34a overexpression on A) cell proliferation (mean ± SEM; n = 3; *p < 0.05) and B-C) tumorigenity (mean + SEM; n = 3; *p < 0.05). [score:6]
Compared to NC group, overexpression of miR-34a significantly inhibited MIBC cell proliferation and vice versa (Figure  4A, P < 0.05), while as shown in Figure  4B-E, elevated miR-34a expression also dramatically decreased the colony and sphere formation ability of all three MIBC cell lines. [score:6]
Among these miRNAs, miR-34a has been described as a “star” miRNAs in cancer research, which commonly functions as a tumor suppressor and is down-regulated in many human cancers [6]. [score:6]
Furthermore, we identified CD44 as being targeted by miR-34a in MIBC cells following cisplatin treatment, and increased CD44 expression could efficiently reverse the effect of miR-34a on MIBC cell proliferation, colongenic potential and chemosensitivity. [score:5]
To investigate the primary target of miR-34a that could be the potential mechanism underlying the association between the increased miR-34a expression and cisplatin chemosensitivity, we initially measured the expression of some well-known targets of miR-34a in 5637, T24 and HT-1376 cells following cisplatin treatment, including MYC, BCL2, NOTCH1, CDK6, SIRT1, E2F1, CDK4, HGF, NOTCH2, SOX2 and CD44 [7, 12, 22, 23, 26, 27]. [score:5]
Elevated miR-34a expression inhibits proliferation and decreases clonogenic potential of MIBC cells in vivo and in vitro. [score:5]
Expression of some well-known targets of miR-34a in 5637, T24 and HT-1376 cells following cisplatin treatment. [score:5]
To investigate the primary target of miR-34a that may help to explain the association between the increased miR-34a overexpression and cisplatin chemosensitivity, we initially measured the expression of some well-known targets of miR-34a in 5637, T24 and HT-1376 cells following cisplatin treatment, including MYC, TP53, BCL2, NOTCH1, CDK6, SIRT1, E2F1, CDK4, HGF, NOTCH2, SOX2 and CD44. [score:5]
Six synthetic, chemically modified short single or double stranded RNA oligonucleotides (miR-34a mimics, mimics NC, miR-34a inhibitor, inhibitor NC, agomir-miR-34a and agomir-NC) were purchased from Ribo Biotech (Guangzhou, China). [score:5]
Vogt et, al indicated that promoter hypermethylation could be an important reason for the decreased miR-34a expression in urothelial cancer [24], in our study, using a more exact method, Massaraay, which could detected up to 500 bp nucleotides containing 14 CpGs, combined with the data of 5-aza-dC demethylation treatment, we clearly demonstrated that promoter hypermethylation caused the decrease of miR-34a expression. [score:5]
Increased miR-34a expression significantly sensitized MIBC cells to cisplatin and inhibited the tumorigenicity and proliferation of cancer cells in vitro and in vivo. [score:5]
E) CD44 protein expression was inhibited in miR-34a transfected MIBC cells. [score:5]
However, it seems that only the expression of CD44 decreased in a doze -dependent manner following cisplatin treatment, which correlated with the expression of miR-34a (Figure  5A and Additional file 1). [score:5]
All these data presented here strongly suggesting that the tumor-suppressive and chemosensitivity effect of miR-34a was mediated by reducing the production of CD44 as its predominant target. [score:5]
Click here for file Expression of some well-known targets of miR-34a in 5637, T24 and HT-1376 cells following cisplatin treatment. [score:5]
We then tested whether miR-34a inhibited MIBC cell proliferation, colongenic potential and chemosensitivity through targeting CD44. [score:5]
It is of note that aberrant expression of microRNA has been linked to chemosensitivity, so we next examined the expression of miR-34a in MIBC cells after cisplatin treatment. [score:5]
As shown in Figure  6D-F, CD44 overproduction appeared to have a dramatic positive effect on tumor cell growth and tumorigenesis, and importantly, miR-34a induced tumor suppression was largely eliminated upon the overexpression of CD44. [score:5]
Cisplatin can upregulate miR-34a by promoter demethylation in MIBC cell lines. [score:4]
D) Cisplatin -induced endogenous miR-34a upregulation caused the decrease of CD44-luciferase activity. [score:4]
Together, these results suggested that miR-34a was down-regulated through promoter hypermethylation in MIBC. [score:4]
Cisplatin -based chemotherapy induced demethylation of miR-34a promoter and increased miR-34a expression, which in turn sensitized MIBC cells to cisplatin and decreased the tumorigenicity and proliferation of cancer cells that by reducing the production of CD44. [score:3]
Furthermore, the aberrant miR-34a expression has been linked to chemotherapy resistance in a variety of cancer [7- 11]. [score:3]
As shown in Figure  4F, at d33 post injection, there were still significant miR-34a expression in derived xenograft. [score:3]
Figure 5 CD44 was a primary target of miR-34a in MIBC cells following cisplatin treatment. [score:3]
Cisplatin induced miR-34a expression in A) doze- and B) time -dependent manner, relative to control group (cisplatin 0 μg/ml group or 0 h group, mean + SEM; n = 3; *p < 0.05). [score:3]
Importantly, statistical analysis demonstrated that the inhibition rate of each group satisfy the formula: (miR-34a + cisplatin) > (miR-34a alone) + (cisplatin alone), which indicated the synergistic effect of miR-34a to cisplatin chemotherapy. [score:3]
B) CD44 [+] cells proportion decreased following miR-34a overexpression or cisplatin treatment. [score:3]
F) Relative miR-34a expression in xenografts; G) Photographs of tumors excised 38 days after inoculation of stably transfected cells into nude mice; Mean xenograft tumor volume H) and weight I) in nude mice groups after indicated treatment (mean + SEM; n = 3; *p < 0.05). [score:3]
Our data presented here demonstrated that increased miR-34a expression could efficiently decrease tumorigenity of MIBC cells. [score:3]
This study aimed to reveal the association of the miR-34a expression and its modulation of sensitivity to cisplatin in muscle-invasive bladder cancer (MIBC). [score:3]
Accumulating evidence suggests a tumor suppressive role for miR-34a in human carcinogenesis. [score:3]
As shown in Figure  5D, luciferase activities decreased significantly in the CD44-luc -transfected 5637 cells when treated with cisplatin and such inhibition of luciferase activities by cisplatin could be abolished when the potential miR-34a binding sites were mutated. [score:3]
Our present results indicated that there was a strong association between cisplatin treatment and miR-34a expression in MIBC. [score:3]
In this study, we determined that miR-34a expression was frequently decreased in MIBC cell lines and tumor tissues, which was similar in other cancers. [score:3]
Taken together, these findings provide evidence that cisplatin induced promoter demethylation could be an important reason for increased miR-34a expression in MIBC cell lines. [score:3]
After treatment, all the five cell lines showed a reactivation of miR-34a expression (Figure  1C). [score:3]
Figure 2 Expression of miR-34a increased in MIBC cell lines via promoter demethylation following cisplatin treatment. [score:3]
Increased miR-34a expression sensitizes MIBC cells to cisplatin in vivo and in vitro. [score:3]
Increased CD44 expression could efficiently reverse the effect of miR-34a on MIBC D) cell proliferation (mean ± SEM; n = 3; *p < 0.05), E-F) colongenic potential and G) chemosensitivity (mean + SEM; n = 3; *p < 0.05). [score:3]
The expression of miR-34a was further analyzed by qPCR in 14 paired MIBC/adjacent normal bladder tissue specimens. [score:3]
CD44 is a primary target of miR-34a in MIBC cells following cisplatin treatment. [score:3]
Then T24 cells, which stably expressing miR-34a or NC, were used to construct the xenograft mo del in nude mice. [score:3]
In this study, we demonstrate that expression of miR-34a is frequently decreased in bladder cancer tissues and cell lines through promoter hypermethylation while it is epigenetically increased in bladder cancer cells following cisplatin treatment. [score:3]
As miR-34a expression increased dramatically following cisplatin treatment, we subsequently assessed in the association between miR-34a and chemosensitivity. [score:3]
miR-34a has been described a tumor suppressive miRNA in many kinds of cancers through inducing tumor cell apoptosis, senescence, cell cycle arrest or repressing metastasis [9, 10, 14, 20- 22, 25]. [score:3]
Increased CD44 expression can efficiently reverse the effect of miR-34a on MIBC cell proliferation, colongenic potential and chemosensitivity. [score:3]
Surprisingly, in spite of a glimpse through qRT-PCR on the level of mRNA, we still observed a clear inverse correlation between miR-34a and CD44 expression, but not others. [score:3]
Figure 3 Overexpression of miR-34a in MIBC cells sensitized tumor cells to cisplatin chemotherapy. [score:3]
Taken together, these results demonstrated that miR-34a functions as a tumor suppressor in MIBC cells through decreasing their clonogenic potential. [score:3]
miR-34a expression was frequently decreased in MIBC tissues and cell lines through promoter hypermethylation while it was epigenetically increased in MIBC cells following cisplatin treatment. [score:3]
As described, the CD44-luc-vector we used just contained the seed regions of the miR-34a potentially target sites in CD44 3′UTR. [score:3]
Result showed that chemotherapy led to increased miR-34a expression in all three MIBC cell lines in a dose- and time -dependent manner (Figure  2A and B). [score:3]
A) The effect of ectopic miR-34a expression on MIBC cell proliferation was investigated by CCK-8. The miR-34a activity was mediated by transfection with miR-34a mimics or inhibitor respectively. [score:3]
Then by luciferase reporter system and immunobloting, we determined CD44 was exactly a target of miR-34a in MIBC cells (Figure  5C and E). [score:3]
Subsequently, increased miR-34a expression induced by cisplatin could in turn improve cisplatin sensitivity of MIBC cells in vitro and in vivo. [score:3]
Further experiments validated that the tumor-suppressive and chemosensitivity effect of miR-34a was mediated by reducing the production of CD44. [score:3]
Figure 1 MiR-34a was epigenetically downregulated in MIBC. [score:3]
Figure 6 The tumor-suppressive and chemosensitivity functions of miR-34a were mediated by reduction the production of CD44. [score:3]
C) The seed regions of the miR-34a target sites in CD44 and the luciferase activity assay. [score:2]
White circle means missing data at a given CpG unit; D) qRT-PCR demonstrated miR-34a expression in four MIBC cell lines after treatment with 5-aza-dC compared to mock -treated cells; Average methylation level of miR-34a promoter region in MIBC E) cell lines and F) patient tissues. [score:2]
Subsequently, these assays were repeated when miR-34a mimics and CD44 expressing vector were co -transfected into 5637 and T24 cells. [score:2]
Synthetic short single or double stranded RNA oligonucleotides and lentiviral vector were used to regulate miR-34a expression in MIBC cells to investigate its function in vitro and in vivo. [score:2]
And when compared with NC group, miR-34a stably expressing tumor xenograft had markedly smaller size and lower weight (Figure  4G-I, P < 0.05). [score:2]
Compared to SV-HUC-1 cells, all of four MIBC cell lines had a significantly lower level of miR-34a expression (Figure  1A, P < 0.05). [score:2]
Figure 4 MiR-34a functioned as a tumor suppressor in MIBC cells. [score:2]
Lenti-miR-34a and Lenti-NC were purchased from Genechem Biotech (Shanghai, China) Primary antibody CD44 (1:1000) and GAPDH (1:10000) were purchased from Sigma-Aldrich, St. [score:1]
To further detect the promoter methylation status of the miR-34a quantitatively, the promoter CpG islands in bladder epithelial cell lines and tissue was determined by quantitative sequencing. [score:1]
Moreover, FACS results showed that the CD44 [+] bladder cancer stem cells in T24 cells decreased either transfected with miR-34a or treated with cisplatin (Figure  5B). [score:1]
Methylation analysis of miR-34a promoter region: C) profiling of the unit-specific methylation of CpG sites in the miR-34a promoter region was presented as an epigram. [score:1]
17 days after injection when appreciable tumor formed subcutaneously, agomir-miR-34a or agomir-NC were injected combined with cisplatin or PBS in tumor. [score:1]
miR-34a was one of such miRNAs, which had been extensively studied in bladder cancer and many other cancers [8, 10, 20- 24]. [score:1]
And a highly significant (Figure  1B, P < 0.01) reduction of miR-34a in 12 of 14 MIBC tissues was observed. [score:1]
The methylation analysis of miR-34a promoter region was performed by MassARRAY. [score:1]
miR-34a expression in MIBC cell lines and patient tissues was investigated using qPCR. [score:1]
Quantitative methylation analysis of the promoter of miR-34a was performed using the Sequenom MassARRAY platform (CapitalBio, Beijing, China) as previous study description [37]. [score:1]
Subsequent epigenetic assessment also proved that cisplatin treatment induced miR-34a promoter demethylation (Figure  2C and D). [score:1]
However, this mechanism seems not fitting well in cisplatin -based bladder cancer chemotherapy as miR-34a chemosensitizes bladder cancer cells to cisplatin treatment regardless of p53-Rb pathway status [9]. [score:1]
miR-34a expression was evaluated by qPCR in MIBC A) cell lines and B) patient tissues, U6 served as an internal control. [score:1]
of cell viability assay clearly showed that overexpression of miR-34a in 5637, T24 and HT1376 cells could efficiently increased their sensitivity to cisplatin as compared to NC group (Figure  3A and B, P < 0.05). [score:1]
Data are plotted as the mean ± SEM of 3 independent experiments relative to mock treatments; B) The IC [50] values for cisplatin of MIBC cell lines after transfected with miR-34a mimics (mean + SEM; n = 3; *p < 0.05); Mean xenograft tumor volume C) and weight D) in nude mice groups after indicated treatment (mean ± SEM; n = 3;). [score:1]
Methylation analysis of miR-34a promoter region: C) profiling of the unit-specific methylation of CpG sites in the miR-34a promoter region was presented as an epigram; D) Average methylation level of miR-34a promoter region in MIBC cell lines (mean + SEM; n = 3; *p < 0.05). [score:1]
miR-34a is frequently decreased in human MIBC tissues and cell lines through promoter hypermethylation. [score:1]
So its luciferase activity could only be affected by miR-34a. [score:1]
Finally, we measured the IC [50] values of cisplatin for these MIBC cell lines in differently treated group (Figure  6G), and the results showed that overexpression of CD44 could also efficiently reverse the effect of miR-34a on chemosensitivity. [score:1]
To evaluate the tumorigenity of miR-34a, two groups of 8 mice each were injected subcutaneously with miR-34a/NC stably expressing T24 cells constructed by lentivector infection and puromycin screening. [score:1]
However, there was still a limit in studies focusing on miR-34a in MIBC. [score:1]
Moreover, we found the epigenetic alteration of miR-34a promoter following cisplatin treatment. [score:1]
In order to investigate the phenotypic consequences of miR-34a in MIBC cells, we overexpressed this miRNA in 5637, T24 and HT-1376 cells via miRNA mimics and Lentivectors. [score:1]
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Finally, suppression of miR-34A expression was found to result in widespread up-regulation of long intergenic non-coding RNA transcripts in association with p53 mutation status (Tables S9–S10). [score:9]
To assess the extent to which miR-34A may regulate these transcripts, through direct seed sequence complementarity, the most statistically significant transcripts (p < 0.001) that were down-regulated secondary to miR-34A over -expression (n [TP53 mutant]= 277, n [TP53 wild-type]=17) were surveyed for the ACTGCC sequence (miR-34A, 3′ugUUGGUCGAUUCUGUGACGGu 5′ – miR-34A) (Tables S7–S8). [score:8]
Studies have shown that miR-34A can induce variable effects on p53 transcriptional activity, either positively by targeting p53 inhibitor transcripts such as MDM4, SIRT1, MTA2, HDAC1 and YY1, or negatively by directly targeting TP53 mRNA [4]. [score:8]
A number of known miR-34A target transcripts were significantly de-repressed in cells transfected with the hsa-miR-34A-5p inhibitor providing further support for the efficacy of this reagent in targeting miR-34A (Figure S18, Table S11). [score:7]
Transcriptional response of TP53 mutant and wild-type cells to miR-34A inhibition is characterized by changes in non-coding RNA transcriptsThe number of differentially expressed transcripts detected in the presence of miR-34A inhibition is higher than in the condition of miR-34A over -expression in both wild-type and mutant lines (Figure S2), indicating that loss of miR-34A may have a more widespread impact on the transcriptome in this cellular context. [score:7]
That exogenous miR-34A expression suppresses many of these replication -dependent transcripts in the presence of TP53 mutations, and to a greater extent than wild-type TP53, suggests that miR- 34A may play an important role in sustaining epigenetic integrity when p53 function is compromised by mutation. [score:7]
Indeed, the number of differentially expressed transcripts in TP53 mutant cells transfected with anti-miR-34A relative to control was much higher than that of TP53 wild-type cells (2631 transcripts and 357 transcripts, respectively), however, 91% (324/357) of transcripts found differentially expressed in wild-type cells were also differentially expressed in the TP53 mutant lines (Figure S2). [score:7]
The number of differentially expressed transcripts detected in the presence of miR-34A inhibition is higher than in the condition of miR-34A over -expression in both wild-type and mutant lines (Figure S2), indicating that loss of miR-34A may have a more widespread impact on the transcriptome in this cellular context. [score:7]
These findings demonstrate that, under basal conditions, expression of non-coding RNAs such as minor spliceosome components and LINC-PINT is maintained at low levels and inhibition of miR-34A leads to marked de-repression of these targets, among other non-coding transcripts. [score:7]
E2F1 – a known miR-34A target gene [12] - is down-regulated in response to miR-34A mimic transfection to a similar extent in TP53 mutant lines and TP53 wild-type lines (TP53 mutant: log [10]fold-change = −0.83, q = 9.2e-6; TP53 wild-type: log [10]fold-change = −0.85, q = 5.5e-4). [score:6]
Down-regulation of GMNN is observed in response to ectopic miR-34A expression in TP53 mutant cell lines, and to a lesser extent in wild-type cell lines (Figure 2E). [score:6]
Unlike non-transformed cells with elevated miR-34A expression, inhibition of miR- 34A results in significant deregulation of non-coding RNA transcripts (Figure 3B–3C). [score:6]
Although this transcript does not contain a sequence complimentary to the miR-34A seed region, miR-34A inhibition is directly correlated with LINC-PINT expression with increasing doses of anti-miR-34A (Figures S12–S13). [score:6]
Similarly, miR-34A is known to influence expression of CDK4 and this transcript is down-regulated in both TP53 mutant and TP53 wildtype cell lines (TP53 mutant: log [10]fold-change= −0.86, q = 8.3e- 12; TP53 wild-type: log [10]fold-change= −0.69, q = 9.4e-5). [score:6]
The transcriptional response to miR-34A inhibition by hsa-anti-miR-34A- 5p oligonucleotides in TP53 mutant and TP53 wild-type non-transformed cells revealed a distinct pattern of TP53 mutation -associated signatures, with a prominent effect of elevated transcript expression in TP53 mutant cells (Figure 3A). [score:6]
In summary, among the repertoire of differentially expressed RNA transcripts, there is a prominent effect of elevated miR-34A expression on critical cell cycle mediators, including regulation of replication -associated transcripts at the 6p22 locus, in the context of mutant TP53. [score:6]
Accordingly, there was no significant effect on suppression of cell proliferation mediated by miR-34A inhibition in this cell line (Figure 5A). [score:5]
Transcriptional response of TP53 mutant and wild-type cells to miR-34A over -expression affects cell cycle mediators and replication -dependent histonesThe transcriptional profile of cells transfected with hsa-miR-34A-5p mimic was systematically assessed in order to determine the impact of ectopic miR-34A expression in TP53-mutant and wild-type cell lines (Figure 2A). [score:5]
To further validate that this effect occurs in response to miR-34A inhibition, despite the absence of putative direct base pairing, transcript levels of one of the U12 spliceosome components (RNU4ATAC) were first validated by qPCR, then replicated in two TP53 mutant cell lines with increasing doses of anti-miR-34A (FB04 [S240G] cell line utilized in RNA-Seq experiment, and an independent cell line harboring a R248Q mutation) (Figures S9–S11). [score:5]
Figure 2(A) Distribution of differentially expressed transcripts in TP53 mutant and wild-type fibroblast cell lines with elevated miR-34A expression relative to controls. [score:5]
qPCR validation of anti-miR -mediated suppression of miR-34A expression was also performed (Figure S17). [score:5]
However, within the TP53 mutant cell lines harboring point mutations, the effects of inhibition of anti-miR-34A on the TP53 R158H cell line is less pronounced than the effect observed on the other LFS lines. [score:4]
Among the most significantly differentially expressed transcripts in both TP53 mutant and wild-type cells transfected with anti-miR-34A are numerous long intergenic non-coding RNAs (lincRNAs), other noncoding RNAs, as well as protein-coding transcripts that have been linked to TP53 and miR-34A regulation, such as SLC7A11 and PPP1R10, respectively [14, 15] (Figures S6–S7). [score:4]
Clarification of the mechanisms by which endogenous miR-34A functions as a tumor suppressor, and the vulnerabilities to tumorigenesis that occur as a result of its deregulation, are therefore needed. [score:4]
U11, U12 and U4atac are all found to be significantly upregulated to similar levels in cells transfected with anti-miR-34A (Figure 3D–3E; Figure 4A–4C; RNU11: q(Mut) = 8.37e-264, q(Wt) = 3.45e-12; RNU4ATAC: q(Mut) = 8.68e-202, q(Wt) = 6.97e-183; RNU12: q(Mut) = 6.97e-183; q(Wt) = 1.6e-19). [score:4]
The identification of novel coding and non-coding miR- 34A targets provides new avenues for intervention of the cellular pathways deregulated by miR-34A in human cancers. [score:4]
By contrast, inhibition of miR-34A in FB03, harboring a non-synonymous mutation at codon 163, resulted in decreased cell proliferation, as detected in the initial TP53 mutant cell lines studied (Figure 5A). [score:4]
The transcriptional response to miR-34A modulation reveals that this miRNA may be a crucial switch that can impact expression of numerous cell cycle regulators and non-coding RNA networks. [score:4]
Distinct transcriptional patterns were observed in cells with elevated miR-34A expression relative to controls in both TP53 mutant and wild-type cell lines (Figure S4). [score:3]
A total of 128 transcripts were commonly differentially expressed in TP53 mutant and TP53 wild-type cell lines transfected with the miR-34A mimic (Table S6). [score:3]
Three key components of the minor (U12) spliceosome are among the most highly differentially expressed transcripts in TP53 mutant cells transfected with anti-miR-34A. [score:3]
Since the G2 checkpoint prevents cells from entering mitosis when DNA is damaged, suppression of miR-34A resulting in G2/M arrest demonstrates that this microRNA is indeed essential to cell cycle progression, and this response may be diminished in the absence of functional p53. [score:3]
Transfection efficiency was validated by concomitant transfection with GFP pMAX fluorescent reporter, as well as qPCR validation in three cell lines to test miR-34A over -expression post-transfection (Figure S16). [score:3]
The transcriptional profile of cells transfected with hsa-miR-34A-5p mimic was systematically assessed in order to determine the impact of ectopic miR-34A expression in TP53-mutant and wild-type cell lines (Figure 2A). [score:3]
Briefly, 200,000 (confluent) cells were seeded into a well of a 6-well plate 24 hours before transfection with 3 ul of Lipofectamine 2000 (Invitrogen) and 75 nM of hsa-miR- 34a-5p/control mirVana mimics or hsa-anti-miR-34a-5p/control mirVana inhibitors (Ambion, Life Technologies). [score:3]
Intriguingly, transcript expression of 6p22 histone cluster 1 transcripts that are otherwise elevated in TP53 mutant cells (as shown in Figure 1E), was significantly decreased in TP53 mutant lines transfected with miR-34A mimic, but were essentially unaffected in wild-type cell lines (Figure 2F). [score:3]
In TP53 wild-type cell lines transfected with anti-miR-34A, the most significantly differentially expressed transcript is LINC-PINT (long intergenic non-coding RNA, p53 -induced transcript) (q = 1.24e-80) (Figure 4D). [score:3]
There was no effect on TP53 transcript levels following miR-34A over -expression. [score:3]
The top 50 significantly differentially expressed transcripts in both TP53 mutant lines and wild-type lines transfected with miR-34A mimic were exclusively protein-coding (Figure 2B–2C). [score:3]
tw/), and expression of these transcripts was determined in the anti-miR-34A RNA-Seq dataset (described below). [score:3]
Transcriptional response of TP53 mutant and wild-type cells to miR-34A over -expression affects cell cycle mediators and replication -dependent histones. [score:3]
These studies provide strong insight into the tumor suppressive capacity of miR-34A, demonstrating why miR-34A inactivation may be pervasive in human malignancies. [score:3]
The horizontal axis depicts the log10-transformed fold-change of transcript expression in cells transfected with anti-miR-34A relative to control. [score:3]
The horizontal axis depicts the log10- transformed fold-change of transcript expression in cells transfected with miR-34A mimic relative to control. [score:3]
Marked induction of key components of the minor (U12) spliceosome occurs in response to miR-34A suppression. [score:3]
Points above and to the left of the dotted line indicate transcripts that are expressed at low levels in cell lines transfected with a control mimic but elevated in cell lines with high relative miR-34A levels. [score:3]
The mature RNA transcripts of these components of the U12 spliceosome do not harbor miR-34A complimentary seed sequences, suggesting either miR- 34A regulates these transcripts indirectly through other transcripts or through global effects on the cell cycle state. [score:3]
The effect of miR-34A modulation on cell cycle regulatory genes is consistent with previous reports of miR-34A regulated transcripts in other cellular contexts [10]. [score:3]
Points above and to the left of the dotted line indicate transcripts that are expressed at low levels in cell lines transfected with a control anti-miR but elevated in cell lines with low relative miR-34A levels. [score:3]
Suppression of miR-34A strongly de-represses many non-coding RNA transcripts. [score:3]
Lastly, miR-34A suppression results in an increased fraction of cells arrested in the G2/M phase. [score:3]
Fraction (%) was determined by comparing the percent of cells in G2M for each cell line in the following miR-34A inhibition relative to untransfected cells for each respective cell line. [score:3]
Finally, cell cycle analysis reveals that suppression of miR-34A results in a shift in a fraction of the cell population into G2/M phase relative to untransfected cells (Figure 5B; Figure S15A–S15B). [score:3]
These findings point to a novel putative role of miR-34A in modulating transcription of the U12 spliceosomal machinery, likely indirectly through its impact on the cell cycle. [score:2]
Primary skin-derived fibroblast cell lines from LFS patients provide the opportunity to study miR-34A modulation in the context of TP53 mutation. [score:2]
miR-34A target transcripts that were validated by Western blot and/or reporter assays were downloaded from the miRTarBase database (http://mirtarbase. [score:2]
Collectively, the findings from this study support a putative role for miR-34A in acting as an essential regulator of broad transcriptional networks that converge on the cell cycle. [score:2]
Akin to p53, miR-34A deregulation is pervasive in human cancer. [score:2]
p53 -dependent regulation of miR-34A is mediated by a canonical p53 binding site that occurs within 30 kb of the miR-34A transcription start site at the 1p36 locus [3]. [score:2]
These findings corroborate the transcriptomic results that demonstrate miR-34A -associated regulation of essential cell cycle mediators. [score:2]
Although it is well known that miR-34A deregulation may be an important driver in cancer, the exact mechanisms of its role in cellular homeostasis have remained elusive [9]. [score:2]
Our findings are the first to identify miR-34A as an important node in the transcriptional regulation of numerous lincRNAs and point to further study of micro -RNA-lincRNA related pathways. [score:2]
Suppression of miR-34A using an anti-miR- 34A oligonucleotide reproducibly diminishes the cell proliferation rate of all three TP53 mutant cell lines used in the transcriptome assays, but to a lesser extent in the TP53 wild-type lines (Figure 5A). [score:2]
To further validate this finding, two additional cell lines harboring TP53 mutations, FB02 (E294fs) and FB03 (Y163C) were transfected with the anti-miR-34A oligonucleotide. [score:2]
These analyses reveal that miR-34A is a central node in numerous p53 -dependent and independent networks, including previously unreported regulation of replication -dependent histone genes, long intergenic non-coding RNAs (lincRNAs) and components of the U12 -dependent spliceosome. [score:2]
By exploring the transcriptional response to miR- 34A modulation in TP53 mutant and TP53 wild- type cell lines, we report the first global profile of the miR- 34A -dependent transcriptome in human non-transformed cells and demonstrate that miR-34A differentially regulates transcripts in the background of mutant and wild-type p53. [score:2]
Previous studies have shown that human cell lines with miR-34A knockouts have unimpaired p53 -mediated responses to genotoxic stress and miR-34A is therefore dispensible for the p53 -mediated response to stress in human cells [4]. [score:2]
In addition to p53 -mediated pathways, miR-34A is found to regulate other novel pathways, as evidenced by the differential effect of miR-34A transcriptional landscape in the context of mutant p53. [score:2]
miR-34A is the first identified microRNA (miRNA) found to be involved in the p53 regulatory network [1, 2]. [score:2]
GO term enrichment analysis of the miR-34A-regulated transcripts in the TP53 mutant cell lines demonstrates that genes are strongly enriched for cell cycle mediators (Figure 2D). [score:2]
To functionally characterize the phenotypic impact of miR-34A on non-transformed cells, we sought to determine the effects of miR-34A suppression on cell viability. [score:1]
In order to assess the transcriptional response of these cells to miR-34A modulation, RNA-seq was performed on RNA harvested from untransfected cells as well as cell lines 24 hours post-transfection with hsa-miR-34A-5p mimic or anti-hsa-miR-34a-5p (antagomir), or control oligonucleotides (Table S2). [score:1]
Briefly, fibroblast cells were washed twice in cold PBS and resuspended in 1 × binding buffer (BD Pharmingen) at a concentration of 1×10 [6] cells/mL, 48 h after miR-34A mimic/anti-miR oligonucleotide transfection. [score:1]
Through its complementary and redundant roles to p53, miR-34A may compensate for the effects of mutant p53 in non-transformed cells. [score:1]
This study describes the first analysis of the miR-34A-p53 axis in a non-transformed cellular context and provides insight into the underlying transcriptional networks in association with mutant TP53 and miR-34A. [score:1]
Moreover, miR-34A has been shown to be repressed in cancer stem cell populations [8]. [score:1]
Moreover, all cell lines transfected with anti-miR-34A differ significantly in their transcriptomic profile relative to all other conditions tested (Figure 1A; Figure S1). [score:1]
The vertical axis depicts the proliferation index for the respective cell line transfected with an anti-miR-34A oligonucleotide, normalized to untransfected cells and given as a fraction of the cell proliferation index of the same cell line transfected with a control anti-miR oligonucleotide. [score:1]
These results provide a framework for understanding the basal function of miR- 34A and demonstrate that miR-34A is essential to the maintenance of cellular homeostasis. [score:1]
miR-34A is essential for cell viability and repression of miR-34A results in G2/M cell cycle arrest. [score:1]
Transcriptional response of TP53 mutant and wild-type cells to miR-34A inhibition is characterized by changes in non-coding RNA transcripts. [score:1]
miR-34A is also is associated with transcriptional repression of a host of lincRNAs, including LINC-PINT, a p53 -induced lincRNA [17]. [score:1]
The numbers correspond to the fibroblast cell line ID (A = untransfected; B = control mimic; C = control anti-miR; D = miR- 34A mimic; E = anti-miR-34A). [score:1]
This study unravels a new layer of complexity in the miR-34A pathway, demonstrating that the impact of miR-34A modulation involves a coordinated network of lincRNAs, RNA components of the U12 -dependent spliceosome and protein-coding histone genes that are essential for cellular maintenance. [score:1]
Black bars indicate untransfected lines and red bars indicate cell lines transfected with miR-34A mimic. [score:1]
Owing to its established role in cancer, synthetic miR-34A mimics are currently in Phase I clinical trials for hepatocellular carcinoma, renal cell carcinoma, melanoma, lung cancers, and a number of hematologic malignancies (NCT01829971). [score:1]
miR-34A inactivation by focal loss of 1p36 or promoter hypermethylation has been reported in a multitude of human malignancies [2, 6, 7] (Table S1). [score:1]
miR-34A-GMNN base complementarity is given below. [score:1]
It is also among the most highly induced transcripts in TP53 mutant lines transfected with anti-miR-34A (q = 2.88e-123) (Figure 4D). [score:1]
Similarly, our findings demonstrate that miR-34A and p53 have overlapping but also autonomous roles, and underscore the importance of this microRNA as a key component of p53 -dependent and p53-independent cellular pathways. [score:1]
Our results suggest that miR-34A, much like p53, is an essential cell cycle mediator and future studies aimed at further characterizing the phenotypic impact of miR-34A suppression using alternate approaches may be warranted. [score:1]
Cells were transfected with anti-miR-34A oligonucleotide or control, as described above. [score:1]
RNA was extracted from fibroblast cell lines transfected with miR-34A mimic or anti-miR or respective controls, 24 hours post-transfection, using standard protocols. [score:1]
The miR-34A seed sequence begins at position 2 from the 5′ end of the transcript. [score:1]
miR-34A mimic and anti-miR transfections. [score:1]
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[+] score: 295
The tumor weight inhibition rate of Hi-miR497/34a (75.47%) was greater than the total inhibition rate (65.33%) induced by Hi-miR497 and Hi-miR-34a, whereas the tumor volume inhibition rate of Hi-miR497/34a was comparable to the total inhibition rate of Hi-miR497 and Hi-miR-34a. [score:9]
Chek1 (putative target of miR-497 identified by the algorithm DIANA miRPath v. 2.0), cdc25a (miR-497) [55], and cdk6 (miR-497and miR-34a) [55, 56] are involved in the indirect or direct regulation of the cyclin E1 downstream genes cdk2, RB and E2f3 (target for miR-34a, [33, 57]). [score:8]
In our previous study [17], we reported that an antitumor agent, resveratrol, upregulates the expression of several miRNAs (including miR-497 and miR-34a), and thus might inhibit the proliferation of lung cancer cells. [score:8]
Downregulation of the expression of miR-34a (Figure S1a) or miR-497 (Figure S1b) with inhibitors had no effect on the growth of A549, H460, and H1299 cells (Figure S1c) because the endogenous levels of these miRNAs in these cells are low. [score:8]
In conclusion, we showed that miR-497 and miR-34a act cooperatively to regulate certain aspects of tumorigenesis, including the growth of lung cancer cell lines, especially through their cooperative effect on the downregulation of cyclin E1 expression. [score:7]
Our data indicated that the tumor-suppressive miR-34a and miR-497 could co-inhibit cyclin E1, suggesting that cyclin E1 is a key target mediating the anti-tumor effect of miR-34a and miR-497 in lung cancer. [score:7]
Our results indicated that miR-497 and miR-34a synergistically inhibit cell proliferation, predominantly by repressing the expression of their cotarget, CCNE1. [score:7]
Knockdown of CCNE1 significantly inhibited cell viability and G [0]/G [1] arrest in A549 cells, whereas miR-497 or miR-34a overexpression did not enhance the effect of CCNE1 knockdown (Figure 4b and 4c). [score:7]
First, expression plasmids transiently expressing miR-497 (Hi-miR497), miR-34a (Hi-miR-34a), and a plasmid coexpressing miR-497 and miR-34a (Hi-miR497/34a) were constructed and verified by DNA sequencing and a TaqMan [®] MicroRNA Assay (Figure S4). [score:6]
These and our results, together with the strong possibility that more miR-497 or miR-34a targets will be discovered soon, suggest that these two miRNAs regulate proliferation-related mRNAs (including CCNE1), and thus function as tumor suppressors. [score:6]
miR-34a is downregulated in lung cancer tissues and cells [23, 24]; however, few reports have examined the expression of miR-497 in lung cancer. [score:6]
This suggests that the coexpression of miR-497 and miR-34a enhanced the effect of each individual miRNA on the modulation of cyclin E1 expression. [score:5]
Three bioinformatics algorithms (Targetscan 5.0, RNAhybrid 2.1, and RNA22) predicted that miR-497 and miR-34a target CCNE1, which encodes the cyclin E1 protein. [score:5]
Previous studies showed that miR-34a targets the oncogenes CDK4, CDK6, CCND1, MET, and BCL2 [36, 43, 44], whereas miR-497 targets the oncogenes CCND2 and BCL2 [37, 45] in various types of cancer. [score:5]
MIR497 and MIR34A were then inserted individually into the pcDNA™6.2-GW/EmGFPmiR vector (Invitrogen) using the BLOCK-iT™ Pol II miR RNAi Expression Vector Kit (Invitrogen), to generate the expression constructs Hi-miR497 and Hi-miR34a, respectively. [score:5]
Several studies have demonstrated that miR-34a retards lung cancer cell growth or induces apoptosis by targeting TGFβR2 [23], Axl [27], Notch-1 [28], or HDM4 [29], whereas miR-497 does so by targeting HDGF [26] in lung cancer. [score:5]
The major finding of our study is that miR-497 and miR-34a synergistically inhibit the expression of the same gene, CCNE1, and the function of its encoded protein, cyclin E1, thereby impeding the growth of lung cancer cells. [score:5]
Synergistic effects of miR-497 and miR-34a on cotargeting CCNE1A549 cells stably expressing cyclin E1 (designated Hi-CCNE1a) were generated (Figure S5). [score:5]
This evidence collectively suggests that miR-497 and miR-34a inhibit cell growth and might function as tumor suppressors. [score:5]
Here, we studied the function of miR-497 in lung cancer cells, and showed that miR-497 and miR-34a inhibit cell growth in vitro and in vivo, supporting our hypothesis that the two miRNAs function as tumor suppressors. [score:5]
Cells were transiently transfected with 50 nM miR-497 mimic, miR-34a mimic, miR-497 inhibitor, miR-34a inhibitor, CCNE1 siRNA (Qiagen, Germany), or siRNA negative control (NC siRNA; Qiagen) using Lipofectamine 2000 (Invitrogen). [score:5]
This indicates that the negative correlation between cyclin E1 expression and miR-497 or miR-34a levels is due to post-transcriptional modulation of cyclin E1 expression. [score:5]
The proliferation inhibition rate of Hi-miR497/34a (55.99%) was almost the same as the total inhibition rate (54.82%) of Hi-miR497 and Hi-miR-34a at 72 h. The colony formation rate of cells transiently transfected with Hi-miR497, Hi-miR-34a, or Hi-miR497/34a decreased by 36.84 ± 7.02%, 41.23 ± 4.09%, or 64.04 ± 2.92% (Figure 5b). [score:5]
The cell viability of A549, H460, and H1299 lung cancer cells was decreased by 66.71 ± 1.65%, 46.36 ± 1.96% and 72.10 ± 4.02 %, respectively, in response to miR-34a overexpression, and by 60.71 ± 4.63%, 74.94 ± 3.58%, and 73.71 ± 6.50%, respectively, in response to miR-497 overexpression (Figure 1b). [score:5]
Based on previous data, we designed a study to test the hypothesis that cyclin E1 expression is coregulated by miR-497 and miR-34a in lung cancer. [score:4]
These data confirm that miR-34a directly targets CCNE1. [score:4]
The loss or deletion of chromosome 17p13.1 or 1p36 has been reported in various types of cancer, including lung cancer [31, 32], suggesting that the downregulation of miRNA-497 or miRNA-34a in these cancers arises from genomic DNA loss or deletion. [score:4]
Because CCNE1 was identified as a direct target of miR-497 and miR-34a, we investigated whether the effects of miR-497 and miR-34a on cell proliferation were mediated by the modulation of CCNE1 expression. [score:4]
CCNE1 is a direct target of miR-497 and miR-34a. [score:4]
mt-CCNEβ contains a seven-base mutation in the miR- 34a target region, abolishing its binding to miR-34a. [score:4]
The protein encoded by CCNE1, a newly identified cotarget for miR-34a and miR-497, plays a key role in regulating the growth of lung cancer cells, and its effect is mediated by the cooperative action of the two miRNAs. [score:4]
CCNE1 knockdown affected the miR-497 and miR-34a induced inhibition of proliferation. [score:4]
However, real-time quantitative polymerase chain reaction (real-time qPCR) showed no changes in CCNE1 mRNA levels in response to miR-497 or miR-34a upregulation (Figure S3c). [score:4]
These results support the assumption that the effects of miR-497 and miR-34a on cell growth are mediated by its modulation of CCNE1 expression. [score:3]
Because CCNE1 is a cotarget of miR-497 and miR-34a, we examined whether the two miRNAs exert synergistic effects on cell growth. [score:3]
The 535-nt 3′ untranslated region (UTR) of CCNE1 was screened for complementarity to the seed sequences of miR-497 and miR-34a. [score:3]
To the best of our knowledge, this is the first study to demonstrate the cooperative effect of miR-497 and miR-34a, which belong to different miRNA families, on inhibiting cancer cell growth. [score:3]
When the mt-CCNEβ plasmid was cotransfected with the miR-34a mimic or an inhibitor, the luciferase activity did not differ from that of the control. [score:3]
However, because the endogenous levels of miR-34a and miR-497 are low in A549 cells, transfection with miR-497 and miR-34a inhibitors did not affect the levels of luciferase activity (Figures 3d and 1f). [score:3]
Our study extends the results of others by showing that miR-34a and miR-497 cotarget CCNE1 in lung cancer cells. [score:3]
Elevated levels of miR-497 or miR-34a inhibit cell proliferation. [score:3]
A549 cells were transfected with Hi-miR497, Hi-miR34a, Hi-miR497/34a, or inhibitors. [score:3]
miR-497 and miR-34a act synergistically by cotargeting CCNE1. [score:3]
CCNE1 is a putative target of miR-497 and miR-34a. [score:3]
Taken together, these data indicate that miR-497 and miR-34a cooperate in inhibiting tumor growth. [score:3]
Therefore, overexpression of CCNE1 abolished the growth retardation induced by miR497 and miR34a in A549 cells, and CCNE1 mediates the synergistic effects of miR-497 and miR-34a. [score:3]
The transfection mixtures contained 100 ng of plasmid and 50 nM synthetic mimic, inhibitor, Hi-miR497, Hi-miR34a, or Hi-miR497/34a. [score:3]
One predicted target sequence for miR-34a was found at nt 226–255 (Figure S2d). [score:3]
Overexpression of miR-497 or miR-34a in A549, H460, and H1299 lung cancer cells by transfection with miR-497 or miR-34a mimics (Figure S3a and S3b) markedly reduced the levels of cyclin E1 protein (Figure 3a). [score:3]
The average volume of tumors expressing Hi-miR497 (149.40 ± 17.84 mm [3]), Hi-miR-34a (190.80 ± 19.36 mm [3]), or Hi-miR497/34a (39.60 ± 14.32 mm [3]) was lower than that of the mock group (458.20 ± 30.64 mm [3]) (Figure 5c). [score:3]
The average weight of tumors expressing Hi-miR497 (456.00 ± 27.20 mg), Hi-miR-34a (554.00 ± 28.80 mg), or Hi-miR497/34a (184.00 ± 28.80) was lower than that of the mock group (750.00 ± 40.00 mg) after 5 weeks (Figure 5d). [score:3]
The miR-34a target sequence at nt 226–255 of the 3′-UTR is highly conserved among nine species (Figure S2f). [score:3]
Synergistic effects of miR-497 and miR-34a on cotargeting CCNE1. [score:3]
miR-497 and miR-34a suppress colony formation and tumorigenesis. [score:3]
Several studies have reported that miR-497 and miR-34a are potential anticancer agents based on their ability to target oncogenes [24, 25, 36, 37]. [score:3]
Hi-CCNE1-a cells transfected with Hi-miR497/34a expressed lower levels of cyclin E1 protein than those transfected with Hi-miR497 or Hi-miR34a (Figure 6a). [score:3]
miR-497 and miR-34a inhibit the proliferation of human lung cancer cells. [score:3]
miR-34a, located at 1p36, has been extensively studied as a microtumor suppressor [33, 34], and miR-497, located at 17p13.1, is deleted in 93% of small-cell lung cancers [35]. [score:3]
Figure 1(a) The relative levels of miR-497 and miR-34a were determined with the TaqMan [®] MicroRNA Assay and are expressed as fold change after normalization to the internal control, U6BsnRNA. [score:2]
Analysis of the expression of miR-497 and miR-34a in lung cancer cells showed that the levels of miR-497 and miR-34a (Figure 1a) were reduced by 24.29 ± 2.50% and 9.43 ± 2.96% in A549, 16.11 ± 5.20% and 4.51 ± 0.34% in H460, 53.55 ± 9.28% and 18.25 ± 2.14% in H1299, 43.00 ± 15.46% and 87.01 ± 27.73% in H446, and 42.17 ± 4.26% and 32.04 ± 4.58% in QG56 lung cancer cells, respectively, compared to those in normal bronchial epithelial 16HBE cells. [score:2]
This suggests that the synergistic effects of miR-497 and miR-34a are correlated with the levels of cyclin E1. [score:1]
To investigate the cooperative activities of miR-497 and miR-34a, we used the isocaudomers BamHI (NEB, Beverly, MA) and BglII (NEB) to ligate both MIR497 and MIR34A into the vector to generate the expression construct Hi-miR497/34a, which was confirmed by DNA sequencing. [score:1]
Transfection with miR-497 or miR-34a mimics caused cell-cycle arrest at G [0]/G [1] phase in A549, H1299, and H460 lung cancer cells (Figure 1c). [score:1]
A549 cells transiently transfected with mock (empty plasmid), Hi-miR497, Hi-miR34a, or Hi-miR497/34a were inoculated s. c. into the bilateral inguino-abdominal flanks of nude mice. [score:1]
To confirm that CCNE1 is targeted by miR-497 and miR-34a, we investigated the effects of miR-497 and miR-34a on cyclin E1 levels by immunoblotting. [score:1]
The effect of miR-497 or miR-34a on tumorigenicity was examined in vivo. [score:1]
However, the relationship between miR-497 and miR-34a is complex and requires further research. [score:1]
Elevated levels of miR-497 and miR-34a retard cell growth in vitro and in vivo. [score:1]
Although the sequence of the miR-34a seed region pairs with G:U complementarity at nt 247, 248, and 253 of the UTR, the seed regions of miR-497 (5′-AGCAGCA-3′) and miR-34a (5′-GGCAGUG-3′) are complementary to the same sequence at nt 247–253 (5′-UGCUGCU-3′) in the UTR. [score:1]
Hi-miR34a). [score:1]
Figure 5(a) The cell-growth curves for A549 cells transfected with Hi-miR497, Hi-miR-34a, or Hi-miR497/34a at 24, 48, 72, and 96 h. Means ± SD, n = 3 (*P < 0.05, at 48 h, Hi-miR497/34a vs. [score:1]
At 5 weeks, the average volumes (588.39 ± 117.34 mm [3]) and weights (308.57 ± 26.53 mg) of tumors in groups of seven mice injected with cells transfected with miR-497 mimics were lower than those (1293.16 ± 198.57 mm [3] and 427.14 ± 365.31 mg) in the control group injected with the NC mimic (Figure 2b), while the average volumes (190.25 ± 67.79 mm [3]) and weights (72.86 ± 31.84 mg) of tumors in mice injected with cells transfected with miR-34a mimics were lower than those (913.14 ± 455.23 mm [3] and 287.20 ± 131.09 mg) in the control group injected with the NC mimic (Figure 2c). [score:1]
miR-497 and miR-34a synergistically retard cell growth. [score:1]
The bilateral inguino-abdominal flanks of nude mice were inoculated subcutaneously (s. c. ) with A549 cells transfected with normal control (NC) (left flank) or miR-497 (right flank) mimics or with NC (left flank) or miR-34a (right flank) mimics. [score:1]
The miR-497 -mimic -transfected or miR-34a -mimic -transfected cells generated tumors with smaller volumes and lower weights, as determined at necropsy, than those of tumors generated with NC -mimic -transfected cells in the contralateral flanks. [score:1]
Therefore, miR-497 and miR-34a, with seed sequences 5′-AGCAGCA-3′ and 5′-GGCAGUG-3′, respectively, can bind to the same sequence (3′-UCGUCGU-5′) in the 3′ UTR of CCNE1; however, there are three-base variations between their seed regions. [score:1]
Therefore, the combination of miR-34a and miR-497 could be superior to each individual miRNA in its ability to retard lung cancer cell growth to some extent. [score:1]
Typical histograms of the cell-cycle arrest induced by miR-497 or miR-34a in A549 cells are shown in Figure 1d. [score:1]
The binding sites between miR-34a and the wild-type or mutant CCNE1 were examined (Figure 3c). [score:1]
Cells transfected with miR-497/miR-34a formed fewer and smaller colonies than cells transfected with Hi-miR-34a or Hi-miR497 alone. [score:1]
Figure 2Elevated levels of miR-497 and miR-34a retard cell growth in vitro and in vivo(a) Colony formation by A549 cells transfected with the miR-497 mimic or the miR-34a mimic was examined in soft agar. [score:1]
The effects of miR-497 (b) or miR-34a (c) on tumor formation were examined in a nude mouse xenograft mo del. [score:1]
Therefore, miR-497 and miR-34a share one binding site (nt 247–253) in the 3′-UTR of CCNE1. [score:1]
However, when the mt-CCNEδ plasmid was cotransfected with Hi-miR497/34a, the luciferase activity did not differ from that of cells cotransfected with either Hi-miR497 or Hi-miR34a alone (Figure 6b). [score:1]
The effect of miR-497 and miR-34a on the colony forming ability of A549 cells was assessed. [score:1]
Synergistic effects of miR-497 and miR-34a on tumor growth retardation. [score:1]
The cooperative effects of miR-497 and miR-34a on tumor formation were examined in a nude mouse xenograft mo del. [score:1]
When the wt-CCNE plasmid was cotransfected with the miR-34a mimic, the luciferase activity of A549 cells was markedly reduced by 34.56 ± 1.13% (Figure 3d). [score:1]
control siRNA + NC mimic, control siRNA + miR-34a mimic vs. [score:1]
The viability of cells transiently transfected with Hi-miR497, Hi-miR-34a, or Hi-miR497/34a was reduced at 48, 72, and 96 h. At 72 h, cell viability decreased from 2.28 ± 0.17 in the mock group to 1.73 ± 0.17, 1.58 ± 0.09, or 1.0 ± 0.02 in cells transfected with Hi-miR497, Hi-miR-34a, or Hi-miR497/34a, respectively, indicating that the Hi-miR497/34a plasmid caused a more marked reduction in cell viability than the Hi-miR497 or Hi-miR-34a plasmids (Figure 5a). [score:1]
Cells transfected with miR-497 or miR-34a mimics showed fewer (31.33 ± 2.44 and 21.00 ± 4.00 colonies per well, respectively) and smaller colonies than those observed in the control groups (71.00 ± 9.33 colonies per well) (Figure 2a). [score:1]
Cotransfection of the wt-CCNE plasmid with Hi-miR497/34a reduced luciferase activity to a greater extent than cotransfection of wt-CCNE with Hi-miR497 or Hi-miR34a alone (Figure 6b). [score:1]
The bilateral inguinoabdominal flanks of nude mice were injected s. c. with NC -mimic -transfected A549 cells (left flank) and miR-497 -mimic -transfected A549 cells (right flank), or with NC -mimic transfected A549 cells (left flank) and miR-34a -mimic -transfected A549 cells (right flank). [score:1]
miRNA-497 and miRNA-34a are located on chromosomes 17p13.1 and 1p36, respectively. [score:1]
Hi-miR497 or Hi-miR34a). [score:1]
Cyclin E1 mediates the effects of miR-497 or miR-34a on cell growth. [score:1]
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[+] score: 289
We detected modest upregulation of cMyc, E2f3, Met and Sirt1 in miR-34 -deficient MEFs, while Bcl2 was expressed at similar levels in wild-type and mutant cells (Figure 3K). [score:6]
Expression of members of the miR-34 family was similarly upregulated in response to p53 stabilization (Figure 3G). [score:6]
In addition, miR-449 expression is not substantially increased in miR-34 -null mice, and activation of the p53 pathway does not lead to significant upregulation of miR-449 (Figure S8). [score:6]
We also examined the consequences of miR-34 loss in MEFs on the expression of a subset of its previously reported direct targets [17], [20], [23], [25]. [score:6]
Thus, although a longer follow-up of miR-34 [T KO/T KO] mice may be needed to uncover very subtle defects in tumor suppression, we conclude that loss of miR-34 expression does not lead to a substantial increase in spontaneous tumorigenesis. [score:5]
Because previous work has relied on the use of miRNA antagonists to inhibit miR-34 function, it is possible that some of the previous observations reflected miR-34-independent off-target effects. [score:5]
With respect to the potential tumor suppressive role of miR-34, our experiments indicate that loss of miR-34 expression does not lead to an obvious increase in tumor incidence in mice and does not cooperate with Myc in the context of B cell lymphomagenesis. [score:5]
Importantly, in these three tissues, miR-34 expression is almost entirely p53-independent (Figure 1B–1D and [58]), a finding that suggests that additional transcription factors control the expression of this family of miRNAs in the absence of genotoxic or oncogenic stresses. [score:5]
Consistent with a possible tumor-suppressor role, loss of expression of members of the miR-34 family has been reported in human cancers. [score:5]
Here, we probe the tumor suppressive functions of the miR-34 family in vivo by generating mice carrying targeted deletion of the entire miR-34 family. [score:5]
The upregulation of Myc and E2f3 might contribute to the increased proliferation rate we have observed in miR-34 deficient MEFs. [score:4]
Consistent with previous reports indicating that miR-34a expression is under the direct control of p53 [13], [17], [18], we detected reduced levels of this miRNA in a subset of p53 -deficient tissues (heart, small and large intestine, liver and kidney), but the levels of both miR-34a and miR-34b∼c remained high in the brains, testes and lungs (Figure 1B–1D) of p53 [−/−] mice, a finding that suggests that p53-independent mechanisms determine basal miR-34 transcription in these tissues. [score:4]
Many of the predicted miR-34 target genes encode for proteins that are involved in cell cycle regulation, apoptosis, and growth factor signaling. [score:4]
Importantly, homozygous deletion of miR-34a did not lead to compensatory up-regulation of miR-34b∼c, and vice versa (Figure 2D and data not shown). [score:4]
Our results show that complete loss of miR-34 expression is compatible with normal development and that the p53 pathway is apparently intact in miR-34 -deficient mice. [score:4]
Canonical p53 -binding sites are located in the promoter regions of both miR-34a and miR-34b∼c, and these miRNAs are bona fide direct transcriptional targets of p53 [13], [17], [18]. [score:4]
miR-34 and tumor suppression in vivo To extend our analysis to an in vivo setting, we next examined whether miR-34 inactivation is sufficient to accelerate spontaneous and oncogene -induced transformation in mice. [score:3]
Targeted deletion of miR-34a and miR-34b∼c. [score:3]
Although our observation that single KO and miR-34 [T KO/T KO] mice produce viable offspring argues against an essential role for miR-34 in these processes, members of the related miR-449 family, that are particularly highly expressed in the testis (Figure S8), could partially compensate for miR-34 loss in this context. [score:3]
Despite the growing body of evidence supporting this hypothesis, previous studies on miR-34 have been done in vitro or using non-physiologic expression levels of miR-34. [score:3]
Complete loss of miR-34a and miR-34c expression was further confirmed in MEFs by qPCR (lower panel). [score:3]
p53 -dependent and p53-independent miR-34 expression in vivo. [score:3]
Consistent with these results, doxorubicin treatment caused similar activation of p53 and of its downstream targets in wild-type and miR-34 [T KO/T KO] MEFs (Figure 3E and 3F). [score:3]
To test whether miR-34 plays a role in this context, we ectopically expressed oncogenic K-Ras in wild-type, miR-34 [T KO/T KO], and p53 [−/−] MEFs. [score:3]
Next, we sought to determine whether loss of miR-34 expression affects the p53 response in vitro. [score:3]
Complete loss of miR-34 expression in miR-34 [T KO/T KO] animals was confirmed by Northern blot and qPCR (Figure 2D). [score:3]
Figure S1 Relative miR-34 expression in mouse tissues upon irradiation. [score:3]
Although these observations point towards an important role for miR-34 members as critical downstream effectors of p53 and potential tumor suppressors, these hypotheses have not been formally tested using miR-34 -deficient animals and cells. [score:3]
Our results show that the miR-34 family is not required for tumor suppression in vivo, and they suggest p53-independent functions for this family of miRNAs. [score:3]
In humans, for example, loss of miR-34 expression has been reported in a large fraction of primary melanomas, prostatic adenocarcinomas and small cell lung cancers [27], [28], among others. [score:3]
However, the tumor suppressive function of miR-34 might be restricted to specific tissues and loss of miR-34 might cooperate with specific oncogenic lesions. [score:3]
Ectopic expression of members of the miR-34 family is sufficient to induce cell cycle arrest or apoptosis, depending on the cellular context [14], [17]– [21]. [score:3]
To determine whether loss of miR-34 expression leads to increased spontaneous tumorigenesis, we aged a cohort of 14 miR-34 [T KO/T KO] and 12 wild-type mice. [score:3]
miR-34 and tumor suppression in vitro. [score:3]
We show that under basal conditions the expression of both miR-34 loci is particularly elevated in the testes and, to a lesser extent, in the brains and lungs of mice. [score:3]
1002797.g002 Figure 2(A) Targeting and screening strategy for the generation of constitutive and conditional miR-34a KO alleles. [score:3]
miR-34 and tumor suppression in vitro The p53 pathway provides a crucial barrier against the neoplastic transformation of primary cells [40]. [score:3]
We have reported the generation of mice carrying targeted deletion of miR-34a, miR-34b and miR-34c, and we have investigated the consequences of loss of miR-34 expression on p53 -dependent responses in vitro and in vivo. [score:3]
In addition, inactivation of miR-34 expression has been recently shown to lead to accelerated neurodegeneration and ageing in Drosophila melanogaster [64]. [score:3]
miR-34 and tumor suppression in vivo. [score:3]
The fragment was cloned into the targeting plasmid pKS-DTA, and a second loxP site was introduced into a unique KpnI site located ∼500 bp upstream of the pre-miR-34a sequence. [score:3]
Members of the miR-34 family (miR-34a, miR-34b, and miR-34c) have been wi dely speculated to be important tumor suppressors and mediators of p53 function. [score:3]
Introducing the miR-34 -null alleles we have generated into mouse mo dels of these types of human cancers will be important to fully explore the tumor suppressive potential of this family of miRNAs. [score:3]
These results show that while miR-34 alone is not required for p53 -mediated tumor suppression in MEFs, its loss might cooperate with inactivation of the Rb pathway in promoting cellular transformation. [score:3]
P53 -dependent cell cycle arrest in miR-34 [T KO/T KO] MEFsNext, we sought to determine whether loss of miR-34 expression affects the p53 response in vitro. [score:3]
Under basal conditions, miR-34a and miR-34b∼c expression is particularly intense in the testis, brain, and lung of adult mice (Figure 1B–1D). [score:3]
p53 -dependent and p53-independent miR-34 expression in vivo To investigate the biological functions of miR-34, we first examined the expression of this family of miRNAs under basal conditions and in response to p53 activation in vivo. [score:3]
However, even in this context complete loss of miR-34 expression was not sufficient to accelerate tumor formation. [score:3]
Consistent with this mo del is our observation that while loss of miR-34 expression alone does not allow the transformation of primary cells by oncogenic K-Ras, it slightly increases the efficiency of transformation when combined with inactivation of the Rb pathway by E1A (Figure 5A, 5B). [score:3]
MiR-34b∼c expression seems largely restricted to these three tissues, while miR-34a is detectable, albeit at lower levels, also in a variety of other organs (Figure 1B–1D). [score:3]
Recent reports have also implicated miR-34 in neuronal development and behavior [60], [61] and a role for miR-34c in learning and memory [62], as well as in stress -induced anxiety [63], has been reported. [score:2]
Although we detected a remarkable induction of miR-34a and miR-34c expression in late-passage wild-type MEFs compared to early-passage MEFs (Figure 3A), miR-34 -deficient MEFs became senescent with a kinetic identical to wild-type MEFs (Figure 3B). [score:2]
It is also possible that other miRNAs sharing sequence similarities with miR-34 may compensate for miR-34 loss in the knock-out animals. [score:2]
However, when MEFs were co-transduced with oncogenic K-Ras and E1A, which binds to and inhibits the retinoblastoma protein (pRb) [42], we observed a slight increase in the number of foci formed in miR-34 [T KO/T KO] MEFs compared to wild-type cells (Figure 5A, 5B). [score:2]
The “recombineering” method [65] was used to modify a BAC clone (RP-23-410P10) containing the miR-34a locus to generate the miR-34a conditional knockout allele. [score:2]
MiR-34 expression in wild-type and p53 [−/−] mouse tissues. [score:2]
First, in the tissues and cells used in our experiments, the expression of miR-449 members is much lower compared to miR-34a and miR-34c, as judged by multiple independent methods including qPCR, Northern blotting and high throughput sequencing (Figure S8 and data not shown). [score:2]
Int J Cancer 23 Yamakuchi M Ferlito M Lowenstein CJ 2008 miR-34a repression of SIRT1 regulates apoptosis. [score:2]
To exclude the possibility that tissue culture conditions may have masked a physiologic role of miR-34 in modulating the p53 response, we next examined the consequences of p53 activation in miR-34 -deficient tissues directly in vivo. [score:2]
Generation of miR-34 constitutive and conditional knockout mice. [score:2]
High contribution chimeras were subsequently crossed to Actin-flpe transgenic mice [66] to excise the frt-Neo-frt cassette and generate the miR-34a conditional knockout allele (miR-34a [fl]) or crossed to CAG-Cre mice [67] to excise the entire region flanked by the loxP sites and obtain the constitutive miR-34a KO allele (miR-34a [Δ]). [score:2]
In particular, three highly related miRNAs—miR-34a, miR-34b, and miR-34c (Figure 1A)—are directly induced upon p53 activation in multiple cell types and have been proposed to modulate p53 function [13]– [20]. [score:2]
Promoter hyper-methylation of miR-34a is observed in non-small-cell lung cancers and melanomas [27], [28], and silencing of miR-34a and miR-34b∼c has been described in human epithelial ovarian cancers [29]. [score:1]
Age range of the cohorts is 359–521 days (mean: 464 days) for wild-type and 359–521 days (mean: 445 days) for miR-34 [T KO/T KO]. [score:1]
Although it will be important to follow a larger cohort of animals over a more prolonged period, these results suggest that miR-34 does not provide a potent barrier to tumorigenesis in response to genotoxic stress in vivo. [score:1]
Although as predicted, p53 -null cells failed to arrest in G1 in response to doxorubicin treatment, the response of miR-34 [T KO/T KO] MEFs was indistinguishable from that of wild-type cells (Figure 3H–3I). [score:1]
Thymocytes were isolated from sex-matched, age-matched wild-type, miR-34 [T KO/T KO], and p53 [−/−] mice and seeded at a density of 1×10 [6] cells/ml in MEF medium. [score:1]
For example, p53 has been proposed to modulate autophagy [55] and stem cell quiescence [56], [57] and we cannot exclude that miR-34 plays an important role in these contexts. [score:1]
Probes specific for miR-34a and miR-34c were used. [score:1]
1002797.g005 Figure 5Oncogene -induced transformation in miR-34 [T KO/T KO] fibroblasts and mice. [score:1]
Age- and sex-matched wild-type, miR-34 [T KO/T KO] and p53 [−/−] mice were exposed to 10 Gy of ionizing radiation and euthanized 6 hours later. [score:1]
Gap-repair was used to retrieve a 9.6 kbp fragment containing the frt-Neo-frt-loxP cassette, ∼4 kb of 3′ homology arm, and ∼3.7 kb 5′ homology arm, and including the pre-miR-34a sequence. [score:1]
P53 -dependent cell cycle arrest in miR-34 [T KO/T KO] MEFs. [score:1]
Both wild-type and miR-34 -deficient mice appeared healthy throughout the follow-up period (Figure S7), in striking contrast with the ∼15 weeks reported median tumor-free survival of irradiated p53 [−/−] mice [52]. [score:1]
To extend our analysis to an in vivo setting, we next examined whether miR-34 inactivation is sufficient to accelerate spontaneous and oncogene -induced transformation in mice. [score:1]
Furthermore, loss-of-function studies using miR-34 antagonists have provided some evidence that this miRNA family is required for p53 function [13], [18], [22]– [24]. [score:1]
The incidence and latency of B cell lymphomas was virtually identical in Eμ-Myc;miR-34 [T KO/T KO] and Eμ-Myc;miR-34 [+/+] mice (Figure 5C) and the resulting tumors displayed similar histopathological features and extent of spontaneous apoptosis (Figure 5D–5E). [score:1]
As expected, p53 [−/−] thymocytes were almost entirely resistant to irradiation -induced apoptosis; however, wild-type and miR-34 -deficient cells were equally sensitive to DNA damage -induced apoptosis, as judged by dose-response and time-course experiments (Figure 4A, 4B). [score:1]
To investigate the physiologic functions of the miR-34 family and to determine the extent to which its induction is required for p53 function, we generated mice carrying targeted deletion of both miR-34a and miR-34b∼c loci (Figure 2A–2C). [score:1]
Representative pictures of miR-34a [−/−] (E), miR-34b∼c [−/−] (F), and miR-34 [T KO/T KO] (G) males at 4 weeks of age. [score:1]
Samples obtained from sex- and age-matched adult (age range 3–16 months) wild-type and miR-34 [T KO/T KO] mice were subjected to a standard panel of serum chemistry tests to determine liver and kidney function (n≥5 per genotype). [score:1]
More difficult, however, is to reconcile our findings with previous reports of impaired p53-function in cells treated with miR-34 antagonists. [score:1]
Figure S5Serum chemistry of age- and sex-matched wild-type and miR-34 -deficient mice. [score:1]
Wild-type, miR-34 [T KO/T KO], p53 [−/−] MEFs were seeded at 70% confluence and infected with virus. [score:1]
For the irradiation experiments, 150,000 wild-type, miR-34 [T KO/T KO] and p53 [−/−] MEFs were seeded into each well of a 6-well culture plate and starved for 72 hours. [score:1]
For the miR-34 [T KO] allele (G), double heterozygous mice were inter-crossed. [score:1]
We next sought to determine whether loss of miR-34 might accelerate tumor formation in response to genotoxic stress. [score:1]
Lastly, miR-34a [+/fl] and miR-34a [+/−] were intercrossed to obtain miR-34a [fl/fl] and miR-34a [−/−] animals. [score:1]
The precursors of these miRNAs are transcribed from two distinct loci: the miR-34a locus on chromosome 1p36 and the miR-34b∼c locus on chromosome 11q23. [score:1]
Based on these results we conclude that miR-34 function is not required for p53 -induced cell-cycle arrest and apoptosis in response to genotoxic stresses. [score:1]
Age range of the cohorts is 298–425 days (mean: 333 days) for wild-type and 387–425 days (mean: 401 days) for miR-34 [T KO/T KO]. [score:1]
An additional issue raised by the results presented in this manuscript relates to possible p53-independent functions of miR-34. [score:1]
A frt-Neo-frt-loxP cassette was first inserted ∼480 bp downstream of the pre-miR-34a sequence. [score:1]
Future studies using the miR-34 -deficient animals we have generated will be needed to test these possibilities. [score:1]
These findings highlight likely redundancies among p53's downstream effectors, show that the miR-34 family is largely dispensable for p53 function in vivo, and suggest possible p53-independent functions. [score:1]
For each tissue, the same membrane was serially probed first for the three members of the miR-449 family and lastly for miR-34a. [score:1]
The animals were monitored for at least 12 months (wild-type = 359 days; miR-34 [T KO/T KO] = 359 days) and up to 17.3 months (wild-type = 521 days; miR-34 [T KO/T KO] = 521 days). [score:1]
Wild-type and miR-34 [T KO/T KO] MEFs were seeded into a 6-well plate (40,000 cells/well) and counted every day for the growth curves. [score:1]
Here we report the generation of mice carrying targeted deletion of all three members of the miR-34 family and systematically investigate the impact of miR-34 loss on the p53 pathway. [score:1]
To examine the consequences of complete loss of miR-34 function, we crossed miR-34a [−/−] and miR-34b∼c [−/−] mice to generate compound mutant animals carrying homozygous deletion of all three family members (miR-34 [T KO/T KO]). [score:1]
One notable exception is a recent elegant paper by Choi and colleagues demonstrating that miR-34 -deficient MEFs are more susceptible to reprogramming [30]. [score:1]
The results presented in this paper do not necessarily conflict with previous experiments showing that ectopic expression of miR-34 can induce many of the most characteristic consequences of p53 activation; here we have tested whether miR-34 is necessary for p53 function and not whether it is sufficient. [score:1]
Epigenetic silencing of miR-34 members has also been reported in human cancers. [score:1]
We next examined the role of miR-34 in the response to the DNA damaging agent doxorubicin. [score:1]
Peripheral blood samples obtained from sex- and age-matched adult (age range 3–16 months) wild-type (WT) and miR-34 -null (T KO) mice were subjected to complete blood cell count (n≥5 per genotype). [score:1]
For BrdU cell cycle analysis, wild-type, miR-34 [T KO/T KO], and p53 [−/−] MEFs were plated in complete medium at 70% confluence, treated with varying doses of doxorubicin for 16 hours or treated at different time points, and pulsed with 10 µM BrdU for one hour. [score:1]
Finally, we sought to determine whether genetic ablation of miR-34 could contribute to tumor formation in cooperation with a defined oncogenic lesion. [score:1]
Response to p53 activation in miR-34 [T KO/T KO] mouse embryonic fibroblasts (MEFs). [score:1]
In particular, members of the miR-449 family (miR-449a, b and c) have the same “seed” sequence as miR-34, and miR-34 antagonists could in principle impair their function as well. [score:1]
The experiments described above were performed on asynchronously growing early-passage MEFs and as such may not be sensitive enough to detect a modest effect of miR-34 loss on the S-phase checkpoint. [score:1]
To allow temporally and spatially restricted deletion, we also generated a conditional miR-34a KO allele (miR-34a [fl], Figure 2A). [score:1]
Oncogene -induced transformation in miR-34 [T KO/T KO] fibroblasts and mice. [score:1]
This interpretation is also consistent with the faster proliferation rate displayed by miR-34 -deficient MEFs (Figure 3B, 3C) and with the observation by Lal and colleagues that miR-34a is involved in modulating the cellular response to growth factors [38]. [score:1]
MiR-34a [−/−] and miR-34b∼c [−/−] single KO mice were viable and fertile and were obtained at the expected Men delian frequency (Figure 2E, 2F). [score:1]
RNAs from miR-34 [T KO/T KO] tissues were included to control for cross-hybridization. [score:1]
Five Eμ-Myc;miR-34 [+/+] tumors and and four Eμ-Myc;miR-34 [T KO/T KO] tumors were analyzed. [score:1]
The most logical interpretation of these results is that miR-34 -deficient MEFs, rather than being more resistant to irradiation -induced cell cycle arrest, possess a slightly faster basal proliferation or more rapid re-entry into the cell cycle following serum starvation. [score:1]
P53 -dependent apoptosis in miR-34 [T KO/T KO] cells and mice Having established that miR-34 is not required for cell cycle arrest in response to genotoxic stress in MEFs, we next sought to determine whether this miRNA family might contribute to p53 -induced apoptosis. [score:1]
Ionizing radiation induced similar activation of the p53 pathway and of its downstream effectors in wild-type and miR-34 [T KO/T KO] mice (Figure 4C). [score:1]
The standard 3T3 protocol was followed to determine the cumulative population doublings of wild-type, miR-34 [T KO/T KO], and p53 [−/−] MEFs. [score:1]
To investigate the biological functions of miR-34, we first examined the expression of this family of miRNAs under basal conditions and in response to p53 activation in vivo. [score:1]
1002797.g003 Figure 3Response to p53 activation in miR-34 [T KO/T KO] mouse embryonic fibroblasts (MEFs). [score:1]
Exposure to ionizing radiation, which leads to p53 stabilization and transcriptional activation, resulted in substantial miR-34a induction in the spleen, thymus, small and large intestine of wild-type mice, but not in the other tissues examined (Figure S1). [score:1]
The sequence similarity between the three miR-34 family members (Figure 1A), which share the same “seed”, suggests that they may be functionally redundant. [score:1]
MiR-34 wild-type and miR-34 [T KO/T KO] MEF lines were also verified by qPCR. [score:1]
However, the consequences of miR-34 loss on p53 function were not examined in detail. [score:1]
Our observation that inactivation of miR-34 does not impair p53 -mediated responses in vitro and in vivo is particularly relevant because a key role for miR-34 in the p53 pathway had been previously proposed by a number of independent groups. [score:1]
The blots were then hybridized with [32]P-labeled probes specific for miR-34a, miR-34c, and U6. [score:1]
Having established that miR-34 is not required for cell cycle arrest in response to genotoxic stress in MEFs, we next sought to determine whether this miRNA family might contribute to p53 -induced apoptosis. [score:1]
Figure S4 Complete blood cell count of age- and sex-matched wild-type and miR-34 -deficient mice. [score:1]
The results are representatitve of two independent experiments performed on a total of four wild-type and four miR-34 [T KO/T KO] MEF lines. [score:1]
Hemizygous deletion of the chromosomal region containing the miR-34a locus has been described in neuroblastomas and pancreatic cancer cell lines [14], [21]. [score:1]
Experiments were performed on three independent wild-type and three independent miR-34 [T KO/T KO] MEF lines. [score:1]
The miR-34a< floxed> mice and the miR-34b∼c−/− mice are available to the research community through The Jackson Laboratory (JAX Stock Numbers 018545 and 018546). [score:1]
Figure S7Overall survival of wild-type and miR-34 [T KO/T KO] cohorts. [score:1]
An analysis of the major myeloid and lymphoid populations of the bone marrow, spleen and thymus also did not reveal any statistically significant difference between wild-type and miR-34 [T KO/T KO] mice (Figure S6). [score:1]
Representative images of hematoxylin and eosin staining of heart, kidney, liver, lung, small intestine, ovary, testis, and spleen (black scale bar, 200 µm), brain (green scale bar, 2000 µm), and colon (red scale bar, 100 µm) from wild-type and miR-34 [T KO/T KO] mice. [score:1]
Analogous to what we observed in thymocytes in vitro, the apoptotic response was equally dramatic in wild-type and in miR-34 -deficient mice, while it was virtually absent in p53 [−/−] animals (Figure 4D–4G). [score:1]
P53 -dependent apoptosis in miR-34 [T KO/T KO] cells and mice. [score:1]
We therefore exposed a cohort of 14 miR-34 [T KO/T KO] and 11 wild-type mice to 1 Gy of ionizing radiation soon after birth and monitored them for 42–60 weeks. [score:1]
Notice the loss of signal for miR-449b in the miR-34 [T KO/T KO] lung and testis samples, which likely reflects cross-hybridization of the miR-449b probe to miR-34. [score:1]
Generation of miR-34 -deficient mice. [score:1]
miR-34 [T KO/T KO] embryos were obtained by intercrossing miR-34 mutant mice. [score:1]
We therefore examined the effects of DNA damage on thymocytes from wild-type, p53 [−/−], and miR-34 [T KO/T KO] mice. [score:1]
Figure S6Bone marrow, spleen and thymus analysis of age- and sex-matched wild-type and miR-34 [T KO/T KO] mice. [score:1]
A conclusive test for this hypothesis will require the generation of compound miR-34 and miR-449 mutant animals, but several lines of evidence suggest that this explanation is not particularly likely. [score:1]
A full histological examination (Figure S3), complete blood cell count (Figure S4), and serum chemistry analysis (Figure S5) did not detect any statistically significant defects in adult miR-34 [T KO/T KO] mice of both sexes. [score:1]
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[+] score: 287
More importantly, other potential miR-34 target genes were inhibited in addition to Bcl-2. As shown in Figure 4, Notch1 and HMGA2 were inhibited by all three miR-34a, b, c mimics, while miR-34b mimic inhibited Notch2 and 4, and miR-34c mimic inhibited Notch1-4. Notch1-2 knockdown by miR-34 mimics has been confirmed by Western blot (data not shown). [score:12]
As shown in Figure 3, revealed that transfection of miR-34 mimics downregulated target gene Bcl-2 expression at the protein level, but had no obvious effect on Bcl-xL and Mcl-1 expression, indicating that the Bcl-2 knockdown by miR-34 mimics was sequence-specific. [score:11]
Figure 4 Quantitative real-time PCR shows that restoration of miR-34 by downregulates target gene expression. [score:8]
Figure 3 Restoration of miR-34 by downregulates target gene Bcl-2 expression. [score:8]
This strategy was explored in the current study, where p53 downstream target miR-34 was restored in p53-mutant gastric cancer Kato III cells with a high level of Bcl-2 and low levels of miR-34, resulting in downregulation of Bcl-2 and Notch/HMGA2, tumor cell growth inhibition and accumulation in G1 phase, and chemosensitization and Caspase-3 activation/apoptosis. [score:8]
Bcl-2 is a direct target of miR-34, and our data have shown that miR-34 restoration inhibits Bcl-2 expression. [score:8]
The expression of miR-34 is dramatically reduced in 6 of 14 (43%) non-small cell lung cancers (NSCLC) and the restoration of miR-34 expression inhibits growth of NSCLC cells [10]. [score:7]
As a target of miR-34, Bax was also downregulated by miR-34. [score:6]
He et al. reported that ectopic expression of miR-34 induces cell cycle arrest in both primary and tumor-derived cell lines, which is consistent with the observed ability of miR-34 to downregulate a program of genes promoting cell cycle progression [12]. [score:6]
The mechanism of miR-34 -mediated suppression of self-renewal appears to be related to the direct modulation of downstream targets Bcl-2, Notch, and HMGA2, indicating that miR-34 may be involved in gastric cancer stem cell self-renewal/differentiation decision-making. [score:6]
The mechanism of miR-34 -mediated suppression of self-renewal might be related to the direct modulation of downstream targets Bcl-2, Notch, and HMGA2, indicating that miR-34 may be involved in gastric cancer stem cell self-renewal/differentiation decision-making. [score:6]
The mechanism of miR-34 -mediated suppression of gastric cancer cell self-renewal might be related to the direct modulation of downstream targets Bcl-2, Notch, and HMGA2, implying that miR-34 may be involved in gastric cancer stem cell self-renewal/differentiation decision-making. [score:6]
Since miR-34 is a downstream target of the p53 pathway and Bcl-2 is a direct target of miR-34, our data with Kato III are consistent with the cells' p53-mutant status, i. e., Kato III has mutant p53, the lowest level of miR-34, and the highest level of Bcl-2. Therefore, we focused on this cell line for the current study of the effect of miR-34 restoration. [score:6]
MicroRNA miR-34 was recently found to be a direct target of p53, functioning downstream of the p53 pathway as a tumor suppressor. [score:6]
Expression of miR-34 and target genes in human gastric cancer cell lines. [score:5]
This multi-mode action of miR-34 provides a therapeutic advantage over other molecular therapies, in that miR-34 has multiple targets and can work on multiple cell signalling pathways simultaneously, leading to synergistic effects that may translate into improved clinical efficacy for gastric cancer patients with p53 deficiency and multidrug resistance. [score:5]
Recently, miRNA miR-34 was identified as a p53 target and a potential tumor suppressor [4, 8- 12]. [score:5]
More significantly, miR-34 potently inhibits tumorsphere formation and growth in p53-mutant human gastric cancer cells, providing the first proof-of-concept that there is a potential link between the tumor suppressor miR-34 and gastric cancer cell self-renewal, which involves the presumed gastric cancer stem cells. [score:5]
We next examined these gastric cancer cell lines for the expression level of miR-34 and target genes using qRT-PCR. [score:5]
Kato III cells were infected with feline immunodeficiency virus (FIV) lentiviral system expressing miR-34a (miR-34a-MIF) or vector control (MIF), and stable cells were obtained by antibiotic selection (Zeocin 50 μg/mL) and validated for miR-34a expression. [score:5]
Kato III cells have the lowest levels of both pri-miR34a and mature miR-34a, and the highest expression levels of target genes Bcl-2, Notch1, and Notch4 (Figure 2). [score:5]
As shown in Figure 5, the transfected miR-34 mimics effectively inhibited luciferase reporter gene expression, which is controlled by Bcl-2 3'UTR in the promoter region. [score:5]
Quantitative real-time PCR was performed to determine the expression levels of potential miR-34 target genes. [score:5]
Human gastric cancer Kato III cells with miR-34 restoration reduced the expression of target genes Bcl-2, Notch, and HMGA2. [score:5]
In this study, we examined the effects of miR-34 restoration on p53-mutant human gastric cancer cells and potential target gene expression. [score:5]
Bommer et al. reported that the abundance of the three-member miRNA34 family is directly regulated by p53 in cell lines and tissues, and the Bcl-2 protein is regulated directly by miR-34 [10]. [score:5]
Our results demonstrate that in p53 -deficient human gastric cancer cells, restoration of functional microRNA miR-34 inhibits cell growth, induces apoptosis, and leads to chemosensitization, indicating that miR-34 may restore, at least in part, the p53 tumor-suppressing function. [score:5]
The results demonstrate that the transfected miR-34a, b, c are functional, and confirm that Bcl-2 is a direct target of miR-34, consistent with earlier reports [8, 10, 16]. [score:4]
Delineating the role of miR-34 in regulation of cell growth and tumor progression, as well as its potential relationship to cancer stem cells, will help us better understand the p53 tumor suppressor signalling network, facilitate our research in carcinogenesis and cancer therapy, and serve as a basis for our exploration of novel strategies in cancer diagnosis, treatment, and prevention. [score:4]
Briefly, Kato III cells were infected with the feline immunodeficiency virus (FIV) lentiviral system expressing miR-34a (miR-34a-MIF) or vector control (MIF), according the manufacturer's instructions, and stable cells were obtained by antibiotic selection (Zeocin 50 μg/mL, Invitrogen). [score:3]
miR-34 targets Notch, HMGA2, and Bcl-2, genes involved in the self-renewal and survival of cancer stem cells. [score:3]
miR-34 restoration chemosensitizes gastric cancer cells with a high level of Bcl-2. miR-34 restoration inhibits gastric cancer cell growth. [score:3]
Another important implication from the current study is that our data provide a potential link between tumor suppressor miR-34 and the presumed gastric cancer stem cells. [score:3]
Fold increase was calculated by dividing the normalized target gene expression of the treated sample with that of the untreated control, with the value from the NC mimic set as 1. For cell cycle analysis by flow cytometry, Kato III cells were transfected with miR-34 mimics or NC mimic in 6-well plates, trypsinized 24 hours later and washed with phosphate-buffered saline, and fixed in 70% ethanol on ice. [score:3]
However, our data indicate that miR-34 restoration inhibits tumorspheres from p53-mutant gastric cancer cells, suggesting that miR-34 might be involved in the self-renewal of the presumed gastric cancer stem cells. [score:3]
Our study suggests that restoration of the tumor-suppressor miR-34 may provide a novel molecular therapy for p53-mutant gastric cancer. [score:3]
Since part of the p53 tumor-suppressing function is via promoting apoptosis [19, 20], we next examined the effect of miR-34 restoration on apoptosis. [score:3]
Our data provide the first evidence that miR-34 is able to inhibit tumorsphere formation and growth in p53-mutant gastric cancer cells, implying that miR-34 might play a role in the self-renewal of gastric cancer cells, presumably gastric cancer stem cells. [score:3]
of the potential miR-34 target protein Bcl-2 48 hours after of Kato III cells (100 pmol per well in 6-well plates). [score:3]
Gastric cancer cells were infected with the FIV lentiviral system expressing miR-34a (miR-34a-MIF) or vector control (MIF), according to the manufacturer's instructions, and stable cells were obtained by antibiotic selection (Zeocin 50 μg/mL, Invitrogen). [score:3]
Taken together, these published studies establish that miR-34 is a new tumor suppressor functioning downstream of the p53 pathway, and provide impetus to explore the functional restoration of miR-34 as a novel therapy for cancers lacking p53 signalling. [score:3]
Our results demonstrate that in p53 -deficient human gastric cancer cells, restoration of functional miR-34 inhibits cell growth and induces chemosensitization and apoptosis, indicating that miR-34 may restore p53 function. [score:3]
Our study suggests that restoration of the tumor suppressor miR-34 may provide a novel molecular therapy for p53-mutant gastric cancer. [score:3]
Restoration of miR-34 inhibits tumorsphere formation and growth, which is reported to be correlated to the self-renewal of cancer stem cells. [score:3]
Figure 9 Restoration of miR-34 by MIF lentiviral system inhibits Kato III tumorspheres. [score:3]
miR-34 restoration inhibits gastric cancer tumorspheres. [score:3]
The feline immunodeficiency virus (FIV) lentiviral system expressing miR-34a (miR-34a-MIF) or vector control (MIF), as well as their lentiviral packaging system, were purchased from System Biosciences (SBI, Mountain View, CA). [score:3]
Over 50% of human cancers have mutant p53 and the expression of miR-34a, b, c appears to be correlated with p53 [10, 12]. [score:3]
miR-34 restoration could thus rebuild, at least in part, the p53 tumor-suppressing signalling network in gastric cancer cells lacking p53 function. [score:3]
It has been reported that miR-34 targets Notch, HMGA2, and Bcl-2, genes involved in the self-renewal and survival of cancer stem cells [10, 12, 14]. [score:3]
miR-34 restoration inhibits tumorsphere formation and growth, which is reported to be correlated to the self-renewal of cancer stem cells. [score:3]
Bcl-2 3'UTR reporter assay showed that the transfected miR-34s were functional and confirmed that Bcl-2 is a direct target of miR-34. [score:3]
Restoration of miR-34 chemosensitized Kato III cells with a high level of Bcl-2, but not MKN-45 cells with a low level of Bcl-2. miR-34 impaired cell growth, accumulated the cells in G1 phase, increased caspase-3 activation, and, more significantly, inhibited tumorsphere formation and growth. [score:3]
However, mutation in the Bcl-2 3'UTR complimentary to the miR-34 root sequence abolished this effect, indicating that the observed reporter activity is miR-34 sequence-specific. [score:2]
Human gastric cancer cells were transfected with miR-34 mimics or infected with the lentiviral miR-34-MIF expression system, and validated by miR-34 reporter assay using Bcl-2 3'UTR reporter. [score:2]
As shown in Figure 9, restoration of miR-34 by MIF lentiviral system inhibited Kato III tumorsphere formation and growth; the stable cells with functional miR-34a restoration had significantly fewer tumorspheres, and the formed tumorspheres were significantly smaller, as compared with that of the MIF control (P < 0.001, Student's t-test, n = 3). [score:2]
The role of miR-34 in gastric cancer has not been reported previously. [score:1]
Briefly, in our study the stable clones from Kato III cells infected with miR-34a-MIF or control vector MIF were plated for tumorsphere culture in ultra-low adhesion plates. [score:1]
The cells were transfected with miR-34a or NC mimics by Lipofectamine 2000. [score:1]
and using human primary gastric cancer tissues to identify the true side population of the assumed gastric cancer stem cells, and to delineate the role of miR-34 in these tumor-initiating cells. [score:1]
To evaluate the long-term effects of miR-34 restoration, we have employed a lentiviral system to express miR-34a and have generated stable cells. [score:1]
Cells in each well were also co -transfected with 100 pmol of each miR-34 mimics or NC mimic as indicated, using Lipofectamine 2000. [score:1]
This effect on cell cycle is similar to that of p53 restoration as we previously reported [18- 23], indicating that miR-34 restoration can restore p53 signalling, at least in part, in the cells lacking a functional p53 pathway. [score:1]
miR-34 mimic transfection. [score:1]
As shown in Figure 8, lentiviral restoration of miR-34a in Kato III cells significantly delayed cell growth (P < 0.001, n = 3), a biological activity similar to that of the p53 restoration we reported previously [19- 22]. [score:1]
A. of Kato III cells after miR-34 restoration. [score:1]
As shown in Figure 6A, the miR-34 mimics induced an accumulation of Kato III cells in G1 phase and a reduction of cells in S phase, consistent with other reports on miR-34 restoration in various tumor mo dels [4, 8, 10, 12, 13, 16, 17]. [score:1]
miRNA miR-34a, b, c mimics, antagonists, and negative control miRNA mimic (NC mimic) were obtained from Dharmacon (Chicago, IL) with the sequences for hsa-miR-34a: 5'- uggcagugucuuagcugguugu-3' ; hsa-miR-34b: 5'- caaucacuaacuccacugccau-3' ; hsa-miR-34c: 5'- aggcaguguaguuagcugauugc-3'. [score:1]
Transfection of miR-34 mimics in p53-mutant gastric cancer cells. [score:1]
Tazawa et al. provided evidence that miR-34a induced senescence-like growth arrest in human colon cancer [13]. [score:1]
miR-34 restoration results in Kato III cell accumulation in G1 phase and caspase-3 activation. [score:1]
For miR-34 restoration, we transfected the Kato III cells with miR-34 mimics. [score:1]
Figure 6 Restoration of miR-34 in Kato III cells resulted in G1 block and caspase-3 activation. [score:1]
Since no cellular markers for gastric cancer stem cells have been wi dely accepted thus far, in the current study we employed tumorsphere culture to explore whether there is any link between miR-34 and tumorsphere-forming cells. [score:1]
Lentiviral miR-34a infection and stable cells. [score:1]
In the current study, we examined the effects of functional restoration of miR-34 by miR-34 mimics and lentiviral miR-34a on human gastric cancer cells, and the effect of miR-34 on tumorsphere formation and growth of p53-mutant gastric cancer cells. [score:1]
was performed 24 hours after transfected with miR-34 mimics or negative control mimic (NC mimic). [score:1]
KATO3 cells were transfected with Bcl-2 3'UTR luciferase reporter plasmid or its mutant, plus the control β-galactosidase plasmid and 100 pmol of each miR-34 mimic or NC mimic. [score:1]
Cells in each well were also co -transfected with 100 pmol of each miR-34 mimic or NC mimic as indicated, using Lipofectamine 2000. [score:1]
Figure 8 Restoration of miR-34 in Kato III cells delays cell growth. [score:1]
Our data suggest that miR-34 may hold significant promise as a novel molecular therapy for human gastric cancer, potentially for gastric cancer stem cells. [score:1]
Briefly, Kato III cells were transfected with miR-34 mimics or NC mimic for 24 h, plated in 96-well plates (5,000 cells/well), and treated with serially diluted chemotherapeutic agents in triplicate. [score:1]
As shown in Figure 6B, transient transfection of miR-34 mimics resulted in significantly increased caspase-3 activation, a key indication of the cells undergoing apoptosis. [score:1]
miR-34a has been reported to be involved in p53 -mediated apoptosis in colon cancer and pancreatic cancer [8, 9]. [score:1]
Kato III cells infected by miR-34a MIF or control vector MIF were plated for tumorsphere formation as described in Materials and. [score:1]
Our data demonstrate that miR-34 restoration can chemosensitize those gastric cancer cells that have high levels of Bcl-2 and low basal levels of miR-34, which are dependent on Bcl-2 for survival and drug resistance. [score:1]
However, for gastric cancer MKN-45 cells that have a low level of Bcl-2 and a high level of miR-34, miR-34 restoration showed no chemosensitization (Figure 7B). [score:1]
Thus far, there is limited study on miRNA and gastric cancer; the link between p53 downstream target miR-34 and gastric cancer has not been established; and the role of miR-34 in gastric cancer remains to be investigated. [score:1]
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Other miRNAs from this paper: hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3
c-MET is a proved target gene of miR-34a [14], [15] and c-MET inhibitor demonstrated a manageable safety profile and preliminary antitumor activity in patients with HCC and Child-Pugh A or B cirrhosis [16], hence we have for the first time investigated the combinatorial effect of miR-34a mimic and c-MET targeting agents (c-MET siRNAs or c-MET kinase inhibitor su11274) in HCC cells. [score:7]
Concerning the relationship between miR-34a expression and clinicopathological features, in the present study, miR-34a expression downregulated in the group with metastasis compared to the group with no metastasis, which is consistent with Li et al [14]. [score:7]
miR-34a was reported to be down-regulated in rat liver during hepatocarcinogenesis induced by a methyl -deficient diet, which is relevant to the hepatocarcinogenesis in humans associated with viral hepatitis C and B infections, alcohol exposure and metabolic liver diseases [18]. [score:6]
When studied the relationship between miR-34a expression and clinical TNM stages, we found that the downregulation of miR-34a was related to the progression of HCC. [score:6]
These pathways were influenced little with miR-34a inhibitor transfection, however, the phospho-ERK1/2 and phospho-stat5 were down-regulated by miR-34a mimic 96 h post-transfection (Figure 6). [score:6]
It was reported that c-MET is a target gene of miR-34a [14], [15], We desired to explore the combinatorial effect of the miR-34a mimic and agents targeting c-MET (siRNA or small molecular inhibitor, su11274), using the colorimetric MTS formazan proliferation assay. [score:6]
The results showed that with the miR-34a inhibitor, caspase-3/7 activity was slightly downregulated than the negative controls, but contained no significant difference. [score:6]
Furthermore, in the group with metastasis, miR-34a expression was down-regulated compared to the group without metastasis (P<0.05). [score:5]
With miR-34a mimic, the cell growth inhibitory effect showed a time dependent manner and the cell growth was significantly inhibited 96 h post-transfection. [score:5]
The next question was therefore whether simultaneous inhibition of c-MET, by RNAi or kinase inhibitor, together with miR-34a, would result in increased biological effects. [score:5]
Re -expression and inhibition of miR-34a in HCC cells. [score:5]
Together with previous reports, the current observations strongly proved that miR-34a is a tumor suppressor miRNA that plays a vital role in the oncogenesis and progression of HCC, by targeting multiple pathways. [score:5]
miR-34a enhanced the growth inhibitory effect of c-MET targeting agents in HCC HepG2 cells. [score:5]
Li et al [14] transfected miR-34a duplex oligoribonucleotides into HepG2 cells up to 48 h and cell proliferation was determined using Cell Counting Kit-8. The results showed that the ectopic expression of miR-34a had no significant inhibition of cell proliferation [14]. [score:5]
On the contrary, Li et al [14] reported that down-regulation of miR-34a expression was highly significant in 19 of 25 (76%) human HCC tissues compared with adjacent normal tissues, using real time. [score:5]
HCC cells were seeded in a 24-well plate (2.5×10 [4] cells per well) or a 96-well plate (2.5×10 [3] cells per well) and incubated at 37°C for 24 h. The cells were then transfected with miR-34a inhibitor, miRNA inhibitor negative control, miR-34a mimic and miRNA mimic negative control, respectively (Ambion, Life Technologies Grand Island, NY 14072 USA) at a final concentration of 200 nmol/L for 96 h using combiMAGnetofection (OZ BIOSCIENCES, Marseille cedex 9 France) in accordance with manufacturer's procedure. [score:5]
The underexpression of miR-34a in HCC indicates that miR-34a may play a critical role in the hepatocarcinogenesis, as a tumor suppressor miRNA. [score:5]
M: mock control; C1: Negative control for miRNA inhibitor; Inhi: miR-34a inhibitor; C2: Negative control for miRNA mimic; Mimi: miR-34a mimic. [score:5]
Thus, we demonstrated that miR-34a mimic enhanced the cell proliferation inhibitory effect by c-MET siRNA and c-MET inhibitor su11274. [score:5]
The mechanisms of miR-34a inhibiting cell growth, metastasis and inducing apoptosis could be correlated to different networks between miR-34a and other target genes. [score:5]
miR-34a is a direct transcriptional target of p53, which is a transcription factor that coordinates cellular responses to stresses such as DNA damage and oncogene activation. [score:4]
The relative expression of miR-34a in HCC tissues was significantly lower than that of their matched adjacent noncancerous liver tissues (P<0.01). [score:3]
0061054.g002 Figure 2HepG2 cells (2.5×10 [4] cells per well using 6-well-plate) were transfected with miR-34a inhibitor, miR-34a mimic and their negative controls up to 96 h. Expression of miR-34a was detected using real time and delta delta Cq was calculated. [score:3]
Effect of miR-34a inhibitor and miR-34a mimic on cell migration and invasion in HepG2 cells. [score:3]
miR-34a expression was determined using real time. [score:3]
Contradictorily, miR-34a was found to be consistently up-regulated in the HCCs as compared to the non-neoplastic liver tissues in a chemical -induced HCC F344 rat mo del [19]. [score:3]
0061054.g003 Figure 3HepG2, HepB3 and SNU449 cells (2.5×10 [3] cells per well in 96-well-plate) were cultured for one day and then transfected with miR-34a inhibitor, miR-34a mimic and their controls (200 nM). [score:3]
The use of miR-34a mimic, together with other treatments, for instance, agents targeting c-MET pathway, might thus be a promising approach to HCC therapies in the future, to be explored in vivo and in clinic. [score:3]
On the other hand, cell growth and invasion inhibition, as well as apoptosis induction by miR-34a mimic appear of great relevance due to its possible therapeutic role. [score:3]
miR-34a expression decreased in the cases that the tumor cells invaded into the portal vein. [score:3]
Hence, our results suggested that miR-34a could not only inhibit cell growth, migration and invasion, but also induce apoptosis. [score:3]
Correlation between the expression of miR-34a and Clinicopathological Parameters in HCC. [score:3]
HepG2 cells (5×10 [4] cells per well using 6-well-plate) were cultured to 50% confluent and transfected with the miR-34a inhibitor, miR-34a mimic and the controls. [score:3]
When studied the relationship between miR-34a expression and other clinicopathological parameters, we found that miR-34a level was correlated with the status of portal vein tumor embolus. [score:3]
miR-34a expression in HCC FFPE tissues and its clinicopathological significance. [score:3]
Pineau et al [13] observed by microarray that miR-34a increased in HCC and was linked to disease progression from normal liver through cirrhosis to full-blown HCC. [score:3]
In human HCCs, there were also discrepant reports on the expression of miR-34a. [score:3]
Similar combinatorial effect was observed when the miR-34a mimic was added to the c-MET small molecular inhibitor su1174. [score:3]
In the current study, we transfected miR-34a inhibitor and mimic by combiMAGnetofection into 3 different HCC cell lines. [score:3]
Other targets of miR-34a involved in cell cycle, cell growth, invasion and apoptosis including Cyclin D1, Cyclin E2, E2F, B-cell CLL/lymphoma 2 (BCL2), CCNE2, CCND1, microtubule actin cross-linking factor 1 (MACF1), cyclin -dependent kinase 6 (CDK6), CDK4, Lamin-A/C, microtubuleactin cross-linking factor, tubulin a-1B chain, Glial fibrillary acidic protein (GFAP), Tropomyosin α-4 chain (TPM4), chaperone protein Endoplasmin (HSP90B1), Lamin-A/C (LMNA), Aldehyde dehydrogenase (ALDH2), Leucine-rich repeat-containing protein (LOC100129335), Cathepsin D, baculoviral IAP repeat-containing 3 (BIRC3), decoy receptor 3 (DcR3 also known as TNFRSF6B) and c-MET [14], [20], [21]. [score:3]
Furthermore, the relationship between the miR-34a expression and clinicopathological parameters in HCC was not fully understood. [score:3]
HepG2, HepB3 and SNU449 cells (2.5×10 [3] cells per well in 96-well-plate) were cultured for one day and then transfected with miR-34a inhibitor, miR-34a mimic and their controls (200 nM). [score:3]
The different methods to re-express miR-34a (miR-34a duplex oligoribonucleotides vs synthesized pre-has-miR-34a-PGCSIL-GFP vs miR-34a mimic) and different time points of transfections (48 vs 72 vs 96 h) might partially explain the discreapancies between other reports and the current results. [score:3]
Relationship between miR-34a expression and clinicopathological parameters. [score:3]
Hence it may be valuable to examine miR-34a expression for the clinical prediction of metastasis and progression of HCC. [score:3]
HepG2 cells (2.5×10 [4] cells per well using 6-well-plate) were transfected with miR-34a inhibitor, miR-34a mimic and their negative controls up to 96 h. Expression of miR-34a was detected using real time and delta delta Cq was calculated. [score:3]
The addition of miR-34a mimic to c-MET siRNAs, led to an greater effect on cell growth inhibition and apoptosis induction. [score:3]
Cell viability, cell proliferation, apoptosis and nuclear morphology and caspase-3/7 activity were performed as described previously [27], [29], [30] to study the effects of miR-34a inhibitor and miR-34a mimic. [score:3]
The expression of miR-34a in the tissues in clinical TNM III and IV stages was significantly lower than that in I and II stages. [score:3]
c-MET is a known target gene of miR-34a [14], [15]. [score:3]
miR-34a is suggested to be an important component of the p53 tumor suppressor network [21]. [score:3]
The miR-34a inhibitor had little effect on the migration and invasion activity. [score:3]
miR-34a mimic enhanced the cell proliferation inhibitory effect of c-MET siRNA and of su11274. [score:3]
0061054.g005 Figure 5HepG2 cells (5×10 [4] cells per well using 6-well-plate) were cultured to 50% confluent and transfected with the miR-34a inhibitor, miR-34a mimic and the controls. [score:3]
In line with this, Cheng et al [20] did not find that the overexpression of miR-34a altered apoptosis in HepG2 cells quantified by Annexin V staining. [score:3]
miR-34a was noted to inhibit migration and invasion in HCC cells [14], [20], which was also confirmed in the present study. [score:3]
Li et al [14] studied the expression of miR-34a of 25 cases, with only 3 females included. [score:3]
The transfection of pre-has-miR-34a-PGCSIL-GFP caused a remarkable inhibition of cell proliferation 72 h post-transfection. [score:3]
Transfection efficiency of miR-34a inhibitor and miR-34a mimic in HepG2 cells. [score:3]
The inhibition of cell proliferation and induction of caspase activity were much stronger with miR-34a mimic in combination with c-MET siRNA or su11274, compared to single drug or single miR-34a mimic in HepG2 cells (Figure 8A, Figure 8B, Figure 8C). [score:2]
With the miR-34a inhibitor, cell viability was slightly increased in HepG2, HepB3 and SNU449 cells 96 h post-transfection compared to negative controls, however the difference was not significant. [score:2]
The miR-34a abundance in each sample was normalized to its references. [score:1]
The miR-34a however had no correlation with other features, such as age, histological differentiation grades, cirrhosis, plasma AFP levels, tumor capsular infiltration, number of the tumor nodes or tumor sizes. [score:1]
The miR-34a mimic led to a moderate decreased migration and invasion rate in HepG2 (Figure 5). [score:1]
Interestingly, we also found that miR-34a level was lower in males than females, which had never been reported previously. [score:1]
The influence of miR-34a on apoptosis in HepG2 cells was also explored previously by two groups. [score:1]
Effect of miR-34a on malignant phenotype in HCC cells. [score:1]
Figure S2 Combinational effect of su11274 and miR-34a mimic in HepG2 cells. [score:1]
Effect of miR-34a on downstream pathway signals in HCC cells. [score:1]
Figure S1 Combinational effect of c-MET siRNA and miR-34a mimic in HepG2 cells. [score:1]
Thus, the result in current study reveals an obvious relation between miR-34a and the migration, invasion and metastasis of HCC. [score:1]
Effect of miR-34a on cell growth in HCC cells. [score:1]
The transfection of miR-34a into HepG2 cell resulted in the c-MET gene silencing [14], which was also confirmed in the present study (Figure 8D). [score:1]
Effect of miR-34a on apoptosis in HCC cells. [score:1]
In the current study, the result with real time confirmed the previous report from Li, et al [14], in a larger size of patients of 83 cases, which showed that HCC had lower miR-34a level than the adjacent non-cancerous liver tissues. [score:1]
Moreover, we found that miR-34a level was correlated with the status of portal vein tumor embolus. [score:1]
The circulating miR-34a level was also revealed to be gradually increased with the progress of hepatocarcinogenesis in the same rat mo del [19]. [score:1]
miR-34a level was also found lower in males than in females. [score:1]
HepG2 cells (2.5×10 [3] cells per well in 96-well-plate) were treated with miR-34a mimic with c-MET siRNA (A) or su11274 (B). [score:1]
Additionally, we performed in vitro experiments to study the effect of miR-34a on the cell growth, apoptosis, caspase-3/7 activity, migration and invasion in HCC cell lines. [score:1]
Cheng et al [20] transfected the chemically synthesized pre-has-miR-34a-PGCSIL-GFP into the same HCC cell line HepG2. [score:1]
Further exploration is needed to investigate target genes of miR-34a of HCC cells. [score:1]
miR-34a was also studied functionally in vitro in HCC cells. [score:1]
There was no significant change of sub-G1 DNA content in HepG2 cells, which was indicative of no effect of apoptosis by miR-34a. [score:1]
However, with the miR-34a mimic, caspase- 3/7 activity significantly enhanced at the 72 and 96 h post-transfection in all three cell lines (Figure 4A). [score:1]
After transfection of miR-34a duplex oligoribonucleotides for 48 h, Li et al [14] subjected HepG2 cells to DNA content analysis by flow cytometry. [score:1]
Among all the HCC-related miRNAs, contradictory relationship between miR-34a levels and HCC was reported [13], [14]. [score:1]
miR-34a was also shown to reduce the phosphorylation of ERK1/2, a key factor influencing the tumor growth, migration and invasion [14]. [score:1]
miR-34a level was lower in the cases with portal vein tumor embolus than those without (P<0.05, Table 1, Figure 1). [score:1]
Additionally, Cheng et al [20] also found that the pre-has-miR-34a-PGCSIL-GFP induced an accumulation of HepG2 cells in G1 phase and reduction of cells in S and G2 phase. [score:1]
The miR-34a expression in FFPE experiment was calculated with the formula 2 [−Δcq], and the change ratio of miR-34a in the in vitro experiments was: (1-1/2 [ΔΔCq])×100% [29]. [score:1]
miR-34a mimic induced the apoptosis and caspase activity from 72 h post-transfection and the influence reached the highest summit at 96 h post-transfection. [score:1]
Sequence of miRNA and references using in current study are : miR-34a (Applied Biosystems Cat. [score:1]
In the present study, we investigated the expression of miRNA-34a in HCC and their matched adjacent noncancerous liver tissues in 83 cases of formalin-fixed paraffin-embedded (FFPE) surgically resected samples, using real time quantitative RT-PCR (RT-qPCR). [score:1]
To verify the additive or synergistic nature of combining c-MET targeted agents with the miR-34a mimic, a CI was calculated [17]. [score:1]
After transfection with the miR-34a mimic, a moderate decreasing in viability was noted at the 96 h in all the three cell lines (Figure 3A). [score:1]
0061054.g008 Figure 8HepG2 cells (2.5×10 [3] cells per well in 96-well-plate) were treated with miR-34a mimic with c-MET siRNA (A) or su11274 (B). [score:1]
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Collectively, these results demonstrated that EGFR knockdown exhibited a similar phenotype as miR-34a upregulation in NSCLC cells, and that the tumor suppressive function of miR-34a is mediated partly by the downregulation of EGFR. [score:10]
showed a significant downregulation of Ki67 and EGFR in the tumor tissues stably pLenti-miR-34a, accompanied by an induction of E-cadherin expression, but a decrease in the expression of N-cadherin (Figures 8e and f). [score:8]
Our xenograft data suggested that suppression of tumor growth and metastasis occurs through the inhibition of the proliferation and the induction of apoptosis of the tumor cells, indicating the potential for miR-34a as a strategy for EGFR -targeted therapy. [score:7]
A549 cells were cultured in 24-well plate and transiently co -transfected with 200 ng of luciferase vector EGFR-3′-UTR and a final concentration of 100 n m of miR-34a inhibitor or NC inhibitor, with 20 ng of plasmid expressing the renilla luciferase gene (pRL, Promega) as a control for transfection efficiency. [score:7]
These results suggested that downregulation of miR-34a may act as tumor suppressor gene in NSCLC. [score:6]
We have demonstrated that in the NSCLC cell lines, A549, SPC-A1 and HCC827 (EGFR-mutated), miR-34a acted as a tumor suppressor through the direct targeting of EGFR. [score:6]
MiR-34a mimic, NC mimic, miR-34a inhibitor, NC inhibitor and siRNA were purchased from Ruibo Company (Guangzhou, China). [score:5]
Furthermore, since miR-34a inhibits in vitro proliferation of NSCLC cells, we explored miR-34a’s potential for in vivo tumor suppression. [score:5]
We also co -transfected EGFR-3′-UTR, pRL vector and miR-34a inhibitor, as well as the control (co -transfected with EGFR-3′-UTR, pRL vector and NC inhibitor) into A549 cell line. [score:5]
We have identified that miR-34a acted as an important tumor suppressor in NSCLC with EGFR as a novel target, both in vitro and in vivo. [score:5]
Furthermore, a significant upregulation in the expression of miR-34a was observed in tumor tissues from the pLenti-miR-34a group, when compared to the controls (Figure 8d). [score:5]
In conclusion, our study shows that miR-34a inhibits NSCLC growth by targeting EGFR. [score:5]
20, 41, 42 Recently, studies have further discovered that ectopic overexpression of miR-34a results in the inhibition of some solid tumors, including prostate cancer, [43] colon cancer, 44, 45 NSCLC, [46] endometrial carcinoma, [47] hepatocellular carcinoma [48] and osteocarcinoma. [score:5]
The level of miR-34a expression was significantly upregulated in A549 and SPC-A1 cells transfected with miR-34a mimic, as compared with normal control (NC) mimic group. [score:5]
The level of miR-34a expression was upregulated 26-fold in A549 pLenti-miR-34a, as compared with pLenti groups (Supplementary Figure S3b). [score:5]
org/) to predict potential targets, and identified EGFR as a potential target for miR-34a. [score:5]
[51] Our previous study verified that in NSCLC, overexpression of miR-34a inhibits proliferation and promotes apoptosis in vitro. [score:5]
Meanwhile, we transfected with miR-34a inhibitor, miR-34a level was downregulated when compared with NC mimic group (Figure 2d). [score:5]
In these samples, the downregulation of miR-34a was closely related to tumor size (Figure 1b), but it was not associated with gender (Figure 1c) and pathological stage (Figure 1d). [score:4]
EGFR is a direct target of miR-34a. [score:4]
Moreover, based on prior studies in our lab, 18, 26, 27, 28 we concluded that miR-34a could be involved in the regulation of several target mRNAs, and also be closely related to the signaling pathways for Kras and nuclear factor-κB. [score:4]
Furthermore, we demonstrated that EGFR was a direct target of miR-34a. [score:4]
In this study we detected that miR-34a was downregulated in NSCLC patient samples and NSCLC cell lines. [score:4]
As a tumor suppressor within this regulation network, miR-34a can affect the proliferation, migration, apoptosis and cell cycle of NSCLC cells (Figure 9). [score:4]
revealed that EGFR reverse the suppressive function of miR-34a overexpression on proliferation and cell apoptosis in A549 cell, compared with the NC group (Figures 6d and f). [score:4]
Here we identified miR-34a as an EGFR -targeting miRNA. [score:3]
Inversely, co-transfection of miR-34a with EGFR-3′-mUTR (pGL3-EGFR mut 3′-UTR) resulted in no significant change in luciferase activity, supporting miRNA/target 3′-UTR specificity (Figure 5b). [score:3]
We also analyzed the expression level of miR-34a in NSCLC cell lines. [score:3]
pLenti A549 and pLenti-miR-34a A549 were transfected with 1000 ng/ml EGFR expression vector p2k7 or p2k7-EGFR using Lipofectamine 2000. [score:3]
We make a hypothesis that miR-34a should regulate EGFR directly or EGFR signaling. [score:3]
We also analyzed the effect of miR-34a overexpression on NSCLC proliferation and cell apoptosis. [score:3]
Nude mice were injected subcutaneously with miR-34a-stably overexpressing (pLenti-miR-34a) A549 cells (5 × 10 [6]) or control (pLenti), and the subsequent tumors were assessed after 6 weeks. [score:3]
18, 27 In brief, HEK293T cells were cultured in 24-well plate and transiently co -transfected with 200 ng of luciferase vector EGFR-3′-UTR or EGFR-3′-mUTR, and a final concentration of 100 n m of miR-34a mimic or NC mimic, with 20 ng of plasmid expressing the renilla luciferase gene (pRL, Promega) as a control for transfection efficiency. [score:3]
With regard to cell cycle, transfection of miR-34a mimic in A549, SPC-A1 and HCC827 cells could partly inhibit cell-cycle progression (Figures 4b and d; Supplementary Figures S2c and d). [score:3]
showed that compared with the normal bronchial epithelium cell, BEAS-2B, miR-34a was significantly downregulated in all five NSCLC cell lines (Figure 1e). [score:3]
The miR-34a expression level in tissue samples from tumors >3 cm in size was significantly lower than that in tissue samples ⩽3 cm in size. [score:3]
This result indicated that miR-34a could inhibit the proliferation of NSCLC cell lines. [score:3]
[30] Meanwhile, there are many verified and predicted targets of miR-34a (Supplementary Table S4). [score:3]
Moreover, we verified that miR-34a inhibits proliferation of lung cancer cells by inducing cell apoptosis and cell-cycle arrest. [score:3]
Together, these results indicated that miR-34a could significantly inhibit cell migration in the A549 and SPC-A1 cell lines. [score:3]
showed that transfection of miR-34a mimic significantly inhibited the proliferation of the A549 (EGFR-wild type), SPC-A1 and HCC827 (EGFR-mutated) cell lines (Figures 2b and c; Supplementary Figure S1). [score:3]
MiR-34a is downregulated in NSCLC. [score:3]
To investigate whether the effects of miR-34a was mediated through EGFR, we transfected EGFR expression vector (p2k7-EGFR) and negative control (p2k7) into A549 cell line, which stably overexpressing miR-34a (pLenti-miR-34a) and negative control (pLenti). [score:3]
org/) were used to predict the target genes of miR-34a. [score:3]
These data indicated that miR-34a could inhibit tumor growth in vivo, complementing the results of our functional in vitro studies. [score:3]
On the basis of the tumor suppressive role of miR-34a in vitro, we further examined the role of miR-34a in vivo, to assess its therapeutic potential. [score:3]
[18] This result may suggest the potential for miR-34a as a tumor suppressor in NSCLC. [score:3]
[25] Therefore, exploring the function of miR-34a and the role of its possible target genes in NSCLC is essential to understanding the molecular mechanism of this miRNA in tumorigenesis. [score:3]
Cells were transfected with 80 n m of chemically synthesized miR-34a mimic, 120 n m miR-34a inhibitor or 100 n m siEGFR-1 (Supplementary Table S2) using Lipofectamine 2000 (Thermo, Life Technologies, New York, NY, USA), according to the manufacturer’s instructions. [score:3]
In addition, there was a negative correlation between the relative EGFR expression and miR-34a in the NSCLC tissues samples, according to statistical analysis using the Pearson correlation coefficient (Figure 5h). [score:3]
To further investigate whether miR-34a reduced EGFR expression at both the transcriptional and translational levels in NSCLC cells, we performed qRT–PCR and western blotting to determine the mRNA and protein levels of EGFR. [score:3]
13, 14, 15 In NSCLC, several deregulated miRNAs, such as miR-34a, let-7, miR-124 and miR-154, have been shown to regulate cell proliferation, apoptosis, migration and invasion. [score:3]
[21] It is well known that miR-34a can significantly suppress tumor progression, such as in NSCLC, [18] breast cancer, [22] glioblastoma multiforme, [23] head and neck squamous cell carcinoma [24] and hepatocellular carcinoma. [score:3]
This analysis revealed that the miR-34a expression level in NSCLC tissue samples was significantly lower than that in corresponding non-tumor tissues samples (Figure 1a). [score:3]
While transfection with miR-34a inhibitor promoted the proliferation of the A549 and SPC-A1 cell lines (Figures 2e and f). [score:3]
Therefore, miR-34a and EGFR might be promising molecular targets not only for the treatment of NSCLC but also as a useful and novel prognostic or progression marker for NSCLC. [score:3]
Our present study demonstrated that miR-34a expression was reduced in NSCLC, when compared with the non-tumor tissues. [score:2]
revealed that miR-34a overexpression significantly promoted apoptosis in A549, SPC-A1 and HCC827 cells, compared with the NC group (Figures 4a and c; Supplementary Figures S2a and b). [score:2]
showed that the levels of EGFR mRNA and protein expression were significantly decreased following transfection with miR-34a mimic in both A549 and SPC-A1 cells, compared with the NC mimic group (Figures 5c–e). [score:2]
The mRNA and protein expression of EGFR were also increased following transfection with pLenti-miR-34a in A549 cell, compared with the NC group (Figures 6a–c). [score:2]
MiR-34a suppresses tumor growth in A549 xenograft and metastatic tumors. [score:2]
[49] MiR-34a has the ability to control the expression of a gene family through combining bioinformatic approaches with experimental validation. [score:2]
The A549 xenografts displayed significant inhibition of tumor growth in pLenti-miR-34a group, compared to the control (Figure 8a). [score:2]
MiR-34a suppresses the proliferation and migration of NSCLC. [score:2]
showed that miR-34a could significantly inhibit colony formation in A549 or SPC-A1 cells with miR-34a mimic, when compared with the NC group (Figures 2g–i). [score:2]
Our previous studies have confirmed that the miRNAs, miR-181a-5p, miR-146a-5p, miR-32, miR-34a and miR-486-5p, have important roles in the progression of NSCLC. [score:1]
pLenti-miR-34a or pLenti vector was co -transfected into HEK293T with psPAX2 and pMD2G. [score:1]
[53] In brief, pri-miR-34a sequence was digested with BamHI and XholI, then cloned into the pLenti vector (Invitrogen, Carlsbad, CA, USA), formed pLenti-miR-34a. [score:1]
Target gene for miR-34a, EGFR, was also measured by immunohistochemistry. [score:1]
To ascertain whether or not miR-34a effected tumor metastasis in vivo, A549 cells (5 × 10 [6]) pLenti-miR-34a or pLenti were injected into the tail vein of nude mice. [score:1]
To date, miR-34a is best understood as a more and more anticancer miRNA, acting primarily on cell-cycle arrest, differentiation, senescence and apoptosis, 20, 40 and it is significant to note that a liposomal formulation of miR-34a (MRX34) has become the first miRNA to reach phase I clinical trials. [score:1]
Furthermore, in A549 and SPC-A1 cells, miR-34a affected migration ability, a significant aspect of cancer progression. [score:1]
MiR-34a, a member of miR-34 family, is located in the region of chromosome lp36.23. [score:1]
Besides, the mechanism of miR-34a in blocking NSCLC tumor growth deserves more exploration. [score:1]
To establish the subcutaneous tumor xenograft mo del, 6–8 weeks’ mice were randomly assigned into two groups (five mice per group), and each nude mice was injected subcutaneously in the right flank with 5 × 10 [6] A549 cells (resuspended in 100 μl DMEM medium) pLenti-miR-34a or pLenti. [score:1]
The cells were transfected with pLenti-miR-34a or pLenti and sorted for green fluorescence via flow cytometry. [score:1]
[31] Moreover, study has shown that the therapeutic potential of miR-34a delivery in combination with radiotherapy may represent a novel strategy for treating NSCLC. [score:1]
The tumor nests derived from control cells exhibited a large area of lung tissue destruction and/or necrosis, whereas pLenti-miR-34a cells formed fewer and smaller tumor nests (Figure 8j). [score:1]
To explore the molecular mechanism by which miR-34a contributes to the apoptosis and cell-cycle progression of NSCLC cells, we used miRWalk 2.0 (http://zmf. [score:1]
To evaluate the expression of miR-34a in NSCLC tissues, we performed quantitative real-time PCR (qRT–PCR) on 60 NSCLC patient samples and corresponding para-carcinoma tissues. [score:1]
To investigate whether or not EGFR expression was inversely correlated with miR-34a in NSCLC tissues, we evaluated the mRNA expression of EGFR in the 60 primary NSCLC tumors and non-tumor tissues using qRT–PCR. [score:1]
We analyzed the effect of miR-34a on cell apoptosis and cell-cycle progression. [score:1]
showed that miR-34a could reduce the migration of A549 and SPC-A1 cells (Figures 3b and d). [score:1]
A549 cells were then infected with the viral particles, transfected with pLenti-miR-34a or pLenti and were sorted for green fluorescence via flow cytometry. [score:1]
The pLenti cells generated fine and scattered metastatic nodes, while the pLenti-miR-34a cells resulted in massive and confluent metastatic nodes (Figures 8g and h). [score:1]
The cells were collected 48 h after transfection and miR-34a levels were detected by qRT–PCR analysis, resulting in ~400- and 100-fold increases for A549 and SPC-A1 cells, respectively (Figure 2a). [score:1]
The pLenti-miR-34a cells also exhibited obvious reduction in both tumor size and weight at 6 weeks post implantation (Figures 8b and c). [score:1]
3′-UTR luciferase plasmids were constructed as follows: the 3′-UTR of EGFR containing the predicted binding site of miR-34a was cloned into the pGL3 vector (Promega, Madison, WI, USA) and designated as EGFR-3′-UTR. [score:1]
To investigate the underlying mechanism of action that miR-34a exerts on the inhibition of cancer cell proliferation, we performed flow cytometry after transfecting A549, SPC-A1 and HCC827 cells with miR-34a mimic and NC mimic. [score:1]
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In addition, we also postulate a counteracting pathway in which maintains p53 expression and, indirectly, the expression of miR-34a, providing a substantial protective axis against the loss of let-7. Further studies will be aimed at identifying the mechanism of promotion of p53 expression and its mechanism of negatively regulating let-7. In 2007, several groups identified the miR-34 family of miRNAs (miR-34a, b, and c) as a direct transcriptional target of the key tumor suppressor p53 [26]– [29], [36], [37]. [score:14]
Because is, itself, a known p53 regulated gene, this points at a connection between apoptosis sensitivity, the expression of miR-34a, and a functional p53 pathway resulting in upregulation of p53 target genes; this is also consistent with the fact that p53 activation increases the surface expression of [32]. [score:11]
When HCT116 p53 [−/−] cells were treated with etoposide they were unable to upregulate miR-34a (Figure 2A ), indicating that both miR-34a and upregulation are dependent on p53 expression. [score:9]
Sensitization was not due to an altered surface expression of or reduced expression of the miR-34a targets c-Met or CD44 or an increase in p53 expression (Figure 1G and H ). [score:9]
In addition, we also postulate a counteracting pathway in which maintains p53 expression and, indirectly, the expression of miR-34a, providing a substantial protective axis against the loss of let-7. Further studies will be aimed at identifying the mechanism of promotion of p53 expression and its mechanism of negatively regulating let-7. All cells were maintained in a humidified incubator at 37°C with 5% CO [2]. [score:9]
In addition, we also postulate a counteracting pathway in which maintains p53 expression and, indirectly, the expression of miR-34a, providing a substantial protective axis against the loss of let-7. Further studies will be aimed at identifying the mechanism of promotion of p53 expression and its mechanism of negatively regulating let-7. CSCs have gained much interest as a likely mechanistic explanation for cancer progression, tumor heterogeneity, emergence of aggressiveness, and drug resistance [16], [57], [58]. [score:9]
Inhibition of methylation and/or histone deacetylase inhibitor in pancreatic cancer stem cells caused marked upregulation of miR-34a and induced both cell cycle arrest and apoptosis [62]. [score:8]
Overexpression of in low expressing MCF7 cells caused a pronounced upregulation of miR-34a upon treatment with etoposide. [score:8]
These data suggest that directly regulates the expression of p53 and its ability to induce miR-34a expression. [score:7]
We also postulate a counteracting pathway in which maintains p53 expression and, indirectly, the expression of miR-34a, providing a substantial protective axis against the loss of let-7. In studying -mediated apoptosis, our laboratory has made extensive use of a collection of 60 human cancer cell lines maintained by the National Cancer Institute's Developmental Therapeutics Program (NCI60). [score:7]
To determine whether sensitivity to -mediated apoptosis and miR-34a expression in the NCI60 cells correlated with the expression of other known p53 targets we performed a COMPARE analysis [31] using the gene array data available on the NCI60 cells. [score:7]
mir-34a might regulate p53 expression by targeting SIRT1 which increases p53's acetylation, as recently shown [46]. [score:6]
In contrast, knock down of in either high expressing CAKI-1 or HCT116 cells, which resulted in reduced expression of, attenuated the ability of the cells to respond to genotoxic stress by inducing p53, p21 or miR-34a (Figure 4B and C ). [score:6]
In 2007, several groups identified the miR-34 family of miRNAs (miR-34a, b, and c) as a direct transcriptional target of the key tumor suppressor p53 [26]– [29], [36], [37]. [score:6]
One possible mechanism for the sensitization of cells to -mediated apoptosis is suggested by data showing that miR-34 targets CD44, which has been shown to inhibit -mediated apoptosis by directly binding the region required forL engagement. [score:6]
expression affects the ability of cells to upregulate miR-34 in response to genotoxic stress. [score:6]
However, we did not find altered expression of CD44 in miR-34a overexpressing cells. [score:5]
The importance of miR-34a and miR-34b is highlighted by their loss of expression in more than a dozen different cancers [37], [38] suggesting a crucial role for miR-34 in suppressing tumorigenesis. [score:5]
It appears that the mere presence of p53 or can affect expression levels of miRNAs, although stimulation through did not have a major effect on the expression of either let-7 or miR-34 (data not shown). [score:5]
Again, we found strong positive correlation between c-Met expression and the expression of miR-34a in renal cancer and glioblastoma (sPCCs were 6.45 and 13.74, respectively). [score:5]
Not surprisingly, direct targets of miR-34a/b/c are enriched in genes controlling key cellular processes such as cell cycle control (cyclins D1, E2, and CDK4/6), cell proliferation (c-Myc, N-myc, E2F3, c-Met, Notch, Ezh2, HMGA2), and apoptosis (Bcl-2, SIRT1) [37], [39]. [score:4]
Loss of stemness was shown to be due to miR-34a directly targeting and repressing CD44, a wi dely used marker for stem cells [64]. [score:4]
In order to determine whether this also applied to miR-34a, we compared the expression of miRNAs significantly expressed in the NCI60 cells between p53 responder and nonresponder cells. [score:4]
This analysis suggested that miR-34a and let-7 inversely correlate with a p53 response which may directly affect the expression of. [score:4]
Consistent with this finding, our analysis revealed that in tumor cells sensitive to -mediated apoptosis the second most highly upregulated miRNA was miR-200b (miR-34a = 2.35 fold higher and miR-200b = 1.71 fold higher) (Figure 1A ). [score:4]
Because miR-34a was a positive regulator of the sensitivity to -mediated apoptosis, we tested whether altering expression levels also affected the response of cells to stress induced by DNA damage (Figure 3B ). [score:4]
We now demonstrate that expression of the p53-regulated miRNA miR-34a correlates with sensitivity of cells to -mediated apoptosis. [score:4]
These analyses are not consistent with c-Met being a strong target for miR-34a in the context of cancer. [score:3]
miR-34a was the only miRNA that was significantly expressed more highly in the p53 responder cells (Figure 3A and data not shown). [score:3]
In fact, similar to miR-200 [23] overexpression of miR-34a alone can induce mesenchymal to epithelial transition (MET) in certain tumor cells [50]. [score:3]
miR-34a and are p53 transcriptional targets that are functionally connected. [score:3]
In fact, transfected pre-miR-34a in prostate cancer cell lines suppressed their stem-like phenotype and decreased tumor load in a xenograft mo del. [score:3]
Moreover a CD44 variant (CD44v6) binds the miR-34 target, c-Met, and this interaction is required for c-Met signaling [42]. [score:3]
p53-independent expression of miR-34a has also been reported through activation of the Erk/Elk1 pathway [40]. [score:3]
contains an intra-intronic p53 enhancer, and is a bona fide p53 target gene [24] similar to miR-34a. [score:3]
Experiments in this paper suggest different activation thresholds for p21 and miR-34a upon altering expression as p21 was equally efficiently induced despite modulation in, whereas miR-34a was responsive to changes. [score:3]
Inset shows real-time PCR analysis of miR-34a expression in the transfected cells. [score:3]
While miR-34a was the miRNA that best correlated with the ability of cells to respond to activation of p53, the most significant correlation between p53 responsiveness and the expression of miRNAs was a negative correlation with the let-7 family of miRNAs. [score:3]
Interestingly, mir-34a expression did not significantly correlate with the sensitivity of cancer cells to apoptosis induced by LzTRAIL (Figure 1C ) and the miRNA that most significantly correlated with TRAIL apoptosis sensitivity was miR-31 (Figure 1D ). [score:3]
A similar set of genes was found to positively correlate with the expression of miR-34a (data not shown). [score:3]
In order to test whether an increase in miR-34a expression would cause an increase in the sensitivity of cells to -mediated apoptosis, HCT116, which are moderately sensitive toL induced apoptosis, were transfected with either a scrambled oligonucleotide or pre-miR-34a. [score:3]
No correlation between expression of miR-34a was found with the sensitivity of cells to TRAIL -induced apoptosis. [score:3]
Similar results were obtained when miR-34a was restored by exogenous miRNAs (pre-miR transfection or lentiviral expression) in another pancreatic cancer mo del resulting in an almost 90% reduction in tumor-initiating CD44+ stem cells [59]. [score:3]
miR-34a is ubiquitously expressed, whereas in most tissues miR-34b and miR-34c are minor species [37]. [score:3]
Our finding that the expression of miR-34a positively correlates with the sensitivity of cells to undergo -mediated apoptosis is in line with the proapoptotic function of both miR-34a and p53. [score:3]
Induction of pluripotency by Oct24, Sox2, Klf4, and c-Myc in mouse embryonic fibroblasts induces all three miR-34 species to cooperatively inhibit reprogramming by repressing Nanog, Sox2, and N-myc [63]. [score:3]
Based on these data we now formulate a hypothesis that links all of these players,, p53, miR-34a, and let-7, in a regulatory network (Figure 6 ). [score:2]
A mo del for the role of the/let-7/p53/miR-34a regulatory network and its potential relevance in cancer stem cells. [score:2]
To further explore this aspect, we have compared the expression of c-Met with that of miR-34a in the panel of the NCI60 cells and a positive correlation, rather than a negative one, was found (data not shown). [score:2]
Together with the recognition that apoptosis sensitivity is in part regulated by the p53 status [30], the identification of miR-34a as a marker for apoptosis sensitive cells pointed at a connection between signaling and the p53 network. [score:2]
In summary, we propose that is part of a novel regulatory network together with p53 and the miRNAs let-7 and miR-34a. [score:2]
We have discovered a p53 regulated network that involves, miR-34a, and let-7. Every component of this novel network has crucial functions in the generation or maintenance of cancer stem cells (CSCs). [score:2]
miR-34 is a selective marker for cancer cells that are sensitive to -mediated apoptosis. [score:1]
These data suggested that miR-34a is not only a marker for cells sensitive to -mediated apoptosis but that it can sensitize cells to this form of apoptosis. [score:1]
Addition of leucine-zipper tagged ligand (LzCD95L) induced cell death in control transfected cells that was significantly enhanced in cells which had received pre-miR-34a (Figure 1E ). [score:1]
Taken together, our data are consistent with a miR-34a -mediated increase in apoptotic signaling. [score:1]
The mechanism by which miR-34a sensitizes cells to mediated apoptposis is therefore currently unknown. [score:1]
Of 136 miRNAs that were detected in at least 30 of the 59 NCI60 cells, miR-34a most significantly correlated with apoptosis sensitive cells (Figure 1A and B ). [score:1]
All let-7 family members are highlighted in yellow and miR-34a in light blue. [score:1]
Such miRNAs include the let-7 [20], miR-200 [60], and miR-34 families of miRNAs [61]– [64]. [score:1]
org) we determined that c-Met mRNA is also slightly positively correlated with miR-34a using a data set based on real time PCR data of the miRNAs (miRConnect-Q). [score:1]
miR-34a has been identified as a downstream effector of p53 mediating a number of its activities including apoptosis induction [25]– [29]. [score:1]
We have now identified miR-34a as a marker of cancer cells that are more sensitive to -mediated apoptosis, and as a sensitizer to apoptosis mediated by. [score:1]
In addition, pancreatic cancer cells frequently display miR-34 loss due to epigenetic silencing by methylation. [score:1]
Let-7. p53 and miR-34. [score:1]
Bottom: Sub-G1 peak analysis using PI staining and flow cytometry of HCT116 treated with pre-miR-34a and LzCD95L minus plus zVAD-fmk. [score:1]
These data suggested that miR-34a is a selective marker for apoptosis sensitivity. [score:1]
For quantitative real-time PCR analysis of miR-34a, the same cells were treated with either control medium (−) or 10 µM etoposide (+) for 12 hrs. [score:1]
Cells were reverse transfected in 6-well plates with 100 nM of either scrambled negative control #2 precursor miRNA or pre-miR-34a oligonucleotides (Ambion) using siPORT NeoFX (Ambion) following the manufacturer's protocol. [score:1]
, p53, and miR-34a are functionally linked. [score:1]
miR-34a and let-7 are functional opposites of p53-responsiveness. [score:1]
Taking into account the role of miR-34 as an effector for p53, it is not surprising that miR-34 also participates in protecting both stem cells themselves and the organism from pluripotent cells that have turned rogue. [score:1]
miR-34a is a marker for cells sensitive to -mediated apoptosis. [score:1]
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[+] score: 261
Other miRNAs from this paper: hsa-mir-34b, hsa-mir-34c
Most lung cancer cells showed low expression of miR-34a and high expression of PDGFR-α/β, whereas the adjacent non-malignant lung expressed PDGFR-α rarely and abundantly expressed miR-34a. [score:9]
0067581.g002 Figure 2(a) qRT-PCR showing PDGFR-α and PDGFR-β mRNAs downregulation in Calu-6 and H1703 cells after miR-34 and miR-34c but not miR-34b enforced expression (b) miR-34a and miR-34c enforced expression decreases endogenous levels of PDGFR-α/β protein levels in H1703 and Calu-6 NSCLC. [score:8]
Moreover, we demonstrate that PDGFR-β is a miR-34a/c direct target while we did not see any significant effect on the expression of PDGFR-α and PDGFR-β after miR-34b enforced expression. [score:8]
In 2007, reports from several laboratories showed that members of the miR-34 family are direct p53 targets, and that their upregulation induced apoptosis and cell-cycle arrest [16, 17]. [score:7]
To identify miR-34a, -34b, and -34c targets, we performed a bioinformatics search (Targetscan, Pictar) for putative mRNA targets. [score:7]
PDGFR-α/β downregulation by miR-34a and miR-34c inhibits migration and invasiveness of NSCLC cells. [score:6]
In this study, we show that miR-34a and miR-34c, are strongly downregulated in NSCLC cells and lung tumors whereas they are highly expressed in normal lung tissues. [score:6]
To verify the downregulation of miR-34a and -34c also in vivo, 9 lung tumors (among adenocarcinoma and squamous cell carcinoma) and the adjacent histologically normal lung tissues were analyzed for miR-34a and -34c expression. [score:6]
Remarkably, our study shows that inhibition or downregulation of PDGFR-α and PDGFR-β by miR-34a/c antagonizes tumorigenicity and increases sensitivity to TRAIL -induced cell death with important therapeutic application for future anti-tumor therapy of lung cancer. [score:6]
Enforced expression of miR-34a and miR-34c downregulated PDGFR-α and PDGFR-β mRNA and protein levels. [score:6]
Since PDGFR-α and PDGFR-β regulate the PI3K/Akt and ERK1/2 pathways [23, 24, 25], we next examined, by immunostaining, the expression and/or activation of some of the proteins involved in these pathways following miR-34a and miR-34c enforced expression or PDGFR-α/β silencing by siRNAs. [score:6]
Indeed, overexpression of miR-34a and miR-34c or downregulation of PDGFR-α/β by siRNAs, highly increased the response of semi-resistant NSCLC cells to TRAIL -induced apoptosis. [score:6]
Enforced expression of PDGFR-α or PDGFR-β partially restored NSCLC migration and invasion supporting that the regulation of the expression of these receptors by miR-34a/c plays an important role in NSCLC tumorigenesis. [score:6]
MiR-34a/c enforced expression reduced migration and invasion of Calu-6 cells but overexpression of PDGFR-α or PDGFR-β, along with the two microRNAs, partially restored the migration and invasion capabilities, suggesting that miR-34a/c regulate NSCLC tumorigenesis, at least in part, through PDGFR-α/ β (Figure 6 d,e). [score:6]
MiR-34a and miR-34c are inversely related to PDGFR-α/β expression in vitro and in vivo Next, we analyzed the consequences of the ectopic expression of miR-34a and -34c in Calu-6 and H1703 cells. [score:5]
Among the miRNAs, miR-34 family members play important tumor suppressive roles, as they are directly regulated by p53 and compose the p53 network [16, 17]. [score:5]
Intriguingly, we observed a significant decrease of the migratory and invasive capabilities of Calu-6 and H1703 cells after miR-34a or miR-34c overexpression (Figure 6a) as well after PDGFR-α and PDGFR-β downregulation (Figure 6b), confirmed also by scratch-wound assay (Figure 6c). [score:5]
While this manuscript was in preparation Silber et al. reported that miR-34a expression was lower in proneural gliomas compared to other tumor subtypes and identified PDGFR-α as a direct target of miR-34a [43]. [score:5]
MiR-34a or miR-34c and not miR-34b forced expression decreases PDGFRβ expression levels and reduces the activation of the ERK1/2. [score:5]
The percentage of tumor cells expressing PDGFR-α, PDGFR-β and miR-34a, was then analyzed with emphasis on co-localization of the respective targets. [score:5]
To directly test the functional role of miR-34a/c in tumorigenesis, we overexpressed these two miRNAs or silenced PDGFR-α/β in Calu-6 or H1703 cells. [score:4]
MiR-34a and miR-34c overcome TRAIL resistance of NSCLC cells through PDGFR-α and PDGFR-β downregulation. [score:4]
Taken together the results indicate that miR-34a and miR-34c and not miR-34b directly target PDGFR-α and PDGFR-β. [score:4]
To verify whether PDGFR-α and PDGFR-β were direct targets of miR-34a, -34b and -34c, PDGFR-α 3’ UTR, containing two miR-34a, -34b and -34c binding sites and PDGFR-β 3’ UTR, containing one miR-34a, -34b and -34c binding site (Figure 1a,b), were cloned downstream of the luciferase open reading frame. [score:4]
Here, we reported that miR-34a and miR-34c overexpression or PDGFR-α/β silencing inhibited the migration and invasion capacity of Calu-6 and H1703 cells, compared to cells transfected with a scrambled miR or siRNA control. [score:4]
Luciferase and western blot experiments demonstrated that PDGFR-α and PDGFR-β are direct targets of miR-34a and miR-34c but not of miR-34b. [score:4]
Here, we report that not only miR-34a but miR-34c also downregulates PDGFR-α in NSCLC cells. [score:4]
Interestingly, increased expression of miR-34a and miR-34c, and not miR-34b, upon transfection, confirmed by qRT-PCR (data not shown), significantly decreased luciferase activity, indicating a direct interaction between the miRNAs and PDGFRα and PDGFRβ 3’ UTRs (Figure 1c,d). [score:4]
Moreover, the promoter region of miR-34a, miR-34b and miR-34c contains CpG islands and aberrant CpG methylation reduces miR-34 family expression in multiple types of cancer [18, 19, 20]. [score:3]
Because PDGFR-α/β regulate the PI3K/AKT pathway, notably involved in migration and invasion of different tumors [27, 28], we investigated if miR-34a/c could influence NSCLC migration and invasion through PDGFR-α and PDGFR-β downregulation. [score:3]
We previously demonstrated that the PI3K/AKT pathway plays a key role in TRAIL -induced apoptosis [26], therefore the effects of miR-34a and miR-34c overexpression on cell survival and TRAIL resistance of NSCLC were examined. [score:3]
Among the candidate targets, 3’ UTR of human PDGFR-α and PDGFR-β contained regions (PDGFR-α nucleotides 2670–2676; 2699-2705; PDGFR-β nucleotides 1535-1541) that matched the seed sequences of hsa-miR-34a, -34b and -34c (Figure 1a). [score:3]
Moreover, miR-34a and miR-34c, by targeting PDGFR-α and PDGFR-β, increase TRAIL -induced apoptosis and decrease invasiveness of lung cancer cells. [score:3]
MiR-34a and miR-34c overexpression or PDGFR-α/β silencing decreases migratory and invasive capacity of NSCLC cells. [score:3]
MiR-34a and miR-34c are inversely related to PDGFR-α/β expression in vitro and in vivo. [score:3]
These serial sections were analyzed for miR-34a expression by in situ hybridization, followed by immunohistochemistry for PDGFR-α/β. [score:3]
However, we recognize that other miR-34a/c targets including c-Met [16] and AXL [42] could also be involved. [score:3]
Increased expression of miR-34a and miR-34c upon transfection was confirmed by qRT-PCR (data not shown) and then the effects on PDGFR-α and PDGFR-β mRNA and protein levels were analyzed by qRT-PCR and western blot. [score:3]
First, we analyzed by Real Time PCR (qRT-PCR) miR-34a, -34b and -34c expression in 5 different NSCLC cell lines with p53 WT, mutant or null (Figure S1a in File S1). [score:3]
MiR-34a and miR-34c target PDGFR-α and PDGFR-β 3’ UTRs. [score:3]
Tables reporting the percentage of miR-34a, PDGFR-α and PDGFR-β expression observed in the 106 (PDGFR-α) and 107 (PDGFR-β) tumor samples analyzed (A case with 10% of the tumor cells + was scored as +). [score:3]
Next, we analyzed the consequences of the ectopic expression of miR-34a and -34c in Calu-6 and H1703 cells. [score:3]
Figure S3, Enforced expression of miR-34a and miR-34c or PDGFR-α /β silencing increases the response to TRAIL -induced apoptosis and reduces tumorigenicity of NSCLC cell. [score:3]
Moreover, we analyzed miR-34a, 34c and PDGFR-α/β mRNA expression in 24 primary human lung tumor specimens in comparison with 24 normal tissues. [score:3]
MiR-34a and miR-34c overexpression or PDGFR-α/β silencing increases the response of NSCLC cells to TRAIL -induced apoptosis. [score:3]
0067581.g005 Figure 5(a) Western blot in Calu-6 cells after miR-34a, -34b and -34c forced expression. [score:3]
To further verify that PDGFR-α and PDGFR–β were involved in tumorigenesis of NSCLC cells, miR-34a and -34c were transfected in Calu-6 cells alone or in combination with a plasmid overexpressing only the coding sequence and not the 3’ UTR of PDGFR-α and PDGFR-β. [score:3]
The expression levels of the four PDGF ligands (PDGFA, PDGFB, PDGFC, PDGFD) after miR-34a and miR-34c enforced expression were also evaluated in both Calu-6 and H1703 cells. [score:3]
In summary, these results supported the bioinformatics predictions indicating PDGFR-α and PDGFR-β 3’ UTRs as targets of miR-34a and -34c. [score:3]
Figure S2, Co -expression analysis of miR-34a and PDGFR-α and PDGFR-β in lung tumor samples. [score:3]
MiR-34a and miR-34c are downregulated in the tumors compared to the normal lung tissues. [score:3]
MiR-34a, -34b and -34c had low or absent expression in all the five cell lines (Figure S1b in File S1). [score:2]
As shown in Figure 5a, phosphorylation levels of ERKs decreased after miR-34a and miR-34c enforced expression compared to cells transfected with the control miR. [score:2]
MiR-34a and -34c were almost undetectable in the tumor and highly expressed in the normal lung samples tested (Figure 3c). [score:2]
First, we performed a proliferation assay on Calu-6 and H1703 TRAIL semi-resistant cells after enforced expression of miR-34a and miR-34c. [score:2]
MiR-34a and -34c enforced expression in Calu-6 and H1703 semi-resistant cells, increases the response to TRAIL -induced apoptosis. [score:2]
MiR-34a and PDGFR-α/β expression in the majority of the 107 different tumors analyzed was basically mutually exclusive (Figure 4a,b and Figure S2 in File S1). [score:2]
MiR-34a and -34c (and not miR-34b) overexpression significantly reduced PDGFR-α and PDGFR-β mRNAs (Figure 2a) and the endogenous protein levels, compared to the cells transfected with a scrambled pre-miR (Figure 2b). [score:2]
0067581.g006 Figure 6(a) MiR-34a and -34c enforced expression reduces migratory and invasive capabilities of H1703 cells. [score:2]
Intriguingly, overexpression of PDGFR-α or PDGFR-β (using two plasmids containing only the coding sequences and not the 3’ UTRs of PDGFR-α/β) along with miR-34a or miR-34c, decreased the sensitivity to TRAIL -induced apoptosis, as assessed by both MTT and caspase 3/7 assay (Figure S4 in File S1). [score:2]
Calu-6 and H1703 cells overexpressing miR-34a and -34c showed a significant lower proliferation rate compared to the control cells (Figure S3a in File S1). [score:2]
As shown in Figure 3a and b, miR-34a and miR-34c expression was lower in the tumor compared to the normal samples (Figure 3a,b). [score:2]
MiR-34a (blue) and PDGFR-α/β (brown/red, respectively in RGB and each fluorescent red in Nuance converted image) expression was inversely related in lung cancers and the adjacent normal lung tissues. [score:2]
MiR-34a/c and PDGFR-α/β co -expression in vivo. [score:2]
Briefly, Calu-6 cells were transfected with pcDNA-PDGFR-α, pcDNA-PDGFR-β and/or hsa-miR-34a and miR-34c, respectively. [score:1]
Briefly, 2x 10 [5] Calu-6 cells transfected with pcDNA-PDGFR-α, pcDNA-PDGFR-β and/or miR-34a and miR-34c in RPMI supplemented with 1% FBS were plated into upper chambers of with a 8-um pore size-polycarbonate membrane. [score:1]
In mammalians, the miR-34 family comprises three processed miRNAs that are encoded by two different genes: miR-34a is encoded by its own transcript, whereas miR-34b and miR-34c share a common primary transcript. [score:1]
A previous study indicated that miR-34 methylation was present in NSCLC and was significantly related to an unfavorable clinical outcome [20]. [score:1]
Remarkably, an inverse correlation between miR-34a/c and PDGFR-α and PDGFR-β was observed (Figure 3d). [score:1]
In the figure the alignment of the seed regions of miR-34a/c with PDGFR-α and PDGFR-β 3’ UTRs is shown. [score:1]
Cells were transfected with either scrambled, miR-34a or miR-34c for 72h. [score:1]
Co-transfection of miR-34a/c with PDGFR-α/β significantly decreases the response to the drug. [score:1]
0067581.g001 Figure 1. (a) PDGFR-α and PDGFR-β 3’ UTRs contain, respectively, two and one predicted miR-34a, -34b and -34c binding sites. [score:1]
Moreover, caspase 3/7 assay revealed an increase in TRAIL sensitivity after miR-34a and -34c enforced expression or PDGFR-α/β silencing, compared to the cells transfected with a scrambled miR or siRNA (Figure 5c,d). [score:1]
MiR-34a and miR-34c reduce PDGFR-α and PDGFR-β mRNA and protein levels. [score:1]
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[+] score: 253
Other miRNAs from this paper: hsa-mir-199a-1, hsa-mir-199a-2, hsa-mir-34b, hsa-mir-34c
To upregulate and downregulate the expression of miR-34a, HPMECs were transfected with hsa-miR-34a mimic, specific inhibitors or miRNA -negative control (Ambion, USA) by using Lipofectamine 2000 (Invitrogen, Carlsbad, USA) following the manufacturer’s instructions. [score:11]
CSE: cigarette smoke extract; NC: negative control mimic; 3p inhibitor: miR-34a-3p inhibitor; 5p inhibitor: miR-34a-5p inhibitor; Cas: caspase. [score:9]
[#] P < 0.05 compared to CSE plus miR negative control To identify target genes of miR-34a, we searched for predicted target genes using the following bioinformatics algorithms: TargetScan (http://targetscan. [score:8]
miR-34a expression was analysed by RT-qPCR after treated with miR-34a-5p inhibitor and miR-34a-3p inhibitor (a). [score:7]
Inhibiting the expression of miR-34a remarkably attenuated the expression of Bax and cleaved caspase-3 which are indicator for cell apoptosis in the presence of CSE. [score:7]
Moreover, dysregulation of miR-34a expression inhibited angiogenesis as well as endothelial cell functions [18]. [score:6]
The expression of the apoptotic proteins caspase-3 and bax significantly decreased after treatment with an inhibitor specific for miR-34a compared to the CSE control group; however, this effect was not observed for p53 expression. [score:6]
CSE upregulated the expression of miR-34a in HPMECs. [score:6]
Current research has revealed that ectopic over -expression of miR-34a can induce cell cycle arrest, apoptosis, and senescence in malignant cells by directly targeting mRNA [16, 17]. [score:6]
HPMECs exposed to 1% CSE were treated with miR negative control (c), miR-34a-3p inhibitor (d), or miR-34a-5p inhibitor (e). [score:5]
Spearman’s correlation test revealed a negative correlation between miR-34a relative expression and Notch-1 expression (r = − 0.935 and r = − 0.950, respectively) (Fig. 4c). [score:5]
The miR-34a inhibitor was able to restore the expression of Notch-1 in CSE -treated HPMECs (Fig. 3c). [score:5]
Computational miRNA target prediction suggested that Notch-1 is a target of miR-34a. [score:5]
We assessed the expression level of miR-34a in cell lines and examined its effects on HPMEC apoptosis by detecting the rate of apoptosis and the expression of apoptotic proteins. [score:5]
3′-UTR 3′-untranslated region COPD Chronic obstructive pulmonary disease CSE Cigarette smoke extract HPMECs Human pulmonary microvascular endothelial cells miR-34a microRNA-34a Not applicable This study was supported by the National Natural Science Foundation of China (81370143, 81270100), the National Key Clinical Specialty Construction Projects of China and the Project of Hunan science and technology department (2015SK20403). [score:5]
Computational miRNA target prediction confirmed that Notch-1 is a target of miR-34a. [score:5]
Conversely, we found that expression of p53 significantly increased in HPMECs exposed to CSE and treated with miR-34a inhibitors. [score:5]
In the present study, an increase in miR-34a expression is associated with a decrease in Notch-1 expression in HPMECs treated with CSE. [score:5]
We found that the inhibitor caused a decrease in miR-34a mRNA expression (Fig.   3a). [score:5]
Furthermore, expression of Notch-1, a receptor protein in the Notch signalling pathway, was decreased and was inversely correlated with miR-34a expression in HPMECs treated with CSE. [score:5]
Fig. 4Effect of CSE on Notch-1 in HPMECs and correlation between miR-34a and Notch-1. Notch-1 expression in HPMECs treated with CSE (a, b) is negatively correlated with miR-34a expression (c). [score:5]
Our results showed that the expression of miR-34a was significantly increased in CSE -treated HPMECs, and inhibiting miR-34a attenuated CSE -induced HPMEC apoptosis. [score:5]
Our results suggest that miR-34a plays a key role in CSE -induced endothelial cell apoptosis by directly regulating its target gene Notch-1 in endothelial cells. [score:5]
The present study reveals that miR-34a may be a key regulator of cellular apoptosis and a potential therapeutic target in future. [score:4]
The expression of the cleaved apoptotic proteins caspase-3 and bax significantly decreased after exposure to miR-34a inhibitor compared to control (Fig. 3c). [score:4]
Fig. 5Luciferase report gene demonstrates that miR-34a directly targets the 3’ UTR of Notch-1. Cells were transfected with pLUC reporter plasmids containing either the wild-type (WT) or mutant type (MT) of the 3′UTR of Notch-1 in the presence of miR-34a mimic or negative control, and cultured for 48 h before being harvested for analysis. [score:4]
These results indicate that Notch-1 is a critical downstream target of miR-34a in regulating the CSE -induced HPMEC apoptosis. [score:4]
MiR-34a belongs to one of several evolutionarily conserved families of miRNAs (the miR-34 family), and was originally identified as a TP53 -targeting miRNA [34]. [score:3]
CSE -treated HPMECs exhibited a significant increase in apoptosis rates, whereas miR-34a inhibitor significantly decreased CSE -induced cell apoptosis (Fig. 3b). [score:3]
[☆] P < 0.05 when comparison to 2.5% of concentration To gain an insight into the role of miR-34a in CSE -treated HPMECs, we first examined the expression profiles of miR-34a-5p and miR-34a-3p in HPMECs treated with CSE using RT-qPCR. [score:3]
Previous studies showed that p53 activates miR-34a expression, and that miR-34a increased the activity of p53, suggesting the existence of a p53-miR34 positive feedback loop [49]. [score:3]
Among other Notch receptors, Notch-2 is approved to be another target of miR-34a using bioinformatics algorithms. [score:3]
HPMECs were incubated with 1% CSE the indicated times (0–24 h) and miR-34a expression was analysed by RT-qPCR. [score:3]
Shiro Mizuno et al. described miRNAs as modulators of smoking -induced gene expression changes in patients with COPD, and reported that microRNA-34a (miR-34a) and miR-199a-5p levels were significantly increased in COPD lung tissues and strongly associated with FEV1% predicted [14]. [score:3]
miR-34a Cigarette smoke extract Apoptosis Vascular endothelial cells Notch-1 Chronic obstructive pulmonary disease (COPD) is a common cause of disability and mortality worldwide. [score:3]
Fig. 3Effect of miR-34a inhibitor on CSE -treated HPMEC apoptosis rate, apoptotic proteins and downstream protein. [score:3]
As shown in Fig.   6a, pLV-EGFP-NICD rescued the increased apoptotic protein expression in HPMECs treated with miR-34a mimic, and led to a corresponding induction of p53 levels. [score:3]
The results showed that the expression of miR-34a-5p and miR-34a-3p significantly increased in HPMECs exposed to 1% CSE in an exposure time -dependent manner (Fig.   2). [score:3]
In the present study, we found that miR-34a expression is significantly increased and is involved in CSE -induced apoptosis of HPMECs. [score:3]
Transient transfection of miR-34a mimic or miR-34a inhibitor. [score:3]
The targeting of miR-34a to the 3′-UTR of Notch-1 mRNA was examined using luciferase constructs that were cloned into the pLUC-control vector. [score:3]
With wild-type Notch-1 3e–UTR, luciferase activity decreased following ectopic miR-34a expression (p < 0.05); however, this effect was not observed in the mutant constructs (Fig.   5). [score:3]
The cells were transfected with miR-34a inhibitor. [score:3]
Notch-1 is a target of miR-34a in HPMECs. [score:3]
Furthermore, we explored the target genes miR-34a and the underlying mechanism of its function. [score:3]
Luciferase reporter assay further confirmed the direct interaction between miR-34a and the 3’-untranslated region (UTR) of Notch-1. Restoration of Notch-1 pathway was able to partially block the effect of miR-34a on HPMEC apoptosis. [score:3]
The miR-34a/Notch-1 axis, which affects cell proliferation and apoptosis, has been wi dely studied for its role in malignant diseases [47, 48]. [score:3]
In TSA (trichostatin A) -treated emphysematous rat lungs and human pulmonary microvascular endothelial cells (HPMEC), miR-34a expression was significantly increased [15]. [score:3]
[☆] P < 0.05 compared to 12 h exposure Finally, we constructed a luciferase reporter assay to verify that Notch-1 is the direct target of miR-34a. [score:2]
[☆] P < 0.05 compared to 12 h exposureFinally, we constructed a luciferase reporter assay to verify that Notch-1 is the direct target of miR-34a. [score:2]
Collectively, these results suggest that Notch-1 is negatively regulated by miR-34a in HPMECs. [score:2]
Previous studies have reported an increased expression of miR-34a in the lungs of COPD patients compared with control groups and a strong correlation between miR-34a and FEV1% [14]. [score:2]
The present study was designed to investigate the expression of microRNA-34a (miR-34a) in human pulmonary microvascular endothelial cells (HPMECs) exposed to cigarette smoke extract (CSE), and the potential regulatory role of miR-34a in endothelial cell apoptosis. [score:2]
However, the mechanism by which miR-34a regulates apoptosis of pulmonary endothelial cells in COPD remains unclear. [score:2]
A direct interaction between Notch-1 and miR-34a has already been reported [37]. [score:2]
Numerous studies have demonstrated that miR-34a is critically involved in regulating cell apoptosis and cell cycle [35, 36]. [score:2]
These results imply that miR-34a is involved in regulating CSE -induced cell apoptosis in vitro. [score:2]
HPMECs treated with miR negative control (a), miR-34a mimic (b), blank vector (c), or NICD (d). [score:1]
Identifying functionally important mRNA targets of miR-34a is essential to unravelling its biological function and is helpful for further investigation. [score:1]
Fig. 2Effect of CSE on miR-34a in HPMECs. [score:1]
Moreover, transfection of the intracellular domain of Notch-1 plasmid nullified the effect of miR-34a on cellular apoptosis, indicating that Notch-1 is involved in miR-34a induced apoptosis in HPMECs. [score:1]
miR-34a mediates CSE induced apoptosis. [score:1]
In summary, our results suggest that miR-34a is involved in CSE -induced apoptosis of HPMECs. [score:1]
Restoration of Notch-1 can rescue the effects of miR-34a in CSE -treated HPMECs. [score:1]
We used RT-PCR, western blot analysis, and the luciferase reporter assay to determine whether Notch-1 was regulated by miR-34a. [score:1]
The seed sequences of miR-34a on Notch-1 were mutated using a PCR -based approach. [score:1]
In the present study, we observed the inverse correlation between miR-34a and Notch-1 in HPMECs treated with CSE. [score:1]
Statistically significant inverse correlation between miR-34a and Notch-1 in HPMECs treated with 1% CSE at the indicated times (0–24 h) (Spearman’s correlation analysis, miR-34a-5p: r = − 0.935; miR-34a-3p: p < 0.01, r = − 0.950; p < 0.01). [score:1]
HPMECs were transiently co -transfected with the 3′-UTR reported constructs (1.5 μg/well in 6 well plates) and either hsa-miR-34a mimic or the negative control (Ambion, USA) using Lipofectamine 2000 (Invitrogen, USA). [score:1]
These results suggest that restoration of Notch-1 can rescue the effect of miR-34a -induced apoptosis in HPMECs. [score:1]
Fig. 6Notch-1 attenuates miR-34a induced apoptosis of HPMECs. [score:1]
miR-34a mimic repressed the activity of the wild-type Notch-1 3′-UTR, but not that of the mutant constructs. [score:1]
These results suggest that miR-34a may contribute to apoptosis of endothelial cells. [score:1]
Our in vitro s