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284 publications mentioning mmu-mir-16-1 (showing top 100)

Open access articles that are associated with the species Mus musculus and mention the gene name mir-16-1. Click the [+] symbols to view sentences that include the gene name, or the word cloud on the right for a summary.

1
[+] score: 524
To explore whether HRG regulation of miR-16 was mediated via c-Myc and Stat3 expression, we silenced the expression of both proteins by the use of siRNAs and found that, comparable to our findings with MPA, silencing of Stat3 or of c-Myc both impair HRG -induced miR-16 downregulation in BT-474 cells (Figure 6G), suggesting that HRG and MPA share some of the signaling molecules in the mechanism of downregulation of miR-16. [score:12]
Among the downregulated miRNAs in leiomyomas, the authors identified miR-16, miR-197 and miR-224, three miRNAs we observed to be downregulated in progestin -induced breast cancer [82], highlighting the importance of miR-16 as a tumor suppressor in different cellular contexts. [score:9]
As shown in Figure 4F, MPA did not induce cyclin E upregulation in miR-16 -overexpressing C4HD cells, indicating that CCNE1 mRNA is indeed a direct target of miR-16. [score:9]
In advanced prostate cancer, for instance, miR-15a and miR-16 are significantly downregulated, whereas the expression of BCL2, CCND1 and WNT3A is concomitantly upregulated [37]. [score:9]
Moreover, overexpression of miR-16 abrogated the ability of progestin to induce cyclin E upregulation, revealing that cyclin E is a novel target of miR-16 in breast cancer. [score:8]
Among the differentially expressed miRNAs, we were particularly interested in miR-16, a previously reported tumor suppressor in leukemia [67, 68], which was downregulated by treatment with MPA. [score:8]
Importantly, the tumor suppressor miR-16 was among the downregulated miRNAs, and the cell cycle promoter protein cyclin E was identified as one of its targets (Figure 7). [score:8]
Our present findings showed that indeed the knockdown of Stat3 expression resulted in a complete abrogation of MPA -induced miR-16 downregulation (Figure 3C). [score:7]
Our present results indicate that the synthetic progestin MPA, a potent mitogen in C4HD and T47D cells, regulates a subset of miRNAs in mammary tumor cells including the tumor suppressor miR-16, which is downregulated. [score:7]
The comparison between control- and MPA -treated cells revealed that 16 miRNAs were significantly modulated by more than two-fold (P < 0.05, Figure 1A), nine miRNAs were upregulated (miR-191*, miR-17*, miR- 470*, miR-451, miR-702, miR-434-3p, miR-493, miR-23a* and miR-485*) and seven were downregulated (miR-378*, miR-376a, miR-224, miR-190b, miR-16, miR-410 and miR-197) (Figure 1B). [score:7]
In the current study, we found that transfection with a precursor of miR-16 (pre-miR-16) resulted in the inhibition of MPA -induced cyclin D1 expression in C4HD cells, indicating that cyclin D1 is also a downstream target of miR-16 in breast tumor cells (Figure 4A, upper panel). [score:7]
Overexpression of miR-16 also inhibited progestin -induced breast tumor growth in vitro and in vivo, demonstrating for the first time, a role for miR-16 as a tumor suppressor in mammary tumorigenesis. [score:7]
To predict novel targets for miR-16, we used miRecords, a publicly available miRNA target prediction tool that integrates the predicted targets of the most commonly used search engines. [score:7]
Our results demonstrate that HRG induces a similar mechanism to the one induced by progestins upstream of miR-16 downregulation, that is, activation of Stat3 and upregulation of c-Myc. [score:7]
To explore the direct involvement of c-Myc in the molecular mechanism of MPA -induced miR-16 downregulation, we silenced c-Myc expression using siRNAs. [score:7]
Treatment of C4HD cells with the anti-progestin RU486 or silencing of PR expression using siRNAs overcame the MPA -induced miR-16 downregulation, indicating that this effect is mediated through the classical PR (Figure 2B). [score:6]
A search for miR-16 targets showed that the CCNE1 gene, encoding the cell cycle regulator cyclin E, contains conserved putative miR-16 target sites in its mRNA 3' UTR region. [score:6]
As demonstrated for C4HD and T47D cells, the miR-16 decrease was preceded by the upregulation of c-Myc oncogene (Figure 6C, left panel) and was coincident with an increase in cyclin E expression levels (Figure 6C, right panel). [score:6]
As shown in Figure 6D, we found that HRG treatment induced a significant downregulation of miR-16 expression in BT-474 cells. [score:6]
Our findings indicate that progestins, acting through the classical PR and via Stat3 and c-Myc, downregulate miR-16, which is a potent tumor suppressor in breast cancer. [score:6]
These inhibitory effects on proliferation may be due, at least in part, to the capacity of miR-16 to inhibit cyclin E. In fact, we observed via immunohistochemistry that pre-miR-16-C4HD tumors expressed significantly lower levels of cyclin E as compared to pre-miR-CTRL-C4HD tumors. [score:6]
Figure 3B (left panel) shows that knockdown of c-Myc resulted in the inhibition of MPA -induced effects on miR-16 expression. [score:6]
The latter event results in the upregulation of the oncogenic transcription factor c-Myc (Step 4), which represses miR-16 expression by binding to E-box response elements and inducing chromatin remo deling (decrease of AcH4 and increase of H3K9me [3], Step 5) [36, 73, 74]. [score:6]
Pro-tumor effects of miR-16 downregulation in breast cancer are mediated by cyclin E. miR-16 acts as a tumor suppressor in both in vivo and in vitro progestin -induced breast cancer growth. [score:6]
miR-16, whose function as a tumor suppressor in leukemia has already been shown, was identified as one of the downregulated miRNAs in murine and human breast cancer cells. [score:6]
In this study, we reveal the first progestin-regulated miRNA expression profile and identify a novel role for miR-16 as a tumor suppressor in progestin- and growth factor -induced growth in breast cancer. [score:6]
We also found that the ErbB ligand heregulin (HRG) downregulated the expression of miR-16, which then participates in the proliferative activity of HRG in breast tumor cells. [score:6]
This decrease in the levels of intracellular miR-16 would result in increased expression of its targets, including cyclin D1 and E, and would lead to cell growth (Step 6). [score:5]
B, C4HD cells were treated with MPA or pretreated with 10 nM RU486 before MPA stimulation, and mRNA expression levels of candidate miR-16 target genes were determined by RT-qPCR. [score:5]
In accordance with the results presented here, overexpression of miR-16 was shown to suppress the self-renewal and growth of mouse mammary tumor stem cells and to sensitize MCF-7 human breast cancer cells to the chemotherapeutic drug doxorubicin [85]. [score:5]
Decreased levels of miR-16 would result in an increased expression of its targets, including cyclin D1 and cyclin E, and would lead to cell growth (Step 6). [score:5]
We found that, similar to the molecular mechanism underlying progestin-modulated miR-16 expression, Stat3 and c-Myc participated in the induction of cyclin E expression by progestin. [score:5]
The above results support a role for MPA as a repressor of miR-16 expression via c-Myc, inducing the recruitment of proteins with activity of chromatin remo delers which modulate gene expression. [score:5]
Furthermore, overexpression of miR-16 resulted in significant inhibition of HRG -induced stimulation of BT-474 cell growth (Figure 6E, left panel). [score:5]
The PR/Stat3 transcriptional complex and possibly also Stat3 bound to GAS sites, induce the expression of c-Myc, which would in turn associate with the DLEU2 promoter and repress the expression of miR-16 (Step 5). [score:5]
Target genes of miR-16 were searched through a bioinformatical approach, and the study was focused on cyclin E. Reporter gene assays were performed to confirm that cyclin E 3'UTR is a direct target of miR-16. [score:5]
As shown in Figure 2D, MPA induces a strong proliferative response in both cell lines, which correlates with its ability to induce the downregulation of miR-16 levels (Figure 2A). [score:4]
Because Stat3 was found to be directly involved [55] in these mechanisms, we reasoned that Stat3 may constitute an interesting gene whose participation in progestin -mediated miR-16 expression was worth studying. [score:4]
This paucity indicates that miR-16 downregulation is most likely driven not by PR loading at the DLEU2 promoter, but rather by nonclassical PR tethering mechanisms. [score:4]
No modulation of miR-16 levels was observed following the sole addition of RU486 or after knockdown of PR expression in unstimulated cells (Figure 2B). [score:4]
Reconstitution of PR-B levels in T47D-Y cells [55] restored MPA capacity to downregulate miR-16 (Figure 2C). [score:4]
Downregulation of miR-16 was also associated with resistance to the chemotherapeutic drug docetaxel in human breast cancer cells [86]. [score:4]
In the past several years, miR-16 has been shown to be frequently downregulated in chronic lymphocytic leukemia [67]. [score:4]
Progestins downregulate miR-16 via the classical PR and a hierarchical interplay between Stat3 and c-Myc. [score:4]
Figure 7 illustrates our proposed mo del of progestin -mediated regulation of miR-16 expression leading to breast cancer growth, based on our previous and present findings. [score:4]
In order to explore whether rapid signaling through PR and/or genomic effects participate in the MPA-downregulation of miR-16, we transfected T47D-Y cells with a PR mutant, PR-BmPro, in which three prolines (P422A, P423A, P427A) were converted to alanines (T47D-Y-PR-BmPro cells). [score:4]
Progestins downregulate miR-16 in breast cancer cells via the classical PR and a hierarchical interplay between Stat3 and c-Myc. [score:4]
These findings demonstrate the participation of both rapid (nongenomic) and transcriptional PR effects in progestin -induced miR-16 downregulation. [score:4]
Figure 3 Progestin induces miR-16 downregulation via c-Myc and Stat3. [score:4]
Figure 2 Progestins induce miR-16 downregulation via the classical PR. [score:4]
In vitro proliferation was abolished by transfection with a miR-16 precursor and, more importantly, in vivo expression of miR-16 resulted in the development of smaller tumors, with a growth rate significantly lower than those of the tumors from the control group. [score:4]
To explore the upstream effectors involved in the MPA -mediated downregulation of miR-16, we first conducted an in silico analysis. [score:4]
In this sense, it is worth mentioning a study which came out during the preparation of this manuscript showing that estradiol induces proliferation and upregulation of survival genes in breast cancer cells, through the repression of several miRNA, among them miR-16 [96]. [score:4]
Remarkably, we demonstrated that miR-16 is significantly downregulated by MPA treatment in an in vivo setting. [score:4]
Our findings show that progestins downregulate miR-16 via the classical PR and a hierarchical interplay between Stat3 and c-Myc. [score:4]
We propose that the requirement for both the rapid and genomic functions of PR during the regulation of miR-16 expression by progestins, as demonstrated in our study, may be explained by the fact that after being rapidly activated by PR, Stat3 is recruited, along with PR, to the PRE at the c-Myc promoter, where it acts as a PR co-activator (Step 3). [score:4]
The role of c-Myc as a transcriptional repressor of miR-16 has previously been shown [36, 73] and we here also demonstrate its involvement in mR-16 downregulation upon progestin treatment. [score:4]
Therefore, we hypothesized that c-Myc, long known to be an immediate early gene for several proliferative signal cascades and whose induction by PR is well acknowledged [13, 69, 70], may also be an upstream effector in MPA -induced miR-16 downregulation. [score:4]
We identified Stat3 as a key player in the downregulation of miR-16 by progestin. [score:4]
Figure 7 Mo del of MPA -induced miR-16 downregulation and cell-cycle control. [score:4]
We showed, for the first time, that miR-16 is involved in progestin -induced tumor growth in vitro and in vivo, having the cell-cycle promoter cyclin E as a target. [score:3]
To generalize our discovery of the role of miR-16 as a tumor suppressor, we decided to explore its involvement in the proliferation of breast cancer induced by growth factors, which along with estradiol and progestin are the major mitogens in breast cancer. [score:3]
In A to C, miR-16 expression levels were determined by RT-qPCR. [score:3]
These findings constitute the first piece of evidence to suggest a role for miR-16 as a tumor suppressor in progestin -induced breast cancer growth. [score:3]
Interestingly, we also demonstrated the involvement of miR-16 in HRG -induced breast cancer cell proliferation, confirming the ability of miR-16 to act as a tumor suppressor during breast cancer cell proliferation. [score:3]
A variety of targets for miR-16 has already been reported, including the cell-cycle promoter cyclin D1 and the anti-apoptotic protein Bcl-2 [37]. [score:3]
The results presented here indicate for the first time a role for miR-16 as a tumor suppressor in breast cancer. [score:3]
miR-16 targets were searched using the search engine miRecords [58, 59]. [score:3]
miR-16 expression was studied by RT-qPCR in cancer cell lines with silenced PR, signal transducer and activator of transcription 3 (Stat3) or c-Myc, treated or not with progestins. [score:3]
The official name and the function of the predicted miR-16 target genes that were assessed by RT-qPCR are shown. [score:3]
As shown in Figures 5A and 5B, transfection with pre-miR-16 significantly inhibited MPA -induced proliferation in C4HD and T47D cells. [score:3]
Validated targets of miR-16 include many genes related to the control of cell-cycle progression, such as cyclin D1 [37] and cyclin E [38], among others [39- 41]. [score:3]
Candidate miR-16 target genes assessed by RT-qPCR. [score:3]
Figure 6 miR-16 is a tumor suppressor in HRG -induced breast cancer growth. [score:3]
A, miR-16 inhibits in vitro progestin -induced breast cancer growth. [score:3]
We consider that the wide range of mRNAs and, therefore, proteins, presumably targeted by miR-16 explains the large effects that a relatively modest decrease in its levels has on cell fate. [score:3]
Inset, levels of pre-miR-16 in pre-miR-CTRL-C4HD and pre-miR16-C4HD tumors were studied by RT-qPCR at day 14; data analysis was performed as described in Figure 2. D, Cyclin E is an in vivo target of miR-16. [score:3]
To validate cyclin E as a target of miR-16 action in breast cancer, we transiently transfected primary cultures of C4HD cells with pre-miR-16. [score:3]
miR-16 levels were studied by RT-qPCR, and data analysis was performed as described in Figure 2. The WB in the right side of the figure shows the effects of siRNAs on c-Myc expression in C4HD cells. [score:3]
In addition, MPA was unable to modulate miR-16 in the T47D-Y cell line, a variant of the parental T47D cell line that lacks PR expression (Figure 2C). [score:3]
The role of miR-16 as a tumor suppressor in HRG -induced breast cancer growth. [score:3]
Figure 4 Cyclin E is a target of miR-16 in breast cancer cells. [score:3]
In order to further elucidate the mechanism of c-Myc induced miR-16 downregulation by MPA, we conducted chromatin immunoprecipitation assays (ChIP) on the DLEU2 promoter. [score:3]
Thus, rapid PR signaling is conceivably required to activate Stat3, which would then modulate the transcriptional function of PR to repress miR-16 expression. [score:3]
CCNE1 mRNA contains two highly conserved target sites for miR-16, one at position 485-491 and the other at position 241-247 of its 3' UTR. [score:3]
A miR-16 -based treatment would have the potential to target multiple genes and pathways, thereby amplifying the antiproliferative response. [score:3]
Moreover, we found that miR-16 is a suppressor not only of progestin -induced breast cancer growth but also of heregulin (HRG) -induced breast cancer cell proliferation. [score:3]
Progestin -mediated modulation of miR-16 expression requires an intact PR-signaling function (Figure 2C, PR-B-mPro -transfected T47D-Y cells), the same as Stat3, and also appears to be modulated by PR -mediated transcriptional tethering mechanisms (Figure 2C, C587A-PR -transfected T47D-Y cells). [score:3]
In the first place, we confirmed that also in BT-474, MPA induced an increase in in vitro cell proliferation at 24 hours of treatment (Figure 6A) and that such increase correlated with a decrease in the expression levels of miR-16 (Figure 6B). [score:3]
Forced expression of miR-16 in tumor cells proved to be an efficient means to slow down tumor growth. [score:3]
The authors showed that the miR-16 target protein responsible for the proliferative effect in ovarian cancer was the oncogenic protein Bmi-1 [95]. [score:3]
In addition, c-Myc recruitment to the DLEU2 triggers a chromatin remo deling program which results in a decrease of AcH4 and an increase in H3K9me, which ultimately translate into repression of the DLEU2 locus and miR-16 decrease. [score:3]
C, miR-16 inhibits in vivo progestin -induced breast cancer growth. [score:3]
Thus, we reasoned that cyclin E might be a true target of miR-16 in breast cancer cells. [score:3]
In the absence of progestin stimulation, steady state levels of miR-16 repress the translation of key mRNAs required for cell-cycle progression, such as cyclin D1 and cyclin E mRNAs (left panel). [score:3]
We performed RT-qPCR to amplify those mRNAs (Figure 4B) and, interestingly, we observed that CCNE1 mRNA, encoding the cell cycle regulator cyclin E, and RAP2C mRNA, encoding a member of the RAS oncogene family [75], showed a profile in response to MPA that mimicked MPA -induced proliferation; these mRNAs were also regulated inversely from miR-16. [score:3]
Click here for file Candidate miR-16 target genes assessed by RT-qPCR. [score:3]
miR-16 expression levels were determined by RT-qPCR, and data analysis was performed as described in Figure 2A. [score:3]
Other authors have already shown that cyclin E is a target of miR-16 [38] in different mo dels. [score:3]
Noticeably, although not responsive to the endogenous changes of miR-16 levels, a higher basal luciferase activity was observed for the luc-3' mTS construct as compared to luc-3' 1 xTS or CCNE1-3'UTR, adding further evidence for a negative role of miR-16 response sites on cyclin E expression. [score:2]
These results suggest that miR-16 is regulated as part of the ligand -induced PR effects observed in breast cancer, but would not be involved in PR modulation of breast cancer growth in the absence of the ligand. [score:2]
Treatment with MPA of luc-3' CCNE1 -transfected C4HD cells resulted in a significant increase of luciferase activity, in line with our hypothesis that miR-16 is a negative regulator of cyclin E (Figure 4G, right panel). [score:2]
Consistent with the role of PR, Stat3 and c-Myc as upstream regulators of miR-16, the MPA -induced cyclin E increase was blocked by the silencing of PR (Figure 4C), Stat3 (Figure 4D) or c-Myc using siRNAs (Figure 4E). [score:2]
In addition to identifying a new mechanism of action for progestin in breast cancer, our results suggest that miR-16 may be considered a candidate for targeted breast cancer treatment. [score:2]
In contrast, pre-miR-16-C4HD tumors stained weakly for cyclin E (Figure 5D, right column and inset, H-index: 61 ± 32), showing miR-16 efficiency in vivo negatively regulated cyclin E. Table 1Tumor growth rates [a]. [score:2]
In addition, we studied miR-16 regulation of cyclin E levels in a system in which miR-16 was not being transfected but modulated endogenously by the presence of MPA. [score:2]
Our results suggest that miR-16 is a common regulator of cell fate in the mechanisms of steroid hormone or growth factor modulation of breast cancer cell proliferation. [score:2]
Our present findings demonstrated that at least from six hours (Figure 2C) to 24 hours (data not shown) later, miR-16 levels were not regulated in response to MPA in T47D-Y-PR-BmPro cells. [score:2]
The full list of primers used to amplify miR-16 candidate target genes is shown in Additional file 2. qPCR was performed with 15 seconds of denaturing at 95°C followed by 40 amplification cycles of annealing and extension at 60°C for one minute. [score:2]
In contrast, pre-miR-16-C4HD tumors stained weakly for cyclin E (Figure 5D, right column and inset, H-index: 61 ± 32), showing miR-16 efficiency in vivo negatively regulated cyclin E. Table 1Tumor growth rates [a]. [score:2]
We chose to explore the regulation of CCNE1 mRNA by miR-16 due to its acknowledged role in breast cancer [76]. [score:2]
Neither pre-miR-CTRL nor pre-miR-16 modified luciferase activity in the luc-3'EMPTY cells. [score:1]
miR-16 levels were studied by RT-qPCR, and data analysis was performed as described in Figure 2. The experiment shown was performed with Stat3 siRNA #1, but the same results were obtained with PR siRNA #3. D, C4HD and T47D cells were transfected with Stat3 siRNAs or CTRL siRNAs before MPA stimulation and WBs were performed with anti-c-Myc antibodies. [score:1]
Cells were co -transfected with pre-miR-16 or pre-miR-CTRL (middle panel) or treated with 10 nM MPA for 24 hours (right panel). [score:1]
We next conducted a preclinical trial to test the role of miR-16 in the MPA -induced growth of C4HD tumors in vivo. [score:1]
Bottom panel, as a control of transfection efficiency, miR-16 levels are shown in pre-miR-16-C4HD and pre-miR-CTRL-C4HD cells. [score:1]
Inset, average H-score, used to quantify the levels of cyclin E in pre-miR-CTRL-C4HD and pre-miR-16-C4HD tumors. [score:1]
miR-16 levels in pre-miR-16-C4HD were augmented two-fold at day 14, in comparison to pre-miR-CTRL-C4HD tumors (Figure 5C, inset). [score:1]
A therapeutic strategy is underway that involves the usage of atelocollagen for the delivery of synthetic miR-16 into advanced prostate tumors [84]. [score:1]
As shown in Figure 2C, MPA had no effect on miR-16 levels in T47D-Y-C587A-PR cells. [score:1]
C4HD cells were transfected with pre-miR-CTRL or pre-miR-16 for 48 hours and then injected subcutaneously (s. c. ) into BALB/c mice at 2 × 10 [6 ]cells/mouse. [score:1]
In the right panel, as a control for transfection efficiency, miR-16 levels are shown in pre-miR-16- and pre-miR-CTRL -transfected BT-474 cells. [score:1]
Moreover, a recent study demonstrates that c-Myc induces the recruitment of the histone deacetylase 3 to the DLEU2 locus, causing the decrease of AcH4 and, hence, repression of miR-16 [74]. [score:1]
The mean volume (Figure 5C) and growth rates (Table 1) of the tumors developed from the pre-miR-16-C4HD cells were significantly lower than those of the tumors from the control group. [score:1]
C4HD cells (2 × 10 [6 ]cells per mouse) were transiently transfected with the precursor of miR-16 (pre-miR-16) or with a control precursor (pre-miR-CTRL) and were then injected s. c. into animals treated with a 40 mg MPA depot in the opposite flank to the cell inoculum. [score:1]
C4HD cells were transfected with a construct carrying the CCNE1 3' UTR cloned downstream of the firefly luciferase reporter gene (luc-3'CCNE1), middle panel, or with a construct that carried a minimal region of CCNE1 3'UTR which comprised only one of the miR-16 responding sites either wild type (luc-3' 1×TS) or mutated (luc-3' mTS), right panel. [score:1]
In vivo tumor growthC4HD cells (2 × 10 [6 ]cells per mouse) were transiently transfected with the precursor of miR-16 (pre-miR-16) or with a control precursor (pre-miR-CTRL) and were then injected s. c. into animals treated with a 40 mg MPA depot in the opposite flank to the cell inoculum. [score:1]
Interestingly, tumors growing in the presence of MPA displayed lower levels of miR-16, which correlated with higher levels of both c-Myc and cyclin E (Figure 5F). [score:1]
After 48 hours, cells were re -transfected with either pre-miR-16 or pre-miR-CTRL following the protocol described above. [score:1]
Interestingly, miR-16 was among the miRNAs repressed by c-Myc. [score:1]
Using experimental mo dels, these authors showed that the restoration of miR-16 in prostate cancer cells results in growth arrest, apoptosis and in a marked regression of prostate tumor xenografts [37]. [score:1]
Briefly, 6 nM pre-miR-16 or a pre-miR-control (pre-miR-CTRL) that does not form any known mammalian miRNA, were transfected using the transfection reagent siPORT NeoFx (Ambion). [score:1]
For this purpose, C4HD cells were transfected with pre-miR-CTRL (pre-miR-CTRL-C4HD) or pre-miR-16 (pre-miR-16-C4HD) cells and 2 × 10 [6 ]cells were injected subcutaneously (s. c. ) into mice treated with MPA. [score:1]
A role for miR-16 has also been shown in ovarian cancer. [score:1]
miR-16 and U6 snRNA qPCR. [score:1]
B, C4HD cells were transfected with pre-miR-CTRL or pre-miR-16. [score:1]
We did not find canonical or half PREs at the proximal promoter of DLEU, the miR-16 host gene. [score:1]
It has been demonstrated that miR-16 is located in a chromosomal region commonly deleted in leukemia and that its deletion correlates with an increase in anti-apoptotic and cell-cycle-promoting proteins [83]. [score:1]
Nevertheless, little is known about the role of miR-16 in solid malignancies. [score:1]
Progestin induced a decrease in miR-16 levels via the classical PR and through a hierarchical interplay between Stat3 and the oncogenic transcription factor c-Myc. [score:1]
RNA from C4HD cells treated for 0 to 24 hours was reverse transcribed and analyzed by RT-qPCR to detect the presence of miR-16. [score:1]
The volume, percentage of growth inhibition, and delay in tumor growth (days) in tumors from mice injected with pre-miR16-C4HD cells relative to mice injected with pre-miR-CTRL-C4HD cells were calculated at day 28, as described in. [score:1]
TreatmentMean tumor volume(mm [3])Growth rate(mm [3]/day)Growth inhibition(%)Delay in tumor growth(days) pre-miR-CTRL-C4HD 683.6 ± 192.2* 26.1* pre-miR-16-C4HD231.1 ± 107.9 [#]9.6 [#]66.2 [b]5 [b] [a]Growth rates were calculated as the slopes of growth curves. [score:1]
However, our study is the first to show the relevance of miR-16 modulation in breast cancer mo dels throughout a stimulus that is relevant to breast cancer pathophysiology. [score:1]
Moreover, we found an inverse relationship between the levels of miR-16 and the proliferative state of C4HD and T47D cells. [score:1]
After one week, tumors were excised and studied for miR-16, c-Myc and cyclin E levels. [score:1]
In line with a role for c-Myc as a repressor of miR-16, the addition of MPA caused a significant decrease of the levels of acetylation of histone H4 (AcH4), a chromatin modification already reported to be an activation mark for the DLEU2 locus [74] (Figure 3E, middle panel). [score:1]
Our findings support the notion that Stat3 integrates the rapid and transcriptional effects of PR, leading to a decrease in miR-16 levels. [score:1]
Other authors demonstrated that transfection of tamoxifen-sensitive MCF-7 cells with a clinically important oncogenic isoform of ErbB-2, HER2Δ16, caused a decrease in miR-16 levels and a concomitant increase in Bcl-2 that rendered cells resistant to the treatment with tamoxifen [41]. [score:1]
C4HD or T47D cells were transfected with pre-miR-CTRL or pre-miR-16. [score:1]
Here, we have also demonstrated the role of miR-16 in progestin -induced breast cancer cell proliferation. [score:1]
The experiment shown was performed with c-Myc siRNA #5, but the same results were obtained with c-Myc siRNA #6. F, C4HD cells were transfected with pre-miR-16 or pre-miR-CTRL before MPA stimulation and WB was performed as in C. G, A scheme depicting the different constructions used is shown in the left panel. [score:1]
A, Upper panel, C4HD cells were transfected with pre-miR-16 or pre-miR-control (CTRL) before MPA stimulation. [score:1]
This decrease in miR-16 levels, from at least 16 to 24 hours after treatment, correlated inversely with the proliferative effects of HRG at this time point (Figure 6E, left panel). [score:1]
Additional constructs carrying the wild-type CCNE1 3' UTR, or a minimal region from the CCNE1 3'-UTR which has a response site for miR-16 (luc-3' 1×TS), or the mutated response site for miR-16 (luc-3' mTS) were kindly provided by Dr. [score:1]
In addition to the constructs described above, we used a construct in which only a minimal region of the CCNE1 3' UTR encompassing a miR-16 responding site was included (luc-3' 1×TS) and another in which the same site was mutated (luc-3' mTS, Figure 4G, left panel) [62]. [score:1]
miR-16 belongs to the miR-15/miR-16 cluster that is located on the noncoding gene deleted in leukemia 2 (DLEU2) [36]. [score:1]
Recently, a few papers suggested a role for miR-16 in breast cancer, although none of them studied its modulation by steroid hormones. [score:1]
This result highlights the importance of miR-16 in progestin-promoted human breast cancer growth in vivo. [score:1]
In this sense, comprehensive characterization of the genes modulated by MPA through miRNAs would be necessary to completely elucidate the mechanism responsible for miR-16 -mediated tumor suppression. [score:1]
In this study, we also showed evidence that HRG modulates miR-16 in the context of HRG -induced breast cancer cell proliferation. [score:1]
miR-16 levels were studied by RT-qPCR, and data analysis was performed as described in Figure 2. The experiment shown was performed with Stat3 siRNA #3 and c-Myc siRNA #5, but the same results were obtained with Stat3 siRNA #1 and c-Myc siRNA #6. Experiments shown in A to G were repeated in triplicate with similar results. [score:1]
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[+] score: 326
These results agree with the in vitro data (Fig. 7A) and suggest that miR-16 inhibits the proliferation of hepatoma cells, among other mechanisms, through downregulation of COX-2. Since miR-16 regulates COX-2 expression by binding to the MRE in the 3′-UTR COX-2 and by inhibition of HuR in HCC cell lines, we evaluated the relationship between miR-16, HuR and COX-2 mRNA/protein expression in individual tumoral (T) and paired non-tumoral (NT) HCC human samples. [score:11]
To further support the hypothesis that miR-16 is involved in the down-regulation of COX-2 translation, we tested the expression of COX-2 in Hep3B cells after transfection with siCOX-2 or miR-16, in the presence of the transcription inhibitor actinomycin-D. We found a decrease of COX-2 protein in both cases (Fig. 4A–B). [score:10]
miRNA-16 is able to inhibit cell proliferation, to promote cell apoptosis and to suppress the ability of WRL68 hepatoma cell line to develop tumors in nude mice partially through targeting COX-2 expression. [score:9]
The present results demonstrate that miR-16 regulates COX-2 expression in HCC cells by binding directly to the MRE response element in the COX-2 3′UTR and this binding inhibits mainly COX-2 translation without affecting significantly mRNA decay. [score:9]
Recent works have reported that miR-101 downregulation is involved in COX-2 overexpression in human colon cancer cells (CRC) [24], miRNA-26b regulates the expression of COX-2 in desferrioxamine -treated carcinoma of nasopharyngeal epithelial cells [25] and binding of miR-16 to AREs of TNF-α, IL-6, IL-9 and COX-2 mRNA transcripts could promote their degradation [20], [26]. [score:9]
Our results show that miR-16 directly silences COX-2 expression in hepatoma cells and indirectly through the downregulation of HuR. [score:8]
Therefore, the reduced expression of miR-16 in those HCC with a high COX-2 expression may contribute to the promotion of cell proliferation and the inhibition of apoptosis and consequently facilitate the development of these types of tumors. [score:8]
Indeed, Dixon et al. [37] reported a direct interaction between HuR and miR-16 promoting the downregulation of miR-16 and targeting COX-2 in colon cancer cells. [score:7]
As shown in Fig. 5E–F, miR-16 inhibited the COX-2 and HuR protein levels in both cellular types; however, in the presence of HuR, the ability of miR-16 to downregulate COX-2 protein levels was partially abolished. [score:6]
HuR antagonizes the downregulation of COX-2 expression caused by miR-16 in hepatoma cell lines. [score:6]
The results suggest that miR-16 could specifically bind to the 3′UTR region of COX-2 and represses COX-2 translation reinforcing the hypothesis that COX-2 mRNA is a direct target for miR-16. [score:6]
WRL68 and Hep3B cells were transfected with: 30 nM siRNA anti-COX2 (siCOX-2) or 50 nM of miR-16, miR-16 inhibitor (In-miR-16), miR negative control (miR-NC) or miR negative control inhibitor (In-miR-NC). [score:5]
Corresponding densitometric analysis is shown and the relative expression of each sample is related to the value at 0 h as 1. F) Hep3B cells were transfected with 50 nM miR-16, miR-16 inhibitor (In-miR-16) or lipofectamine and permeabilized with digitonine to obtain soluble and pellet fractions enriched in PB as described in Methods. [score:5]
Our data are in agreement with the proposed interaction between miR-16 and HuR mRNA in HCC cells and suggest two different mechanisms for miR-16 to inhibit COX-2: by binding directly to the MRE response element in the COX-2 3′-UTR and by decreasing the levels of HuR through a direct interaction. [score:5]
The miRNAs (miR-16, miR-26b, miR-101, miR-199a, miR-122 and miR-21) were selected by using miRWalk computational analyses, that covers miRNA-targets interactions information produced by 8 established miRNA prediction programs on 3' UTRs of all known genes of Human, Mouse and Rat, i. e., RNA22, miRanda, miRDB, TargetScan, RNAhybrid, PITA, PICTAR, and Diana-microT, and comparing the obtained results with data collected from the literature. [score:5]
Furthermore, when Hep3B cells were treated with digitonin and fractionated after transfection with miR-16 in order to localize COX-2 mRNA in soluble or P-bodies (PB) fractions [34], the amount of COX-2 mRNA present in PB was more than 90%, suggesting inhibition of translation. [score:5]
Our results clearly show that COX-2 mRNA was located in P-bodies (>90%) after transfection with miR-16, inhibiting its translation. [score:5]
Young et al. [36] demonstrated that miR-16 binds the COX-2 3′UTR and inhibits COX-2 expression by promoting mRNA decay in colon cancer. [score:5]
Moreover, miR-16 is also present in the HuR immunoprecipitated and the analysis of miR-16 predicted target genes determined by using the algorithms miRWalk showed that among miR-16 target genes one is HuR. [score:5]
0050935.g002 Figure 2WRL68 and Hep3B cells were transfected with: 30 nM siRNA anti-COX2 (siCOX-2) or 50 nM of miR-16, miR-16 inhibitor (In-miR-16), miR negative control (miR-NC) or miR negative control inhibitor (In-miR-NC). [score:5]
miR-16 binds COX-2 mRNA and inhibits its translation. [score:5]
The miR-16 precursor (PM10339), which was a double-stranded RNA mimicking the endogenous mature miRNA, the miR-16 inhibitor (In-miR-16, AM10339) which was a single stranded nucleic acid designed to specifically bind to and inhibit endogenous microRNA molecule, their negative controls (miR-NC, AM17110; In-miR-NC, AM17010) and anti-COX-2 siRNA (siCOX-2) (positive control, forward 5′- GGGCUGUCCCUUUACUUCAtt -3′and reverse 5′- UGAAGUAAAGGGACAGCCCtt-3′) were purchased from Ambion (Austin, TX, USA). [score:5]
To determine whether miR-16 -mediated COX-2 protein loss was due in part to a decrease in HuR expression, Hep3B and WRL68 cell lines were cotransfected with miR-16 and HuR expression vectors. [score:5]
miR-16 Binds COX-2 mRNA and Inhibits its Translation. [score:5]
It has been described by Huang et al. that miR-16 decreased the association of its target mRNA with polysomes in 293T and HeLa cells by mediating the association of mRNA with processing bodies (P-bodies), since localization of mRNAs to these structures is a consequence of translational repression [34]. [score:5]
miR-16 regulates COX-2 expression in HCC cell lines. [score:4]
To further analyze the effect of miR-16 on hepatoma cell growth in vivo, the WRL68 cells were transiently transfected with miR-16, miR-NC or miR-16 together with a human COX-2 expression vector that lacks the 3′ UTR and, therefore, it cannot be regulated by miR-16. [score:4]
Figure S1 miR-16 downregulates COX-2 by binding its 3′UTR. [score:4]
From a functional point of view, COX-2 down-regulation by miR-16 increased apoptosis and decreased cell proliferation in human hepatoma cell lines. [score:4]
HuR Antagonizes miR-16 Activity in Regulating COX-2 Expression in Hepatoma Cell Lines. [score:4]
Among the six miRNAs analyzed, the expression of miR-16 showed the highest inverse correlation with the COX-2 protein/mRNA ratio (R [2] = 0.858, p = 0.016) (Fig. 1B), suggesting that miR-16 is involved in COX-2 regulation in hepatoma cell lines. [score:4]
Our results show that miR-16 silences COX-2 expression in hepatoma cells by two mechanisms: by binding directly to the MRE motif in the COX-2 3′-UTR and by decreasing the levels of HuR. [score:4]
Downregulation of COX-2 by miR-16 increases apoptosis in HCC cells. [score:4]
miR-16 Regulates COX-2 Expression in HCC Cell Lines. [score:4]
In almost all HCC lines analyzed, miR-16 expression was lower than in control hepatocytes (HH), whereas COX-2 protein levels were higher (Fig. 1A). [score:3]
These results suggest that miR-16 may exert its pro-apoptotic function partially through decreasing COX-2 expression. [score:3]
We performed a similar experiment in the presence of the protein synthesis inhibitor, cycloheximide (CHX) and the results obtained reveal that both miR-16 and CHX induced a rapid decay of COX-2 protein with a synergistic effect (Fig. 4D–E). [score:3]
WRL68 cells were transfected in vitro with 50 nM miR-NC or miR-16 and pPyCAGIP-hCOX-2 ORF (hCOX-2 expression vector lacking COX-2 3′ UTR) by using lipofectamine 2000. [score:3]
mir-16 Suppresses the Growth of Hepatoma Cells in vitro and in vivo. [score:3]
The results demonstrate that miR-16 interacts with COX-2 mRNA and promotes COX-2 protein decrease mostly through a translational repression mechanism. [score:3]
miR-16 suppresses growth of hepatoma cells in vitro and tumorigenicity in vivo. [score:3]
Inverse Correlation between miR-16 and COX-2 Expression is Observed in HCC Human Biopsies. [score:3]
As a further control, the effect of both miR-16 and miR-NC inhibitors were analyzed. [score:3]
Overexpression of miR-16 promoted apoptosis in Hep3B hepatoma cells. [score:3]
Our data show that the ectopic expression of miR-16 repressed cell proliferation of hepatoma cells in vitro and tumor growth in vivo, and these effects were partially reverted by treatment with PGE [2]. [score:3]
As shown in Fig. 5C–D, overexpression of miR-16 in WRL68 and Hep3B cell lines led to a substantial decrease in HuR protein levels. [score:3]
To establish whether the effect of miR-16 on COX-2 expression was mediated through a direct miRNA:mRNA interaction, we performed a RNA immunoprecipitation (RNA-IP) assay in WRL68 cells transfected with miR-16. [score:3]
COX-2 mRNA amounts (white bars), normalized to the expression of 36b4 mRNA, and miR-16 expression (grey bars), normalized against U6 RNA levels, were calculated. [score:3]
Our data suggest an important role for miR-16 in HCC and implicate the potential therapeutic application of miR-16 in those HCC with a high COX-2 expression. [score:3]
The expression profile of six miRNAs (miR-16, miR-26b, miR-101, miR-199a, miR-122 and miR-21) was analyzed in HCC cell lines (Table 1). [score:3]
Furthermore, COX-2 inhibition mediated by miR-16 promoted apoptosis in HCC cells by increasing apoptotic proteins such as caspase-3. Various cytoplasmic proteins have been observed to bind to the COX-2 ARE. [score:3]
Moreover, a reduced miR-16 expression correlates with high levels of COX-2 in liver from HCC patients. [score:3]
Moreover a reduced miR-16 expression tends to correlate to high levels of COX-2 protein in liver from patients affected by HCC. [score:3]
As shown in Fig. 3A, COX-2 mRNA was present in the Argo2 immunoprecitation samples where miR-16 was expressed whereas capture of the negative control actin mRNA was unchanged. [score:3]
We overexpressed miR-16 in HCC cell lines and examined whether it decreases endogenous COX-2 levels. [score:3]
In the present report, miR-16 target site prediction for COX-2 was performed using RNAhybrid [35] and we found one predicted MRE for miR-16 at positions 1195–1217 taking as position 1 the beginning of the 3′ UTR region. [score:3]
miR-16 interacts with HuR mRNA in the 3′UTR and represses HuR translation in human breast cancer cells [39]. [score:3]
To further establish a functional relationship between miR-16 and COX-2, we tested whether COX-2 expression was required to miR-16 -dependent induction of apoptosis. [score:3]
COX-2 mRNA and miR-16 expression were analyzed by real-time PCR. [score:3]
COX-2 mRNA and miR-16 expression were normalized against 36b4 mRNA and U6 RNA levels, respectively. [score:3]
RNA immunoprecipitation (RNA-IP) was performed to determine whether HuR would associate with COX-2 and whether there is a direct interaction between HuR and miR-16 in WRL68 cell line. [score:2]
The expression of intratumoral miR-16, measured by real-time PCR, increased in tumors injected with cells transfected with miR-16 compared with miR-NC without being modified by COX-2 overexpression (Fig. 7D). [score:2]
mir-16 Suppresses the Growth of Hepatoma Cells in vitro and in vivo We sought to determine whether miR-16 affects the growth of hepatoma cell lines assessed by the MTT reduction assay. [score:2]
To ensure that miR-16 can bind to this predicted region and cause translational repression, we performed a luciferase reporter gene assay in HuH-7 and HepG2 cells with low levels of miR-16. [score:2]
Figure S2COX-2 correlates inversely with miR-16 and directly with HuR in HCC human biopsies. [score:2]
NT samples (E) COX-2 protein levels were compared to miR-16 expression in T samples. [score:2]
miR-16 Down Regulation of COX-2 Sensitizes HCC Cells to Apoptosis. [score:2]
However, the functional consequences of miR-16 associated with HCC progression have not been established. [score:1]
We cloned the 3′UTR region of COX-2 containing the miR-16 putative binding site (seed region) and a mutant variant downstream the Luc gene in pGL3-vector (pGL3-seed and pGL3-mut, respectively) (Fig. 3B). [score:1]
A fragment of 3′UTR COX-2 mRNA (region 1195–1217, from NM_000963) which include the MRE binding site for miR-16, and a mutant variant were cloned into pGL3-Promoter vector (pGL3-empty, Promega, USA) downstream firefly luciferase gene (SacI, HindIII sites) to obtain the luciferase reporter constructs (pGL3-seed and pGL3-mut, respectively). [score:1]
A similar distribution of COX-2 mRNA was observed upon transfection of Hep3B cells with In-miR-16 (Fig. 4F–G). [score:1]
Moreover, the transfection of In-miR-16 induced an increase of COX-2 protein mainly in Hep3B cells. [score:1]
The binding of miR-16 to COX-2 mRNA, HuR to COX-2 mRNA and the binding of HuR to miR-16 were analyzed by immunoprecipitation and PCR analysis. [score:1]
the miR-16+ COX-2 condition. [score:1]
In pGL3-mut this region was mutated in order to avoid the binding between miR-16 and Luc mRNA. [score:1]
Using several programs (RNAhybrid, PITA, and RNA22), miR-16 was predicted to associate with the 3′UTR region of COX-2 to different MRE motifs (Table S1). [score:1]
miR-16 caused a decrease in COX-2 protein levels within 48 h of transfection in both cell lines (Fig. 2A–B). [score:1]
org), miR-16 was predicted to associate with the 3′UTR region of COX-2 to different MRE motifs. [score:1]
Next, we investigated the effect of COX-2 -mediated inhibition by miR-16 in hepatocarcinogenesis. [score:1]
miR-16 and COX-2 Correlate Inversely in Hepatoma Cell Lines. [score:1]
These results provide further evidence that COX-2 mRNA is post-transcriptionally controlled by miR-16. [score:1]
COX-2 -dependent production of PGE [2] increased the volume and the weight of tumors comparing with miR-16 (Fig. 7B–C). [score:1]
Moreover, we did not observe variations of the luciferase activity in cells cotransfected with pGL3-mut and miR-16, in comparison to cells transfected only with pGL3-mut (Fig. 3C–D). [score:1]
Transfection of the cells with miR-16 decreased cell growth up to 40%, being restored to 70% in the presence of PGE [2]. [score:1]
In pGL3-seed, the putative binding site of miR-16 on COX-2 mRNA 3′-UTR region (as detected by RNAhybrid software) was introduced downstream luciferase gene. [score:1]
miR-16 and COX-2 correlate inversely in HCC cell lines. [score:1]
Table S1 Several binding sites for miR-16 wihtin COX-2 3′UTR, predicted by diferent algorithms. [score:1]
To study the relationship between miR-16 and HuR in HCC cell lines, we determined whether HuR levels were altered by miR-16 transfection. [score:1]
However, a recent work [39] has demonstrated that miR-16 inversely correlates with HuR protein levels in human breast carcinoma. [score:1]
Hep3B cell lines were transfected with miR-16 or In-miR16 48 hours prior to harvesting at a final concentration of 50 nM. [score:1]
Effect of miR-16 on COX-2 mRNA and protein stability. [score:1]
Using several programs (RNAhybrid, PITA, and RNA22), miR-16 was predicted to associate with the 3′UTR region of COX-2 to different MRE motifs (Table S1) and we found one predicted MRE for miR-16 at positions 1195–1217 taking as position 1 the beginning of the 3′ UTR region. [score:1]
However, the effect of miR-16 on apoptosis was partially attenuated by treatment of cells with PGE [2] (Fig. 6A). [score:1]
As indicate in Fig. 7A, the growth of Hep3B cells transfected with miR-16 was significantly decreased relative to control cells. [score:1]
Western blot analysis of active caspase-3 showed an increase in the pro-apoptotic protein by the effect of miR-16 and this effect was also reverted in the presence of PGE [2] (Fig. 6B). [score:1]
The 3′UTR sequence of human COX-2 was retrieved using Ensembl Data base, and miR-16 sequence for Homo Sapiens was downloaded from mirBase website. [score:1]
miR-16 (C) Tumor weight and a representative picture of the tumors. [score:1]
the miR-16 transfection condition. [score:1]
We found that miR-16 led to a significant reduction in the volume and weight of the tumor comparing with the mice injected with miR-NC. [score:1]
miR-16 condition. [score:1]
Hep3B cells were transfected with 50 nM miR-16 or miR-NC, or 30 nM siCOX-2. 5 µg/ml actinomycin-D (Act D) or 10 µg/ml cycloheximide (CHX) were added after transfection. [score:1]
The transfection of In-miR-16 increased the luciferase activity while the transfection of miR-NC had no effects. [score:1]
The presence of COX-2 mRNA in WRL68 cell transfected with miR-16 or Lipofectamine after Ago2 immunoprecipitation was assessed, and fold differences were plotted. [score:1]
Moreover, when RNA-IP was performed, miR-16 was also present in the HuR immunoprecipitates (Fig. 5B). [score:1]
0050935.g004 Figure 4Hep3B cells were transfected with 50 nM miR-16 or miR-NC, or 30 nM siCOX-2. 5 µg/ml actinomycin-D (Act D) or 10 µg/ml cycloheximide (CHX) were added after transfection. [score:1]
Cells (3×10 [4] cells/well) were seeded in 24-wells plate and transfected for 6–12 h with pGL3-empty (750 ng), pGL3-seed (750 ng), pGL3-mut (750 ng), pGL3-UTR (750 ng), pGL3-UTR mut (750 ng), pRL-SV40 vector (50 ng, Promega, USA), miR-16 (50 nM), In-miR-16 (50 nM) or miR-NC (50 nM) or a different combinations of them using lipofectamine 2000 reagent protocol. [score:1]
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[+] score: 321
Other miRNAs from this paper: mmu-mir-16-2
SW480 cells were untreated (mock), infected with a lentivirus to overexpress miR-16, transfected with a plasmid to overexpress KRAS, or co -transfected with a lentivirus to overexpress miR-16 and a plasmid to overexpress KRAS. [score:9]
We also provided evidence that the restoration of KRAS expression could partly rescue miR-16 -suppressed cell proliferation and invasion and miR-16 -induced apoptosis, suggesting that the targeting of KRAS is an important mechanism by which miR-16 exerts its tumor-suppressive function. [score:9]
More importantly, tumors with both miR-16 and KRAS overexpression exhibited significantly higher levels of KRAS than tumors with miR-16 overexpression (Fig. 5E and F), suggesting that KRAS overexpression could rescue the KRAS suppression caused by miR-16. [score:9]
SW480 cells were infected with the miR-16 overexpression lentivirus alone, transfected with the KRAS overexpression plasmid alone, or co -transfected with the miR-16 overexpression lentivirus and the KRAS overexpression plasmid. [score:9]
Subsequently, SW480 cells were infected with the miR-16 -overexpressing lentivirus, or transfected with the KRAS overexpression plasmid, or co -transfected with the miR-16 -overexpressing lentivirus and the KRAS overexpression plasmid, and then the untreated or transfected cells were subcutaneously injected into C57BL/6J nude mice. [score:9]
Cells transfected with pre-miR-16 and the KRAS overexpression plasmid showed significantly higher proliferation rates than cells transfected with pre-miR-16 alone (Fig. 4C), suggesting that the overexpression of KRAS rescued the miR-16 -mediated downregulation of the proliferation rates of SW480 cells. [score:8]
Considering that miR-16 is an upstream regulator of KRAS, it is possible to restore miR-16 expression to inhibit KRAS expression in vivo. [score:8]
More importantly, this study identified miR-16 as a novel link between the KRAS regulatory pathway and CRC and pointed the important role of miR-16 as a tumor suppressor in CRC through the inhibition of KRAS translation. [score:8]
Liang et al. reported that miR-16 showed an inverse correlation with the protein expression of FEAT in breast cancer, lung cancer and hepatocellular cancer tissues and that the overexpression of miR-16 promoted the apoptosis of cancer cells by targeting FEAT 45. [score:7]
To overexpress KRAS, an expression plasmid designed to specifically express the full-length KRAS ORF without the miR-16-responsive 3′-UTR was constructed. [score:7]
The expression of mature miR-16 was observed to be 8-10-fold higher after infection (Supplementary Fig. 3A), and KRAS protein expression was consequently inhibited (Supplementary Fig. 3B and C). [score:7]
Additionally, KRAS overexpression attenuated the suppressive effect of miR-16 on tumor growth (Fig. 5A and B), suggesting that miR-16 may inhibit tumor growth by silencing KRAS. [score:7]
Ke et al. found that miR-16 was downregulated in non-small cell lung cancer cells and had an inverse correlation with the protein expression of hepatoma-derived growth factor 44. [score:6]
To determine whether the negative regulatory effect of miR-16 on KRAS expression was mediated by the binding of miR-16 to the predicted target sites in the 3′-UTR of the KRAS mRNA, the KRAS 3′-UTR containing the predicted miR-16 binding site was inserted the downstream of the firefly luciferase gene in a reporter plasmid. [score:6]
Thus, the inhibition of cell invasion by KRAS knockdown was similar to that elicited by miR-16 overexpression, further indicating that miR-16 and KRAS have opposite effects on cell invasion. [score:6]
Subsequent studies demonstrated that miR-16 is downregulated and serves as a tumor suppressor in a variety of cancer types. [score:6]
Likewise, KRAS overexpression attenuated the anti-proliferative effect caused by miR-16 overexpression (Fig. 5G and I). [score:5]
To generate a viral expression construct, a 300-bp fragment containing the genomic sequences of miR-16 was obtained by PCR amplification of human genomic DNA and cloned into a lentiviral expressing vector. [score:5]
For example, Rivas et al. showed that downregulation of miR-16 via progestin -mediated oncogenic signaling contributed to breast cancer development 42. [score:5]
Moreover, the overexpression of miR-16-resistant KRAS remarkably attenuated the anti-invasion effect of miR-16 when SW480 cells were co -transfected with pre-miR-16 and the KRAS overexpression plasmid (Fig. 4D and E). [score:5]
A mammalian expression plasmid designed to specifically express the open reading frame (ORF) of human KRAS without the miR-16-responsive 3′-UTR was purchased from GeneCopoeia (Germantown, MD, USA). [score:5]
miR-16 inhibits cell proliferation and invasion and promotes cell apoptosis by targeting KRAS. [score:5]
Likewise, KRAS mRNA levels were unchanged in tumors from the miR-16 -overexpressing group but increased in tumors from the KRAS -overexpressing group (Fig. 5D). [score:5]
Immunohistochemical staining also revealed the presence of lower levels of KRAS in the tumors from mice implanted with miR-16 -overexpressing cells, whereas the tumors from the KRAS -overexpressing mice showed increased KRAS protein levels (Fig. 5G and H). [score:5]
Furthermore, experiments using tumor xenografts in mice validated the tumor-suppressive role of miR-16 in CRC tumorigenesis through the targeting of KRAS. [score:5]
Xenografts with both miR-16 and KRAS overexpression exhibited more cell mitosis than xenografts with miR-16 overexpression (Fig. 5G). [score:5]
The mutated luciferase reporter was unaffected by the overexpression or knockdown of miR-16 (Fig. 3E). [score:4]
Because the detailed roles that miR-16 plays in the initiation and progression of CRC remain not fully understood, the results of this study may explain, at least in part, why the downregulation of miR-16 during CRC carcinogenesis can promote cancer progression. [score:4]
Moreover, tumors from the miR-16 -overexpressing group displayed reduced KRAS protein levels compared to tumors from the control group, whereas tumors from the KRAS -overexpressing group showed elevated KRAS protein levels (Fig. 5E and F). [score:4]
The results indicate that miR-16 may modulate cell apoptosis by downregulating KRAS. [score:4]
As anticipated, the overexpression of miR-16 dramatically reduced the KRAS protein levels in SW480 and HT29 cells, whereas the knockdown of miR-16 significantly increased the KRAS protein levels in Caco2 cells (Fig. 3B and C). [score:4]
These results demonstrate that miR-16 specifically regulates KRAS expression at the post-transcriptional level. [score:4]
We also determined the level at which miR-16 regulates KRAS expression. [score:4]
Overexpression or knockdown of miR-16. [score:4]
A marked reduction in the size and weight of the tumors was observed in the miR-16 -overexpressing group compared to the control group, whereas the size and weight of the tumors in the KRAS -overexpressing group was dramatically increased. [score:4]
These results were consistent with the findings of the in vitro assays, which firmly validated the tumor-suppressive role of miR-16 in CRC tumorigenesis through the targeting of KRAS. [score:4]
Validation of KRAS as a direct target of miR-16. [score:4]
Tumors from the miR-16 -overexpressing group showed a significant increase in the expression of mature miR-16 compared to tumors from the control group (Fig. 5C). [score:4]
KRAS is a direct target of miR-16. [score:4]
MiR-16 overexpression was achieved by transfecting SW480 and HT-29 cells with pre-miR-16, whereas miR-16 knockdown was achieved by transfecting Caco2 cells with anti-miR-16. [score:4]
Ma et al. reported that miR-16 could inhibit proliferation and induce the apoptosis of CRC cells by regulating the P53/survivin signaling pathway 43. [score:4]
The efficient overexpression of miR-16 in SW480 and HT-29 cells and knockdown of miR-16 in Caco2 cells was shown in Fig. 3A. [score:4]
Taken together, the results indicate that miR-16 can inhibit cell proliferation by silencing KRAS. [score:3]
The SW480 cells were cultured in 12-well plates and transfected with pre-miR-16, KRAS siRNA or the KRAS overexpression plasmid and the Caco2 cells transfected with anti-miR-16 to induce apoptosis. [score:3]
In this study, we identified a novel regulatory network that employs miR-16 and KRAS to regulate cell proliferation, invasion and apoptosis in CRC cells. [score:3]
Subsequently, we investigated whether the overexpression of miR-16-resistant KRAS (KRAS ORF) was sufficient to rescue the suppression of KRAS by miR-16 and attenuated the anti-proliferative effect of miR-16 in CRC cells. [score:3]
Additionally, H&E staining of xenograft tissues showed less cell mitosis in the group implanted with the miR-16 lentivirus, whereas more cell mitosis was observed in the KRAS overexpression group (Fig. 5G). [score:3]
The mean expression levels of miR-16 in the CRC and NAT samples are shown in Supplementary Fig. 1C. [score:3]
Moreover, when SW480 cells were co -transfected with pre-miR-16 and the KRAS overexpression plasmid, KRAS dramatically attenuated the miR-16 -induced apoptosis effect (Fig. 4E and F), suggesting that KRAS might reverse the pro-apoptosis effect of miR-16. [score:3]
SW480 cells were infected with the miR-16 overexpression lentivirus. [score:3]
In SW480 cells with enhanced miR-16 expression, the percentage of apoptotic cells was significantly higher than in the control cells, whereas anti-miR-16 had the opposite effect on cell apoptosis in Caco2 cells (Fig. 4F and G). [score:3]
In addition, we showed for the first time that We also provided evidence that miR-16 could inhibit the proliferation and invasion and induce the apoptosis of CRC cells by silencing KRAS. [score:3]
The reactions were incubated at 95 °C for 5 min, followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s. Overexpression of miR-16 was achieved by transfecting cells with pre-miR-16 (a synthetic RNA oligonucleotide mimicking the miR-16 precursor). [score:3]
Firstly, we examined the inherent expression levels of miR-16 in three CRC cell lines, SW480, HT-29 and Caco2. [score:3]
In summary, these results suggest that miR-16 may suppress cell invasion by silencing KRAS. [score:3]
We next analyzed the biological consequences of the repression of KRAS expression caused by miR-16 in CRC cells. [score:3]
All three algorithms predicted miR-16 as a candidate miRNA that targets KRAS. [score:3]
The minimum free energy value of the hybridization between miR-16 and KRAS mRNA was −24.2 kcal/mol, which is well within the range of genuine miRNA target pairs 34 35. [score:3]
Prediction of KRAS as the target of miR-16. [score:3]
How to cite this article: You, C. et al. Deregulation of the miR-16-KRAS axis promotes colorectal cancer. [score:2]
The correlation between miR-16 and KRAS was examined by evaluating the levels of KRAS in human CRC cell lines after the overexpression or knockdown of miR-16. [score:2]
Taken together, this study highlights an important role for miR-16 in the regulation of KRAS in CRC cells and may open new avenues for future CRC therapy. [score:2]
For luciferase reporter assays, SW480 or Caco2 cells were seeded in 24-well plates and co -transfected with 1 μg of the firefly luciferase reporter plasmid, 1 μg of the β-galactosidase (β-gal) expression plasmid (Ambion), and equal amounts (100 pmol) of pre-miR-control, pre-miR-16 or anti-miR-control, anti-miR-16 using Lipofectamine 2000 (Invitrogen). [score:2]
Furthermore, we introduced point mutations into the corresponding complementary sites in the 3′-UTR of KRAS to eliminate the predicted miR-16 binding site. [score:2]
Furthermore, we assessed the effect of miR-16 and KRAS on the invasion ability of SW480 and Caco2 cells. [score:1]
By measuring the expression of KRAS mRNA after transfecting CRC cells with pre-miR-16 or anti-miR-16, we found that miR-16 did not significantly affect the mRNA levels of KRAS (Fig. 3D). [score:1]
Effect of miR-16 and KRAS on the proliferation, invasion and apoptosis of CRC cells. [score:1]
The seed region of miR-16 and the seed-recognizing site in the KRAS 3′-UTR are indicated in red, and all nucleotides in seed-recognizing site are completely conserved in several species. [score:1]
Thus, miR-16 and KRAS have opposite effects on cell proliferation. [score:1]
The resulting plasmid was transfected into SW480 cells along with either pre-miR-16 or pre-miR-control, or into Caco2 cells along with either anti-miR-16 or anti-miR-control. [score:1]
We then investigated whether the expression levels of miR-16 were inversely correlated with the levels of the KRAS protein in CRC tissues. [score:1]
Firefly luciferase reporters containing either the wild-type (WT) or mutant (MUT) form of the human KRAS 3′-UTR were co -transfected into SW480 cells along with pre-miR-16 or pre-miR-control, or into Caco2 cells along with anti-miR-16 or anti-miR-control. [score:1]
The predicted interaction between miR-16 and KRAS mRNA is illustrated in Fig. 2A. [score:1]
This result suggests that the putative miR-16 binding site of KRAS strongly contributes to the miRNA-mRNA interaction. [score:1]
The effect of miR-16 and KRAS on the growth of CRC cells in vivo. [score:1]
Synthetic pre-miR-16, anti-miR-16 and scrambled negative control RNA (pre-miR-control and anti-miR-control) were purchased from GenePharma (Shanghai, China). [score:1]
We measured the miR-16 levels in the same paired CRC and NAT samples and found that miR-16 was significantly decreased in CRC samples (Fig. 2B), which is consistent with the notion that the levels of miRNAs are opposite to that of their targets. [score:1]
Prediction of conserved miR-16 binding sites within the 3′-UTR of KRAS. [score:1]
Thus, we selected SW480 and HT-29 cells to perform the gain of function experiments and selected Caco2 cells to perform the loss of function experiments for miR-16. [score:1]
At the same time, Caco2 cells were transfected with anti-miR-16 or anti-miR-control and then subjected to Transwell analysis. [score:1]
Effects of miR-16 and KRAS on the growth of CRC xenografts in mice. [score:1]
The inverse correction between miR-16 and KRAS protein levels (Fig. 2C) and the disparity between the miR-16 and KRAS mRNA levels (Fig. 2D) were further illustrated using Pearson’s correction scatter plots. [score:1]
Greater research emphasis is needed to characterize the feasibility of targeting miR-16 in CRC therapy and develop simplified and cost-effective manipulation methods. [score:1]
At the same time, Caco2 cells were transfected with anti-miR-16 or anti-miR-control and then subjected to apoptosis analysis. [score:1]
Furthermore, the miR-16 binding sequence in the KRAS 3′-UTR was highly conserved across species. [score:1]
miR-16 was first found to be associated with chronic lymphocytic leukemia 40 41. [score:1]
In each well, 100 pmol of pre-miR-16, pre-miR-control, anti-miR-control and anti-miR-16 were used. [score:1]
The result indicated that miR-16 levels were significantly lower in SW480 and HT-29 cells than in Caco2 cells. [score:1]
Firstly, SW480 and Caco2 cells were transfected with pre-miR-control or anti-miR-16, respectively, and then the changes in cell proliferation were analyzed. [score:1]
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Other miRNAs from this paper: mmu-mir-16-2, rno-mir-16
Our results have suggested a role of the STAT3/c-Myc pathway in miR-16 down-regulation during hypertrophy, as pharmacological inhibition of either STAT3 or c-Myc prevented the down-regulation of miR-16 and subsequent hypertrophy. [score:9]
Overexpression of miR-16 or siRNA knockdown of CCND1, CCND2 or CCNE1 inhibited ANP up-regulation and size increases of cardiomyocytes. [score:9]
We next determined whether the expressions of CCND1, CCND2 and CCNE1 genes were down-regulated by miR-16 overexpression. [score:8]
Our data have also revealed that miR-16 inhibits hypertrophic phenotype in cardiomyocytes through down-regulation of CCND1, CCND2 and CCNE1 expression and inactivation of cyclin/Rb pathway (Fig. 6). [score:8]
In this study, we have shown that down-regulation of miR-16 was accompanied by up-regulations of CCND1, CCND2 and CCNE1 in hypertrophic myocardium and cell mo dels of cardiac hypertrophy. [score:7]
Notably, our findings showing that down-regulation of miR-16 results in cardiac hypertrophy through up -regulating the expressions of CCND1, CCND2 and CCNE1 have raised many other questions. [score:7]
In this study, we have, for the first time, shown that miR-16 was down-regulated in hypertrophic cardiomyocytes in vivo and in vitro, which was accompanied by up-regulation of CCND1, CCND2, CCNE1 and activation of cyclin/Rb pathway. [score:7]
As expected, overexpression of miR-16 can ameliorate the hypertrophic phenotype in vitro and in vivo through targeting the expression of CCND1, CCND2 and CCNE1. [score:7]
Importantly, viral expression of miR-16 inhibited ANP mRNA and protein expressions in NRVCs induced by PE treatment (Fig. 3C). [score:7]
Taken together, our results have revealed that miR-16 was down-regulated in cardiomyocytes during cardiac hypertrophy, and overexpression of miR-16 could ameliorate hypertrophic phenotype in cardiomyocytes. [score:6]
miR-16 inhibits cardiac hypertrophic phenotype in vivo and in vitroAfter establishing that miR-16 was down-regulated in hypertrophic myocardium, we investigated the effect of restoring miR-16 expression on the cardiac hypertrophic phenotype in vivo. [score:6]
These results suggest that either miR-16 overexpression or knockdown of CCND1, CCND2 or CCNE1 can similarly inhibit PE -induced hypertrophic phenotype of NRVCs. [score:6]
Overexpression of miR-16 down-regulated CCND1, CCND2 and CCNE1 at the post-transcriptional level. [score:6]
We have provided further evidence to support that miR-16 inhibited cardiac hypertrophy through targeting CCND1, CCND2, CCNE1 and cyclin/Rb pathway. [score:5]
More specifically, several lines of evidence derived from our studies support that miR-16 inhibits cardiac hypertrophy through targeting CCND1, CCND2 and CCNE1. [score:5]
miR-16 inhibitor could increase ANP and β-MHC mRNA expressions in NRVCs. [score:5]
Previous study revealed that STAT3/c-Myc pathway mediated progestin -induced suppression of miR-16 expression in mammary tumour cells 27. [score:5]
Cell sizes and the levels of newly transcribed RNA in NRVCs were increased in response to treatment with PE, which were significantly inhibited by miR-16 overexpression (Fig. 3D and E). [score:5]
Our data showed that the expressions of CCND1, CCND2, CCNE1 proteins and phosphorylated pRb were significantly reduced (Fig. 4F– b) and the cell population of S phase was also decreased (Fig. S3B) in NRVCs following overexpression of miR-16, but mRNA levels of CCND1, CCND2 and CCNE1 remained unchanged (Fig. 4F– a). [score:5]
ANP protein expression was shown increased in miR-16 inhibitor -modified NRVCs. [score:5]
Second, enforced expression of miR-16 through adenovirus inhibited cardiac growth in rat AAC mo del. [score:5]
Given that growth of cardiac myocytes results from activation of various signalling pathways and increases in gene transcription and protein synthesis 36, 37, our results showing that overexpression of miR-16 reduced the newly transcribed RNA in PE -treated NRVCs further support that miR-16 inhibits cardiac hypertrophy. [score:5]
Our results have also suggested a role of the STAT3/c-Myc pathway in miR-16 down-regulation during cardiac hypertrophy. [score:4]
Our results demonstrated that treatment with either 10058-F4 or cryptotanshinone prevented the down-regulation of miR-16 induced by treatment with PE (Fig. 5C). [score:4]
Taken together, these data indicate that miR-16 down-regulation resulted in an increase in CCND1, CCND2 and CCNE1, and activation of cyclin/Rb pathway in hypertrophic myocardium. [score:4]
Down-regulation of miR-16 in hypertrophic myocardium. [score:4]
Importantly, knockdown of endogenous miR-16 in cardiomyocytes increased the expression of ANP and β-MHC. [score:4]
Compared with the control vector, adenovirus -mediated overexpression of miR-16 markedly decreased the levels of CCND1, CCND2, CCNE1 proteins and phosphorylated pRb, with no effect on mRNA expressions of CCND1, CCND2 and CCNE1(Fig. 4D– a and D– b). [score:4]
Restoration of miR-16 resulted in attenuation of PE -induced ANP up-regulation in NRVCs. [score:4]
First, miR-16 is highly expressed in cardiomyocytes 30, and negatively regulates cellular growth and cell cycle progression 31. [score:4]
Our data have shown that miR-16 is down-regulated in hypertrophic myocardium and cardiomyocytes. [score:4]
After establishing that miR-16 was down-regulated in hypertrophic myocardium, we investigated the effect of restoring miR-16 expression on the cardiac hypertrophic phenotype in vivo. [score:4]
For example, what triggers down-regulation of miR-16 during hypertrophy? [score:4]
As expected, significant down-regulation of miR-16 was also observed in hypertrophic myocardium in mice in both two mouse mo dels (Fig. 1B and C). [score:4]
To further validate down-regulation of miR-16 in hypertrophic myocardium, we utilized two other mouse mo dels of cardiac hypertrophy through subcutaneous injection of PE (Fig. S1B) and TAC surgery (Fig. S1C). [score:4]
Our results showed that ANP mRNA was down-regulated consistently in PE -treated NRVCs in response to transfection with miR-16 mimic, CCND1 siRNA, CCND2 siRNA and CCNE1 siRNA respectively (Fig. 4G). [score:4]
Fig 5The STAT3/c-Myc pathway mediates miR-16 down-regulation during cardiac hypertrophy. [score:4]
Activation of the STAT3/c-Myc signalling pathway negatively regulates miR-16 expression during cardiac hypertrophy. [score:4]
Down-regulation of miR-16 in hypertrophic cardiomyocytes. [score:4]
We have also demonstrated that the pathway involving STAT3/c-Myc activation mediates down-regulation of miR-16 in hypertrophic cardiomyocytes. [score:4]
Collectively, miR-16 down-regulation in hypertrophic cardiomyocytes results from activation of the STAT3/c-Myc pathway. [score:4]
miR-16 is down-regulated through the STAT3/c-Myc pathway. [score:4]
org), we found that the target sites for miR-16 have been observed on the following genes: CCND1, CCND2 and CCNE1. [score:3]
Therefore, we conclude that miR-16 might be a potential target for prevention and treatment of cardiac hypertrophy. [score:3]
Our results revealed that ANP mRNA was highly up-regulated, and miR-16 level was significantly reduced in the hypertrophic NRVCs and NMVCs compared with their control cells, respectively (Fig. 2B and  D). [score:3]
In addition, we determined expressions of CCND1, CCND2, CCNE1 and pRb in rat hypertrophic myocardium after 2-week infection with recombinant miR-16 adenovirus. [score:3]
miR-16 was overexpressed in the left ventricle, contributing to decreased LV mass. [score:3]
To overexpress miR-16, we first prepared recombinant miR-16 adenovirus and control adenovirus (Fig.  S2). [score:3]
All these results have further indicated that miR-16 post-transcriptionally modulates CCND1, CCND2 and CCNE1 expressions, which is consistent with previous reports 32, 34, 35. [score:3]
of in silico analysis suggesting the presence of miR-16 target sites on the genes of CCND1, CCND2 and CCNE1. [score:3]
Consistently, rats were given the same anaesthesia and euthanasia as above in an in vivo study of overexpression of miR-16. [score:3]
miR-16 targets CCND1, CCND2 and CCNE1, contributing to its repressing effect on cardiac hypertrophy. [score:3]
Collectively, miR-16 reduces the expression of CCND1, CCND2 and CCNE1 in NRVCs at the post-transcriptional level. [score:3]
Furthermore, we transfected NRVCs with miR-16 mimic, siRNA for CCND1, CCND2 or CCNE1, respectively, followed by examining ANP mRNA expression and morphologies of NRVCs. [score:3]
miR-16 inhibits cardiac hypertrophic phenotype in vivo and in vitro. [score:3]
The matching positions for miR-16 within 3′UTR of the targeted mRNAs are shown in Figure 4A. [score:3]
Data on luciferase reporter activities show the interaction between miR-16 and 3′UTRs of target genes. [score:3]
Quantification of miR-16 showed that miR-16 expression was restored in NRVCs in response to infection with recombinant miR-16 adenovirus (Fig. 3B). [score:3]
In addition, a previous study reported that the STAT3/c-Myc pathway mediated miR-16 suppression by progestin in mammary tumour cells 27, further supporting our conclusion. [score:3]
Recombinant adenovirus vector expressing miR-16 was generated by cloning miR-16 DNA template into pAdTrack-CMV (Stratagene, La Jolla, CA, USA), which was further recombined with pAdEasy-I (Stratagene) for the construction of recombinant miR-16 adenovirus in human embryonic kidney 293 cells. [score:3]
miR-16 mimic and inhibitor, siRNAs for CCND1, CCND2, CCNE1 and the cell-light™ EU detection kit were provided by RiboBio (Guangzhou, China). [score:3]
To further confirm the effects of miR-16 on the hypertrophic phenotype in NRVCs, we transfected cells with 100 nM synthesized miR-16 inhibitor using oligofectamine reagent (Invitrogen). [score:3]
Interestingly, in contrast to the positive control with PE treatment, blockage of miR-16 function resulted in significant increase in ANP and β-MHC expressions at both mRNA and protein levels (Fig. 3F and G). [score:3]
Our results showed that the ratio of the LV weight to heart weight and cell size was significantly reduced in the hypertrophic myocardium with forced expression of miR-16 (Fig. 3A– c and  A– d). [score:3]
Activities of firefly luciferase (FL) and renal luciferase (RL) were measured 24 hrs after transfection, and the relative ratio of the FL/RL was used to determine miR-16 -mediated knockdown of target genes. [score:2]
The role of miR-16 was also previously implicated in cell cycle regulation of cancer cells 32, neural differentiation 33 and mesenchymal stem cell differentiation towards myogenic phenotype 34. [score:2]
Therefore, we investigated whether miR-16 down-regulation in cardiac hypertrophy was also mediated through activation of the STAT3/c-Myc pathway. [score:2]
Using a site-directed mutagenesis kit (TransGen, Beijing, China), the GCUGCU in the binding site for miR-16 in pGl3-TG-bs was replaced with GAAAAU to construct pGl3-TG-bs-MUT. [score:2]
In this study, it has been suggested that microRNA-16 (miR-16) modulates the cell cycle regulatory proteins and cyclin/Rb pathway during cardiomyocyte hypertrophy. [score:2]
Figure S2 Preparation of recombinant miR-16 adenovirus. [score:1]
The negative control mimic (NC) were transfected into NRVCs (a), meanwhile, NC (b), miR-16 mimic (c), CCND1 siRNA (d), CCND2 siRNA (e) and CCNE1 siRNA (f) were also transfected in NRVCs followed with PE incubation, respectively. [score:1]
Cells were cotransfected with 200 ng of pGl3-TG-bs or pGl3-TG-bs-MUT, 50 nM mir-16 mimic, and 20 ng of pRL-TK as an internal control (Promega). [score:1]
MUT, indicates the mutated miR-16 binding site sequence GAAAAU instead of GCUGCU. [score:1]
In addition, attenuation of miR-16 derepresses the cyclins D1, D2 and E1 and activates cyclin/Rb pathway to provoke cardiomyocyte hypertrophy. [score:1]
As indicated, NRVCs were transduced with Adeno-empty or Adeno-miR-16 adenovirus (MOI 4). [score:1]
DNA template for miR-16 precursor was amplified from rat genomic DNA using PCR technique. [score:1]
As expected, high miR-16 level was confirmed in rat myocardium after 2-week infection with recombinant miR-16 adenovirus, but not the control virus (Fig. 3A– b). [score:1]
To investigate miR-16 expression in hypertrophic cardiomyocytes, we established a NRVC mo del of 20 mM PE -induced hypertrophy and a NMVC mo del of 10 nM Ang-II -induced hypertrophy. [score:1]
We next measured miR-16 expression in rat hypertrophic myocardium using qRT-PCR. [score:1]
After 2 weeks, rats with and without miR-16 overexpression were killed for further investigations (Fig. 3A– a). [score:1]
Mature miR-16 level was detected using Bulge-Loop miRNA qRT-PCR kit (Ribobio). [score:1]
The seed sequence of miR-16 is AGCAGCA, and the complementary nucleotide sequences are shown in bold words. [score:1]
We measured ANP mRNA and miR-16 expression in the hypertrophic cardiomyocytes using qRT-PCR. [score:1]
Hence, NRVCs were infected without (empty vector) and with recombinant miR-16-encoding adenovirus for 24 hrs. [score:1]
As our previous report 26, the double-stranded DNA fragments containing the potential miR-16 binding site sequences of CCND1, CCND2 and CCNE1 genes were prepared. [score:1]
Fig 6 Schematic diagram of the mechanism whereby miR-16 attenuation contributes to myocardial hypertrophy. [score:1]
Next, we investigated the effect of forced expression of miR-16 on the PE -induced hypertrophic phenotype in NRVCs. [score:1]
FITC-phalloidin staining results demonstrated that cell size changes of PE -treated NRVCs were also significantly reversed after transfection with miR-16 mimic, or siRNA for CCND1, CCND2 or CCNE1, respectively (Fig. 4H). [score:1]
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Despite high levels of FGFR-1, miR-16 still markedly reduced the secretion of HGF (Fig.   3h), suggesting the presence of other miR-16 targets which control HGF expression downstream FGFR-1. Interestingly, miR-16 transfection abrogated ERK1/2 activation also in the presence of FGFR-1 over -expression and this correlated with the downregulation of the MEK1/2 kinases (Fig.   3g), which are responsible for ERK1/2 phosphorylation and could therefore represent additional targets of miR-16. [score:12]
Altogether, our data show that miR-16 acts as potent inhibitor of HGF by targeting directly and indirectly several mediators of the FGFR-1 pathway which in turn controls HGF expression. [score:9]
According to our data, miR-16 reduces fibroblast HGF secretion by direct targeting of HGF itself and by inhibiting the FGFR-1 pathway, which in turn promotes HGF expression. [score:8]
This effect is due to a pleiotropic activity of miR-16 which prevents HGF expression through direct inhibition of FGFR-1 signaling and targeting of HGF mRNA. [score:8]
In fact, even if not directly targeting the FGFR-1 mRNA, miR-16 reduces FGFR-1 protein levels and directly targets MEK1, which is crucial for FGFR-1 downstream signaling. [score:7]
e The direct targeting of miR-16 on the 3′UTR of the HGF mRNA was determined by luciferase assay performed by transfection of 293 T cells with 100 nM miRNAs together with the pMirTarget HGF 3′UTR and a Renilla -expressing plasmid. [score:7]
Similar to what was observed for FGFR-1, ectopic expression of MEK1 is not sufficient to prevent the miR-16 -dependent downregulation of HGF (Fig.   4e, f). [score:6]
Importantly, mouse lung analysis revealed that HGF inhibition also reduced the capacity of A549 cells to metastasize to lungs (Fig.   6e) in a significant manner while ectopic expression of miR-16 in CAF154-hTERT fibroblasts reduced the metastasizing potential of cancer cells, albeit not significantly (Fig.   6e, f). [score:5]
We identified miR-16 as a master regulator of fibroblast secretome and showed that its upregulation reduces HGF secretion by fibroblasts, impairing their capacity to promote cancer cell migration. [score:5]
a CAF154-hTERT cells were transfected with miR-16 and control miR-C, a non -targeting siRNA, and siRNAs targeting FGFR-1 or HGF. [score:5]
MEK1 is a direct target of miR-16 and regulates HGF levels in fibroblast CM. [score:5]
b CAF154-hTERT fibroblasts were transfected with the non -targeting miR-C, miR-16, a control siRNA, or a siRNA targeting FGFR-1 or HGF. [score:5]
Over -expression of miR-16 in fibroblasts and inhibition of soluble HGF hinder cancer aggressiveness in vivo. [score:5]
MiR-16 expression indirectly decreases FGFR-1 levels and inhibits its signaling pathway further contributing to HGF reduction. [score:5]
Importantly, also, miR-497 and miR-15b, which target the same seed region of miR-16, were found among the top candidates (cluster #1, Table  2), and the other miRNAs belonging to the same family (miR-195, miR-15a, and miR-424), even if less efficiently, all showed an inhibitory effect on A549 cells (0.902, 0.837, and 0.834 normalized A549 cell number, respectively). [score:5]
Furthermore, transfection with miR-16 abrogated the pro-migratory effect of cells with elevated HGF production strengthening even further the link between miR-16 and HGF in lung fibroblasts and highlighting the potential role of this mechanism in regulating aggressiveness of this deadly disease. [score:4]
Nonetheless, in our case series, we found a correlation between smoke exposure (and COPD) and reduced expression of miR-16 in lung fibroblasts derived from normal tissue raising the intriguing hypothesis that smoke -induced stromal modifications could pave the way for cancer development by stimulating the growth of incipient lesions. [score:4]
In conclusion, we show here that miR-16 is a crucial mediator of HGF production by lung fibroblasts through regulation of different downstream targets including FGFR-1 and MEK1. [score:4]
We demonstrated a direct targeting of the HGF transcript by miR-16, but this could not fully explain the extent of HGF reduction. [score:4]
Fig. 3The FGFR-1 receptor regulates HGF secretion, and it is targeted by miR-16. [score:4]
To rule out the possibility that miR-16 regulates HGF levels in a cell-type-specific manner, we transfected a number of patient-derived primary fibroblasts with miR-16 and consistently found a striking reduction of HGF levels in the CM (Fig.   2d), while miR-16 inhibition reproducibly increased HGF levels (Additional file  4: Figure S4). [score:4]
The pMirTarget 3′UTR HGF was mutagenized using QuikChange II XL Site-Directed Mutagenesis Kit (Agilent, Santa Clara, CA) to delete the putative binding site for miR-16. [score:4]
Of note, downregulation of miR-16 levels in the circulation is also a feature of our recently validated circulating miRNA -based signatures with predictive and prognostic value in heavy smokers undergoing spiral CT screening for lung cancer [35, 49]. [score:4]
We further identify MEK1 as a novel direct target of miR-16. [score:4]
In fact, miR-16 upregulation reduced FGFR-1 levels, and this further decreased HGF levels. [score:4]
Importantly, when the sequence corresponding to the seed region of miR-16 was mutated in the HGF 3′UTR (Additional file  3: Figure S3), the effect of the miRNA was almost completely abrogated (Fig.   2e), indicating a direct targeting of miR-16 on HGF mRNA. [score:4]
Among the identified candidates, we focused on miR-16, one of the prominent negative regulators of the pro-tumorigenic activity of fibroblasts in our assay and previously shown to have direct oncosuppressive properties in tumor cells [15, 16, 41] and to mediate tumor-stroma interactions in prostate cancer [17]. [score:4]
e, f Cells transduced as described in Fig.   3g were further transduced with lentiviral particles to ectopically express MEK1 or GFP, as a control, and transfected with control miR-C and miR-16. [score:3]
We then asked whether the restoration of FGFR-1 levels was sufficient to prevent the effect of miR-16 on HGF and therefore transduced CAF154-hTERT cells to ectopically express FGFR-1 (Fig.   3g). [score:3]
We checked whether miR-16 directly targets the HGF 3′UTR by luciferase assay and found a partial, yet significant, reduction of the luciferase activity when cells were transfected with miR-16 (Fig.   2e). [score:3]
c A549 cells (5 × 10 [5] cells) were injected in the flanks of immunosuppressed nude mice after 24 h culturing in CM (1:2) collected from CAF154-hTERT fibroblasts transfected with mir-C, with or without a neutralizing anti HGF antibody (HGFi), and miR-16 (n = 6). [score:3]
Importantly, inhibition of miR-16 resulted in an opposite effect and caused the accumulation of HGF both in the CM (Fig.   2c, left) and intracellularly (Fig.   2c, right). [score:3]
Moreover, inhibition of HGF in CM by means of a neutralizing antibody showed an effect comparable to miR-16 transfection (Fig.   6c). [score:3]
Importantly, we provide also evidence that tobacco smoking may promote the reduction of miR-16 expression in lung fibroblasts and favor the increase of systemic HGF levels. [score:3]
Accordingly, transfection of CAF154-hTERT cells with miR-16 resulted in the inhibition of both fibroblast and adjacent A549 cell proliferation (Fig.   1e, upper panel) in our HTS, and the effect on A549 cells co-cultured with transfected fibroblasts was further confirmed in independent experiments (Fig.   1e (lower panel), f). [score:3]
Together with FGFR-1, other downstream mediators of its signaling pathway, including MEK1, were also identified as targets of miR-16 (Fig.   7). [score:3]
Fig. 2Ectopic expression of miR-16 affects the CAF secretome and reduces HGF levels. [score:3]
For fibroblast transfection, the negative control miRNA #1 (miR-C, #4464058) and hsa-miR-16-5p (miR-16, MC10339) miRNA mimics, the negative control miRNA #1 (miR-C inh, #4464076) and hsa-miR-16-5p (miR-16 inh, MC10339) miRNA inhibitor (Thermo Fisher Scientific), control siRNA (siCtr; 5′-CGUACGCGGAAUACUUCGATT-3′, Eurofins), siFGFR-1 #SI02224677 and #SI02224684, siHGF #SI03046946, siMEK1#6 #SI00300699, and siMEK1#7 #SI02222955 (Qiagen, Hilden, Germany) were used. [score:3]
Consistently, chemical or genetic inhibition of FGFR-1 mimics miR-16 activity and prevents HGF production. [score:3]
This notion was confirmed in vivo where miR-16 expression in fibroblasts reduced their ability to induce cancer cells to form subcutaneous tumors and disseminate to lungs. [score:3]
Interestingly, miR-16 also suppresses the fibroblast growth factor-2 (FGF-2)/FGF receptor-1 (FGFR-1) axis [17], and therefore, the loss of this miRNA can eventually enhance cancer cell survival, proliferation, and migration. [score:3]
Expression of miR-16 affects CAF secretome and massively decreases HGF levels. [score:3]
showing the levels of HGF and cMet in CAF154-hTERT fibroblasts 72 h after transfection with miR-16 and non -targeting miR-C. Actin is shown as a loading control. [score:3]
d Luciferase assay performed as described in Fig.   2e to test direct targeting of MEK1 3′UTR by miR-16 (** p = 0.0084). [score:3]
Fig. 4MEK1 regulates HGF secretion and is regulated by miR-16. [score:3]
Primary fibroblast cell lines were transfected with control miRNA (miR-C inh) and miR-16 inhibitor (miR-16 inh) and CM collected 72 h later (four cell lines in two independent experiments, paired t test p = 0.0430). [score:3]
Finally, in vivo experiments confirmed that restoration of miR-16 expression in fibroblasts reduced their ability to promote tumor growth and that HGF plays a central role in the pro-tumorigenic activity of fibroblasts. [score:3]
By using a luciferase 3′UTR reporter assay, we confirmed that MEK1 is a direct target of miR-16 (Fig.   4d) and it represents a determinant of HGF secretion in the CM (Fig.   4b). [score:3]
In fact, by analyzing the levels of this miRNA in 47 primary cell lines of lung fibroblasts established from fresh tumor biopsies obtained from lung cancer patients (including 21 matched samples from both cancer tissue—cancer -associated fibroblasts (CAFs)—and non-involved lung parenchyma—normal fibroblasts (NFs)—for a total of 26 CAF lines and 21 NF lines), we explored the correlations between miR-16 expression levels and clinical parameters (Table  3). [score:3]
Mechanistically, miR-16 targets FGFR-1 downstream mediator MEK1, thus reducing ERK1/2 activation. [score:3]
Interestingly, strongly reduced levels of miR-16 were also detected in fibroblasts from patients with chronic obstructive pulmonary disease (p = 0.048), in particular in NF (p = 0.004). [score:3]
c HGF levels in the CM and western blot of CAF154-hTERT fibroblasts transfected as in c with a control miRNA (miR-C inh) and miR-16 inhibitor (miR-16 inh; n = 3, p = 0.0358). [score:3]
Potential miR-16 target regions in HGF, FGFR-1, and MEK1 mRNA. [score:3]
d The effect of miR-16 ectopic expression was determined in CM collected from a number of primary patient-derived fibroblasts (CAF154, n = 3, p = 0.0259; CAF226, n = 2; NF221, n = 3, p = 0.0316). [score:3]
g CAF154-hTERT fibroblasts were transduced with lentiviral particles to stably express FGFR-1 and transfected with control miR-C and miR-16. [score:3]
The concentration of each soluble factor after miR-16 transfection is expressed as a percentage compared of miR-C -transfected CM. [score:2]
Of note, we report that primary fibroblast cell lines derived from lungs of heavy smokers express reduced miR-16 levels compared to those from lungs not exposed to smoke and that HGF concentration in heavy smokers’ plasma correlates with levels of tobacco exposure. [score:2]
Overall, these results uncover a central role for miR-16 in regulating HGF production by lung fibroblasts, thus affecting their pro-tumorigenic potential. [score:2]
Correlation between smoking exposure and miR-16 levels could provide novel clues regarding the formation of a tumor-proficient milieu during the early phases of lung cancer development. [score:2]
Importantly, the effects observed in our experiments could not be attributed to a direct effect of miR-16 on cancer cells, but rather to a modulation of fibroblast secretome. [score:2]
To test the specificity of miR-mRNA interaction, the assay was performed also with a mutated version of the pMirTarget HGF 3′UTR in which the putative binding site of miR-16 was mutagenized (Additional file  3: Figure S3; ** p = 0.002; ns, non-significant). [score:2]
Two experiments were performed by culturing the A549 cells with the CM collected from CAF154-hTERT fibroblasts transfected with miR-16 and control miR-C (Fig.   6a, b) before mouse engrafting (Fig.   6c) or by directly co-injecting the A549 cells with the transfected fibroblasts in nude mice (Fig.   6d). [score:2]
normal), histology, and stage or grade of disease, but lower levels of miR-16 were detected in fibroblasts from smoke-exposed lungs compared to fibroblasts from non-exposed lungs [median intensity value 860 (IQ range 310–1437) vs. [score:2]
Fibroblast miR-16 levels regulate motility features of adjacent cancer cells. [score:2]
After performing a genome-wide functional screening analysis, we focused specifically on miR-16, demonstrating that it regulates HGF secretion by fibroblasts in an FGFR-1 -dependent manner. [score:2]
Our observation that fibroblast-derived HGF promotes cancer cell migration in vitro and tumor development in vivo supports the notion that both cancer and stromal miR-16 levels affect cancer aggressiveness. [score:2]
To confirm the involvement of MEK1 in the miR-16 -dependent regulation of HGF, we transfected CAF154-hTERT cells with this miRNA and found that MEK1 was indeed reduced (Fig.   4a). [score:2]
FGFR-1 3′UTR was mutagenized to delete to potential miR-16-directed region. [score:2]
MiR-16 inhibition results in increased HGF levels in primary fibroblasts. [score:2]
We identified HGF as one of the most affected factors, strongly depleted in the CM of cells upon miR-16 transfection. [score:1]
However, with experiments on transfected fibroblasts conditioned medium (CM), we estimated a concentration of released miR-16 ranging between 0.1 and 0.05 nM and found that similar amounts of miRNA did not influence A549 cell proliferation (data not shown). [score:1]
Even if correlation between smoke exposure and miR-16 levels in clinical samples is at present interesting, future work should address the mechanistic basis of this correlation. [score:1]
Levels of FGF-1 and FGF-2 in the CM of CAF154-hTERT fibroblasts were transfected with control miR-C and miR-16, collected 72 h after the transfection, and analyzed by multiplex analysis. [score:1]
CAF154-hTERT fibroblasts were transfected with control miRNA (miR-C) or miR-16, and CM analyzed after 72 h by multiplex array to quantify 91 unique soluble factors, belonging to three human protein sets: (i) cytokines, (ii) MMPs and TIMPs, and (iii) angiogenetic factors. [score:1]
e, f Lungs collected from mice described in c and b, respectively, were collected and analyzed by FACS for the presence of metastatic human cells (e miR-C vs miR-16, p = 0.2997; miR-C vs HGFi * p = 0.0312; f miR-C vs miR-16, p = 0.0735) In both cases, transfection with miR-16 of CAF154-hTERT fibroblasts delayed the ability of the A549 cancer cells to form nodules. [score:1]
Smoking correlates with reduced levels of miR-16 in lung fibroblasts. [score:1]
Finally, we reasoned that the microenvironmental changes related to miR-16 reduction, which we previously observed in lung-derived fibroblasts (Table  3), could also potentially influence systemic levels of HGF. [score:1]
Tumor microenvironment Lung cancer miR-16 Cancer cell migration FGFR-1 signaling Lung cancer is the leading cause of cancer deaths worldwide due to its high incidence and mortality [1]. [score:1]
e Effect of miR-16 on A549 and CAF154-hTERT cell proliferation in the HTS (upper panel; values normalized to controls) and validation of the effect of miR-16 transfection in CAF154-hTERT cells on co-cultured A549 cell growth (lower panel). [score:1]
This is the case, for example, of miR-16 which, together with miR-15, was the first miRNA described to be deleted in cancer, specifically in chronic lymphocytic leukemia (CLL) cells [14]. [score:1]
Fig. 5The CM derived from miR-16 -transfected fibroblasts displays reduced pro-tumorigenic properties. [score:1]
We next investigated therefore whether miR-16 ectopic expression in CAF154-hTERT fibroblasts could affect the secretome profile of the transfected cells, thereby influencing the growth of A549 cells. [score:1]
f Correlation between circulating HGF concentration and smoke exposure (pack-years), r = Spearman correlation p value (left), and circulating HGF concentration and COPD, p = Wilcoxon test p value (right) in healthy heavy smokers (n = 90)Among the perturbed factors, HGF was the most affected by miR-16 transfection and was almost completely depleted in the CM (Fig.   2a, b). [score:1]
CM collected from CAF154-hTERT cells transfected with miR-16 displayed a reduced capacity to promote proliferation of A549 cancer cells (Fig.   5b, upper panel) and also induced a small, but significant, difference in proliferation in another cell line previously shown to be responsive to microenvironment cues [30] (LT73, Fig.   5b, bottom panel). [score:1]
Stimulation of this receptor by means of its cognate ligand FGF-2 resulted in the accumulation of HGF in the CM (Fig.   3a) while miR-16 transfection strongly reduced the levels of FGFR-1 (Fig.   3b), but not of its cognate ligands FGF-1 and FGF-2 (Additional file  5: Figure S5), and it hindered the activation of the FGFR-1 downstream mediators ERK1/2 (Fig.   3b). [score:1]
f CAF154-hTERT fibroblasts were analyzed by real-time PCR 72 h after transfection with miR-C or miR-16 to evaluate miR-16 levels The 60 miRNA candidates displaying the strongest stimulatory effect on co-cultured A549 cells and the top 60 miRNAs which resulted in an inhibitory effect were then clustered according to their predicted ability to interact with the same mRNAs. [score:1]
b Concentration of HGF in CM collected 72 h after transfection of 1.5 × 10 [5] CAF154-hTERT fibroblasts with miR-C and miR-16 (n = 3; p < 0.0001). [score:1]
The FGFR-1 3′UTR displays two putative miR-16 binding sites (Additional file  3: Figure S3). [score:1]
FGF-1 and FGF-2 levels are not affected by miR-16. [score:1]
Curiously, in this respect, maternal-smoke -associated reduction of miR-16 in the placenta has been described [46]. [score:1]
f CAF154-hTERT fibroblasts were analyzed by real-time PCR 72 h after transfection with miR-C or miR-16 to evaluate miR-16 levelsThe 60 miRNA candidates displaying the strongest stimulatory effect on co-cultured A549 cells and the top 60 miRNAs which resulted in an inhibitory effect were then clustered according to their predicted ability to interact with the same mRNAs. [score:1]
Nonetheless, the transfection of CAF154-hTERT cells with miR-16 or an FGFR-1-specific siRNA both resulted in HGF reduction (Fig.   3d). [score:1]
Biological triplicates were prepared for each condition, i. e., transfection with miR-C and miR-16. [score:1]
Taking into account the strong effect of miR-16 on HGF secretion (Fig.   2a, b) and the only partial reduction of the luciferase activity in the HGF 3′UTR reporter assay (Fig.   2e), we also considered additional inhibitory mechanisms and focused on the FGFR-1 receptor. [score:1]
f Correlation between circulating HGF concentration and smoke exposure (pack-years), r = Spearman correlation p value (left), and circulating HGF concentration and COPD, p = Wilcoxon test p value (right) in healthy heavy smokers (n = 90) Among the perturbed factors, HGF was the most affected by miR-16 transfection and was almost completely depleted in the CM (Fig.   2a, b). [score:1]
Of note, miR-16 -induced reduction of FGFR-1 apparently did not stem from a direct effect of this miRNA on FGFR-1 mRNA, as judged by the lack of effect of miR-16 on FGFR-1 3′UTR in a luciferase reporter assay (Fig.   3c). [score:1]
For co-injection experiments, A549 cells (1 × 10 [3]) were injected s. c. with fibroblasts transfected with miR-C or miR-16. [score:1]
Here, we show that miR-16 levels in lung fibroblasts control HGF production in an FGFR1 -dependent manner unraveling a novel mechanism of communication between stromal and neoplastic cells in lung cancer, with potential clinical implications. [score:1]
a HGF levels in the CM medium employed and b miR-16 expression in the CAF154-hTERT fibroblasts were evaluated by ELISA and real-time PCR, respectively. [score:1]
The miR-16 binding site in the MEK1 3′UTR is shown (Additional file 3: Figure S3). [score:1]
d Levels of HGF in CM of CAF154-hTERT fibroblasts transfected with the control miR-C and miR-16 or a control siRNA and siRNA specific for FGFR-1 or HGF. [score:1]
When miR-16 is reduced or lost, HGF is secreted by fibroblasts and contributes to cancer cell aggressiveness through the stimulation of cMet pathway. [score:1]
In detail, miR-16 levels in fibroblasts were not statistically associated with sex or age of patients, tissue of origin (cancer vs. [score:1]
e, f Lungs collected from mice described in c and b, respectively, were collected and analyzed by FACS for the presence of metastatic human cells (e miR-C vs miR-16, p = 0.2997; miR-C vs HGFi * p = 0.0312; f miR-C vs miR-16, p = 0.0735)In both cases, transfection with miR-16 of CAF154-hTERT fibroblasts delayed the ability of the A549 cancer cells to form nodules. [score:1]
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Previously, Myc has been reported to be upregulated in mutated FLT3 expressing cells postulating reduced expression of miR-16 is possibly associated with up-regulated Myc [9], [78]. [score:11]
Thus, suppressed miR-16 in FLT3/ITD expressing cells appears to be highly associated with activated FLT3 signalling with ITD mutation, and suppression of miR-16 can be reversed by FLT3 inhibition. [score:10]
As such, it can be postulated that suppressed miR-16 in FLT3/ITD expressing cells may contribute to up-regulation of Pim-1. To prove our hypothesis of direct interaction of miR-16 and Pim-1, we first performed luciferase reporter assay to determine whether miR-16 can bind to 3′UTR of Pim-1. Our data have shown that miR-16 binds to two 3′-UTR regions of Pim-1 demonstrating miR-16 interacts with Pim-1. We next performed QPCR and immunoblotting to quantify Pim-1 expression upon miR-16 mimic transfection in FD-FLT3/ITD cells. [score:10]
Several miRNAs including miR-16 and miR-223 were down-regulated after transformation whereas several miRNAs including miR-21 were up-regulated after transformation of FDC-P1 cells by expression of FLT3/ITD. [score:9]
We discovered that miR-16 is down regulated in FD-FLT3/ITD cells and it is highly upregulated upon FLT3 inhibition in FLT3/ITD expressing FD-FLT3/ITD, MV4-11, and MOLM-14 cells. [score:9]
Taken together, miR-16 appears to bind to the 3′ UTR of Pim-1 and thus mediate negatively the regulation of Pim-1 expression at a posttranscriptional level, and suppressed miR-16 may contribute to continuous growth of FD-FLT3/ITD cells through up-regulation of anti-apoptotic molecules including Pim-1. 10.1371/journal. [score:9]
In this study, miR-16 was found to be one of most significantly down-regulated genes in FD-FLT3/ITD cells, and was up-regulated upon FLT3 inhibition. [score:9]
Taken together, miR-16 appears to bind to the 3′ UTR of Pim-1 and thus mediate negatively the regulation of Pim-1 expression at a posttranscriptional level, and suppressed miR-16 may contribute to continuous growth of FD-FLT3/ITD cells through up-regulation of anti-apoptotic molecules including Pim-1. 10.1371/journal. [score:9]
We discovered that miR-16 expression was increased in each FLT3/ITD expressing cell we tested (FD-FLT3/ITD, MV4-11 and MOLM-14 cells) with increasing time points of incubation with FLT3 inhibitors (lestaurtinib or sunitinib) (Fig. 4). [score:7]
Thus, our results in this study identified miR-16 is a dysregulated miRNA in FLT3/ITD expressing cells, and suggest that suppressed miR-16 may be associated with regulation of Pim-1 levels in FLT3/ITD signalling. [score:7]
Indeed, deletion or down-regulation of miR-15 and miR-16 in CLL is inversely correlated to BCL2 expression, and both miRNAs have been shown to negatively regulate BCL2 at a posttranscriptional level [17]. [score:7]
Pim-1 protein also exhibited an approximate 2-fold decrease in FD-FLT3/ITD cells indicating Pim-1 is a direct target of miR-16 and miR-16 induces translational repression of Pim-1 (Fig. 6A, inset). [score:6]
Significantly, this provides further evidence that miR-16 expression is an important biomarker of this transformation and provides an alternative target for future drug development. [score:6]
For this reason, regulation of Pim-1 level and modulation of miR-16 expression may be important for FLT3/ITD expressing AML. [score:6]
Combined with previous published reports, we hypothesise that Pim-1 may be down-regulated not only by deactivation of its possible transcription factors such as STAT5, but also by increment of miR-16 after treatment of FLT3 inhibitors. [score:6]
Following transfection, miR-16 was quantified using TaqMan miRNA assay kit (Applied Biosystems, CA) to confirm over -expression or reduction of miR-16 expression in target cells. [score:6]
A few transcriptional regulators for miR-16 such as p53 [75], NF-κB [76] and Myc [77] have been suggested to be involved in regulating the expression of miR-16. [score:5]
miR-16 expression increases in response to FLT3 inhibition in FD-FLT3/ITD, MV4-11, and MOLM-14 cells. [score:5]
However, only a handful of the potentially hundreds of miR-16 target genes have been identified to date, including CCND1, WNT3A, CAPRIN1, HMGA1, BMI1, WIP1, and SERT (serotonin transporter), though this does suggest an important role for miR-16 in regulating biological processes such as cell cycle regulation, apoptosis, and proliferation [31], [32], [33], [34], [35]. [score:5]
To validate increased miR-16 expression can reduce Pim-1 mRNA levels, we analysed expression of Pim-1 by QPCR using total RNA extracted from FD-FLT3/ITD cells upon miR-16 mimic transfection. [score:5]
We then confirmed that a selection of these including miR-16, miR-223, and miR-21 are differentially expressed in FLT3/ITD expressing cells. [score:5]
To validate microarray data, we performed QPCR using TaqMan miRNA assays on these three miRNA and confirmed that miR-16 and miR-223 were suppressed, whilst miR-21 was up-regulated in FD-FLT3/ITD cells correlating strongly with data from the microarray experiments. [score:5]
There is an emerging body of research to suggest that miRNAs play an important role in the pathology of haematological malignancies [23], first suggested with the deletion or down-regulation of miR-15 and miR-16 in a large proportion of chronic lymphocytic leukemia (CLL) cases [24]. [score:4]
The putative target site of miR-16 in 3′-UTR of Pim-1 and luciferase reporter assay results suggested the possibility of inverse correlation of Pim-1 expression by miR-16. [score:4]
Our results identified that miR-16 appears to bind to the 3′ UTR of Pim-1 and mediate negative regulation of Pim-1 expression. [score:4]
Our data indicated a miR-16–specific regulation of reporter gene expression of both Pim1_16_1 and Pim1_16_2 constructs (Fig. 5D). [score:4]
Pim-1 is a Direct Target of miR-16. [score:4]
Our data show that enforced expression of miR-16 could not completely deplete Pim-1 expression and reduces both mRNA and protein levels to approximately 50% compared to control cells. [score:4]
Taken together, miR-16 appears to bind to the 3′ UTR of Pim-1 and mediate negative regulation of Pim-1 expression. [score:4]
Pim-1 is Down-regulated by miR-16 Mimic. [score:4]
Pim-1 protein expression has been reduced in FD-FLT3/ITD cells upon miR-16 mimic transfection (lane 2) when compared to control Pim-1 protein expression (lane 1). [score:4]
Thus, miR-16 possesses two MREs within the Pim-1 3′ UTR that it can bind to regulate Pim-1 expression levels. [score:4]
Pim-1 is a direct target of miR-16. [score:4]
Our results in this report suggest that Pim-1 may be a potential target of miR-16. [score:3]
We also found that FD-FLT3/ITD cell growth was reduced upon miR-16 mimic transfection showing miR-16 may interact with anti-apoptotic molecules including Pim-1. Recently, Pim-1 has been reported to be targeted by miR-33a [79]. [score:3]
U6 small nuclear RNA was used as an internal control to normalize the level of miR-16 expression. [score:3]
In our study, we more focused on miR-16 to further analyse its role in FLT3/ITD expressing cells. [score:3]
We next examined whether enforced expression of the miR-16 mimic might result in changes of cell growth. [score:3]
mir-16 Increases in Response to FLT3 Inhibition. [score:3]
To test whether miR-16 targets Pim-1, two predicted MREs from Pim-1 were cloned into the pMIR-REPORT multiple cloning site to generate reporter constructs. [score:3]
Since miR-16 has been shown to target Bcl-2 [17], the RNA level of Bcl-2 was also analysed as a positive control. [score:3]
We selected miR-16 for further analysis by QPCR and FLT3 inhibitors as it has been reported to be involved in anti-apoptosis in leukaemic cells. [score:3]
Mir-16 mimic down-regulates Pim-1 and decelerates FD-FLT3/ITD cell growth. [score:3]
The RNA level of Bcl-2 paralleled Pim-1 expression upon miR-16 mimic transfection confirming Pim-1 mRNA levels were reduced due to an interaction with miR-16. [score:3]
Numerous target mRNAs of miR-16 have been identified so far [31], [32], [33], [34], [35]. [score:3]
Interestingly, we discovered through bioinformatic computer mo dels that miR-16 may target the 3′ UTR region of both human and mouse Pim-1 (Fig. 5A and B). [score:3]
This suggested that miR-16 is acting like a tumour suppressor gene in FLT3/ITD -mediated leukemic transformation. [score:3]
Additionally, in the mouse Pim-1 3′ UTR, putative target sites for miR-16 were located in two different locations whilst alignment scores of both sites are equally high (Fig. 5B). [score:3]
Finally, we focused on miR-16 for further study and found the Pim-1 oncogene, a regulator of FLT3/ITD signalling, is itself potentially regulated by miR-16. [score:3]
We confirmed miR-16 and miR-223 were down-regulated: greater than two-fold lower in FD-FLT3/ITD cells compared to control cells. [score:3]
Using quantitative real-time RT-PCR and immunoblotting, we confirmed that reduced Pim-1 mRNA and protein levels were the outcome of miR-16 mimic transfection demonstrating that binding of miR-16 on the 3′ UTR of Pim-1 results in the negative regulation of Pim-1 at a posttranscriptional level. [score:2]
To verify Pim-1 as a target of miR-16, we performed luciferase reporter assays with both putative miR-16 binding sites from the Pim-1 3′ UTR (Pim1_16_1 3′ UTR and Pim1_16_2 3′ UTR, Fig. 5C). [score:2]
Interestingly, we found that computer mo dels from public bioinformatic resources predicted a potential regulatory mechanism between miR-16 and Pim-1 mRNA. [score:2]
Increment of miR-16 expression by 50 nM lestaurtinib or sunitinib with increasing time of treatment compared to control (fold = 1) is presented. [score:2]
RNA was then harvested and used for miR-16 expression analysis by QPCR using TaqMan miRNA assay. [score:2]
The graph is presented to show relevant gene expression fold differences in FD-FLT3/ITD cells after miR-16 mimic transfection compared to scrambled miR-16 transfection (fold = 1) as a control. [score:2]
The Pim-1 mRNA and protein were decreased after transfection of the miR-16 mimic demonstrating miR-16 possibly regulates Pim-1 at a posttranscriptional level. [score:2]
A quantity of 1×10 [6] FD-FV and FD-FLT3/ITD cells were cultured after transfection with synthetic miR-16 mimics for 4 days. [score:1]
0044546.g003 Figure 3The three miRNAs (miR-16, miR-223, and miR-21) selected from Fig. 2 were analysed by QPCR on total RNA from FD-FLT3/ITD cells. [score:1]
Lipofectamine 2000 reagent (Invitrogen) was used to transiently co-transfect HEK-293 cells with 4 ng of reporter construct (Pim1_16_1 or Pim1_16_2) and 20 ng of pRL-TK renilla luciferase construct; with 30 nM synthetic miRNA (miR-16 mimic or miR-16 scramble control) or 100 nM anti-miR LNA -modified oligonucleotide (anti-miR-16 or anti-miR scramble control) (Table 1). [score:1]
The three miRNAs (miR-16, miR-223, and miR-21) selected from Fig. 2 were analysed by QPCR on total RNA from FD-FLT3/ITD cells. [score:1]
The inset shows the results from immunoblotting of Pim-1 protein upon transfection of miR-16 mimic. [score:1]
Among these miRNAs, we selected miR-16, miR-21, and miR-223 to validate the microarray data by quantitative real-time RT-PCR (QPCR), showing a high degree of correlation. [score:1]
Predicted miR-16 binding site is depicted. [score:1]
We selected miR-16, miR-223 and miR-21 from the set of miRNAs for further analysis on the basis of relevancy to the leukemia phenotype and published literatures. [score:1]
The responsiveness of each reporter-MRE to co -transfected miR-16 or anti-miR-16 was determined by their relative firefly luciferase activities with respect to Renilla luciferase activity (transfection control), normalised against the relative activity with their respective scrambled controls. [score:1]
The numbers (+487–508) represent the nucleotides (relative to Pim-1 termination codon) that are predicted to base pair with the miR-16 seed sequence. [score:1]
Viable cells were counted daily based on trypan blue exculsion after transfection of synthetic miR-16. [score:1]
0044546.g006 Figure 6(A) Using QPCR, Pim-1 mRNA level was quantified from FD-FLT3/ITD cells upon miR-16 mimic transfection. [score:1]
We selected miR-16, miR-21 and miR-223 on the basis of published reports relevant to the leukaemic phenotype. [score:1]
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Restoration of these downregulated miRNAs in mouse primary sarcoma cell lines showed that miR-16, but not other downregulated miRNAs, was able to significantly suppress both migration and invasion in vitro, without altering cell proliferation. [score:9]
Therefore, just as we recently demonstrated that miR-182 regulates metastasis by targeting multiple genes (Sachdeva et al., 2014), miR-16 may also regulate metastasis through a number of targets, which are differentially regulated in immunocompromised versus immunocompetent mice. [score:8]
Previous studies have shown that miR-16 can suppress cell cycle progression by targeting multiple G1 cyclins (Bandi et al., 2009) and that miR-223 can indirectly regulate cyclin E (Xu et al., 2010). [score:7]
miR-16 overexpression suppresses lung metastasis in a nude mouse transplant mo del in vivoTo determine whether miR-16 can suppress metastasis in vivo, we transplanted sarcoma cells into the muscle of nude mice so that the tumor cells would grow at an orthotopic site. [score:7]
miR-16, but not other miRNAs, suppresses both migration and invasion in vitroGlobal downregulation of miRNAs has been shown to correlate with both high-grade tumors and poor patient survival (Lu et al., 2005; Martello et al., 2010). [score:6]
We subsequently focused on proteins whose mRNA has a putative binding site for miR-16 using Targetscan and found that only 11 of the ∼170 upregulated proteins were encoded by genes containing predicted miR-16 binding sites (supplementary material Table S4). [score:6]
Hypoxia-microRNA-16 downregulation induces VEGF expression in anaplastic lymphoma kinase (ALK) -positive anaplastic large-cell lymphomas. [score:6]
miR-15a and miR-16 are implicated in cell cycle regulation in a Rb -dependent manner and are frequently deleted or down-regulated in non-small cell lung cancer. [score:5]
In addition, in an orthotopic amputation mo del, miR-16 overexpression in a sarcoma cell line (KP1) suppresses both the rate and number of lung metastases. [score:5]
This might explain the apparent discordance with the orthotopic transplant experiments in which miR-16 overexpression decreased metastasis, indicating that high levels of miR-16 suppress metastasis. [score:5]
Fig. 3. miR-16 overexpression suppresses lung metastasis in a nude mouse transplant mo del in vivo. [score:5]
miR-16 overexpression suppresses lung metastasis in a nude mouse transplant mo del in vivo. [score:5]
d. (B) Cell growth assay showing that ectopic expression of miRNAs does not affect cell proliferation in vitro with the exception of miR-223, which enhances the growth at days 2 and 3, and miR-146a, which suppresses cell growth at days 2 and 3. (C,D) Quantification of Matrigel assay demonstrating that miR-16, but not any of the other miRNAs, suppresses both migration and invasion, respectively. [score:5]
Restoration of miR-16 expression in mouse primary sarcoma cell lines significantly suppressed both migration and invasion in vitro and metastasis to the lung of sarcoma cells transplanted into the muscle of immunocompromised mice (an orthotopic mouse mo del of STS). [score:5]
For injection, we utilized an STS cell line derived from a primary tumor that had a high base rate of metastasis (KP1), allowing us to test whether miR-16 overexpression can suppress metastasis. [score:5]
The proteomic screen revealed that ∼300 proteins were differentially regulated at least 2-fold between the miR-16 -expressing and miR-16- deleted sarcoma cells. [score:4]
By comparing the downregulated miRNAs in metastatic sarcomas from human and mouse, we found six miRNAs common to both: miR-16, miR-103, miR-146a, miR-223, miR-342 and miR-511 (Fig.  1D,E). [score:4]
miR-16 was first reported to be frequently deleted and/or downregulated in chronic lymphocytic leukemia (CLL) at the 13q14.3 locus (Calin et al., 2002). [score:4]
They report that miR-16 is downregulated in both human and mouse metastatic STS. [score:4]
To our knowledge, our study reports for the first time that miR-16 is downregulated in both human and mouse metastatic STS. [score:4]
miR-15a and miR-16-1 down-regulation in pituitary adenomas. [score:4]
This finding suggests that restoration of miR-16 may downregulate gene(s) responsible for cellular migration and invasion. [score:4]
To search for potential targets of miR-16 that can regulate metastasis, we performed a liquid chromatography/mass spectrometry (LC/MS) proteomic screen of cell lines derived from primary sarcomas with (n=3) or without (n=3) miR-16 deletion, as previously described (Sachdeva et al., 2014). [score:4]
Therefore, one potential mechanism by which tumors might downregulate miR-16 to promote metastasis is via hypoxia. [score:4]
However, it does not rule out the possibility that, in concert with downregulation of other miRNAs, loss of miR-16 could contribute to metastasis. [score:4]
For example, stable overexpression of miR-16 suppresses both migration and invasion of cells in a Matrigel assay without affecting proliferation of the cells. [score:4]
Of note, immunostaining of sarcomas with an antibody directed against Ki-67, which is a marker of cellular proliferation because it is expressed during interphase (G1, S, G2) and mitosis (M), but not in resting G0 cells (Scholzen and Gerdes, 2000), showed no difference between the two groups suggesting that miR-16 deletion had no significant impact on proliferation of sarcoma cells in vivo (Fig.  4C,D). [score:4]
Similar analysis in mouse metastatic sarcomas revealed overlap for several downregulated miRNAs including miR-16, miR-103, miR-146a, miR-223, miR-342 and miR-511. [score:4]
In this mo del, primary sarcoma cells with or without miR-16 overexpression are injected into the muscle of immunocompromised mice and after tumors developed they were resected and the mice were followed for the development of lung metastasis. [score:4]
Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. [score:4]
Although we did not observe any deletion of the miR-16 locus in primary metastatic mouse sarcomas by comparative genomic hybridization (data not shown), there could be other factors that regulate miR-16 expression within primary sarcomas. [score:4]
Using unbiased genomic profiling, miR-16 was found to be lost in human osteosarcoma specimens and was further shown to suppress tumor growth by activating caspase-3 in xenograft studies in nude mice. [score:3]
Unlike the other miRNAs tested, overexpression of only miR-16 significantly decreased both migration and invasion of KP cells (Fig.  2C and D, respectively). [score:3]
For example, 9/10 mice developed lung metastasis in the vector control group, but only 4/10 mice developed lung metastasis from the sarcoma cells overexpressing miR-16. [score:3]
Taken together, these in vitro and in vivo results suggest that miR-16 can act as a metastasis suppressor in sarcoma. [score:3]
The discordant results reported here between the effects of miR-16 overexpression in an orthotopic transplant mo del in immunocompromised mice and miR-16 deletion in a primary tumor mo del in immunocompetent mice demonstrate the importance of utilizing complementary gain-of-function and loss-of-function approaches and primary tumor mo del systems for the study of metastasis. [score:3]
In addition, we demonstrate that miR-16 can suppress in vitro migration and invasion of primary STS cells. [score:3]
To determine whether miR-16 can suppress metastasis in vivo, we transplanted sarcoma cells into the muscle of nude mice so that the tumor cells would grow at an orthotopic site. [score:3]
Because miR-16 has been shown to suppress cell proliferation in various tissues, we next determined whether deletion of miR-16 had any effect on proliferation of sarcoma cells. [score:3]
We generated primary sarcomas by injecting an adenovirus expressing Cre recombinase (Ad-Cre) into KP mice (miR-16 WT) and into KP miR-16 [flox/ flox] mice to generate sarcomas with deletion of miR-16 (miR-16 F/F) (Fig.  4A). [score:3]
miR-16, but not other miRNAs, suppresses both migration and invasion in vitro. [score:3]
In addition, qRT-PCR confirmed that miR-16 expression is lost at the transcript level in the primary cell lines (Fig.  4B). [score:3]
To further test the functional significance of miR-16 as a metastasis suppressor gene, we used a loss-of-function approach in primary sarcomas. [score:3]
In addition, orthotopic transplantation of a sarcoma cell line stably expressing miR-16 into the muscle of immunocompromised mice revealed that restoration of miR-16 can significantly decrease lung metastasis in vivo. [score:3]
The miR-15a-miR-16-1 cluster controls prostate cancer by targeting multiple oncogenic activities. [score:3]
Mice with tumors overexpressing miR-16 were more likely to survive without metastasis to the lung. [score:3]
The lungs from mice in which orthotopic tumors overexpressed miR-16 had a decreased number of lung metastases. [score:3]
This finding suggests that decreasing miR-16 expression is not sufficient to promote lung metastasis in primary sarcomas in KP mice. [score:3]
Tumors were generated by intramuscular injection of an adenovirus expressing Cre recombinase, as previously described (Kirsch et al., 2007) into the hind limb of mice with genotype LSL-Kras [G12D/+]; p53 [fl/fl] (KP) or LSL-Kras [G12D/+]; p53 [fl/fl]; miR-15a/16-1 [fl/fl] (KP miR-16 F/F). [score:3]
These results demonstrate that miR-16 can suppress migration and invasion in vitro in multiple KP sarcoma cell lines without altering cell proliferation. [score:3]
A recent study found that miR-16 acts as a tumor suppressor in osteosarcoma (Jones et al., 2012). [score:3]
Overexpression of miR-16 significantly decreased both migration and invasion in both of these cell lines in vitro, without altering their cell proliferation (Fig.  1F,G and data not shown). [score:3]
For example, expression of miR-16 is decreased by hypoxia (Dejean et al., 2011), and hypoxia also correlates with metastasis in STS (Brizel et al., 1996; Eisinger-Mathason et al., 2013). [score:3]
These results are consistent with the in vitro experiments in which overexpression of miR-16 decreased metastatic phenotypes of invasion and migration (Fig.  2). [score:3]
However, these orthotopic transplant experiments do not address whether decreased miR-16 expression is sufficient to promote metastasis. [score:3]
Taken together, these results indicate that miR-16 can have metastasis-suppressing properties both in vitro and in vivo. [score:3]
We further demonstrate that overexpression of miR-16 decreases metastasis in an orthotopic mouse mo del in immunocompromised mice, but deletion of miR-16 in primary sarcomas fails to increase metastasis. [score:3]
Using miR-16 [flox/ flox] mice, we were able to delete two alleles of miR-16 during sarcomagenesis and then follow the mice for the development of lung metastases. [score:2]
There is no impact of miR-16 deletion on time to sarcoma development (N=18 control KP miR-16 WT mice and N=26 KP-miR-16 F/F mice). [score:2]
Later, many investigators found downregulation of miR-16 in multiple tumor types, including prostate cancer, pituitary adenomas and gastric cancer (Bonci et al., 2008; Bottoni et al., 2005). [score:2]
Overexpression of miR-16 in sarcoma cells was sufficient to limit metastasis in an orthotopic assay in immunodeficient mice, which suggests that decreased miR-16 may be necessary for metastasis in some sarcomas. [score:2]
We thank Riccardo Dalla-Favera at Columbia University for providing miR-16 [flox/+] mice. [score:1]
To our surprise, we did not observe any change in the rate of lung metastasis after deleting miR-16. [score:1]
As shown in Fig.  3A, there was no change in the growth of primary tumors between vector control and miR-16-infected cells, but a significant difference was observed in the rate and number of lung metastases (Fig.  3B,C). [score:1]
However, the loss-of-function experiments in autochthonous tumors indicate that loss of miR-16 is not sufficient to promote metastasis in vivo. [score:1]
We crossed KP mice to mice in which miR-16 was flanked by loxP sites (i. e. miR-16 [flox/ flox] mice) so that Cre recombinase can delete miR-16 (Fig.  4A). [score:1]
This loss-of-function experiment suggests that deletion of miR-16 is not sufficient to promote metastasis. [score:1]
Confirming that miR-16 is efficiently deleted in primary sarcomas and with no effect on sarcoma proliferation, we expanded a cohort of KP miR-16 [flox/ flox] and KP miR-16 [wt/wt] mice to generate primary sarcomas to study metastasis. [score:1]
However, deletion of miR-16 failed to promote metastasis in a primary tumor mo del system, which suggests that loss of miR-16 is not sufficient to promote metastasis in vivo. [score:1]
PCR showing Cre -mediated excision of miR-16 with the recombined allele from primary sarcomas (bottom panel). [score:1]
Fig. 4. Deletion of miR-16 fails to promote lung metastasis in a mouse mo del of primary sarcoma. [score:1]
PCR analysis on genomic DNA from primary cell lines generated from sarcomas with or without miR-16 deletion demonstrated efficient recombination of the miR-16 allele in KP sarcomas (Fig.  4A). [score:1]
The lentiviral vectors (System Biosciences) encoding miR-16 and other miRNAs were packaged and used to infect cell lines KP1-KP3, as described previously (Sachdeva et al., 2012). [score:1]
However, no change in the rate of lung metastasis was observed when miR-16 was deleted in mouse primary sarcomas at sarcoma initiation. [score:1]
We did not observe a significant change in tumor onset or tumor growth kinetics after deletion of miR-16 in KP tumors (Fig.  4E). [score:1]
Alternatively, the results from the primary tumor experiment might simply reflect the fact that there was an unexpectedly high rate of lung metastasis in the control KP mice, which could be a consequence of the mixed genetic background of the miR-16 [flox/ flox] mice. [score:1]
The cell lines derived from independent tumors are numbered 1-5 (red, miR-16 WT; blue, miR-16 F/F). [score:1]
Deletion of miR-16 does not increase metastasis in a mouse mo del of primary STS. [score:1]
Therefore, to better define the role of miR-16 in metastasis, we utilized a primary mo del of STS in immunocompetent mice. [score:1]
Notably, however, there was no change in the rate of lung metastasis when miR-16 was deleted in autochthonous tumors (a tumor that forms where it is found rather than being transplanted from elsewhere) in a mouse mo del of primary STS previously developed by the authors in which sarcomas develop in a spatially and temporally restricted manner and can be surgically resected so that the true metastatic potential of the primary tumor can be determined. [score:1]
Fifty thousand exponentially growing KP1 vector or KP1 miR-16 cells were injected into the hind limb muscle of the nude mice. [score:1]
Mice with tumors with deletion of miR-16 have similar survival without metastasis. [score:1]
Three cell pellets each from either miR-16 WT or miR-16 F/F cells were washed with 50 mM ammonium bicarbonate and solubilized by sonication in 200 µl of 0.2% Rapigest-SF (w/v). [score:1]
First, the primary tumor mo del system examined miR-16 deletion. [score:1]
Red asterisks denote samples with either partial recombination of the miR-16 flox allele or stromal contamination in the primary sarcoma cell line. [score:1]
The average number of lung metastases per mouse was 4.5 with vector-infected cells and 1 with miR-16-infected cells (Fig.  3C). [score:1]
After Ad-Cre injection, KP mice with miR-16 deletion developed primary sarcomas with similar kinetics to the mice with wild-type miR-16. [score:1]
Therefore, deletion of miR-16 in this experimental system may have been unable to further promote metastasis. [score:1]
To further characterize the role of miR-16 on migration and invasion of KP cells and to exclude the possibility that this miR-16 phenotype was restricted to KP cell line 1 (KP1), we stably expressed miR-16 in two additional KP cell lines (KP2 and KP3; Fig.  1E). [score:1]
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In addition, the target gene’s expression level of miR-16, BCL-2, was inhibited after H/R under C/EBP-β overexpression (Fig. 5C III). [score:9]
In our study on miR-16 targeted genes, we mainly focused on apoptosis signaling pathway gene, BCL-2. However, using integrative analysis of targetome (1,195 targets), TGF-β, PI3K-Akt, p53, GnRH, MAPK and ubiquitin mediated proteolysis signaling pathway were also found in the miR-16 regulatory network 31. [score:8]
Figure 1A shows that 30 miRNAs were significantly modulated by more than two-fold, within these seven miRNAs were upregulated (let-7d, life-26-3p, miR-16, miR-451, miR-486-5p, miR-518e*, miR-720) and twenty one were downregulated. [score:7]
High expression of miR-16 regulated by NFκ-β in gastric cancer has been observed 45, therefore, we cannot rule out the possibility that miR-16 regulated by NFκ-β may target BCL-2 during kidney I/R. [score:7]
Therefore, whether C/EBP-β is expressed in the kidney during I/R, meaning that C/EBP-β blocks BCL-2 protein expression through upregulation of pri-miR-16 and that this is necessary for kidney dysfunction homeostasis, was investigated. [score:6]
But the results both from in vitro overexpression with lenti-miR-16 (Fig. 3E) and mice infused with shRNA-miR-16 (Fig. 4) definitely demonstrated the inhibition effect of miR-16 to BCL-2 and protected kidney function effect from I/R suggestion miR-16 actually plays an important pathophysiology role in kidney. [score:5]
Whether the pathophysiological role of C/EBP-β induced miR-16 in I/R injury is via inhibition of the BCL-2 pathway, the gain-of-function of lentivirus -mediated gene transfer was used to overexpress C/EBP-β in the kidneys of sham or I/R mice. [score:5]
But in gastric adenocarcinoma 29 and human nasopharyngeal carcinoma 30, miR-16 played an inhibitory role in cancer activation, suggesting that miR-16 targets different genes on various organs resulting in differential clinical outcomes. [score:5]
Furthermore, both BCL-2 mRNA and protein expression at 2 and 4 hrs post-reoxygenation were further inhibited by miR-16 transfection and abolished by anti-sense miR-16 (Fig. 3E middle and right panel). [score:5]
These observations confirm that miR-16 binds to BCL-2 3′UTR and inhibits BCL-2 transcription and subsequent translation after H/R in vitro. [score:5]
The intrinsic relationship between BCL-2 and miR-16 in 293T cells after hypoxia/reoxygenation (H/R) also showed that the appearance of up -expression of miR-16, with maximum increase detected at 2 hrs post-reoxygenation, was accompanied by down -expression of BCL-2 after H/R. [score:5]
MiRNA plasmid was constructed using miExpress Precursor miRNA Expression clone (GeneCopoeia, MD, USA) containing murine precursor miR-16 DNA under the pEGFP plasmid. [score:5]
In the present study, we identified miR-16 as a novel direct target of C/EBP-β in tubular epithelium cells (Fig. 5). [score:4]
In this report, we show that C/EBP-β upregulates miR-16, and miR-16 blocks one of the anti-apoptotic genes, BCL-2, after I/R. [score:4]
Pri-miR-16 is transcriptionally regulated directly by C/EBP-β and targets BCL-2 by the in vitro assay. [score:4]
Whether epigenetic modification of C/REBP-β is involved in miR-16 transcription, previous emerging evidence indicates that C/REBP-β can activate EF2-regulated gene by recruiting coactivator p300 46 and acetylation by p300 resulting in C/EBP-β- mediated IL-6 and TGF-1 expression 47. [score:4]
As shown in Fig. 4A, overexpression of miR-16 significantly increased the mRNA level of miR-16 in the kidney without or with I/R compared to lentivirus GFP injection alone and overexpression of miR-16 also induced renal dysfunction (Fig. 4B), increased numbers of apoptotic TUNEL -positive tubular epithelial cells (Fig. 4C), elevated activity of the cleaved, active form of caspase-3 (Fig. 4D). [score:4]
In breast cancer and esophageal squamous cell carcinoma, miR-16 suppressed cell apoptosis while promoted growth by regulating RECK, SOX6 or RPS6KB1 27 28. [score:4]
C/EBP-β upregulated miR-16 transcription level. [score:4]
The 3′UTR of BCL2 was a direct target of miR-16. [score:4]
We cannot rule out the possibility of miR-16 targeting other proapoptotic and antiapoptotic genes like programmed cell death 11 (PDCD11) and SOCS6. [score:3]
In situ hybridization also revealed that the expression of miR-16 was predominantly within the tubular epithelial cytosol after reperfusion for 3 hrs (Fig. 2F). [score:3]
Enhanced renal dysfunction with overexpression of C/EBP-β was accompanied by increased numbers of apoptotic TUNEL -positive tubular epithelial cells, elevated activity of the cleaved, active form of caspase-3 and urinary miR-16 level (Fig. 7D,E). [score:3]
Thus, overexpression of miR-16 can induce kidney dysfunction which is accompanied by elevated miR-16 level in the urine. [score:3]
During CEBP-β alteration in kidney after I/R (bottom panel), low activity of CEBP-β fails to transactivate miR-16, which results in lack of BCL-2 inhibition and results in block of I/R induced kidney injury. [score:3]
These results suggest that inhibition of anti-apoptotic gene BCL-2 by C/EBP-β induced miR-16 pathway together with increasing urinary miR-16 level may play important pathophysiological roles in the kidneys after I/R. [score:3]
As shown in Fig. 7A,B, both C/EBP-β and pri-miR-16 expression were increased in the kidney tissue infused with lentivirus containing C/EBP-β compared to the lenti-pSin (self-inactivating vector) indicating that miR-16 was indeed transcriptionally regulated by C/EBP-β. [score:3]
In addition, renal cell carcinoma related signaling pathways molecules, like PI3kinase, VEGFR2, VEGF, EIF4E and RAS, all are miR-16 target genes 32. [score:3]
High expression level of miR-16 was observed only in acute kidney injury mice (Fig. 2E,F). [score:3]
In addition, in Fig. 3E transfected with shRNA of miR-16 only in vitro did not effect on BCL-2 expression may also be effected by theses miRs except for miR-16. [score:3]
Our in vitro H/R experiments revealed that elevated miR-16 level was accompanied by a decrease in BCL-2 protein expression (Fig. 3D right panel). [score:3]
Because anti-apoptotic effects of PI3kinase, VEGFR2, VEGF, EIF4E, RAS may result from its anti-apoptosis or anti-oxidative effect after I/R, inhibiting these genes by miR-16 would result in greater apoptosis and hence larger damage to the kidneys. [score:3]
Lorenzenz et al. also demonstrated that the levels of circulating miRs including miR-16, 24, 1244, 620, 320, 30d, let 7f and let 7b all were upregulated in patients with AKI compared to healthy controls or patients with acute myocardial infarction (non-AKI) 41. [score:3]
In our study, results from clinical patients definitely indicated that up -expression of urinary miR-16 may be served as a biomarker for AKI patients. [score:3]
Therefore, these miR-16 related targeted genes may also involve in I/R induced nephropathy. [score:3]
After I/R, increasing amount of mature miR-16 are cleaved from pri-miR-16 by Dicer, which then inhibit one of the anti-apoptotic protein, BCL-2, leading to severe kidney injury (Fig. 8). [score:3]
Using semi-quantitative qPCR, we found that miR-16 was expressed highly in the heart, moderately in the kidneys, testes and lung, and lowest in the liver, brain and spleen (Fig. 2A). [score:3]
The other possibility of high urinary miR-16 may result from increased miR-16 expression in the podocytes, which perturbs the actin cytoskeleton, and increases the release of exosomes containing miR-16 as previously reported 43 (Fig. 8). [score:3]
Overexpression of epigenetic C/EBP-β by lentivirus can increase urinary miR-16 after I/R in the kidney. [score:3]
Finally, urinary miR-16 is stable and expressed earlier than creatinine, which makes it a useful indicator for AKI patients. [score:3]
In this study, we demonstrated that miR-16 was regulated transcriptionally by C/EBP-β and whether other miRs are regulated by C/EBP-β resulting in kidney dysfunction will be evaluated by comparing the microRNA array analysis from the knock down of C/EBP-β in 293T cells after H/R 24 hours. [score:2]
These results suggest that C/EBP-β interacts with the miR-16 regulatory region. [score:2]
To study the direct interaction between miR-16 and BCL-2 transcription, the 3′UTR of BCL-2 downstream of the fluorescent reporter gene was cloned into the pRFP-C1 vector (RFP-BCL-2-3′UTR); precursors of miR-16 was constructed into pEGFP plasmids (pEGFP-premiR-16). [score:2]
Our results demonstrated a novel mechanism for C/EBP-β to regulate miR-16 in the kidney pathophysiology. [score:2]
In contrast, overexpression of antisense-miR-16 in mice attenuated I/R -induced renal dysfunction compared to lenti-pSin controls (Fig. 4F). [score:2]
To further address the role of miR-16 in I/R injury, we compared the renal function (as assessed by blood urea nitrogen and creatinine) of mice with or without overexpression of either miR-16 or antisense-miR-16 in the presence or absence of renal I/R. [score:2]
We were particularly interested in miR-16, a previously reported tumor suppressor in leukemia 19, which showed the largest increase in AKI patients compared to normal subjects and also revealed consistent result with real time polymerase chain reaction (qPCR) (Fig. 1B). [score:2]
To quantify the amount of mRNA (BCL-2 and CEBP-β) and pri-miR-16, we used TaqMan Gene Expression Assay using GAPDH as an internal control. [score:2]
We also showed that miR-16 was expressed at higher levels in the urines of AKI patients in the ICU compared to patients without AKI or normal individuals. [score:2]
Figure 5D indicates that overexpression of C/EBP-β increased the amount of C/EBP-β protein recruited to the miR-16 promoter region compared to the scrambled control. [score:2]
For the detection of miR-16 expression, we used TaqMan MicroRNA assays (Life Technologies) using small nuclear U6B (RNU6B) RNA as an internal standard. [score:2]
Schematic representation of regulation of kidney function by CCAAT enhancer binding protein beta (C/EBP-β)–microRNA-16(miR-16)-B cell lymphoma 2 (BCL-2) axis. [score:2]
Several putative transcriptional factor binding sites including ATF3/CRE, PPARα-RXRα, NF-κB, C/EBP-β were found in the upstream promoter regions of miR-16 gene (Fig. 5A). [score:1]
Thus, increased urinary miR-16 level reflects the condition of AKI. [score:1]
Increased urinary miR-16 concentration reflected AKI condition. [score:1]
During ischemia-reperfusion (I/R), C/EBP-β (top panel) transactivates miR-16 which, in turn, leads to BCL-2 repression and activation of epithelium cells apoptosis, resulting in kidney function loss. [score:1]
However, the urinary level of miR-16 of ICU patients with AKI was significantly higher than those without AKI and normal volunteers (Fig. 1D, left). [score:1]
Normally, C/EBP-β presents in the cytosol, and after I/R, it translocates into the nucleus and binds to the promoter region of pri-miR-16 genome resulting in the elevated level of pri-miR-16 in the nucleus and its subsequent translocation into the cytosol. [score:1]
The putative promoters of the Homo sapiens miR-16 were predicted by miRstart (http://mirstart. [score:1]
The serum miR-16 levels among the healthy volunteers, ICU patients with and without AKI did not differ significantly (Fig. 1C). [score:1]
Dicer and associated miRs have been reported to involve in I/R injury in the kidney 20, and I/R injury is a major cause of AKI meaning miR-16 may come from immature miR-16, pri-miR-16, after I/R injury. [score:1]
We also demonstrated that miR-16 can be detected by using capped gold nanoslit SPR in a microfluidic chip after freeze-throw four times (Supplemental Fig. S2). [score:1]
Paraffin-fixed sections of mouse kidney were hybridized with the digoxigenin-labeled miR-16 probe and nuclei staining was stained by Contrast green. [score:1]
Figure 5E shows that significantly reduced luciferase activity was observed in the miR-16 promoter −1.0 to −0.5 kb region, but not in the −0.5 to −0.0 kb region (Fig. 5E). [score:1]
Twenty-four hours after Lenti-pSin, Lenti-miR16, or Lenti-CEBPβ transfection, HEK293T cells were incubated under conditions of normoxia or hypoxia (1% O [2]) for 4 hrs and reperfusion for the indicated time. [score:1]
Urinary miR-16 was induced earlier than traditional kidney injury marker, urea or creatinine, in a mouse I/R renal failure mo del. [score:1]
Promoter analysis indicated that upstream genome region of miR-16 contains NFκ-β, PPARα-RXRα, ATF3/CRE and C/REBP-β binding sites, but except for NFκ-β and C/REBP-β, the other transcription factors failed to induce miR-16 (Supplemental Fig. S1). [score:1]
To further confirm the relationship between C/EBP-β and miR-16 after H/R, both C/EBP-β and miR-16 were all increased in 293T cells after 24 hrs of reoxygention (Fig. 5B). [score:1]
In vitro hypoxia-reoxygenation experimentTwenty-four hours after Lenti-pSin, Lenti-miR16, or Lenti-CEBPβ transfection, HEK293T cells were incubated under conditions of normoxia or hypoxia (1% O [2]) for 4 hrs and reperfusion for the indicated time. [score:1]
After 24 hrs, the cells were divided into four groups and each was transfected separately with the following four vectors including pRFP-BCL-2 3′UTR, scramble negative control, EGFP-miR-16 and miR-16 antisense. [score:1]
293T cells were transfected with pGL4, pGL4-miR16 promoter luciferase reporter vectors (pGL4-miR16-p0.5k, pGL4-miR16-p1k, pGL4-miR16-p2k) and Renilla vector using Lipofectamine 2000 (Invitrogen). [score:1]
Without (left) or with infused miR-16 (right) mice underwent a sham operation (top) or 45 mins of renal clamping to induce ischemia, followed by 6 hours of reperfusion (bottom). [score:1]
org), we identified that nucleotides 2455–2462 of the 3′UTR of mouse BCL-2 and 2529–2536 of the 3′UTR of human BCL-2 are complementary to seed sequences of miR-16 (Fig. 3A). [score:1]
How to cite this article: Chen, H. -H. et al. Urinary miR-16 transactivated by C/EBPβ reduces kidney function after ischemia/reperfusion–induced injury. [score:1]
To our knowledge, this is the first report that shows the mechanism for urinary miR-16 levels enhancement by C/EBP-β after I/R in the kidney. [score:1]
Likewise, urinary miR-16 level was reduced by in vivo lentivirus -mediated antisense-miR-16 gene transfer into the kidneys (Fig. 4G). [score:1]
It has been shown that miR-16-5p may be a prospective biomarker for gastric cancer and its progression by the variation of its plasma level 37. [score:1]
Whole-mount in situ hybridizationLocked nucleic acid -modified miR-16 oligonucleotide probe (Exiqon, Vedbaek, Denmark) was labeled with digoxigenin. [score:1]
The fluorescent activity was reversed and returned to control level by miR-16 anti-sense transfection (Fig. 3B). [score:1]
Locked nucleic acid -modified miR-16 oligonucleotide probe (Exiqon, Vedbaek, Denmark) was labeled with digoxigenin. [score:1]
The elevated urinary miR-16 appeared earlier than the elevated serum urea and creatinine (Fig. 2C). [score:1]
In addition, Mall et al. showed that miR-16 and miR-21 are relatively stable in the urine under a variety of storage conditions, which supports their utility as urinary biomarkers 40. [score:1]
Whether urinary miR-16 may exist as extracellular vesicle form called microparticle miR-16 (Fig. 8) and inhibit BCL-2 activity by endocytosis will be evaluated later. [score:1]
However, most of these miR-16 studies failed to determine whether it is related to kidney dysfunction. [score:1]
Figure 2B shows that serum urea and creatinine level were increased after ischemia followed by reperfusion for 3 hrs, however, urinary miR-16 was significantly increased after reperfusion for 1 hr. [score:1]
Serum miR-16 values differed significantly between the control and ICU patients with AKI or between ICU patients with and without AKI. [score:1]
High level of urinary miR-16 in AKI patients may come from the breakdown of tubular cells mediated by decreased BCL-2, resulting in apoptosis or necrosis 42, which may explain why high level of urinary miR-16 were found in our clinical AKI patients (Fig. 1A,B). [score:1]
Role of miR-16 in I/R -induced renal function and apoptotic response. [score:1]
293T Cells (4 × 106) were seeded into 10-cm [2] culture dishes in 5.5 ml of the medium and transfected the following day with 2 μg of pMD-G plasmid, 8 μg of pCMV8.9 plasmid, and 12 μg of Lenti-miR16 vector plasmid (GeneCopoeia, MD, USA), antisense-miR-16 (SBI CA, USA) or Lenti-CEBβ. [score:1]
Proportions of TUNEL -positive renal epithelial nuclei to total nuclei in mice infused with or without miR-16 and subjected to the sham operation or I/R injury are shown. [score:1]
Figure 2E (left) indicates that pri-miR-16 was rapidly declined from 1 hr lasting to 6 hrs after reperfusion, in contrast, the level of miR-16 was increased from 1 hr to 6 hrs after reperfusion in mice (Fig. 2E right). [score:1]
These results suggest that miR-16 plays important pathophysiological roles in the kidneys after I/R and urinary miR-16 level may be a biomarker for kidney injury. [score:1]
Most miR-16 studies have been focused on cancer and conflicting results have been reported. [score:1]
Our findings provide new insights for the C/EBPβ -mediated microRNA induced kidney dysfunction progression, which is the key step to disrupt renal epithelium cells resulting in elevated urinary miR-16 level. [score:1]
Briefly, 293T cells (5 × 10 [5]) were seeded into 6-well plates and transfected with pEGFP and pEGFP-miR-16 plasmids respectively. [score:1]
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Other miRNAs from this paper: mmu-mir-16-2
In addition, we validated VEGF as a direct target gene for miR-16, and we found that miR-16 negatively regulates VEGF expression by directly targeting the 3′UTR of VEGF mRNA in HTR-8/SVNEO and JEG-3 cells. [score:10]
mir-16 inhibits placentation and angiogenesis in vitro and in vivoConsidering the inhibitory effect of miR-16 on VEGF expression, which is related to placental angiogenesis, maintenance and stabilization, we assessed the anti-angiogenic activity of miR-16 by transfecting Human Umbilical Vein Endothelial Cells (HUVECs) with a miR-16 mimic/inhibitor in vitro. [score:9]
Immunohistochemistry also showed similar results in which miR-16 overexpression inhibited placental angiogenesis, whereas the inhibition of miR-16 expression induced placental angiogenesis (Fig. 3I,J). [score:9]
miR-16 directly targeted VEGF 3′UTR and down-regulates its expression. [score:9]
To verify whether miR-16 directly suppresses VEGF expression, we analyzed the potential miR-16 seed sequence in the 3′UTR of VEGF using the online publically available algorithms (TargetScan andmiRanda) and cloned a wild-type or mutant fragment containing the binding sequence (376 bp) into the luciferase reporter gene system (Fig. 5B). [score:8]
The overexpression of miR-16 in HUVECs significantly inhibited HUVEC proliferation and migration, whereas the reduction of miR-16 expression promoted HUVEC proliferation and migration (Fig. 2B–D). [score:7]
In vitro, miR-16 overexpression in HUVECs suppresses proliferation, migration and tube formation, suggesting that miR-16 may be a novel suppressor of and may play a critical role in angiogenesis. [score:7]
Considering the inhibitory effect of miR-16 on VEGF expression, which is related to placental angiogenesis, maintenance and stabilization, we assessed the anti-angiogenic activity of miR-16 by transfecting Human Umbilical Vein Endothelial Cells (HUVECs) with a miR-16 mimic/inhibitor in vitro. [score:7]
One study suggested that miR-16 inhibits angiogenesis, trophoblast cell proliferation, and migration, and inhibits angiogenesis through VEGF suppression 21. [score:7]
Overall, the current study found that miR-16 expression is upregulated in the villi and decidua of RSA patients. [score:6]
However, the inhibition of miR-16 upregulated VEGF levels (Fig. 5C,D). [score:6]
Western blot analysis revealed that VEGF expression was significantly downregulated by miR-16 mimic transfection in HTR-8/SVNEO and JEG-3 cells. [score:6]
However, injection with miR-16 inhibitors significantly upregulated the PCNA protein levels (Fig. 4E,F). [score:6]
Immunohistochemistry and western blotting confirmed that the VEGF protein expression level was significantly decreased by miR-16 mimic injection but significantly elevated by miR-16 inhibitor injection (Fig. 4A–D). [score:5]
In addition, overexpression due to injection of the placenta with miR-16 mimics significantly decreased the protein expression of Proliferating Cell Nuclear Antigen (PCNA). [score:5]
In the clinical specimens, the VEGF levels in the decidua with high miR-16 expression were significantly lower than those in the decidua with low miR-16 expression (Fig. 5A). [score:5]
control mimic/inhibitor group, n = 5. (A) An inverse relationship between the expression of miR-16 and VEGF was found in clinical decidual specimens of RSA patients (scale bar = 50 μm). [score:5]
Subsequent histologic analysis revealed that the total placental vasculature and the number of capillaries were significantly inhibited by miR-16 mimic injection but significantly promoted by miR-16 inhibitor injection (Fig. 3G,H, Table 1). [score:5]
Moreover, we discovered that miR-16 overexpression significantly repressed HUVEC tube formation, whereas transfection with the miR-16 inhibitor increased tube formation (Fig. 2E,F). [score:5]
VEGF is a direct target of mir-16. [score:4]
miR-16 regulates VEGF expression and placental proliferation in vivo. [score:4]
How to cite this article: Zhu, Y. et al. MicroRNA-16 inhibits foeto-maternal angiogenesis and causes recurrent spontaneous abortion by targeting vascular endothelial growth factor. [score:4]
A recent study indicated that miR-16 inhibits the proliferation, migration and angiogenesis -regulating potential of mesenchymal stem cells 22. [score:4]
The results showed that overexpression of miR-16 in both cell lines led to significantly reduced luciferase activity for the wild-type, whereas the miR-16 knockdown increased wild-type luciferase activity. [score:4]
The higher expression of miR-16 was confirmed in the villi of RSA cases (Fig. 1C). [score:3]
All placentas in a single litter received the same miRNA (control or miR-16 mimic/inhibitor) injection. [score:3]
Importantly, quantification of the data indicated that miR-16 was expressed at significantly higher levels in the villi and decidua of the RSA patients than that in the controls. [score:3]
We found that the expression of miR-16 in the decidua of the RSA group was significantly greater than that in the decidua of the control group (Fig. 1B). [score:3]
In contrast, the activity of the luciferase reporter gene linked to the 3′UTR of mutant VEGF did not change in the presence of the miR-16 mimic/inhibitor (Fig. 5E,F). [score:3]
To demonstrate whether miR-16 affects the expression of VEGF, we first evaluated the correlation between VEGF expression and the miR-16 level in placentas of pregnant mice. [score:3]
In contrast, transfecting HUVECs with a miR-16 inhibitor significantly decreased the miR-16 levels (Fig. 2A). [score:3]
HUVECs were transfected with control or miR-16 mimic/inhibitor for 48 h. (A) qPCR analysis was performed to examine miR-16 levels. [score:3]
Tumor cell-conditioned medium was prepared from 1 × 10 [6] JEG-3 cells transfected with a miR-16 mimic or inhibitor, which were cultured in the same conditions for 48 h. Images showing the formation of capillary-like structures were obtained after 12 h with an inverted microscope (Olympus, Tokyo, Japan) at 50 ×  magnification. [score:3]
Therefore, these results suggested that miR-16 is overexpressed in RSA. [score:3]
Aberrantly high miR-16 expression in RSA cases. [score:3]
mir-16 inhibits placentation and angiogenesis in vitro and in vivo. [score:3]
The miR-16 mimic/inhibitor and the redesigned luciferase reporter plasmid were then co -transfected into HTR-8/SVNEO and JEG-3 cells. [score:3]
In the present study, we initially detected the expression level of miR-16 in samples of RSA patients and control women. [score:3]
Taken together, the identification of miR-16 provides a potential diagnostic marker and therapeutic target for RSA patients. [score:3]
Our data are consistent with a previous study demonstrating that miR-16 exerts inhibitory biological features during placental angiogenesis. [score:3]
Our data revealed that the important molecular mechanism by which miR-16 exerts its negative effects on placental angiogenesis occurs via VEGF suppression. [score:3]
These results indicate that the status of miR-16 expression should be determined in RSA patients. [score:3]
Although miR-16 overexpression did not affect the number of embryos, the number of progeny was significantly reduced after miR-16 injection (Fig. 3B,C). [score:3]
In this study, we demonstrated that miR-16 mediates the reduction of VEGF and found that normal VEGF expression is a notable feature and may be one of the many critical events that occur in placental angiogenesis. [score:3]
Aberrant angiogenesis may be partially attributed to overexpressed miR-16 in the placenta of RSA patients. [score:3]
Moreover, the weights of the placenta and embryo were significantly decreased in the miR-16 overexpression group compared with the control group (Fig. 3D,E). [score:2]
qPCR analysis demonstrated that transfecting HUVECs with the miR-16 mimic increased the miR-16 levels by approximately 14-fold over the endogenous transcription level. [score:1]
To identify the effect of miR-16 on placental angiogenesis in vivo, cholesterol-conjugated miRNAs were locally injected into the placenta using a percutaneous ultrasound -guided approach on day 7.5 of pregnancy. [score:1]
Impact of miR-16 on placentation and angiogenesis. [score:1]
The miR-16 mimics and control mimics were purchased from Ambion (Austin, TX, USA). [score:1]
These data imply that miR-16 may play a critical role in placental angiogenesis. [score:1]
Moreover, injection with miR-16 in the placenta of pregnant mice caused remarkably decreased placental vasculature and microvascular density, leading to aberrant placentation and spontaneous abortion. [score:1]
The abundance of mir-16 in the villi and deciduais elevated in RSA patients. [score:1]
The effect of miR-16 on HUVEC proliferation, migration and tube formation. [score:1]
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10
[+] score: 209
Other miRNAs from this paper: mmu-mir-150, mmu-mir-16-2, mmu-mir-29c, mmu-mir-221
In conclusion, our current study show miR-16-5p is significantly downregulated in breast carcinoma, and its overexpression contributes to growth inhibition in vitro and in vivo, cell apoptosis and the decrease of invasion ability, which is at least in part achieved by directly targeting VEGFA. [score:11]
To verify whether miR-16-5p overexpression contributes to growth inhibition of breast carcinoma, lentiviral vector armed with miR-16-5p was used to transfect breast carcinoma cell lines, we found lentiviral vector carrying miR-16-5p significantly increased miR-16-5p level in breast carcinoma cells, and further investigation found miR-16-5p overexpression markedly inhibited tumor growth in vitro and in vivo, suggesting miR-16-5p may be a potential molecular target for the patients with breast carcinoma. [score:9]
To deeply understand the molecular mechanisms of miR-16-5p in vivo, we found that miR-16-5p overexpression evidently downregulated HIF-α and VEGF protein expression in nude mice tumor tissues, which were an accepted fact that these two proteins play an essential role in the development and progression of many tumors by multiple different mechanisms [36– 46]. [score:9]
Most notably, our current results confirmed miR-16-5p mediated biology function may be tightly related to HIF-α and VEGFA expressions, which was directly correlated with tumor development and progression, and thus manipulation of miR-16-5p may be a novel molecular target for the patients with breast carcinoma. [score:7]
These data suggest that miR-16-5p suppresses VEGFA protein expression by directing binding to its 3′-UTR region. [score:6]
Cells with different treatments (2000/well) were seeded into 96-well plates, CCK-8 kits were was added to corresponding wells and absorbance value at 450 nm were determined using microplate reader at different time points including days 1, 2, 3, 4 and 5. (E) Overexpression of miR-16-5p markedly inhibited colony formation in MCF-7 and MDA-MB-231 cells. [score:5]
The results showed that expressions of HIF-α and VEGFA proteins in miR-16-5p treatment groups were obviously lower than those in blank and NC groups; however, there were no differences in expressions of HIF-α and VEGFA proteins between blank group and NC group (Figure 7A and 7B, Figure 8A–8D). [score:5]
Overexpression of miR-16-5p contributed to the suppresses of cell proliferation and colony formation in MCF-7 and MDA-MB-231 cells. [score:5]
Overexpression of miR-16-5p significantly suppressed tumor growth in MCF-7 and MDA-MB-231 xenografts. [score:5]
Cells with different treatments (2000/well) were seeded into 96-well plates, CCK-8 kits were was added to corresponding wells and absorbance value at 450 nm were determined using microplate reader at different time points including days 1, 2, 3, 4 and 5. (D) Overexpression of miR-16-5p significantly inhibited the proliferation of MDA-MB-231 cells. [score:5]
Overexpression of miR-16-5p suppressed cell invasion ability in MCF-7 and MDA-MB-231 cells. [score:5]
Overexpression of miR-16-5p reduced HIF-α and VEGFA expression in MCF-7 and MDA-MB-231 xenografts. [score:5]
The downstream target genes of miR-16-5p were predicted by three online programs with different databases involved in various algorithms, such as TargetScan (http://www. [score:5]
These data support that miR-16-5p functions as tumor suppressor in the development and progression of breast carcinoma. [score:4]
However, miR-16-5p was significantly upregulated in Kaposi's sarcoma [32]. [score:4]
The results demonstrated that miR-16-5p overexpression significantly suppressed cell proliferation and colony formation ability both in MCF-7 cells and MDA-MB-231 cells, compared with blank and NC groups (Figure 2C–2E). [score:4]
VEGFA is a direct target of miR-16-5p. [score:4]
VEGFA as a direct target of miR-16-5p. [score:4]
To verify whether VEGFA was the direct target of miR-16-5p, VEGFA-3′-UTR-WT and VEGFA-3′-UTR-MUT were constructed (Figure 5A). [score:4]
Zhang J, et al. found miR-16-5p was downregulated in gastric carcinoma [31], which was consistent with our results in breast carcinoma. [score:4]
MiR-16-5p overexpression contributed to the inhibition of proliferation and colony formation ability in breast carcinoma cells. [score:4]
To confirm the roles of miR-16-5p in the development and progression of breast carcinoma, was utilized to detect the expression of miR-16-5p in breast carcinoma tissues. [score:4]
These findings suggest that miR-16-5p may be tightly associated with the development and progression of breast carcinoma, and thus miR-16-5p will provide a novel molecular target for therapy of the patients with breast carcinoma. [score:4]
Finally, tumor growth curve was made to determine the effect of miR-16-5p expression on tumor growth. [score:3]
To verify the relationship between miR-16-5p and HIF-α as well as VEGFA expressions in breast carcinoma, immunohistochemistry and were employed to investigate the HIF-α and VEGFA expressions in nude mice tumor tissues. [score:3]
ORG and miRDB were used to investigate the potential target of miR-16-5p, and preliminarily confirmed VEGFA was a potential target of miR-16-5p (Figure 5A). [score:3]
These findings highlight the potential therapeutic value of miR-16-5p in breast carcinoma, and combination of miR-16-5p with the related signaling pathway of HIF-α and VEGFA may be an effective molecular target for the patients with breast carcinoma in future. [score:3]
These findings suggest that miR-16-5p may be a potential molecular target for the patients with breast carcinoma. [score:3]
We found that the invasive cell numbers in miR-16-5p overexpression group was significantly lower than those in blank and NC groups (P < 0.05) (Figure 4A and 4B). [score:3]
Figure 174 cases of breast carcinoma tissues and corresponding normal breast tissues were collected from the First Affiliated Hospital of Zhengzhou University, total RNA and proteins were extracted from the tissues above, and andting were used to detect the miR-16-5p as well as HIF-α and VEGFA protein expression, respectively. [score:3]
We found that the expression of miR-16-5p in breast carcinoma tissues was significantly lower than that in paired normal tissues (P < 0.05) (Figure 1A). [score:3]
These findings suggest that antitumour efficacy of miR-16-5p in breast carcinoma may be partly achieved by reducing HIF-α and VEGFA expressions. [score:3]
MCF-7 and MDA-MB-231 cells were infected using LV1-miR-16-5p and LV1-NC viruses, and clones stably expressing miR-16-5p and NC were selected using puromycin (Sigma-Aldrich, USA) according to manufacturer's instructions. [score:3]
To further verify the roles of miR-16-5p in cell apoptosis as well as invasion of breast carcinoma, we found that miR-16-5p overexpression significantly induced cell apoptosis, meanwhile, reduced invasion ability in breast carcinoma cells. [score:3]
Overexpression of miR-16-5p induced apoptosis in breast carcinoma cells. [score:3]
74 cases of breast carcinoma tissues and corresponding normal breast tissues were collected from the First Affiliated Hospital of Zhengzhou University, total RNA and proteins were extracted from the tissues above, and andting were used to detect the miR-16-5p as well as HIF-α and VEGFA protein expression, respectively. [score:3]
Overexpression of miR-16-5p reduced cell invasion ability in breast carcinoma cells. [score:3]
In vivo experiment suggests that miR-16-5p may be a novel tumor molecular target for breast carcinoma. [score:3]
Therefore, miR-16-5p may be a potential molecular target for breast carcinoma. [score:3]
Expression profiles of miR-16-5p as well as HIF-α and VEGFA proteins in breast carcinoma. [score:3]
To further elucidate the molecular mechanisms mediated by miR-16-5p, TargetScan, MicroRNA. [score:3]
miR-16-5p significantly suppressed tumor growth in MCF-7 and MDA-MB-231 xenografted tumors. [score:3]
Overexpression of miR-16-5p induced cell apoptosis in MCF-7 cells and MDA-MB-231 cells. [score:3]
More detailed and new insights into molecular mechanisms of miR-16-5p in the development and progression of breast carcinoma are urgently needed to be elucidated, which will build a solid foundation for the clinic transformation of miR-16-5p in the future. [score:2]
To construct VEGFA-3′-UTR-wild type (VEGFA-3′-UTR-WT) vector, human VEGFA 3′-UTR region with miR-16-5p binding sequences was amplified, which was ligated to the pGV126 vector (GeneChem, China), VEGFA-3′-UTR-mutation (VEGFA-3′-UTR-MUT) vector with a substitution of 12 bp in miR-16-5p binding region. [score:2]
The results revealed that miR-16-5p significantly reduced the luciferase activity of VEGFA-3′-UTR-WT, but didn't affect that of VEGFA-3′-UTR-MUT in MCF-7 and MDA-MB-231 cells (Figure 5B and 5C), implying miR-16-5p directly binds to the 3′-UTR region of VEGFA. [score:2]
Further bioinformation assay revealed that VEGFA may be the potential target gene of miR-16-5p. [score:2]
The current results revealed that overexpression of miR-16-5p markedly induced apoptosis in MCF-7 and MDA-MB-231 cells, compared with blank and NC groups (Figure 3A and 3B). [score:2]
Lentiviral vector carrying miR-16-5p significantly increased miR-16-5p level in breast carcinoma cells. [score:1]
Stepwise investigation revealed that the expression of miR-16-5p in breast carcinoma cells were significantly lower than that in benign non-tumorigenic MCF10A cells (P < 0.05) (Figure 1C), in which MCF-7 and MDA-MB-231 exhibited lowest endogenous miR-16-5p level (P < 0.01) (Figure 1C). [score:1]
Figure 3(A) miR-16-5p induced apoptosis of MCF-7 cells. [score:1]
Figure 2Lentiviral vector armed with miR-16-5p and control empty vector were transfected to breast carcinoma cell lines MCF-7 and MDA-MB-231 cells, and stable clones were selected using G418. [score:1]
Tumor growth curve was made to determine the effects of miR-16-5p on tumor growth. [score:1]
To explore the antitumor efficacy of miR-16-5p in breast carcinoma, MCF-7 and MDA-MB-231 exnografts were established by injecting different doses MCF-7 and MDA-MB-231 cell numbers with different treatment. [score:1]
These results reflect the facts that miR-16-5p level may depend on tumor types, and further exerts different biological role in various tumors. [score:1]
To investigate the function of miR-16-5p in breast carcinoma, lentiviral vector carrying miR-16-5p and control vector were transfected to breast carcinoma cells, and real-time quantitative PCR was employed to determine the expression of miR-16-5p in MCF-7 and MDA-MB-231 cells. [score:1]
Lentiviral vector armed with miR-16-5p and control empty vector were transfected to breast carcinoma cell lines MCF-7 and MDA-MB-231 cells, and stable clones were selected using G418. [score:1]
Reduced miR-16-5p as well as HIF-α and VEGFA levels in breast carcinoma. [score:1]
MCF-7 and MDA-MB-231 cells were co -transfected using reporter plasmids (400 ng per 20 ng internal control renilla luciferase plasmid pRL-SV40) and LV1-miR-16-5p or LV1-NC by Lipofectamine 2000 (Invitrogen, USA). [score:1]
In this study, real-time quantitative PCR was used to investigate the miR-16-5p in 74 cases breast carcinoma and matched normal tissues, we found miR-16-5p exhibited lower expression in breast carcinoma tissues than in normal breast tissues, which was supported by the results from different breast carcinoma cells. [score:1]
Investigation from the other groups showed that miR-16-5p expression was exhibited at similar levels in most tissues [26], and was recommended as an internal control in breast tissues or serum and plasma [27– 30]. [score:1]
These findings suggest the essential role of miR-16-5p in the apoptosis of breast carcinoma cells. [score:1]
Our current investigation revealed that miR-16-5p was downregulated in breast carcinoma in 74 cases of breast carcinoma tissues and paired normal breast tissues by real-time quantitative PCR, which will impel us to further investigate the biological function of miR-16-5p in the development and progression of breast carcinoma, which was not so far reported in the world. [score:1]
Further investigation demonstrated that miR-16-5p overexpression reduced VEGFA and HIF-α protein levels in MCF-7 and MDA-MB-231 (Figure 5D and 5E). [score:1]
To further confirm the role of miR-16-5p in cell invasion ability in breast carcinoma, transwell chamber was used to detect cell invasion ability in different treatment MCF-7 and MDA-MB-231 cells. [score:1]
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[+] score: 173
Up-regulation of exosomal miR-16 by EGCG treatment down-regulates IKKα and subsequently induces IκB accumulation in TAM, and inhibits M2 polarization. [score:9]
Together, the results suggest that EGCG treatment leads to up-regulation of miR-16, which might be transferred by exosomes to TAMs, and contributes to the suppression of NF-κB activity by down -regulating IKKα and subsequently accumulating Iκ-B, and inhibition of TAM infiltration and M2 polarization. [score:9]
To address this issue, we first screened the miRNAs whose expressions are modulated in 4T1 cells by miRNA microarray analysis using both total cellular miRNA and exosomal miRNA after treatment with 100 μM of EGCG for 24 h. In brief, a set of miRNAs including let-7, miR-16, miR-18b, miR-20a, miR-25, miR-92, miR-93, miR-221, and miR-320 were up-regulated, and dozens of miRNAs including miR-10a, miR-18a, miR-19a, miR-26b, miR-29b, miR-34b, miR-98, miR-129, miR-181d were down-regulated in both total cellular and exosomal fraction by EGCG treatment (data not shown). [score:9]
Figure 4 Exosomes derived from EGCG -treated tumor cells suppresses NF-κB pathway and inhibits M2 polarization of tumor -associated macrophages, which is dependent on up-regulated miR-16 in EGCG -treated tumor exosomes. [score:8]
MiR-16 expression was not elevated in exosomes from EGCG -treated and miR-16 inhibitor -transfected 4T1 cells (data not shown), unlike exosomes from EGCG -treated 4T1 cells where miR-16 was up-regulated by EGCG (Figure 3). [score:8]
In this study, we have revealed for the first time that EGCG modulates the miRNA profile within tumor exosomes and upregulates miR-16, which was responsible for EGCG -treated exosome down -regulating IKKα and inhibiting M2 polarization of TAM. [score:7]
MiR-15a and miR-16 have been known to act as a negative regulator of NF-κB activity by regulating IKKα expression, which contributes to the ability of miR-15 and niR-16 as a tumor suppressor. [score:7]
Considering that miR-16 can function as a tumor suppressor, it is possible that up-regulated exosomal miR-16 might also have had an effect on the survival and proliferation of tumor cells in our in vivo experiment. [score:6]
Our data demonstrate that EGCG up-regulates miR-16 in tumor cells, which can be transferred to TAM via exosomes and inhibits TAM infiltration and M2 polarization. [score:6]
Therefore, we tested whether exosomes derived from EGCG -treated 4T1 cells could suppress IKKα expression and, if so, whether that process occurs through exosomal miR-16. [score:5]
Overexpression of miR-16 was shown to suppress the self-renewal and growth of mouse breast tumor stem cells and to sensitize MCF-7 human breast cancer cells to the chemotherapeutic drug doxorubicin [47]. [score:5]
In fact, a study has reported that during monocyte-macrophage differentiation, expressions of miR-15a and miR-16 were decreased with higher expression of the IKKα [30]. [score:5]
Web resources was used to predict miR-16 targets, including a viewer for browsing potential target sites, conserved with or without positional constraints, on aligned UTRs, with periodic updates (http://www. [score:5]
The scramble miRNA inhibitor or miR-16 inhibitor (100 nM) were obtained from Shanghai GenePharma Co (Shanghai, China). [score:5]
These cells were transfected with either scramble miRNA inhibitor or miR-16 inhibitor using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). [score:5]
EGCG up-regulated miR-16 in 4T1 cells and in the exosomes. [score:4]
EGCG up-regulates cellular and exosomal miR-16 levels in murine breast cancer cells, 4T1. [score:4]
Moreover, we revealed that EGCG modulates miRNAs, particularly up-regulates miR-16, which is transferred to adjacent tumor cells and TAM via tumor-derived exosomes and which has an influence on macrophages in tumor microenvironment. [score:4]
Among the miRNAs by modulated by EGCG, we focused on miR-16 because it had been identified as one of the down-regulated miRNAs in murine and human breast cancer cells. [score:4]
Additionally, targets of miR-16 include many genes related to the control of cell-cycle progression, such as cyclin D1, cyclin E, and the anti-apoptotic protein, Bcl-2 [43- 45]. [score:3]
Figure 3 EGCG increases cellular and exosomal miR-16 expression in murine breast cancer cell line, 4T1. [score:3]
Moreover, when TAM was incubated with exosomes from EGCG -treated and miR-16 inhibitor -transfected 4T1 cells, IKKα levels had recovered to the that of the control (4T1 exosome treated TAM) (Figure 4B). [score:3]
In addition to the role of tumor suppressor, miR-16 plays a role in macrophages. [score:3]
We verified that the levels of miR-16 are reduced by transfection of miR-16 inhibitor (Additional file 1). [score:3]
Significant up-regulation of miR-16 in both EGCG -treated cells and exosomes was observed with a 1.45 and 2.54 folds change compared with the levels of controls (p < 0.05 and p < 0.005, respectively) (Figure 3). [score:3]
We found that miR-16 mimics significantly suppressed LPS -induced IL-1β and IL-6 production in these RAW264.7 cells (Additional file 2). [score:3]
For example, IKKα mRNA is a target for miR-15 and miR-16. [score:3]
More importantly, this alteration of cytokines was restored when TAM was incubated with exosomes from EGCG -treated and miR-16 inhibitor -transfected 4T1 cells (Figure 4C). [score:3]
Click here for file 4T1 cells were incubated with EGCG (100 μM) and simultaneously (24 hours) transfected with scramble or miR-16 inhibitor for 24 hours. [score:3]
com/1471-2407/13/421/prepub 4T1 cells were incubated with EGCG (100 μM) and simultaneously (24 hours) transfected with scramble or miR-16 inhibitor for 24 hours. [score:3]
Tumor cells or TAM isolated from murine tumor graft were incubated with exosomes derived from EGCG -treated and/or miR-16 inhibitor -transfected 4T1 cells. [score:3]
To test whether exosome from EGCG -treated 4T1 cells could inhibit M2 polarization of TAM through exosomal miR-16, we treated TAM with exosomes either from EGCG -treated 4T1 cells or from from EGCG -treated and miR-16 inhibitor -transfected 4T1 cells, and measured the level of cytokines including IL-6, TGF-β, and TNF-α. [score:3]
As already known, we identified the possible target sequence in the 3′-UTR of IKKα for miR-16. [score:3]
Finally, we observed a 1.68-fold increase of miR-16 expression in tumor cells from mice treated with EGCG compared to control (Figure 4D). [score:2]
Treatment of tumor cells or TAM with exosomes derived from EGCG -treated and miR-16-knock-downed 4T1 cells restored the above effects on chemokines, cytokines, and NF-κB pathway elicited by EGCG -treated exosomes. [score:2]
Click here for file RAW264.7 cells were stimulated with 5 μg/ml LPS in the transfection scramble or miR-16 mimics, and total cellular RNA was extracted and submitted to RT-qPCR analysis for IL-1β and IL-6. Histogram shows the relative expression of molecules compared to GAPDH as internal control. [score:2]
Histogram shows the relative expression of miR-16 compared to U6 as an internal control. [score:2]
RAW264.7 cells were stimulated with 5 μg/ml LPS in the transfection scramble or miR-16 mimics, and total cellular RNA was extracted and submitted to RT-qPCR analysis for IL-1β and IL-6. Histogram shows the relative expression of molecules compared to GAPDH as internal control. [score:2]
The scramble miRNA mimics or miR-16 mimics (100 nM) were obtained from Genolution (Seoul, Korea). [score:1]
The restoration of miR-16 in prostate cancer cells results in growth arrest, apoptosis and in marked regression of prostate tumor xenografts [45]. [score:1]
Quantification of miR-16 by RT-qPCR. [score:1]
A therapeutic strategy is underway that involves the delivery of synthetic miR-16 into advanced prostate tumors [46]. [score:1]
Then, total RNA from cells or secretory exosomes was extracted and subjected to RT-qPCR to assess the miR-16 level. [score:1]
Specifically, the miR-16 was selected because it was elevated by EGCG treatment and has been known to be associated with immune cell function. [score:1]
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[+] score: 132
Other miRNAs from this paper: mmu-mir-16-2
increases miR-16 expression and downregulates cell proliferative miR-16 targets in mice. [score:8]
One prospective pathway through which may be upregulating miR-16 expression levels involves the tumor suppressor p53. [score:8]
As evidenced in the results produced in this manuscript, upregulates miR-16 expression in mice through PAD inhibition. [score:8]
If miR-16 expression is increased with treatment (Figure 3), then we expect to see the downregulation of these cell proliferation targets of miR-16. [score:8]
When miR-16 is downregulated, this relieves its inhibition of cell proliferative targets, like Cyclin D1 and E1. [score:8]
Figure 5 As evidenced in the results produced in this manuscript, upregulates miR-16 expression in mice through PAD inhibition. [score:8]
Although other miRNAs have been found associated with either mouse or human UC, we focused here on miR-16 because our previous in vitro data showed that induces miR-16 expression and decreases the expression of several miR-16 targets (i. e. Cyclins D1, D2, D3, E1, and CDK6) involved in the progression through the cell cycle [17]. [score:7]
These results are consistent with the hypothesis that is suppressing tumorigenesis in our mouse mo del by inhibiting cell proliferation via increased miR-16 expression. [score:7]
At this point, the cell proliferation targets (Cyclin D1 and E1) of miR-16 are downregulated, accounting for the decreased tumorigenesis found at the endpoint of our study. [score:6]
Likewise, treatment decreases protein expression of the miR-16 targets, Cyclins D1 and E1, and the cell proliferation marker, Ki67 (Figure 4). [score:5]
Because our results demonstrate that can suppress these targets of miR-16, this verifies the activity of miR-16 throughout our mo del. [score:5]
Since the inhibition of cell proliferation is a goal of many anti-cancer drug therapies, we hypothesized that is preventing tumorigenesis by increasing miR-16 expression in vivo. [score:5]
miR-16 has multiple cell proliferation targets, such as Cyclin D1 and Cyclin E1; supporting the premise that it is a tumor suppressor miRNA [17, 22- 25]. [score:5]
Furthermore, the lower level of miR-16 expression in epithelial cells from the higher dosage group (0.25 mg/mL), compared to the lower dosage group (0.05 mg/mL), is highly suggestive of a direct correlation between the expression level of miR-16 and tumor incidence; however, the cause of this variability between treatment groups is currently unknown. [score:5]
Overall, this study presents as a viable cancer preventative therapy against colitis -associated colorectal cancer and provides an innovative mechanism of action involving the upregulation of miR-16, ultimately leading to decreased cell proliferation and prevention of tumorigenesis in vivo. [score:4]
The relative fold change (FC) of miR-16 expression, as compared to U6 expression, was determined based on the comparative threshold cycles (Ct) of miR-16 and U6. [score:4]
Our current in vivo study is a substantial extension of our previous mechanistic in vitro data and suggests that increased miR-16 expression in mice treated with results in decreased tumor formation [17]. [score:3]
Future studies will explore the mechanism(s) by which is increasing miR-16 expression and will determine optimum dosages for preventing tumorigenesis. [score:3]
IRS of colons at day 35 stained with A. Cyclin D1, B. Cyclin E1, both known targets of miR-16, and C. Ki-67, a cell proliferation marker. [score:3]
To uncover the mechanism by which is acting, we revealed that increases miR-16 expression in colon epithelial cells (Figure 3). [score:3]
p53 boosts the post-transcriptional maturation of miR-16 and is found to increase miR-16 expression in a p53 -dependent manner in vitro [17, 37- 39]. [score:3]
We have previously shown that increases miR-16 expression in a p53 -dependent manner resulting in a cell cycle arrest in vitro [17]. [score:3]
Consistent with our previously published in vitro data [17] and our current hypothesis, miR-16 expression in the AOM + DSS only group was significantly lower than the AOM only group and both treated groups. [score:3]
Interestingly, the IRS values of Cyclin D1 and Ki67 staining at day 35 revealed a similar trend to the tumor incidence and miR-16 expression levels in colon epithelial cells. [score:3]
MiR-16 expression. [score:2]
Then, as described in the methods, total RNA was extracted and primed to measure miR-16 expression using qPCR. [score:1]
Figure 3 shows the relative fold change in miR-16 levels in the epithelial cells. [score:1]
To test this, we measured miR-16 expression levels in isolated colon epithelial (CD45-) cells at day 35 (see methods). [score:1]
Likewise, miR-16 is found at lower levels in CRC than in normal tissue [36]. [score:1]
We chose to investigate the miR-16 expression levels at the day 35 time point because we were interested in the mechanism preventing tumorigenesis at day 70. [score:1]
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[+] score: 104
Considering the signaling pathway which induces the miRNA transcription directly upon LPS stimulation and suitable number of miRNA targets for biomarkers, expression of miRNAs in whole blood following LPS injection was quantified using real-time RT-PCR to verify selected up-regulated, but not down-regulated, miRNA targets with at least 4-fold increase in expression (let-7d, miR-15b, miR-16, miR-25, miR-92a, miR-103, miR-107, and miR-451). [score:16]
Notably, LTA did not up-regulate the expression of the other 7 miRNA targets, and decreased the expression levels of let-7d, miR-15b, miR-16, miR-103, and miR-107 at the various concentrations tested (Figure 5). [score:10]
Only 3 miRNAs with upregulated expression (miR-16, miR-103, and miR-107) in whole blood showed high expression in the lung, and no miRNA in whole blood showed high expression in other tissues, including brain, liver, or spleen. [score:10]
Upregulated expression of the miRNA targets (let-7d, miR-15b, miR-16, miR-25, miR-92a, miR-103, miR-107 and miR-451) following LPS injection on real-time RT-PCR was dose- and time -dependent. [score:8]
Among the miRNAs with upregulated expression levels in the whole blood, only 4—miR-16, miR-103, miR-107, and let-7a—showed high expression in the lung. [score:8]
With a dose- and time -dependentupregulated expression of the miRNA targets (let-7d, miR-15b, miR-16, miR-25, miR-92a, miR-103, miR-107 and miR-451) following LPS injection, these whole blood-derived miRNAs are promising as biomarkers for LPS exposure. [score:8]
In our study, miR-16 expression was upregulated by approximately 5-fold following LPS treatment, preventing its use as an internal control. [score:6]
Expression of representative up-regulated miRNAs (let-7d, miR-15b, miR-16, miR-25, miR-92a, miR-103, miR-107, and miR-451) identified using miRNA microarray of whole blood using real-time RT-PCR. [score:6]
Following injection with 10 μg of LPS, increased expression was observed in the 8 miRNAs, but expression levels of only let-7d, miR-16, and miR-103 were significantly higher than the control (Figure 2A). [score:5]
In addition, following exposure to LPS, expression of 3 miRNAs (miR-15b, miR-16, and miR-451) was not significantly lower in TLR4 receptor knockout mice. [score:4]
Figure 5 Expression of let-7d, miR-15b, miR-16, miR-25, miR-92a, miR-103, miR-107, and miR-451 of whole blood from C57BL/6 mice using real-time RT-PCR experiment 6 h after exposure to 10, 100, and 1000 μg LTA originating from Staphylococcus aureus ; *, P  < 0.05 vs. [score:3]
Figure 3 Expression of let-7d, miR-15b, miR-16, miR-25, miR-92a, miR-103, miR-107, and miR-451 from whole blood of C57BL/6 mice based on real-time RT-PCR experiments 6 h after exposure to 100 μg LPS originating from different bacteria, including Escherichia coli serotype 026:B6, Klebsiella pneumonia, Pseudomonas aeruginosa, Salmonella enterica, serotype Enteritidis, and Serratia marcescens. [score:3]
In this study, we demonstrated that expression of multiple miRNAs (let-7d, miR-15b, miR-16, miR-25, miR-92a, miR-103, miR-107, and miR-451) is significantly altered in the whole blood of mice after exposure to LPS in a dose- and time -dependent fashion. [score:3]
To investigate the role of the TLR4 receptor in inducing expression of the miRNA targets, expression of let-7d, miR-15b, miR-16, miR-25, miR-92a, miR-103, miR-107, and miR-451 in the whole blood of Tlr4 [−/−] mice 6 h after intraperitoneal injection of 100 μg LPS (L3755) was measured against that from the whole blood of Tlr4 [−/−] mice injected with PBS. [score:3]
Although previous studies have reported the use of miR-16 and miR-142-3p, which show relatively stable expression in the serum, as endogenous controls [39, 40], it is unknown whether they are stable in the circulation. [score:3]
In contrast, upon 100 ug and 1000 ug of LPS injection, all these 8 miRNAs (let-7d, miR-15b, miR-16, miR-25, miR-92a, miR-103, miR-107, and miR-451) had a significant expression than the control (Figure 2A). [score:3]
Figure 4 Expression of let-7d, miR-15b, miR-16, miR-25, miR-92a, miR-103, miR-107, and miR-451 of whole blood from C57BL/6 and Tlr4 [−/−] (C57BL/10ScNJ) mice from real-time RT-PCR experiments 6 h after exposure to 100 μg LPS; **, P  < 0.01 vs. [score:3]
To investigate that whether lipoteichoic acid (LTA) originating from gram -positive bacteria induces expression of let-7d, miR-15b, miR-16, miR-25, miR-92a, miR-103, miR-107, and miR-451, whole blood was drawn at 6 h following intraperitoneal injections of 10, 100, or 1000 μg LTA from S. aureus for real-time PCR. [score:1]
60 miR-16 1.01             miR-590-5p 1.17 miRNA microRNA; LPS lipopolysaccharide. [score:1]
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14
[+] score: 100
miR-16 is implicated in induction of apoptosis by targeting Bcl-2 [25], and is involved in cell cycle regulation by targeting CDK6, cell division cycle protein 27 (CDC27), the caspase recruitment domain-containing protein 10 (CARD10), cyclin D1 and cyclin E [26- 28]. [score:6]
The expression of miR-16, let-7a and miR-34a was consistently upregulated in neural differentiation mo dels. [score:6]
In contrast, proapoptotic miRNAs are usually downregulated in cancer, and include miR-15, miR-16, the let-7 family and members of the miR-34 family. [score:4]
Therefore, a possible role for miR-16 in the context of differentiation may be associated with cell cycle control in downregulating the proliferation potential of differentiating cells. [score:4]
miR-16 was shown to be implicated in cell cycle regulation, as well as apoptosis induction by targeting Bcl-2 [25- 28]. [score:4]
In conclusion, the identification of miR-16, let-7a and miR-34a, whose expression patterns are conserved in mouse, rat and human neural differentiation, implicates these specific miRNAs in mammalian neuronal development. [score:4]
Interestingly, and similar to miR-34a/b/c, miR-16 was upregulated at both 3 and 8 days of neural differentiation. [score:4]
C) Expression of miR-16, Let-7a and miR-34a at 4 and 8 days of ES cell differentiation and in control (DMSO -treated) and LY411575 -treated rosette cultures at 8 days. [score:3]
Indeed, miR-16 expression was markedly increased throughout mouse, rat and human neural differentiation. [score:3]
Figure 6 Differentiation of PC12 and NT2N cells were associated with modulated levels of miR-16, let-7a and miR-34a expression. [score:3]
A and B) miR-16, let-7a and miR-34a expression during PC12 and NT2N differentiation, respectively. [score:3]
Nevertheless, here-to-fore unrecognized miR-16 targets may exert distinct, yet crucial functions during cell differentiation. [score:3]
Therefore, it is possible that additional mechanisms exist to antagonize let-7a and miR-16 expression during NS cell differentiation. [score:3]
Importantly, although highly modulated during cell differentiation, both let-7a and miR-16 were significantly expressed in neurospheres (data not shown). [score:3]
In NT2N, miR-16 expression was increased by 3.6-fold at day 14 (p < 0.001) and almost 4-fold at day 21 (p < 0.05) of differentiation (Figure 6B). [score:3]
Nevertheless, the onset of miR-16 expression during mouse NS cell differentiation was not associated with the appearance of any specific cell type. [score:3]
Nevertheless, no difference in miR-16 expression levels was detected at 7 days, under different NGF treatments. [score:3]
miR-16 and let-7a expressions in cells treated with 5 and 50 ng/ml NGF were also significantly different from NGF untreated cells at day 4 (p < 0.05) and day 2 (p < 0.01), respectively. [score:3]
Similarly to miR-16 and let-7a, a decrease in miR-34a expression occurred from day 6 to day 8. Figure 2 Apoptosis -associated miRNAs are modulated during mouse NS cell differentiation. [score:3]
Our results showed that the differential expression of miR-16, let-7a and miR-34a during mouse NS cell differentiation was not associated with cell death. [score:3]
In conclusion, our results demonstrate that apoptosis -associated miRNAs are differentially expressed during neural differentiation, in the absence of cell death, and identify miR-16, let-7a and miR-34a as important players. [score:3]
Indeed, miR-16 expression increased by 1.8- (p < 0.01) and 1.4-fold (p < 0.05) at 2 and 4 days, respectively. [score:3]
Similarly to miR-16 and let-7a, a decrease in miR-34a expression occurred from day 6 to day 8. Figure 2 Apoptosis -associated miRNAs are modulated during mouse NS cell differentiation. [score:3]
In fact, at this stage of differentiation, expression levels of miR-16, let-7a and miR-34a were increased when compared with day 4 (Figure 5B). [score:2]
Expression of specific proapoptotic (miR-16, let-7a and miR-34a) and antiapoptotic miRNAs (miR-20a and miR-19a) were analyzed by quantitative Real Time-PCR from 10 ng of total RNA using specific Taqman primers and GAPDH for normalization. [score:2]
Our results demonstrated that treatment of PC12 cells with 50 ng/ml of NGF markedly induced miR-16 expression at 2 and 4 days, compared with NGF-untreated cells (Figure 6A). [score:2]
To further validate the role of the proapoptotic miRNAs, miR-16, let-7a and miR-34a in neural cell differentiation, we investigated whether they were upregulated in other neural differentiation mo dels, including mouse ES cells, PC12 and NT2N cell lines. [score:2]
Surprisingly, increased differentiation after replating was associated with a significant decrease in both miR-16 and let-7a expression by ~ 2 (p < 0.05) and 5-fold (p < 0.001), while miR-34a increased by 4.5-fold (p < 0.05), compared with non-replated cells. [score:2]
Notably, miR-16 expression levels were significantly increased from 12 hours to 3 days of differentiation, when compared with undifferentiated cells (p < 0.05) (Figure 2). [score:2]
The involvement of miR-16 in cell differentiation has not been previously reported. [score:1]
Notably, LY411575 -induced neurogenesis resulted in a significant increased of miR-16 and miR-34a, by 3- and 2-fold (Figure 5B), respectively (p < 0.05), supporting the potential involvement of both miRNAs in neuronal differentiation. [score:1]
Next, we characterized the expression of proapoptotic miRNAs, including miR-16, let-7a and miR-34a in distinct mo dels of neural differentiation, including mouse embryonic stem cells, PC12 and NT2N cells. [score:1]
Based on a possible link between miR-16, let-7a and miR-34a with known apoptotic molecules that have already been associated with differentiation, we decided to validate microarray data for the three proapoptotic miRNAs throughout mouse NS cell differentiation by quantitative real time-PCR (Figure 2). [score:1]
In contrast to let-7a and miR-16, miR-34a was barely detected in undifferentiated cells, supporting its specific involvement in cell differentiation. [score:1]
Nevertheless, an involvement of miR-16 in cell differentiation is virtually unknown. [score:1]
Figure 5 miR-16, let-7a and miR-34a are increased during mouse ES cell differentiation. [score:1]
These results strongly suggested that modulation of miR-16, let-7a and miR-34a was most likely due to cell differentiation rather than cell death. [score:1]
[1 to 20 of 37 sentences]
15
[+] score: 88
Figure 5 The loss of the retinoblastoma tumor suppressor (RB ) expression plays a role in Smurf2 downregulation in triple -negative breast cancer (TNBC) cells, via upregulation of miR-15, miR-16 and miR-128. [score:11]
We also have revealed that microRNAs such as miR-15a, miR-15b, miR-16 and miR-128, whose expression is increased by inactivating mutations of the retinoblastoma (RB) gene, downregulate translation of Smurf2 protein in TNBC cells. [score:9]
Studies using quantitative PCR and specific microRNA inhibitors indicated that increased expression of miR-15a, miR-15b, miR-16 and miR-128 was involved in Smurf2 downregulation in those triple -negative cancer cell lines, which have mutations in the retinoblastoma (RB) gene. [score:9]
To further delineate the role of the miRNAs in Smurf2 downregulation observed in BT549, MDA-MB-436 and DU4475 cells, cells were transfected with miRNA inhibitors (antagomirs) against miR-15a, miR-15b, miR-16 or miR-128 (Figure  4). [score:6]
Low expression of Smurf2 protein was also observed in several TNBC cell lines, which had RB mutations and high expression of miR-15a, miR-15b, miR-16 and miR-128. [score:6]
Forced expression of GFP-RB resulted in a significant increase in cellular levels of Smurf2 protein, accompanied by substantial decreases in the expression of miR-15a, miR-15b, miR-16 and miR-128b (Figure  5C). [score:5]
DU4475 cells showed increased expression of miR-15b, miR-16 and miR-128, relative to their expression in MCF-10A cells. [score:5]
Whereas deletion of miR-15a and miR-16 was reported in some non-small cell lung cancers [19], miRNA expression profiling in human breast cancer subtypes showed that basal-like TNBCs expressed higher levels of miR-15b than other subtypes [20]. [score:5]
Figure 4 MicroRNAs such as miR-15, miR-16 and miR-128 are involved in downregulation of Smurf2 protein in triple -negative breast cancer. [score:4]
A recent study demonstrated that miR-15 and miR-16 are direct targets of the E2F transcription factors [16]. [score:4]
It was previously demonstrated that miR-15 and miR-16 are direct transcriptional targets of E2F-1, and these miRNAs in turn restrict E2F activities [16, 19]. [score:4]
Figure 3 Expression levels of miR-15a, miR-15b, miR-16 and miR-128 in breast cancer cell lines. [score:3]
Human triple -negative breast cancer cell lines, BT549, MDA-MB-436 and DU4475 cells, were transfected with microRNA inhibitors against miR-15a, miR-15b, miR-16 and miR-128, or nonspecific ssRNA as negative control (NC), and cellular levels of Smurf2 protein were determined at 24 h (A, B) or 48 h (C) post-transfection by immunoblotting. [score:3]
Cells were transfected with Ambion® Anti-miR™ miRNA Inhibitors specifically against miR-15a, miR-15b, miR-16 and miR-128 (Ambion/Invitrogen, Carlsbad, CA), using the Lipofectamine® RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. [score:3]
Also in MCF-7 cells, the levels of miR-15a, miR-15b and miR16 were low, whereas the expression of miR-128 was modestly higher. [score:3]
MDA-MB-436 cells had increased expression of miR-15b, miR-16, and miR-128. [score:3]
BT549 cells exhibited increased expression of miR-15a, miR-15b and miR-16. [score:3]
Thus, we measured the expression of miR-15a, miR-15b, miR-16 and miR-128b in the breast cancer cell lines (Figure  3). [score:1]
The analysis led us to candidates such as miR-128 (binding to Smurf2 3′UTR, 5′-CACUGUGA-3′) and the miR-15 family miRNAs including miR-15a, miR-15b and miR-16 (binding to Smurf2 3′UTR, 5′-GCUGCUA-3′). [score:1]
[1 to 20 of 19 sentences]
16
[+] score: 79
Significant expression of these 10 miRNA targets was detected at 8 and 24 h after CLP, and 6 targets (miR-16, miR-17, miR-20a, miR-20b, miR-26b, miR-106a) showed remarkable upregulation of up to 50- and even 100-fold at 24 h (Fig. 4B). [score:10]
The exosomes showed marked expression of 6 (miR-16, miR-17, miR-20a, miR-20b, miR-26a, and miR-26b) of the 10 miRNA targets at 8 h after CLP; the expression of the remaining 4 miRNA targets (miR-106a, miR-106b, miR-195, and miR-451) increased; however, the increase was not significant (Fig. 5B). [score:9]
In addition, miR-16 expression is up-regulated following LPS treatment [11], and it was shown to be up-regulated following CLP in this study, which precludes the use of miR-16 as internal control. [score:9]
The miRNA targets that were significantly up-regulated in the CLP experiment, as shown by the microarray experiments, are shown in Table 1. The expressions of 2 (miR-16 and miR-17), 6 (miR-20a, miR-16, miR-17, miR-451, miR-106a, and miR-106b), and 7 miRNAs (miR-26b, miR-20b, miR-17, miR-20a, miR-106a, miR-26a, and miR-195) increased significantly in the whole blood of mice at 4, 8, and 24 h after CLP, respectively. [score:8]
Expression of circulating miR-16 was up-regulated ∼5-fold following LPS treatment [11] and ∼70-fold at 24 h following CLP treatment, as demonstrated in this study. [score:6]
In this study, the mice with CLP experienced bacterial infection first and then septicemia, therefore, the results clearly showed that 8 miRNA targets (miR-16, miR-17, miR-20a, miR-26a, miR-26b, miR-106a, miR-106b, and miR-451) were up-regulated in both the CLP alone group and the E. coli infection group. [score:6]
Previously, we had demonstrated a dose- and time -dependent up-regulation of 8 (let-7d, miR-15b, miR-16, miR-25, miR-92a, miR-103, miR-107, and miR-451) and 4 (miR-451, miR-668, miR-1902, and miR-1904) circulating miRNA targets in mice following injection of LPS [11] and LTA [12], respectively. [score:6]
Among the circulating miRNAs that are up-regulated following CLP, miR-16 has been suggested to regulate pro-apoptotic pathways in circulating lymphocytes of patients with bacterial infections [30]. [score:5]
Although in some previous studies miR-16 and miR-142 showed relatively stable expression in the serum, owing to which these were used as endogenous controls [34], [35], studies have also shown that miR-16 and miR-451 expressed in red blood cells were the major source of variation in the estimated levels of circulating miR-16 and miR-451 [36]. [score:5]
However, in the CLP -based mo del of sepsis, only miR-16 was up-regulated. [score:4]
In this study, we demonstrated that experimental sepsis induced by CLP caused time -dependent upregulation of the circulating miRNAs miR-16, miR-17, miR-20a, miR-20b, miR-26a, miR-26b, miR-106a, miR-106b, miR-195, and miR-451. [score:4]
These results show that 8 miRNAs (miR-16, miR-17, miR-20a, miR-26a, miR-26b, miR-106a, miR-106b, and miR-451) were up-regulated after both CLP and subcutaneous injection of E. coli. [score:4]
Serum levels of both miR-15a and miR-16 were significantly higher in patients with sepsis that in healthy individuals [29] or cirrhotic patients [30], and the levels of these miRNAs were significantly elevated in patients with systemic inflammatory response syndrome (SIRS) [29]. [score:1]
Among these miRNAs, the levels of miR-195 and miR-451 detected in the Ago2 immunoprecipitates were more than 20-fold that in the controls and the levels of miR-16, miR-20a, miR-26a, and miR-106b were more than 10-fold that in the control. [score:1]
Circulating miR-15a and miR-16 [29], [30], miR-150 [31], miR-146a, and miR-223 [32] have been identified as potential biomarkers of sepsis. [score:1]
[1 to 20 of 15 sentences]
17
[+] score: 61
Thus, the result was the same with VEGF -overexpressing transgenic mice mo del that miR-16 can inhibit lung cancer growth by suppressing VEGF expression. [score:9]
Hua et al. [29] showed that VEGF is predicted to be targeted by multiple miRNAs, including miR-15b, miR-16, miR-20a and miR-20b, and transfection of these miRNAs into CNE cells (a human nasopharyngeal carcinoma cell line) can inhibit VEGF expression [29]. [score:7]
We further demonstrated that the intranasal administration of miR-16 could inhibit lung cancer growth by significantly suppressing VEGF expression. [score:7]
Using the lung-specific adenocarcinoma transgenic mouse mo del and the A549-pCAG-iRFP-2A-Verus orthotopic lung tumor mouse mo del, we further investigated the novel approaches for microRNA target therapy and demonstrated that miR-16 effectively inhibits lung cancer growth by suppressing VEGF expression via the intrinsic and extrinsic apoptotic pathways (Figure 8). [score:7]
Thus, miR-16 can inhibit lung cancer growth by suppressing VEGF expression. [score:7]
Figure 9A showed that miR-16 lowered pulmonary tumorigenesis via decreased the expression of VEGF in both lung tissues (Figure 9A, lower panel) and circulation blood (Figure 9B), and also decreased the expression of CD105 tumor marker in serum (Figure 9C), affected the formation of lung tumors in human orthotopic non-small cell lung cancer xenograft mo del. [score:5]
After three intranasal administrations of 20 μg miR-16 or mock miR per mouse once a week, we found that miR-16, which lowered the expression of VEGF in both lung tissues (Figure 8B and 8C) and circulation blood (Figure 8D) (p < 0.01), affected the formation of lung tumors in > 12-month-old lung-specific hVEGF-A [165] overexpressing transgenic mice (Figure 8A). [score:5]
The results of the western blot analyses revealed that the cleaved forms of caspase 3, 8, 9, and poly (ADP-ribose) polymerase (PARP) were activated after miR-16 treatment in the hVEGF-A [165] overexpressing transgenic mice (Figure 8E). [score:3]
Therefore, we further investigated the inhibitory effect of microRNA-16 (miR-16) on lung tumors (Figure 8) because recent reports have linked the expression of specific microRNAs with tumorigenesis and metastasis. [score:3]
MicroRNA-16 reduces pulmonary tumorigenesis in VEGF -overexpressing transgenic mice. [score:2]
MicroRNA-16 reduces pulmonary tumorigenesis in vascular endothelial growth factor (VEGF) -overexpressing transgenic mice. [score:2]
The mice were then sacrificed after four weeks of miR-16 administration, and lung tissues were collected for pathological histology, immunohistochemistry staining, and protein extraction. [score:1]
The mice were then sacrificed after miR-16 administration, and lung tissues and sera were collected for pathological histology, immunohistochemistry staining, serum VEGF and CD105 detections. [score:1]
Based on these results, miR-16 may induce apoptosis via both the intrinsic and extrinsic pathways. [score:1]
The transgenic mice were randomly assigned to the following two groups for treatment: Tg/Mock (in vivo-jetPEI in 5% glucose solution) and Tg/miR-16 (miR-16 mixed with in vivo-jetPEI in 5% glucose solution). [score:1]
[1 to 20 of 15 sentences]
18
[+] score: 59
These targets are downregulated during satellite cell activation, consistent with increased expression of miR-16, miR-93, and miR-106b in proliferating satellite cells as compared to quiescent satellite cells (Figure 8). [score:7]
Many predicted targets of miR-16 and the miR-93/106b family inhibit cell cycle progression and cell growth. [score:5]
Comparing relative levels in muscle tissue and satellite cells revealed that miR-124 is likely only expressed in satellite cells, while miR-16, miR-93, and miR-106b are most likely expressed in satellite cells and in differentiated muscle fibers. [score:5]
The four candidate miRNAs (miR-16, miR-93, miR-106b, and miR-124) that displayed dynamic expression in satellite cells were inhibited in myofiber -associated satellite cells prior to the first cell division. [score:5]
Inhibition of miR-16 elevated the total number of Pax7+ cells at 5 days of culture and inhibition of miR-93 did not have any detectable effect (Figure 7-F). [score:5]
Moreover, four of the six miRNAs were expressed in satellite cells (miR-16, miR-93, miR-106b, and miR-124), while two were likely present only in differentiated muscle (miR-107 and miR-200b). [score:3]
In contrast, miR-106b remained elevated following injury while miR-16 trended to slightly lower expression (Figure 6B-E). [score:3]
200nM miRIDIAN hairpin inhibitors (Dharmacon) against miR-16, miR-93, miR-106b, and miR-124 were co -transfected with pEGFP-C1-H2B. [score:3]
We further examined the role of miRNAs in satellite cell activation using Ingenuity® System’s IPA and identified PTEN signaling and Cell Cycle Regulation by BTG Family Proteins as the top canonical pathway regulated by miR-16, miR-93, miR-106b, and miR-124 in the transition of a quiescent satellite cell to a proliferating myoblasts (Figure 8). [score:3]
The same four micro RNAs (miR-16, miR-93, miR-106b, and miR-124) exhibit dynamic changes in relative expression when comparing activated satellite cells (G, J) to proliferating satellite cells (H, J) and quiescent satellite cells (I, J). [score:3]
This increase in satellite cells following at 5 days was observed in both proliferating satellite cells (D) and quiescent satellite cells (F) for miR-16, miR-106b, and miR-124 while inhibition of miR-93 resulted in a specific increase in proliferating satellite cells at 5 days (D). [score:3]
Here, we report global gene expression profiles and candidate miRNAs associated with quiescent and activated satellite cells as well as identify a novel function for miR-16, miR-106b, and miR-124 in satellite cell fate determination. [score:3]
The predicted target genes of miR-16, miR-93, miR-106b, and miR-124 were identified using Ingenuity® Systems (http://www. [score:3]
miR-16, miR-106b, and miR-124 regulate satellite cell fate. [score:2]
The expression level of miR-16 is maintained in proliferating cells, while miR-93 declines and miR-106b is dramatically induced in proliferating satellite cells as compared to freshly isolated and quiescent satellite cells (Figure 6G-I). [score:2]
In contrast, miR-16 enhanced the relative numbers of Pax7+ cells but did not appear to alter the percentage of Pax7+/MyoD + myoblasts or Pax7+/MyoD- ‘reserve’ cells relative to a scrambled control. [score:1]
In contrast to miR-124, miR-16 and miR-93 are present at low to undetectable levels in quiescent satellite cells and are induced in freshly isolated satellite cells (Figure 6G, I). [score:1]
miR-16, miR-93, miR-106b, miR-107, miR-124, and miR-200b are detected in satellite cells by in either primary satellite cells or in the satellite cell derived MM14 cell line. [score:1]
The changes in relative levels of miR-16, miR-93, miR-106b, and miR-124 in satellite cells following a muscle injury suggests that these four miRNAs may play a role in the transition from a quiescent satellite cell to a proliferating myoblast. [score:1]
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19
[+] score: 44
Highest-ranking miRNAs included miR-16/15a (46 targets), miR-27b (44 targets), let-7f (35 targets), miR-26b (33 targets), and miR-25 (30 targets). [score:11]
Both upregulated and downregulated genes predicted to be regulated by miR-16/15a were included in the analysis. [score:8]
This miRNA belongs to the miR-16/15 superfamily [41] and is predicted to regulate a large number of mRNA transcripts (476 conserved targets according to TargetScan. [score:6]
Interestingly, PI3K/Akt and mTOR signaling, involved in translational control and protein output, are among the pathways potentially regulated by miR-16/15. [score:4]
Detailed analysis of miR-16/15-regulated genes (ranked first) showed an association with biological terms such as neurological disease (P<0.001), cell death and survival (P<0.001), and PIK3/Akt signaling (P<0.001) (Fig. 3C). [score:4]
Furthermore, we highlighted potential pathologically-relevant networks (genes) regulated by miR-16/15. [score:2]
This was exemplified by the generation of networks related to the miRNA family involved in the regulation of the highest number of genes, miR-16/15. [score:2]
Changes in miR-16/15a levels were confirmed by qRT-PCR. [score:1]
A representative amplification curve of miR-16 by real-time qRT-PCR shows a significant enrichment (∼500 fold) of this miRNA pulled down by RIP-Ago2. [score:1]
miR-16/15 are also involved with mitochondrial dysfunction and apoptosis [33], [42]. [score:1]
These observations were validated by qRT-PCR on “positive” miRNAs (i. e., miR-16, miR-15a, and miR-25) (Fig. 4D) in an independent set of animals (n = 3 per group). [score:1]
The top-ranking miRNAs are miR-16 and miR-15a, which harbour the same seed sequence (GCTGCT), and thus functional mRNA binding site. [score:1]
Here, a representative qRT-PCR using miR-16 is shown. [score:1]
These predictions are in agreement with the literature suggesting a role for miR-16/15 in cell survival [32], [33]. [score:1]
[1 to 20 of 14 sentences]
20
[+] score: 42
In prion disease, we saw an up-regulation of miR-16-5p during early disease and the expression of this miRNA decreased with disease progression. [score:12]
Upon a short term infusion of miR-16-5p into the brains of diseased mice, the authors found a decrease in APP accumulation, further suggesting that miR-16-5p has an inhibitory effect on APP protein formation [137] and therefore, acts as a protective molecule in the disease. [score:7]
We further confirmed the expression levels of 7 miRNAs using qRT-PCR, miR-16-5p, miR-26a-5p, miR-29a-3p, miR-132-3p, miR-140-5p, miR-124a-3p and miR-146a-5p, all of which were up-regulated in infected samples prior to 130 DPI (Figure 6B, C and D ). [score:6]
In total, 17 miRNAs were identified as significantly dysregulated between 70–110 DPI and we successfully validated the expression levels of 6 of these miRNAs: miR-16-5p, miR-26a-5p, miR-29a-3p, miR-132-3p, miR-140-5p and miR-146a-5p. [score:4]
It would be an interesting area of study to determine whether miR-16-5p has a similar neuroprotective role in prion diseased mice. [score:3]
Recent publications have, however, revealed one interesting gene target of miR-16-5p. [score:3]
We analyzed the expression levels of 7 miRNAs (miR-16-5p, miR-26a-5p, miR-29a-3p, miR-140-5p, miR-132-3p, miR-146a-5p and miR-124a-3p) using a multiplex qRT-PCR approach. [score:3]
One of these is amyloid protein precursor (APP) and in a murine mo del of early-onset Alzheimer's disease the authors found a decrease in miR-16-5p levels while APP was increased [137]. [score:3]
Furthermore, miR-146a-5p, miR-16-5p and miR-140-5p, although lower in abundance than miR-124a-3p, were also present in mouse hippocampus in this study. [score:1]
[1 to 20 of 9 sentences]
21
[+] score: 41
Fig. 5. Atm expression is upregulated in Dgcr8 and Dicer c KOs and a target of germline-expressed miR-18, as well as miR-183 and miR-16. [score:10]
The target sites of two other miRNAs, miR-183 and miR-16, clustered in the same region of the Atm 3′UTR as the miR-18 target sites; moreover, the miR-183 and miR-16 target sites were predicted as the second and third strongest sites, respectively, within Atm (Fig.  5C). [score:7]
Taken together, these results indicate that miR-18, miR-183, and miR-16 target sites in Atm are functional and effective at eliciting downregulation in response to very low levels of miRNA. [score:6]
Many miRNAs are predicted to target the Atm 3′UTR, but only three are also expressed in spermatocytes and show depletion in Dgcr8 and Dicer c KOs: miR-18, miR-183 and miR-16. [score:5]
Therefore, miR-18, miR-183 and miR-16 are the strongest candidates for miRNA -mediated regulation of Atm expression in mammalian spermatogenesis. [score:4]
Like the miR-18 target sites, the single site for miR-183 (Fig.  5F) and combined miR-16 sites (Fig.  5H) were able to mediate an ∼2-fold repression of the reporter construct at high-to-moderate concentrations (25-nM–1-nM) of miR-183 and miR-16 mimetic. [score:3]
We also identified alterations in many small RNAs, including miR-18, miR-183 and miR-16, among whose targets is the mRNA encoding ATM. [score:3]
Our results underscore the significance of specific miRNAs in ensuring the fi delity of gametogenesis, and point to miR-18, miR-183 and miR-16 as miRNAs playing an important role in male fertility. [score:1]
Reporter constructs were transfected into A549 cells along with varying levels of siRNA duplexes corresponding to miR-18 (using RNA oligonucleotides: 5′-UAAGGUGCAUCUAGUGCA-GAU-3′ and 5′-CUGCACUAGAUGCACCUUAAU-3′), miR-183 (5′-UA-UGGCACUGGUAGAAUUCACU-3′ and 5′-UGAAUUCUACCAGUG-CCAGAUA-3′), miR-16 (5′-UAGCAGCACGUAAAUAUUGGCG-3′ and 5′-CCAAUAUUUACGUGCUGUUAUU-3′), or, as a control, miR-124 (Lim et al., 2005). [score:1]
Sites were mutated as follows: the miR-18 site (GCACCUUA) was mutated to GCAggaUA for both miR-18 sites; the miR-183 site (GUGCCAUA) was mutated to GUGaCgUA, the miR-16 sites (GCUGCU) were mutated to either GCcGaU, GaUGgU, GCcGaU (corresponding to the order of the sites within the reporter construct). [score:1]
[1 to 20 of 10 sentences]
22
[+] score: 40
Figure 4. The ‘extended VCR’ of stratum 2 (shared by Homo and Pelodiscus sequences): (a) miR-16 target site (also shown in Fig. 2e) and nearby target sites for miR-376a, miR-335-3p, miR-493 and miR-379 (the Xenopus sequence contains a 44-bp insertion at the site of the asterisk that includes two target sites for miR-335-3p are shown in red); (b) conserved pair of target sites for miR-320a and miR-182; (c) conserved triplet of target sites for miR-378, miR-99a and miR-30aA notable feature of stratum 2 is a pair of complementary sequences, 800 nucleotides apart, that are predicted to form the stems of a strong double helix (18 bp, –32.3 kcal/mol). [score:11]
Figure 4. The ‘extended VCR’ of stratum 2 (shared by Homo and Pelodiscus sequences): (a) miR-16 target site (also shown in Fig. 2e) and nearby target sites for miR-376a, miR-335-3p, miR-493 and miR-379 (the Xenopus sequence contains a 44-bp insertion at the site of the asterisk that includes two target sites for miR-335-3p are shown in red); (b) conserved pair of target sites for miR-320a and miR-182; (c) conserved triplet of target sites for miR-378, miR-99a and miR-30a A notable feature of stratum 2 is a pair of complementary sequences, 800 nucleotides apart, that are predicted to form the stems of a strong double helix (18 bp, –32.3 kcal/mol). [score:11]
The 4.8-kb gigaloop is a putative structure formed by pairing of VCR and a complementary sequence (cVCR) Figure 2. Conserved sequences of stratum 1 (shared by Homo and Callorhinchus IGF1R 3'-UTRs): (a) the 3' end of the long IGF1R transcript; (b) a miR-7-3p target site that has been lost from the Pelodiscus sequence; (c) let-7-3p target site; (d) miR-186 target site; (e) The VCR with predicted binding sites for miR-376c, miR-675 (derived from the imprinted H19 RNA) and miR-16. [score:7]
The VCR contains a target site for miR-675 [13], a target site for the miR-16 family of microRNAs [24, 25] and a target site for miR-376c [26]. [score:7]
The miR-675 and miR-16 target sites are proximal to the polyadenylation site and thus included within the short transcript. [score:3]
Although the facile alignment of Homo and Callorhinchus 3’-UTRs ends shortly after the miR-16 binding site of the VCR at the location of the polyadenylation site for the short human transcript (Fig.  2e), strong similarity continues beyond this point for Homo, Mono delphis, Pelodiscus and Xenopus sequences. [score:1]
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[+] score: 38
Finally, the fact that consistent expression of five genes in circulating miRNA expression profiles exists from multiple mouse strains, at different ages, and with different disease mo dels provides strong evidence that miR-146a, miR-16, miR-195, miR-30e, and miR-744 are useful as circulating miRNA endogenous references. [score:7]
As shown in Figure 2, these five serum miRNAs, miR-146a, miR-16, miR-195, miR-30e, and miR-744 were stably expressed in mouse regardless of strain, age, and disease condition. [score:5]
We preformed normalization of single qRT-PCR targets from the related mouse disease mo del using miR-146a, miR-16, and miR-195. [score:5]
As shown in Figure 2, miR-146a and miR-16 are highly expressed in serum, while miR-30e, miR-195, and miR-744 have lower expression levels. [score:5]
Furthermore, miR-146a and miR-16 are highly expressed in serum, while miR-30e, miR-195, and miR-744 have relatively lower expression. [score:5]
Interestingly, two recently published papers reporting on serum miRNA biomarkers for specific diseases have used miR-16 as an internal control to normalize qRT-PCR data [16], [36]. [score:3]
We have found five miRNAs, miR-146a, miR-16, miR-195, miR-30e, and miR-744 to be stably expressed in all tested strains across different ages and conditions. [score:3]
Cross normalization of these references to each other revealed that miR-16 and miR-195 are extremely stable endogenous references in this particular mouse mo del (our unpublished data). [score:1]
−ΔC [T] values of miR-146a, miR-16, miR-195, miR-30e, and miR-744 show stability across all samples (w = weeks). [score:1]
Notably, these five miRNAs, miR-16, miR-744, miR-195, miR-146a, and miR-30e share a 100% identity between human and mouse [32], [33], [34], [35]. [score:1]
0031278.g002 Figure 2−ΔC [T] values of miR-146a, miR-16, miR-195, miR-30e, and miR-744 show stability across all samples (w = weeks). [score:1]
These 72 miRNAs were candidate endogenous controls, following a series of statistical analyses only five genes (miR-146a, miR-16, miR-195, miR-30e, and miR-744) passed the criteria of ANOVA p>0.3, SD<1, and pair-wise |ΔΔ C [T]|<0.5. [score:1]
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[+] score: 34
Other miRNAs from this paper: hsa-let-7c, hsa-let-7d, hsa-mir-16-1, hsa-mir-21, hsa-mir-24-1, hsa-mir-24-2, hsa-mir-28, hsa-mir-29a, hsa-mir-30a, hsa-mir-31, hsa-mir-99a, hsa-mir-101-1, hsa-mir-16-2, mmu-let-7g, mmu-let-7i, mmu-mir-27b, mmu-mir-30a, mmu-mir-99a, mmu-mir-101a, mmu-mir-125b-2, mmu-mir-126a, mmu-mir-128-1, mmu-mir-9-2, mmu-mir-142a, mmu-mir-144, mmu-mir-145a, mmu-mir-151, mmu-mir-152, mmu-mir-185, mmu-mir-186, mmu-mir-24-1, mmu-mir-203, mmu-mir-205, hsa-mir-148a, hsa-mir-34a, hsa-mir-203a, hsa-mir-205, hsa-mir-210, hsa-mir-221, mmu-mir-301a, mmu-let-7d, hsa-let-7g, hsa-let-7i, hsa-mir-27b, hsa-mir-125b-1, hsa-mir-128-1, hsa-mir-142, hsa-mir-144, hsa-mir-145, hsa-mir-152, hsa-mir-9-1, hsa-mir-9-2, hsa-mir-9-3, hsa-mir-125b-2, hsa-mir-126, hsa-mir-185, hsa-mir-186, mmu-mir-148a, mmu-mir-200a, mmu-let-7c-1, mmu-let-7c-2, mmu-mir-16-2, mmu-mir-21a, mmu-mir-24-2, mmu-mir-29a, mmu-mir-31, mmu-mir-34a, mmu-mir-148b, mmu-mir-339, mmu-mir-101b, mmu-mir-28a, mmu-mir-210, mmu-mir-221, mmu-mir-9-1, mmu-mir-9-3, mmu-mir-125b-1, mmu-mir-128-2, hsa-mir-128-2, hsa-mir-200a, hsa-mir-101-2, hsa-mir-301a, hsa-mir-151a, hsa-mir-148b, hsa-mir-339, hsa-mir-335, mmu-mir-335, hsa-mir-449a, mmu-mir-449a, hsa-mir-450a-1, mmu-mir-450a-1, hsa-mir-486-1, hsa-mir-146b, hsa-mir-450a-2, hsa-mir-503, mmu-mir-486a, mmu-mir-542, mmu-mir-450a-2, mmu-mir-503, hsa-mir-542, hsa-mir-151b, mmu-mir-301b, mmu-mir-146b, mmu-mir-708, hsa-mir-708, hsa-mir-301b, hsa-mir-1246, hsa-mir-1277, hsa-mir-1307, hsa-mir-2115, mmu-mir-486b, mmu-mir-28c, mmu-mir-101c, mmu-mir-28b, hsa-mir-203b, hsa-mir-5680, hsa-mir-5681a, mmu-mir-145b, mmu-mir-21b, mmu-mir-21c, hsa-mir-486-2, mmu-mir-126b, mmu-mir-142b, mmu-mir-9b-2, mmu-mir-9b-1, mmu-mir-9b-3
Of the miRNAs found to be down-regulated in the metastatic xenografts, miR-16, showing a >17-fold decrease in expression, has been reported to be down-regulated in prostate cancer [23], [24] and to have a metastasis-suppressing function. [score:11]
Of the down-regulated miRNAs a number have been reported to be down-regulated in prostate cancer relative to benign prostate tissues, i. e. miR-16 [23]– [25], miR-24 [26]– [28], miR-29a [26], miR-145 [23], [24], [27], [29], [30], and miR-205 [24], [31], [32]. [score:7]
The down-regulation of miR-16 [25], miR-34a [33], miR-126* [34], miR-145 [35] and miR-205 [36] correlated with the development of prostate cancer metastasis. [score:5]
Thus some of the miRNAs have already been linked to this phenomenon, in particular down-regulated miRNAs such as miR-16, miR-34a, miR-126*, miR-145 and miR-205, supporting the validity of our analytical approach. [score:4]
A number of these miRNAs (21/104) have previously been reported to show similar down- or up-regulation in prostate cancers relative to normal prostate tissue, and some of them (e. g., miR-16, miR-34a, miR-126*, miR-145, miR-205) have been linked to prostate cancer metastasis, supporting the validity of the analytical approach. [score:4]
Moreover, metastatic prostate tumor growth in vivo could be inhibited by systemic delivery of synthetic miRNA-16 [25]. [score:3]
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25
[+] score: 33
Wang Y. Jiang L. Ji X. Yang B. Zhang Y. Fu X. D. Hepatitis B viral RNA directly mediates down-regulation of the tumor suppressor microRNA miR-15a/miR-16–1 in hepatocytesJ. [score:7]
Wu G. Yu F. Xiao Z. Xu K. Xu J. Tang W. Wang J. Song E. Hepatitis B virus X protein downregulates expression of the miR-16 family in malignant hepatocytes in vitroBr. [score:6]
Ofir M. Hacohen D. Ginsberg D. MiR-15 and miR-16 are direct transcriptional targets of E2F1 that limit E2F -induced proliferation by targeting cyclin EMol. [score:6]
Both miR-15b and miR-16–2, which are in the same cluster and have a common promoter, are down-regulated by HBx. [score:4]
Bonci D. Coppola V. Musumeci M. Addario A. Giuffrida R. Memeo L. D’Urso L. Pagliuca A. Biffoni M. Labbaye C. The miR-15a-miR-16–1 cluster controls prostate cancer by targeting multiple oncogenic activitiesNat. [score:3]
Cimmino A. Calin G. A. Fabbri M. Iorio M. V. Ferracin M. Shimizu M. Wojcik S. E. Aqeilan R. I. Zupo S. Dono M. miR-15 and miR-16 induce apoptosis by targeting BCL2Proc. [score:3]
Xia L. Zhang D. Du R. Pan Y. Zhao L. Sun S. Hong L. Liu J. Fan D. miR-15b and miR-16 modulate multidrug resistance by targeting BCL2 in human gastric cancer cellsInt. [score:3]
The miR-15/16 family is composed of miR-15a, miR-15b and miR-16. [score:1]
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[+] score: 32
Candidate target genes regulated by the upregulated miRNAs (mmu-miR-199a-5p, mmu-miR-329-3p, mmu-miR-136-5p and mmu-miR-16-1-3p). [score:7]
Of these miRNAs, four were upregulated (mmu-miR-199a-5p, mmu-miR-329-3p, mmu-miR-136-5p, and mmu-miR-16-1-3p), and one was downregulated (mmu-miR-212-3p). [score:7]
Of the five differentially expressed miRNAs, four miRNAs, namely, mmu-miR-199a-5p, mmu-miR-329-3p, mmu-miR-136-5p, and mmu-miR-16-1-3p, were significantly upregulated in the vitrified blastocysts. [score:6]
Among the upregulated miRNAs, 292, 178, 164, and 243 candidate target genes were predicted for mmu-miR-199a-5p, mmu-miR-329-3p, mmu-miR-136-5p, and mmu-miR-16-1-3p, respectively (S2 Table). [score:6]
Mmu-mir-16 is reported to be related to apoptosis in mouse embryos [33], and miRNA profiling suggested that genes targeted by this miRNA are important in spindle organization in mouse oocytes [34]. [score:3]
In this study, 5 of the 760 identified miRNAs, namely, mmu-miR-199a-5p, mmu-miR-329-3p, mmu-miR-136-5p, mmu-miR-16-1-3p, and mmu-miR-212-3p, showed significantly different expression between the vitrified and fresh mouse blastocysts. [score:3]
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[+] score: 31
Other miRNAs from this paper: hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3, hsa-let-7b, hsa-let-7c, hsa-let-7d, hsa-let-7e, hsa-let-7f-1, hsa-let-7f-2, hsa-mir-15a, hsa-mir-16-1, hsa-mir-17, hsa-mir-18a, hsa-mir-21, hsa-mir-24-1, hsa-mir-24-2, hsa-mir-25, hsa-mir-27a, hsa-mir-30a, hsa-mir-31, hsa-mir-29b-1, hsa-mir-29b-2, hsa-mir-103a-2, hsa-mir-103a-1, hsa-mir-16-2, mmu-let-7g, mmu-let-7i, mmu-mir-15b, mmu-mir-29b-1, mmu-mir-30a, mmu-mir-30b, mmu-mir-125a, mmu-mir-125b-2, mmu-mir-126a, mmu-mir-127, mmu-mir-9-2, mmu-mir-141, mmu-mir-145a, mmu-mir-155, mmu-mir-10b, mmu-mir-24-1, mmu-mir-205, mmu-mir-206, hsa-mir-30c-2, hsa-mir-30d, mmu-mir-30e, hsa-mir-10b, hsa-mir-34a, hsa-mir-205, hsa-mir-221, mmu-mir-290a, mmu-mir-34c, mmu-mir-34b, mmu-let-7d, mmu-mir-106b, hsa-let-7g, hsa-let-7i, hsa-mir-15b, hsa-mir-30b, hsa-mir-125b-1, hsa-mir-141, hsa-mir-145, hsa-mir-9-1, hsa-mir-9-2, hsa-mir-9-3, hsa-mir-125a, hsa-mir-125b-2, hsa-mir-126, hsa-mir-127, hsa-mir-206, mmu-mir-30c-1, mmu-mir-30c-2, mmu-mir-30d, mmu-let-7a-1, mmu-let-7a-2, mmu-let-7b, mmu-let-7c-1, mmu-let-7c-2, mmu-let-7e, mmu-let-7f-1, mmu-let-7f-2, mmu-mir-15a, mmu-mir-16-2, mmu-mir-18a, mmu-mir-21a, mmu-mir-24-2, mmu-mir-27a, mmu-mir-31, mmu-mir-34a, mmu-mir-103-1, mmu-mir-103-2, mmu-mir-322, hsa-mir-200c, hsa-mir-155, mmu-mir-17, mmu-mir-25, mmu-mir-200c, mmu-mir-221, mmu-mir-29b-2, mmu-mir-9-1, mmu-mir-9-3, mmu-mir-125b-1, hsa-mir-106b, hsa-mir-30c-1, hsa-mir-34b, hsa-mir-34c, hsa-mir-30e, hsa-mir-373, hsa-mir-20b, hsa-mir-520c, hsa-mir-503, mmu-mir-20b, mmu-mir-503, hsa-mir-103b-1, hsa-mir-103b-2, mmu-mir-145b, mmu-mir-21b, mmu-let-7j, mmu-mir-21c, mmu-mir-30f, mmu-let-7k, mmu-mir-126b, mmu-mir-290b, mmu-mir-9b-2, mmu-mir-9b-1, mmu-mir-9b-3
The miR-16 family act as tumor suppressors that induce cell cycle arrest at the G1 phase by targeting several cyclin-CDK genes including CDK6, cyclin D1, cyclin D3, E2F3 and WEE1 and all the miRNAs in this family are downregulated in a wide variety of tumors [136]. [score:8]
Wang F. Fu X. D. Zhou Y. Zhang Y. Down-regulation of the cyclin e1 oncogene expression by microrna-16–1 induces cell cycle arrest in human cancer cells BMB Rep. [score:5]
This investigation revealed that miR-21 and miR-29b were significantly up-regulated and miR-15b, miR-16 were significantly down-regulated in breast cancers in both species [131]. [score:5]
Takeshita F. Patrawala L. Osaki M. Takahashi R. U. Yamamoto Y. Kosaka N. Kawamata M. Kelnar K. Bader A. G. Brown D. Systemic delivery of synthetic microrna-16 inhibits the growth of metastatic prostate tumors via downregulation of multiple cell-cycle genes Mol. [score:5]
Calin G. A. Dumitru C. D. Shimizu M. Bichi R. Zupo S. Noch E. Aldler H. Rattan S. Keating M. Rai K. Frequent deletions and down-regulation of micro- rna genes mir15 and mir16 at 13q14 in chronic lymphocytic leukemia Proc. [score:4]
Linsley P. S. Schelter J. Burchard J. Kibukawa M. Martin M. M. Bartz S. R. Johnson J. M. Cummins J. M. Raymond C. K. Dai H. Transcripts targeted by the microrna-16 family cooperatively regulate cell cycle progression Mol. [score:3]
Liu Q. Fu H. Sun F. Zhang H. Tie Y. Zhu J. Xing R. Sun Z. Zheng X. Mir-16 family induces cell cycle arrest by regulating multiple cell cycle genes Nucleic Acids Res. [score:1]
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Description miR-451[39] Upregulated in heart due to ischemia miR-22[40] Elevated serum levels in patients with stablechronic systolic heart failure miR-133[41] Downregulated in transverse aortic constrictionand isoproterenol -induced hypertrophy miR-709[42] Upregulated in rat heart four weeks after chronicdoxorubicin treatment miR-126[43] Association with outcome of ischemic andnonischemic cardiomyopathy in patients withchronic heart failure miR-30[44] Inversely related to CTGF in two rodent mo delsof heart disease, and human pathological leftventricular hypertrophy miR-29[45] Downregulated in the heart region adjacent toan infarct miR-143[46] Molecular key to switching of the vascular smoothmuscle cell phenotype that plays a critical role incardiovascular disease pathogenesis miR-24[47] Regulates cardiac fibrosis after myocardial infarction miR-23[48] Upregulated during cardiac hypertrophy miR-378[49] Cardiac hypertrophy control miR-125[50] Important regulator of hESC differentiation to cardiacmuscle(potential therapeutic application) miR-675[51] Elevated in plasma of heart failure patients let-7[52] Aberrant expression of let-7 members incardiovascular disease miR-16[53] Circulating prognostic biomarker in critical limbischemia miR-26[54] Downregulated in a rat cardiac hypertrophy mo del miR-669[55] Prevents skeletal muscle differentiation in postnatalcardiac progenitors To further confirm biological suitability of the identified miRNAs, we examined KEGG pathway enrichment using miRNA target genes (see ). [score:31]
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[+] score: 30
Both miR-21-5p and miR-16-5p were downregulated significantly in our study, and this is associated with increased mRNA levels of BCL2 (at 18 h post exposure, Fig.   5b) that may contribute to inhibition of apoptosis and cell proliferation in SP-A2 males, but not in KO males, where the expression of BCL2 did not change (Fig.   5c). [score:8]
This mRNA is targeted by miR-9-5p [47], miR-21-5p, miR-16-5p (TargetScan), miR-183-5p [47], miR-486b-5p [82], and miR-153-3p [47]. [score:5]
miR-195a-5p that has the same seeding sequence with miR-16-5p, is predicted to bind BCL2 mRNA, and this was also downregulated in our study and may increase further the anti-apoptotic effects of miR-21-5p and miR-16-5p. [score:4]
Both miR-21-5p and miR-16-5p were significantly downregulated in our study. [score:4]
miR-195a-5p that has the same seeding sequence with miR-16-5p and is predicted to bind BCL2 mRNA was also downregulated while miR-153-3p that was also found to bind BCL2 experimentally (by Western blot, qRT-PCR, and LUC) [45] was increased. [score:4]
FOXO1 is targeted by a multitude of miRNAs that are changed in our study miR-9-5p, miR-21-5p, miR-16-5p, miR-183-5p [47], miR-486b-5p, and miR-153-3p. [score:3]
miR-21-5p is predicted to bind both, BCL2 and STAT3 mRNAs (TargetScan), and miR-16-5p has been shown to bind BCL2 by several experimental approaches (Western blot, qRT-PCR, and luciferase reporter assays) [69, 70]. [score:2]
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30
[+] score: 29
mRNA targets that showed inversely correlated expression with miRNAs (Additional file 3) include previously validated miRNA/target pairs such as Mef2c with miR-223 [14], Bcl2 with miR-15 or miR-16 [38], Mybl2 with miR-29 or miR-30 family members [39], and Ezh2 with miR-26a [40]. [score:7]
Several miRNAs that were upregulated during granulopoiesis (miR-15a, miR-16 and miR-29) have previously been shown to be downregulated in acute myeloid leukemia [46, 47]. [score:7]
More recently, mouse-specific miR-709 was found to be enriched in the nucleus to target pri-miR-15a and pri-miR-16, thus regulating the expression of mature miR-15a and miR-16 [31]. [score:6]
However, the transcription factor Myb was downregulated with concurrent overexpression of miR-15b, miR-16, miR-150 and miR-195 (Figure 3 and Additional file 3). [score:6]
Amongst these are those differentially regulated in our study including miR-16, miR-19a, miR-26a, miR-26b, miR-139, miR-195 and miR-223. [score:2]
Of these, we observed that several had been previously identified as key modulators of granulopoiesis in human and mouse, including miR-223, miR-16, and miR-29a [14, 35, 36]. [score:1]
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Other miRNAs from this paper: hsa-mir-15a, hsa-mir-16-1, hsa-mir-16-2, mmu-mir-15a, mmu-mir-16-2
A T → A point mutation and G deletion on the negative strand in the 3’ flanking region of mir-16-1 was discovered NZB mice (de novo mouse mo del of CLL) and was associated with 50% reduction in expression of mature miR-15a/16-1 [11– 13]. [score:4]
In summary, the results presented here show that the alterations found in the mir-15a/16-1 loci of NZB lead to decreased processivity resulting in decreased expression of mature miR-15a and miR-16-1, which in turn gives rise to B-1 expansion. [score:3]
While there was no difference in the free energy between the wild-type and the mutated mo deled structures, the presence of the mutation alters the structure of the pre-miR-16-1 perhaps reducing accessibility to Drosha and decreasing the processivity of this microRNA precursor. [score:2]
Raveche et al reported the discovery of a germline point mutation and deletion (T → A and G deletion on the negative strand) in the 3’ flanking region of miR-16-1 of NZB mice [12]. [score:2]
Calin et al also reported a similar point mutation (G→A on negative strand) in the 3’ flanking region of miR-16-1 in a small population of CLL patients [13]. [score:2]
Based on mo deling analysis, there is a significant potential structural alteration in pre-miR-16-1 due to the mutation present in NZB mice (Fig 1C). [score:2]
Additionally no difference in the free energy value of pre-miR-16-1 was observed with and without the mutation using the RNA mfold program (Fig 1C). [score:2]
Reduced mature miR-15a/16-1 in DBA congenic mice (D [miR-/-]) and its reverse in NZB congenic mice (N [miR+/-]) is a further proof that the NZB mir-15a/16-1 locus is the cause for reduction in mature miR-15a/16-1. Given the synteny between mouse and human in this loci, it is likely that a similar processivity block is present in the CLL patients with germline mutations in miR-16-1 as reported by Calin et al [13, 44]. [score:2]
Computer predicted structure of pre-miR-16-1. Statistics. [score:1]
B) RQ values for pri-miR-16-1, pre-miR-16-1 and mature miR-16-1 in NZB (light) and non-NZB (dark) cell lines. [score:1]
Decreased pre-miR16-1 and Mature miR16-1 in NZB. [score:1]
Computational folding of pre-miR-16-1 with the NZB mutation gave rise to a potential altered structure when compared to the wild-type sequence predicted structure. [score:1]
B) The level of mature miR-16 in DBA (non-NZB) versus NZB (CLL) spleen cells in sorted B-1 (IgM+CD5dull, B220dull) and B-2 (IgM+, CD5-, B220+) subpopulations. [score:1]
However, the amount of pre-miR-16-1 and mature miR16-1 was significantly reduced in the NZB cell line (Fig 2B). [score:1]
In addition, NZB mice have a significant decrease in the levels of mature miR-15a and miR-16 (Fig 1B). [score:1]
C) Predicted pre-miR-16-1 structure using the stem loop ± 11 nt sequence of wild type (left) and NZB (right) sequence using RNA mFold software. [score:1]
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Similarly, expressions of miR-16 and miR-143 inhibit cell proliferation and suppress tumorigenesis, and miR-143 has been observed to be down-regulated in cervical cancer [36]. [score:10]
For bats, 3 out of 4 up-regulated miRNA (miR-101-3p, miR-16-5p, miR-143-3p) likely function as tumor suppressors against various kinds of cancers, while one down-regulated miRNA (miR-221-5p) acts as a tumorigenesis promoter in human breast and pancreatic cancers. [score:9]
The summary of the six DE miRNA common to all species is described in Fig.   5. Briefly, all DE candidates were single copy miRNA across all libraries, and 4 DE miRNA (miR-101-3p, miR-16-5p, miR-143-3p and miR-155-5p) were up-regulated in bats while 2 (miR-125-5p and miR-221-5p) were down-regulated. [score:7]
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[+] score: 25
39, 40, 41 Overexpression of either miR-15b or miR-16 repressed the expression of the target protein VEGF and the proliferation of early EPC (endothelial progenitor cells), while the opposite phenomenon was observed upon knockdown of these miRNAs. [score:8]
Using qPCR, we found that miR16-5p and miR17-5p significantly inhibited VEGFR2 mRNA expression but that miR322-5p and miR497-5p had no significant effect. [score:5]
Increasing miR-16 expression might be a promising strategy for tumor therapy by repressing tumor angiogenesis and inducing tumor cell death through targeting VEGF and BCL2. [score:5]
The progression of the cell cycle from the G [0] to S phase is believed to be mediated by some members of the miR-16 family, thereby inducing cell-cycle arrest and acting as potent tumor suppressors. [score:3]
VEGFR2 3′-UTR including miR-16-5p/322-5p/497-5p target sequence was subcloned into the psicheck2 -vector RiboBio (Guangzhou, Guangdong, China) using the touchdown PCR method and the following primers: 3′-UTR-VEGFR2 forward, 5′-TCGAGTGAAATAGCAAACCCGAGTTTCTTCCTC TGCTGCTGGCCATTTCCTAAACAGC-3′ 3′-UTR-VEGFR2 reverse, 5′-GGCCGCTGTTTAGGAAATGGCCAGCAGCAGAGGAAGAAACTCGGGTTTGCTATTTCAC-3′ 3′-UTR-VEGFR2 mutant forward, 5′-TCGAGTGAAATAGCAAACCCGAGTTTCTTCCTC TCTCGTCGGCCATTTCCTAAACAGC-3′ 3′-UTR-VEGFR2 mutant reverse, 5′-GGCCGCTGTTTAGGAAATGGCCGACGAGAGAGGAAGAAACTCGGGTTTGCTATTTCAC-3′. [score:2]
VEGFR2 3′-UTR including miR-16-5p/322-5p/497-5p target sequence was subcloned into the psicheck2 -vector RiboBio (Guangzhou, Guangdong, China) using the touchdown PCR method and the following primers:3′-UTR-VEGFR2 forward, 5′-TCGAGTGAAATAGCAAACCCGAGTTTCTTCCTC TGCTGCTGGCCATTTCCTAAACAGC-3′ 3′-UTR-VEGFR2 reverse, 5′-GGCCGCTGTTTAGGAAATGGCCAGCAGCAGAGGAAGAAACTCGGGTTTGCTATTTCAC-3′ 3′-UTR-VEGFR2 mutant forward,5′-TCGAGTGAAATAGCAAACCCGAGTTTCTTCCTC TCTCGTCGGCCATTTCCTAAACAGC-3′ 3′-UTR-VEGFR2 mutant reverse, 5′-GGCCGCTGTTTAGGAAATGGCCGACGAGAGAGGAAGAAACTCGGGTTTGCTATTTCAC-3′. [score:2]
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[+] score: 25
Other miRNAs from this paper: hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3, hsa-let-7f-1, hsa-let-7f-2, hsa-mir-16-1, hsa-mir-17, hsa-mir-19a, hsa-mir-19b-1, hsa-mir-19b-2, hsa-mir-23a, hsa-mir-26a-1, hsa-mir-26b, hsa-mir-27a, hsa-mir-29a, hsa-mir-30a, hsa-mir-31, hsa-mir-100, hsa-mir-29b-1, hsa-mir-29b-2, hsa-mir-16-2, mmu-mir-23b, mmu-mir-27b, mmu-mir-29b-1, mmu-mir-30a, mmu-mir-30b, mmu-mir-127, mmu-mir-128-1, mmu-mir-132, mmu-mir-133a-1, mmu-mir-188, mmu-mir-194-1, mmu-mir-195a, mmu-mir-199a-1, hsa-mir-199a-1, mmu-mir-200b, mmu-mir-205, mmu-mir-206, hsa-mir-30c-2, hsa-mir-30d, mmu-mir-122, mmu-mir-30e, hsa-mir-199a-2, hsa-mir-199b, hsa-mir-205, hsa-mir-211, hsa-mir-212, hsa-mir-214, hsa-mir-217, hsa-mir-200b, hsa-mir-23b, hsa-mir-27b, hsa-mir-30b, hsa-mir-122, hsa-mir-128-1, hsa-mir-132, hsa-mir-133a-1, hsa-mir-133a-2, hsa-mir-127, hsa-mir-138-1, hsa-mir-188, hsa-mir-194-1, hsa-mir-195, hsa-mir-206, mmu-mir-19b-2, mmu-mir-30c-1, mmu-mir-30c-2, mmu-mir-30d, mmu-mir-200a, mmu-let-7a-1, mmu-let-7a-2, mmu-let-7f-1, mmu-let-7f-2, mmu-mir-16-2, mmu-mir-23a, mmu-mir-26a-1, mmu-mir-26b, mmu-mir-29a, mmu-mir-29c, mmu-mir-27a, mmu-mir-31, mmu-mir-351, hsa-mir-200c, mmu-mir-17, mmu-mir-19a, mmu-mir-100, mmu-mir-200c, mmu-mir-212, mmu-mir-214, mmu-mir-26a-2, mmu-mir-211, mmu-mir-29b-2, mmu-mir-199a-2, mmu-mir-199b, mmu-mir-19b-1, mmu-mir-138-1, mmu-mir-128-2, hsa-mir-128-2, mmu-mir-217, hsa-mir-194-2, mmu-mir-194-2, hsa-mir-29c, hsa-mir-30c-1, hsa-mir-200a, hsa-mir-30e, hsa-mir-26a-2, hsa-mir-379, mmu-mir-379, mmu-mir-133a-2, mmu-mir-133b, hsa-mir-133b, mmu-mir-412, mmu-mir-431, hsa-mir-431, hsa-mir-451a, mmu-mir-451a, mmu-mir-467a-1, hsa-mir-412, hsa-mir-485, hsa-mir-487a, hsa-mir-491, hsa-mir-503, hsa-mir-504, mmu-mir-485, hsa-mir-487b, mmu-mir-487b, mmu-mir-503, hsa-mir-556, hsa-mir-584, mmu-mir-665, mmu-mir-669a-1, mmu-mir-674, mmu-mir-690, mmu-mir-669a-2, mmu-mir-669a-3, mmu-mir-669c, mmu-mir-696, mmu-mir-491, mmu-mir-504, hsa-mir-665, mmu-mir-467e, mmu-mir-669k, mmu-mir-669f, hsa-mir-664a, mmu-mir-1896, mmu-mir-1894, mmu-mir-1943, mmu-mir-1983, mmu-mir-1839, mmu-mir-3064, mmu-mir-3072, mmu-mir-467a-2, mmu-mir-669a-4, mmu-mir-669a-5, mmu-mir-467a-3, mmu-mir-669a-6, mmu-mir-467a-4, mmu-mir-669a-7, mmu-mir-467a-5, mmu-mir-467a-6, mmu-mir-669a-8, mmu-mir-669a-9, mmu-mir-467a-7, mmu-mir-467a-8, mmu-mir-669a-10, mmu-mir-467a-9, mmu-mir-669a-11, mmu-mir-467a-10, mmu-mir-669a-12, mmu-mir-3473a, hsa-mir-23c, hsa-mir-4436a, hsa-mir-4454, mmu-mir-3473b, hsa-mir-4681, hsa-mir-3064, hsa-mir-4436b-1, hsa-mir-4790, hsa-mir-4804, hsa-mir-548ap, mmu-mir-3473c, mmu-mir-5110, mmu-mir-3473d, mmu-mir-5128, hsa-mir-4436b-2, mmu-mir-195b, mmu-mir-133c, mmu-mir-30f, mmu-mir-3473e, hsa-mir-6825, hsa-mir-6888, mmu-mir-6967-1, mmu-mir-3473f, mmu-mir-3473g, mmu-mir-6967-2, mmu-mir-3473h
Out of these 25 miRNAs, 18 miRNAs were differentially expressed in a consistent manner between the 2 groups (Figure 4A, highlighted); 8 miRNAs were downregulated in both groups (miR-16, miR-200, miR-205, miR-3064, miR-379, miR-431, miR-485 and miR-491) and 10 miRNAs were upregulated in both groups (miR-194, miR-1894, miR-211, miR-3072, miR- 3077, miR-4436, miR-5128, miR-669a, miR-669c and miR-6967). [score:9]
In the microarray data, 12 miRNAs were consistent in their change either with HHcy or diabetes; of which 4 miRNAs were downregulated in both groups (miR-16, miR-1983, miR-412 and miR-487) and 8 miRNAs (miR-194, miR-188, miR-1896, miR-467e, miR-504, miR-5110, miR-669k and miR-696) were upregulated in both groups. [score:7]
A triple comparison was also done that included cbs [–/–], cbs [+/–] and STZ retinas, which revealed 6 miRNAs (miR-194, miR-16, miR-212, miR-30c, miR-5128 and miR-669c) that were commonly changed among cbs [–/–], cbs [+/–] and diabetes; 2 of these miRNAs were consistently changed among the three groups (miR-194 was upregulated and miR-16 was downregulated). [score:7]
Interestingly, miR-16-5p has been reported to be tissue protective and to be decreased in a diabetic rat's kidney [92]. [score:1]
Among those miRNAs, 2 miRNAs (miR-16-5p and miR-194) were consistently changing among the three different groups. [score:1]
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[+] score: 24
1 + p53 Conditional knockout in hematopoietic cells Aggressive AML Nras:Bcl-2 Conditional transgenic Myelodysplastic syndrome Bcl-2 inhibitors TERC Conditional knockout Leukemia stem cell maintenance AML-ETO Inducible transgenic APL RARα fusion Transgenic, variable AML Transretinoic acid CML BCR-ABL1 Humanized mice transplanted with retroviral vector Chronic myeloproliferative syndrome Conditional transgenic in hematopoietic cells CML Tyrosine kinase inhibitors Transposon -based insertional mutagenesis Acute blast crisis Acute lymphoblastic leukemia (ALL) ETV6–RUNX1 Transgenic using Ig heavy chain enhancer Block in B-cell differentiation E2A–PBX1 Conditional transgenic using Lck enhancer, TCR Vβ promoter B-cell ALL NOTCH1 Tumor-derived engraftment of NOD/SCID Xenograft T-ALL Monoclonal antibody against Notch1 PRDM14 Inducible transgenic Rapid onset T-ALL Monoclonal antibody against Notch1 Chronic lymphocytic leukemia (CLL) miR-16 Spontaneous in New Zealand Black Clonal CD5+ B cell disease T-cell leukemia 1 Serial transfer transgenic Rapid progression CLL PD-1 immune checkpoint inhibitor BCR NSG™ with orthotopic splenic engraftment CLL Ibrutinib efficacy Homozygous deletion of the upstream regulatory element of PU. [score:12]
A common genomic aberration in CLL leads to increased expression of anti-apoptotic protein BCL-2, which is negatively regulated by miR-15a and miR-16-1. The expression of these miRs is lost via deletion of a region on chromosome 13, 13q14.3 (59, 60). [score:6]
To mo del a common genetic alteration in the human disease, a transgenic mouse lacking the chromosomal region 13q14 encoding for DLEU-2, miR-15, and miR-16 were developed (67). [score:3]
These mice also have reduced expression of miR-16-1 (62). [score:3]
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[+] score: 24
The commonly upregulated miRNAs included those of 34 known tumor suppressor genes (e. g., miR-16, miR-96, miR-150, miR-183, miR-186, miR-194, miR-320, and miR-371), nine miRNAs of oncogenes (e. g. miR-454), and 14 miRNAs that show both tumor suppressive and oncogenic function(Supplementary Table S1). [score:8]
The HDACIs upregulated well-known tumor suppressive miRNAs including miR-16, miR-22, miR-192-194-215 cluster, and miR-320 family. [score:6]
Moreover, when we consider our previous findings that downregulation of miR-16 was occurred even in early stage of MF that was also restored by vorinostat [18], dysregulated miRNAs by HDACs should be crucially involved in the pathogenesis of MF throughout the early and advanced stages. [score:5]
Along with inhibition of cell proliferation, miR-16 induced apoptosis or cellular senescence [18]. [score:3]
In this study, we also demonstrated that the induction of apoptosis or senescence was dependent on p53 status: miR-16 induced apoptosis in p53-mutated CTCL, but senescence in the p53 wild-type. [score:1]
In recent our study, we demonstrated that miR-16 was repressed in early to advanced CTCL. [score:1]
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[+] score: 23
0022586.g001 Figure 1Comparison of circulating miRNAs predicted to target Bmal1 or other genes regulating Bmal1 expression with a highly abundant miRNA in serum, miR-16. [score:6]
Comparison of circulating miRNAs predicted to target Bmal1 or other genes regulating Bmal1 expression with a highly abundant miRNA in serum, miR-16. [score:6]
In mice exposed to LD 12∶12, diurnal fluctuations were observed in the relative expression of miR-494 and miR-152 (normalized to miR-16) in the serum (Fig. 3). [score:3]
These synthetic miRNAs were used in a dilution series ranging from 1 molecule/µl to 10 [10] molecules/µl to generate standard curves for quantification of molecules of miR-16 and miR-152. [score:1]
miR-16 was also amplified from the same samples using identical parameters to control for differences in sample RNA content and reverse-transcription efficiencies because: 1) this miRNA has provided a good standard for normalization and comparisons of relative abundance in previous studies [21], [22]; and 2) ANOVA analysis indicates that miR-16 levels in the serum exhibit no significant variation (p = 0.19) over the 24-hour time course for sampling (data not shown). [score:1]
Quantitative analysis of miR-16 and miR-152 levels revealed that the estimated concentrations of these miRNAs were 408,000–749,000 and 3,400–6,800 copies/µl serum (Fig. 1B), respectively. [score:1]
Standard curves derived from concentrations yielding Ct values within the linear range were used to estimate the number of copies of miR-16 and miR-152 in the input RNA from ZT7 serum samples that were simultaneously reverse-transcribed, PCR-amplified and analyzed on the same plate. [score:1]
Symbols denote determinations (in duplicate) of the number of copies/µl serum in each sample that were extrapolated by comparing the Ct values for experimental samples with standard curves consisting of a dilution series of known quantities of synthetic miR-16 and miR-152 analyzed on the same plate. [score:1]
The plotted values correspond to the ratios of miR-494 (top left), miR-152 (top right) and miR-142-3p (bottom) signal normalized to miR-16 levels in each sample and are represented as a percentage of the maximal value obtained for each miRNA. [score:1]
To estimate the number of copies of representative miRNAs in serum samples, synthetic single-stranded RNA oligonucleotides encoding the mature miRNA sequences for miR-16 and miR-152 were purchased from Integrated DNA Technologies, Inc. [score:1]
Consistent with previous observations on its circulating levels in humans [9], miR-16 was highly abundant relative to other miRNAs detected in mouse serum. [score:1]
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[+] score: 23
Even by considering the common predicted targets from four existing miRNA target prediction databases (i. e. setting the database filter equal to four), PPM1D is still hidden in 883 predicted targets of miR-16-5p, suggesting that applying the database filter alone is not an efficient way to reduce the non-functional miRNA targets. [score:9]
On the contrary, if using existing miRNA target prediction databases (e. g. miRecords [16], miRWalk [22], miRSystem [19] and starBase [23]), researchers will have difficulty to pick out PPM1D among hundreds or even thousands of predicted targets of miR-16-5p (see Table 3). [score:5]
CSmiRTar was run with the settings shown in Table 2. - [a]151 (6) means that in miRecords, miR-16-5p has 151 target genes (including PPM1D) predicted by at least 6 different algorithms. [score:3]
Note that in miRecords, 6 is the most stringent setting of the algorithm filter for still reporting PPM1D as a predicted target gene of miR-16-5p. [score:3]
For example, human miR-16-5p is known to regulate the gene PPM1D in breast cancer cells [32]. [score:2]
Researchers then have a high chance to pick out the functional targets (e. g. PPM1D) of miR-16-5p for further experimental investigation. [score:1]
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[+] score: 21
Other miRNAs, such as miR-23a, miR-31, miR-132, or miR-16, were also significantly downregulated with cardamonin treatment for 24 h compared to that of treatment for 3 h. Since most of miRs have been downregulated and miR-21 was strongly suppressed by cardamonin, we used miR-21 mimics and miR-21 inhibitors to test the function of cardamonin on HUVECs. [score:10]
It is reported that miR-132 plays an important role in angiogenesis in infectious ocular disease [32] and miR-16 affects the angiogenesis by targeting VEGF [33, 34]. [score:5]
It is noted that other miRNAs, for example, miR-132 and miR-16, were also obviously downregulated after treatment with 50  µM cardamonin. [score:4]
We selected 14 of these miRs which might have been involved in regulation of angiogenesis, including miR-17-5p, miR-19a, miR-23a, miR-24, miR-31, miR-34a, miR-126, miR-130a, miR-132, miR-16, miR-21, miR-217, miR-221, and miR-378 for our study. [score:2]
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[+] score: 21
We demonstrate that HMGA1P7 overexpression increases H19 and Igf2 levels inhibiting their mRNA suppression by miRNAs that target HMGA1P7 gene, namely, miR-15, miR-16, miR-214, and miR-761. [score:9]
As proposed by our mo del, siRNA- Igf2 transfection induces a significant H19 downregulation, that is reverted by the transfection with the Anti miR-16 oligonucleotide, suggesting that both H19 and Igf2 transcripts can talk each-other through miRNAs mediation (Fig. 4D). [score:4]
For transfection of Anti miR-16 oligonucleotides, cells were transfected with 50 nmol/ml of Anti miR-16 or with a control no -targeting scrambled oligonucleotides (Thermo Fisher Scientific Inc). [score:3]
To this aim, we transfected miR-15, miR-16, miR-214 and miR-761 (already reported to target HMGA1P7) 17 into NIH3T3 cells, and analyzed H19 and Igf2 mRNA levels by qRT-PCR. [score:3]
The luciferase signal was considerably lower after transfection with miR-15, miR-16, miR-214 and miR-761 in comparison with the cells transfected with the scrambled oligonucleotide (Fig. 4B). [score:1]
Relative luciferase activity in HEK293 cells transiently transfected with miR-15, miR-16, miR-214, miR-761 and a control scrambled oligonucleotide. [score:1]
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[+] score: 20
This included seven (87.5%; miR-16-5p, miR-29b-3p, miR-29a-3p, miR-503-5p, miR-15a-5p, miR-155-5p, and miR-425-5p) that were significantly upregulated and one (12.5%; miR-880-3p) that was downregulated (>2 folds, P < 0.05). [score:7]
This study observed that expression of the miRNAs miR-155-5p, miR-425-5p, miR-15a-5p, miR-503-5p, miR-16-5p, miR-29a-3p, and miR-29b-3p in the liver of Cmah -null mice may downregulate components of the insulin/PI3K-AKT signaling pathway in concert with other genes. [score:6]
Among them, miR-155-5p, miR-425-5P, miR-15a-5p, miR-503-5p, miR-16-5p, miR-29a-3p, and miR-29b-3p were significantly upregulated in the liver and pancreas of Cmah -null mice. [score:4]
As shown in Figure 4(b) miR-155-5p miR-15a-5p, and miR-425-5p in the case of insulin signaling and miR-29b-3p, miR-29a-3p, miR-16-5p, and miR-503-5p in the case of PI3K-AKT1-mTOR signaling were significantly dysregulated. [score:2]
Among them, we found two major signal pathways such as insulin signaling (miR-155-5p, miR-425-5p, and miR-15a-5p) and PI3K-AKT signaling (miR-503-5p, miR-16-5p, miR-29a-3p, and miR-29b-3p) pathways (Table 2). [score:1]
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[+] score: 20
Other miRNAs from this paper: mmu-mir-122, dme-mir-277, dme-mir-289, dme-bantam, mmu-mir-16-2
We note that miR-16, proposed by Jing et al. to recognize AREs in the mammalian system [8], is different from miR-369-3 identified by Vasudevan et al. to enhance translation of an ARE-containing reporter mRNA [10]. [score:3]
In contrast, wt MEFs show prominent expression of mature miR-16. [score:3]
A) Total RNA was extracted from wt and dicer ko MEFs, and the expression of miR-16 was examined by Northern blot analysis. [score:3]
0028907.g001 Figure 1A) Total RNA was extracted from wt and dicer ko MEFs, and the expression of miR-16 was examined by Northern blot analysis. [score:3]
These MEFs fail to produce mature miR-16 and show accumulation of the precursor pre-miR-16 (Fig. 1A). [score:1]
For detection of mature miR-16 and its precursors, RNA was isolated from Dicer -deficient and wildtype MEF clones with the RNeasy Plus Mini Kit (QIAGEN). [score:1]
In a previous report, TTP function was suggested to depend on the microRNA miR-16 [8]. [score:1]
This result shows that neither Dicer nor miR-16 are required for ier3 mRNA decay, strongly suggesting that AREs do not genereally depend on the miRNA pathway to trigger mRNA. [score:1]
After UV cross-linking (2×120 mJ), the membrane was hybridized for 1 h at 37°C with a digoxigenin-labeled DNA probe specific for miR-16. [score:1]
In a first approach to assess the contribution of miR-16, and miRNAs in general, to the decay activity of TTP, we used Dicer -deficient mouse embryonic fibroblasts (MEFs) [15]. [score:1]
Probes were generated with the DIG Oligonucleotide 3′-End Labeling Kit, 2nd Generation (Roche) using the following oligonucleotides: 5-CGCCAATATTTACGTGCTGCTA-3′ for miR-16 and 5′- ATCTTCTCTGTATCGTTCCAATTTTAGTAT-3′ for U6 snRNA. [score:1]
dicer ko MEFs show an accumulation of the precursor pre-miR-16, whereas mature miR-16 is detected only in the wt MEFs. [score:1]
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[+] score: 20
The specificity of the RT-qPCR assay was further supported by the biodistribution patterns obtained for the tissue-specific miRNAs miR-122 (liver), miR-208a (heart) and miR-124-3p/-5p (brain), and the ubiquitously expressed miRNAs miR-191 and miR-16, using the method (Figure 3a). [score:2]
For this, two groups of mice, each consisting of five animals, were dosed intravenously with either AMO-miR-16 (80 mg/kg) or PBS. [score:1]
Despite the rapid plasma clearance, on sections obtained 24 h post dosing revealed a broad biodistribution pattern of AMO-miR-16, suggesting extensive uptake of AMO-miR-16 in a wide variety of tissues (Figure 4c). [score:1]
The quantification of AMO-miR-16 was performed in a two-step reaction. [score:1]
AMO-miR-16 injection. [score:1]
Indeed, quantification of AMO-miR-16 by CL-qPCR confirmed the presence of compound in the kidney, liver, lung and spleen but not in the brain of the treated animals. [score:1]
Figure 4. Validation of the biodistribution obtained for AMO-miR-16 using CL-qPCR. [score:1]
The highest levels of AMO-miR-16 could be observed in the kidney whereas no signal could be detected in samples co-localizing with the brain. [score:1]
AMO-miR-16 was serially diluted in either Poly(A) (10 ng/μl diluted in RNAse-free water/GE Healthcare, #27-4110-01) or in diluted (1:750 in RNAse-free water) tissue lysates from a PBS -treated mouse. [score:1]
Quantification of AMO-miR-16. [score:1]
As expected, AMO-miR-16 was rapidly cleared from the plasma reaching undetectable levels within 1 h post dosing (Figure 4b and Supplementary Figure S9). [score:1]
Plasma samples were collected at various time points over a period of 24 h, and AMO-miR-16 levels were quantified using the CL-qPCR method. [score:1]
AMO-miR-16 standard curves preparation. [score:1]
AMO-miR-16 synthesis. [score:1]
The biodistribution of genomic 18S, on the other hand, was very similar between the two animals and did not suggest that lack of miR-16 could be due to poor extraction efficiencies. [score:1]
The statistical relevance of the results obtained for the quantification of miR-16 and AMO-miR-16 was tested by Mann–Whitney Rank Sum Test. [score:1]
Mice were treated intravenously with 80 mg/kg AMO-miR-16 (dissolved in PBS) via tail vein injection. [score:1]
For example, a 2′- o-Methyl -modified anti-miRNA-16 oligonucleotide (AMO-miR-16) is readily detectable by RT-qPCR (30) whereas a 2′- o-(2-methoxyethyl) -modified sequence is not (Supplementary Figure S6). [score:1]
The presence of AMO-miR-16 also significantly reduced miR-16 levels in these tissues whereas miR-191 levels (Figure 4f and Supplementary Figure S12) remained unaffected, suggesting that the loss of miR-16 could indeed be the result of AMO binding. [score:1]
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[+] score: 18
According to previous studies in cancer (MCF-7) cells EGCG up-regulates the expression of miR-16, a member of the miR-15b family (family of miR-16/miR-15a/miR-497/miR-322/miR-195) and consequently, EGCG down-regulates Bcl-2 expression level and thus counteracts cancer progression [25]. [score:11]
For instance, EGCG treatment of MCF-7 and hepatocellular carcinoma HepG3 cancer cells up-regulates miR-16, which in turn contributes to triggering of tumor cell death [10, 15, 18, 25]. [score:4]
A. Cartoon showing the murine mmu-miR-15b (family of miR-16/miR-15a/miR-497/miR-322/miR-195) with STIM2 3’-untranslated region (3’-UTR) with seed sequence. [score:3]
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[+] score: 18
We demonstrate that overexpression of these HMGA1 pseudogenes increases HMGA1 protein levels, and inhibits the suppression of HMGA1 protein synthesis by miRNAs that target the HMGA1 gene, namely, miR-15, miR-16, miR-214, and miR-761 [31- 34]. [score:9]
Figure 2 HMGA1P6 and HMGA1P7 are targeted by HMGA1 -targeting miRNAs (A) qRT-PCR analysis of HMGA1P6 (left), HMGA1P7 (middle) and HMGA1 (right) mRNA from the MCF7 cells transfected with scrambled-oligonucleotide, miR-15, miR-16, miR-214 and miR-761. [score:5]
Within the high homology regions, we found perfectly conserved seed matches for miRNAs that have been predicted (miR-103, miR-142-3p, miR-370, and miR-432) or already demonstrated (miR-15 [31], miR-16 [31], miR-26a [32], miR-214 [33], miR-548c-3p [34] and miR-761 [33]) to target the HMGA1 gene (Figure 1B and 1C). [score:3]
Relative luciferase activity in HEK293 cells transiently transfected with miR-15, miR-16, miR-214, miR-761 and a control scrambled oligonucleotide. [score:1]
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[+] score: 18
We have found that miR-16 is down-regulated, and its predicted targets, including BDNF, apoptosis regulator Bcl-2 (BCL-2), Serotonin transporter (SERT), and Wnt2, have been shown to regulate hippocampal response to serotonin reuptake inhibitor (SRI) antidepressants [27, 58, 59, 60]. [score:10]
Zhang B. Chen C. F. Wang A. H. Lin Q. F. MiR-16 regulates cell death in Alzheimer’s disease by targeting amyloid precursor proteinEur. [score:5]
The involvement of miRNAs in the response to fluoxetine is only beginning to be explored, for example, miR-16 mediated the action of fluoxetine by acting as a micromanager of hippocampal neurogenesis [27, 28, 29]. [score:1]
Yang Y. Hu Z. Du X. Davies H. Huo X. Fang M. miR-16 and Fluoxetine Both Reverse Autophagic and Apoptotic Change in Chronic Unpredictable Mild Stress Mo del RatsFront. [score:1]
Baudry A. Mouillet-Richard S. Schneider B. Launay J. M. Kellermann O. miR-16—A key for adaptive responses of neurons to fluoxetineMed. [score:1]
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[+] score: 18
miR-16 the epithelial marker, expressed in these cells [36] together with wi dely expressed nucleotide markers, miR-141 and miR-200c were studied. [score:5]
miR-16 is a wi dely expressed miRNA across cell types, whereas miR-141 is associated with epithelial cells [31, 33, 34]. [score:3]
MicroRNAs, expressed as relative quotient (RQ) indicative of fold change (Fig 2C, 2D and 2E), also showed significant increases at 30 min by when compared to baseline (0 min) for all the three miRNAs studied (miR-16: 23-fold increase, p = 0.0001; miR-141: 23-fold increase, p<0.0001; miR-200c: 20-fold increase, p<0.0001) and whereas there was no significant change in miRNA levels for the control group within the same time (miR-16: 3-fold increase, p = 0.99; miR-141: 4-fold increase, p<0.86; miR-200c: 3-fold increase, p<0.93). [score:2]
MicroRNAs, miR-16 and miR-141, were also analyzed. [score:1]
The data was significant within 10 min for 0.7 and 1 W/cm [2] for miR-16 (0.7 W/cm [2]: 10 min: p = 0.04; 30 min: p<0.0001; 1 W/cm [2]: 10 min: p<0.0001, 30 min: p<0.0001) and miR-141 (0.7 W/cm [2]: 10 min: p = 0.017, 30 min: p<0.0001; 1 W/cm [2]: 10 min: p<0.0001, 30 min: p<0.0001). [score:1]
There was a significant (*p≤0.05) increase in CEA (A), CA19-9 (B), miR-16 (C) and miR-141 (D) at 0.7 and 1 W/cm [2] for 10 and 30 min of treatment. [score:1]
There was a significant (*p≤0.05) increase in PSA (A), miR-16 (B), miR-141 (C) and miR-200c (D) at 0.7 and 1 W/cm [2] for 10 and 30 min of treatment. [score:1]
Media was collected at 0, 10, 30 min was analyzed for PSA, miR-16, miR-141 and miR-200c (n = 2). [score:1]
The levels reached significance when comparing the treated mice to the untreated controls for miR-141 (p = 0.035) and miR-200c (p = 0.027) and did not reach significance for miR-16 (p = 0.3), even though it showed an increase over the controls (Fig 4C, 4D and 4E). [score:1]
A significant increase was also observed with all the miRNA levels by 10 min for 0.7 (miR-16: 10 min: 11-fold increase, p = 0.0002, 30 min: 26-fold increase, p<0.0001; miR-141: 10 min: 13-fold increase, p = 0.007, 30 min: 30-fold increase, p<0.0001; miR-200c: 10 min: 8-fold increase, p<0.0001, 30 min: 15-fold increase, p<0.0001) and 1 W/cm [2] (miR-16: 10 min: 38-fold increase, p<0.0001, 30 min: 59-fold increase, p<0.0001; miR-141: 10 min: 32-fold increase, p<0.0001, 30 min: 39-fold increase, p<0.0001; miR-200c: 10 min: 21-fold increase, p<0.0001, 30 min: 31-fold increase, p<0.0001) (Fig 3B, 3C and 3D). [score:1]
This release was seen to increase relative to pre-treatment baseline (0-min) levels, from 12-fold (0.1 W/cm [2]) to 568-fold (1.0 W/cm [2]) for miR-16 and 60-fold (0.1 W/cm [2]) to 212-fold (1.0 W/cm [2]) for miR-141, following 30 min of treatment (Fig 1C and 1D). [score:1]
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[+] score: 17
In addition, miR-16 overexpression could regulate APP expression in vivo in the mouse brain [50]. [score:6]
Interestingly, TargetScan predicts more than 1000 human target genes for miR-16, several of which are associated with networks related to cell death, cellular organization, and molecular transport (S. S. Hébert, unpublished observations). [score:5]
Taken together, these observations highlight the potential importance of the miR-16 family in AD development by regulating cell survival, amyloid production, and tau phosphorylation (Figure 1). [score:3]
It will be interesting to see whether loss of miR-16 family members in vivo recapitulates, at least in past, the observed effects on ERK and, most importantly, tau phosphorylation in vivo. [score:1]
Of mention, both endogenous miR-16 and tau are enriched in distal axons of sympathetic neurons [44]. [score:1]
Our functional assays provided the validation that several members of the miR-16 family (miR-16, -15, -195, -497) could directly modulate endogenous ERK1 and tau phosphorylation in neuronal cells in vitro, including rat primary neurons. [score:1]
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[+] score: 17
Other miRNAs from this paper: hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3, hsa-let-7b, hsa-let-7c, hsa-let-7d, hsa-let-7e, hsa-let-7f-1, hsa-let-7f-2, hsa-mir-15a, hsa-mir-16-1, hsa-mir-17, hsa-mir-18a, hsa-mir-20a, hsa-mir-21, hsa-mir-29a, hsa-mir-33a, hsa-mir-29b-1, hsa-mir-29b-2, hsa-mir-107, hsa-mir-16-2, mmu-let-7g, mmu-let-7i, mmu-mir-1a-1, mmu-mir-29b-1, mmu-mir-124-3, mmu-mir-126a, mmu-mir-9-2, mmu-mir-132, mmu-mir-133a-1, mmu-mir-134, mmu-mir-138-2, mmu-mir-145a, mmu-mir-152, mmu-mir-10b, mmu-mir-181a-2, hsa-mir-192, mmu-mir-204, mmu-mir-206, hsa-mir-148a, mmu-mir-143, hsa-mir-7-1, hsa-mir-7-2, hsa-mir-7-3, hsa-mir-10b, hsa-mir-34a, hsa-mir-181a-2, hsa-mir-181b-1, hsa-mir-204, hsa-mir-211, hsa-mir-212, hsa-mir-181a-1, mmu-mir-34c, mmu-mir-34b, mmu-let-7d, mmu-mir-106b, hsa-let-7g, hsa-let-7i, hsa-mir-1-2, hsa-mir-124-1, hsa-mir-124-2, hsa-mir-124-3, hsa-mir-132, hsa-mir-133a-1, hsa-mir-133a-2, hsa-mir-138-2, hsa-mir-143, hsa-mir-145, hsa-mir-152, hsa-mir-9-1, hsa-mir-9-2, hsa-mir-9-3, hsa-mir-126, hsa-mir-134, hsa-mir-138-1, hsa-mir-206, mmu-mir-148a, mmu-mir-192, mmu-let-7a-1, mmu-let-7a-2, mmu-let-7b, mmu-let-7c-1, mmu-let-7c-2, mmu-let-7e, mmu-let-7f-1, mmu-let-7f-2, mmu-mir-15a, mmu-mir-16-2, mmu-mir-18a, mmu-mir-20a, mmu-mir-21a, mmu-mir-29a, mmu-mir-29c, mmu-mir-34a, mmu-mir-330, hsa-mir-1-1, mmu-mir-1a-2, hsa-mir-181b-2, mmu-mir-107, mmu-mir-17, mmu-mir-212, mmu-mir-181a-1, mmu-mir-33, mmu-mir-211, mmu-mir-29b-2, mmu-mir-124-1, mmu-mir-124-2, mmu-mir-9-1, mmu-mir-9-3, mmu-mir-138-1, mmu-mir-181b-1, mmu-mir-7a-1, mmu-mir-7a-2, mmu-mir-7b, hsa-mir-106b, hsa-mir-29c, hsa-mir-34b, hsa-mir-34c, hsa-mir-330, mmu-mir-133a-2, mmu-mir-133b, hsa-mir-133b, mmu-mir-181b-2, hsa-mir-181d, hsa-mir-505, hsa-mir-590, hsa-mir-33b, hsa-mir-454, mmu-mir-505, mmu-mir-181d, mmu-mir-590, mmu-mir-1b, mmu-mir-145b, mmu-mir-21b, mmu-let-7j, mmu-mir-21c, mmu-let-7k, mmu-mir-126b, mmu-mir-9b-2, mmu-mir-124b, mmu-mir-9b-1, mmu-mir-9b-3
Downregulation of the tumor-suppressor miR-16 via progestin -mediated oncogenic signaling contributes to breast cancer development. [score:7]
Tumor-suppressor miR-16 (downregulated in some cancers) and miR-132 (which is methylation-silenced in prostate cancer) have been identified as putative endogenous modulators of neuronal tau phosphorylation and tau exon 10 splicing, respectively (Bottoni et al., 2005; Hébert et al., 2010; Formosa et al., 2012; Rivas et al., 2012). [score:6]
miR-15a and miR-16-1 down-regulation in pituitary adenomas. [score:4]
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[+] score: 17
We chose to profile the ubiquitously expressed miR-16, five ESC-specific miRNAs (miR-290, miR-291-3p, miR-292-3p, miR-294, and miR-295) [23], [24], and two miRNAs that are upregulated in ESCs undergoing differentiation (miR-21 and miR-22) [23], [24]. [score:6]
Using RT-PCR, we tested for the presence of a ubiquitous miRNA, miR-16, which is expressed in ESCs. [score:3]
The ubiquitously expressed miR-16 (and the small nuclear RNA, RNU6b) did not transfer and remained near baseline at all time points tested (Figure 7). [score:3]
Alternatively, the abundance of miRNAs may be tightly regulated by specific nucleases such that miR-16 levels inside MEFs are kept within a specific range, but the ESC specific miRNAs are not. [score:2]
Using equivalent amounts of starting total RNA template, we detected a miR-16 signal only with the RNA samples containing small RNAs (not shown). [score:1]
The difference in Ct values between the negative control (MEFs alone) and each experimental group (miR-290, miR-291-3p, miR-292-3p, miR-294, miR-295, miR-16, and RNU6b) is shown. [score:1]
The miRNAs tested include miR-16 (lane 1), miR-21 (lane 2), miR-22 (lane 3), miR-290 (lane 4), miR-291-3p (lane 5), miR-292-3p (lane 6), miR-294 (lane 7), miR-295 (lane 8), and the small nuclear RNA, RNU6b (lane 9). [score:1]
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[+] score: 17
The expression of each miRNA was represented as relative values normalized by the expression of miR-16 and cel-miR-39 used as an internal control and spike-in miRNA, respectively. [score:5]
Ubiquitously expressed miR-16, miR-21, and miR-212 were used as positive controls, and did not show any significant differences between wt, mdx and tg serum, whereas miR-122a and miR-323 which expressed specifically in liver and brain, respectively, and miR-302 did not dectect in all of EVs (S3B Fig). [score:5]
miR-16 were used as ubiquitous-expressed marker (S3A Fig). [score:3]
miR-16, miR-21, and miR-212 were used as ubiquitous-expressed miRNAs. [score:3]
S3 Fig(A) miR-16 level in the EVs separated by immunoprecipitation with anti-caveolin-3 (Cav3), anti-CD63, anti-CD81, anti-flotillin-1 (Flot1), or anti-MHC class II (MHC II) antibodies from the sera of DMD patients and controls (n = 5 and 4, respectively). [score:1]
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[+] score: 17
Numbers below miR-16 panel indicate relative expression. [score:3]
As seen in Fig. 5B, seven and thirteen days post infection with the recombinant virus, cellular miR-16 expression was diminished (1.9-fold at day 7 and 2.6-fold at day 13) reflecting the expected defect in miRNA maturation. [score:3]
However, the TargetScan algorithm [19] only predicted binding sites for members of the miR-16 and miR-21 families. [score:3]
B. Northern blot experiments showing, miR-16 and MCMV miR-M23-2 expression in peritoneal macrophages isolated from Dicer [flox/ flox] mice 7 days and 13 days upon MCMV-Cre infection. [score:3]
It remains of course possible that other miRNAs, such as miR-210, miR-135a/b or miR-16, could also be involved in the regulation of this chemokine. [score:2]
Mean Ct values were used for data analysis; n = 3. Northern blotting of miRNAs was performed as described previously [16] using 2 µg of total RNA and radiolabeled antisense oligodesoxyribonucleotides complementary to miR-16, miR-M23-2 or part of U6 snRNA as probes. [score:1]
In addition, the putative binding site of miR-21 is more extensive than the miR-16 one as assessed using RNAhybrid [20] (Fig. 3E). [score:1]
Concomitant increase of the precursor form (pre-miR-16) was also observed (data not shown). [score:1]
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[+] score: 17
A known tumor suppressor-like miRNA, miR-16, binds to versican 3′UTR and has a hypothetical target site in Rb1 mRNA. [score:5]
miR-16, which potentially targeting Versican 3′UTR, also has a potential target site on Rb1. [score:5]
In the 3′UTR of mouse PTEN, additional sites are found to be targeted by several Let-7 family members and miR-16, which are known miRNAs contributing to development of cancer. [score:4]
Besides miR-144 and miR-136, two other miRNAs, miR-16 and let-7 miRNA, could potentially interact with versican 3′UTR and have hypothetical target site on the PTEN mRNA (SI Figure S5b). [score:3]
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[+] score: 16
In our study, we revealed that mIGF-1 up-regulated miR-16, which in turn stimulates the degradation of TNF-α and the inhibition of MCP-1 expression. [score:8]
miR-16 was indeed strongly downregulated in the diaphragm of 4-week-old mdx compared to wild type, while its expression was rescued in mdx/mIGF-1 diaphragm (Figure 4D). [score:5]
To further support this hypothesis we analyzed miR-16 expression, which induces TNF-α mRNA degradation (Jing et al., 2005). [score:3]
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[+] score: 16
Using the partial sciatic nerve injury mo del we also observed a sustained downregulation of miR-1 and -206, but not miR-16, in DRG. [score:4]
Moreover, capsaicin induced a significant upregulation of miR-1 and miR-16, but not miR-206, in DRG (Figure 4B). [score:4]
Conversely, miR-16 showed no difference in the expression level over the entire period of study. [score:3]
On the other hand, miR-1 showed a significant decrease in the spinal dorsal horn from day 1 to day 7 whereas no change was observed in the expression of miR-16 and -206 (Figure 3B). [score:3]
No significant change was detected for miR-16 (gray bars). [score:1]
Therefore, we investigated the temporal, spatial and stimulus -dependent specificity of miRNAs by monitoring the time-course expression of miR-1, miR-16, miR-122a, and miR-206 in mouse DRG and spinal cord dorsal horn under inflammatory and neuropathic pain states as well as after acute nociceptive stimulation. [score:1]
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[+] score: 16
For example, miR-223 is reported to be expressed in myeloid cells [7, 23]; miR-125 and 128 are highly expressed in the brain [13, 14]; and miR-16 is expressed in a wide variety of tissues [7, 14, 23] (see also heat map of expression in Figure 5a). [score:9]
1-/- precursor cells and fully differentiated Th1 cells, but it's expression was not detectable in the other cell types tested (Table 1); in contrast, miR-16 was expressed in all cell types analyzed, but it's expression was relatively variable both in arrays and Northern blots, so quantification was not attempted (data not shown). [score:7]
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[+] score: 15
As expected, normalization to miR-16 and miR-223 produced similar expression ratios to the average Cq method. [score:3]
The differences between normalization methods did not fundamentally change the interpretation of our previous results for miRNAs which show large, statistically significant differences in expression such as miR-1 (Fig. 2E ) (although the significance was lower when normalizing to miR-16). [score:3]
We have also observed considerable natural variation in the abundance of the proposed endogenous control miRNAs (miR-16 and miR-223) with these miRNAs showing variable expression across the time course samples, and between experimental groups (Figs. 5, 6 ). [score:3]
The effects of normalization to the average Cq, RNA spike control, miR-223 or miR-16 are shown for (E) miR-1, (F) miR-15b. [score:1]
All three endogenous miRNAs showed positive and highly significant correlations with the spike-in control (miR-16: r = 0.691 and P = 5×10 [−18], miR-31 r = 0.734 and P = 3.2×10 [−21], miR-223 r = 0.637 and P = 8.5×10 [−15]). [score:1]
Plot of raw Cq values from the time course study for cel-miR-39 against (A) miR-16, (B) miR-31, and (C) miR-223. [score:1]
Conversely, miR-16 has a lower stability value (ranking 65 [th]). [score:1]
These miRNAs were included because miR-16 is commonly used as a reference miRNA in cell and tissue studies [15], [24], miR-223 has been previously used as a normalizer by our group [15] and others [13], [25], and miR-31 has been recently been proposed as a novel normalizer [26]. [score:1]
0089237.g004 Figure 4 Plot of raw Cq values from the time course study for cel-miR-39 against (A) miR-16, (B) miR-31, and (C) miR-223. [score:1]
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[+] score: 14
Expression of E3 ubiquitin-protein ligase DXT4 (DTX4) (a), leiomodin-1 (LMOD1) (b), a disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1 motif, 19 (ADAMTS19) (c) and neuron navigator 1 (NAV1) (d) was assessed by RT-PCR in HK2 cells treated or not with antagomirs against let-7a, miR-125b-5p, miR-16-5p, miR-26a-5p or miR-29b-3p. [score:3]
Expression of let-7a (a), miR-125b-5p (b), miR-16-5p (c), miR-26a-5p (d) and miR-29b-3p (e) was assessed by RT-PCR in HK2 cells treated or not with antagomirs. [score:3]
On the other hand, to our knowledge, this is the first time that miR-16 and let-7a are associated to the development of kidney disease, as these miRNAs were mostly characterized to be involved in cancer [29– 34]. [score:2]
MiRNAs let-7a-5p, miR-125b-5p, miR-16-5p, miR-26a-5p and miR-29b-3p were consistently modified in mice and humans. [score:1]
In addition, a slight but significant inverse correlation with pelvic diameter was also observed for miR-let-7a-5p and miR-125-5p and with hydronephrosis grade for miR-let-7a-5p and miR-26a-5p, and a slight but significant positive correlation with age for miR-let-7a-5p and miR-16-5p (Table  4). [score:1]
Antagomirs for miR-125b-5p, miR-16-5p and miR-29b-3p showed no effect on DTX4, LMOD1 and ADAMTS19, respectively (Fig.   2a–c). [score:1]
no 67488990) hsa-miR-16-5p (ref no. [score:1]
In the presence of antagomirs, the detected signal of let-7a, miR-16-5p, miR-125b-5p, miR-26a-5p and miR-29b-3p was significantly decreased (Fig.   1). [score:1]
These five miRNAs were let-7a-5p miR-16-5p, miR-29b-3p, miR-125b-5p and miR-26a-5p (Table  3). [score:1]
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[+] score: 14
Other miRNAs from this paper: hsa-mir-16-1, hsa-mir-16-2, mmu-mir-16-2
In order to demonstrate miRNA accessibility in a wide range of tissues, we studied the ubiquitously expressed miRNA-16 in 58 routinely obtained and processed tissues from a variety of organs, consisting of benign and malignant tissues (table 1). [score:3]
miR-122a was chosen as a liver-specific miRNA, while miR-16, known to be ubiquitously expressed, was considered as a representative miRNA of all other organs and tissues. [score:2]
Error bars indicate standard deviation and asterisks indicate a significant decrease of miR-16 level after 17 and 27 years of storage (p<0.01). [score:1]
Level of miR-16 microRNA in different formalin-fixed paraffin-embedded tissues. [score:1]
Median of miR-16 level in different organs determined by real-time PCR of 10 ng RNA for each sample. [score:1]
A 10 ng quantity of total RNA for each sample was used and miR-16 levels were taken from a standard curve. [score:1]
Level of miR-16 microRNA in archived formalin-fixed paraffin-embedded samples from three decades. [score:1]
PCR analysis revealed some variation in miRNA-16 level; this was expected because of the unique nature of each sample and the wide morphological differences. [score:1]
Fixation kinetics of mouse livers and experiments with different fixatives were normalised using miR-16 as a reference, and this was followed by calculating the specific calibrated mirR-122a microRNA expression of each mouse liver sample. [score:1]
To determine the amount of miR-16 microRNA, a dilution series of total RNA in five steps was performed. [score:1]
miR-16 levels were determined in formalin-fixed paraffin-embedded human lymph nodes by real-time PCR. [score:1]
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[+] score: 14
Other miRNAs from this paper: hsa-let-7a-2, hsa-let-7c, hsa-let-7e, hsa-mir-15a, hsa-mir-16-1, hsa-mir-21, hsa-mir-22, hsa-mir-23a, hsa-mir-24-2, hsa-mir-100, hsa-mir-29b-2, mmu-let-7i, mmu-mir-99b, mmu-mir-125a, mmu-mir-130a, mmu-mir-142a, mmu-mir-144, mmu-mir-155, mmu-mir-183, hsa-mir-196a-1, mmu-mir-199a-1, hsa-mir-199a-1, mmu-mir-200b, hsa-mir-148a, mmu-mir-143, hsa-mir-181c, hsa-mir-183, hsa-mir-199a-2, hsa-mir-199b, hsa-mir-181a-1, hsa-mir-200b, mmu-mir-298, mmu-mir-34b, hsa-let-7i, hsa-mir-124-1, hsa-mir-124-2, hsa-mir-130a, hsa-mir-142, hsa-mir-143, hsa-mir-144, hsa-mir-125a, mmu-mir-148a, mmu-mir-196a-1, mmu-let-7a-2, mmu-let-7c-1, mmu-let-7c-2, mmu-let-7e, mmu-mir-15a, mmu-mir-21a, mmu-mir-22, mmu-mir-23a, mmu-mir-24-2, rno-mir-148b, mmu-mir-148b, hsa-mir-200c, hsa-mir-155, mmu-mir-100, mmu-mir-200c, mmu-mir-181a-1, mmu-mir-29b-2, mmu-mir-199a-2, mmu-mir-199b, mmu-mir-124-1, mmu-mir-124-2, mmu-mir-181c, hsa-mir-34b, hsa-mir-99b, hsa-mir-374a, hsa-mir-148b, rno-let-7a-2, rno-let-7c-1, rno-let-7c-2, rno-let-7e, rno-let-7i, rno-mir-21, rno-mir-22, rno-mir-23a, rno-mir-24-2, rno-mir-29b-2, rno-mir-34b, rno-mir-99b, rno-mir-100, rno-mir-124-1, rno-mir-124-2, rno-mir-125a, rno-mir-130a, rno-mir-142, rno-mir-143, rno-mir-144, rno-mir-181c, rno-mir-183, rno-mir-199a, rno-mir-200c, rno-mir-200b, rno-mir-181a-1, rno-mir-298, hsa-mir-193b, hsa-mir-497, hsa-mir-568, hsa-mir-572, hsa-mir-596, hsa-mir-612, rno-mir-664-1, rno-mir-664-2, rno-mir-497, mmu-mir-374b, mmu-mir-497a, mmu-mir-193b, mmu-mir-466b-1, mmu-mir-466b-2, mmu-mir-568, hsa-mir-298, hsa-mir-374b, rno-mir-466b-1, rno-mir-466b-2, hsa-mir-664a, mmu-mir-664, rno-mir-568, hsa-mir-664b, mmu-mir-21b, mmu-mir-21c, rno-mir-155, mmu-mir-142b, mmu-mir-497b, rno-mir-148a, rno-mir-15a, rno-mir-193b
The predicted genomic coordinates of pri-miRNAs are provided in Additional file 1. Here, we describe in detail the annotation of the pri-miRNA containing miR-15a and miR-16-1. The structure of a polycistronic transcript expressing miR-15a and miR-16-1 is strongly supported by all seven types of transcriptional features. [score:3]
The predicted genomic coordinates of pri-miRNAs are provided in Additional file 1. Here, we describe in detail the annotation of the pri-miRNA containing miR-15a and miR-16-1. The structure of a polycistronic transcript expressing miR-15a and miR-16-1 is strongly supported by all seven types of transcriptional features. [score:3]
These data agree with previous annotation by the VEGA project of non-protein-coding transcripts (accessions: OTTHUMT00000044959 and OTTHUMT00000044961) expressing miR-15a and miR-16-1 in human, called DLEU2 [39]. [score:3]
The structure of a polycistronic transcript expressing miR-15a and miR-16-1 is strongly supported by all seven types of transcriptional features. [score:3]
PolyA signals 'AATAAA', 'ATTAAA' and 'TATAAA' are predicted at an average distance of 4,695 bp and 4,595 bp from the 3' end of miR-16-1 in human and mouse respectively. [score:1]
The 3' end is also supported by ditags in human (U_144334, U_1281401 and U_141201), 4,208 bp from the 3' end of miR-16-1, and by ESTs and cDNAs in both human and mouse (Figure 6). [score:1]
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Notably, 23 circulating miRNAs (mmu-miR-16, mmu-let-7i, mmu-miR-26a, mmu-miR-17, mmu-miR-107, mmu-miR-195, mmu-miR-20a, mmu-miR-25, mmu-miR-15b, mmu-miR-15a, mmu-let-7b, mmu-let-7a, mmu-let-7c, mmu-miR-103, mmu-let-7f, mmu-miR-106a, mmu-miR-106b, mmu-miR-93, mmu-miR-23b, mmu-miR-21, mmu-miR-30b, mmu-miR-221, and mmu-miR-19b) were significantly downregulated in DIO mice but upregulated in DIO + LFD mice. [score:7]
As shown in the Venn diagram in Fig.   7, notably, 23 of the 28 upregulated miRNAs in DIO + LFD mice (mmu-miR-16, mmu-let-7i, mmu-miR-26a, mmu-miR-17, mmu-miR-107, mmu-miR-195, mmu-miR-20a, mmu-miR-25, mmu-miR-15b, mmu-miR-15a, mmu-let-7b, mmu-let-7a, mmu-let-7c, mmu-miR-103, mmu-let-7f, mmu-miR-106a, mmu-miR-106b, mmu-miR-93, mmu-miR-23b, mmu-miR-21, mmu-miR-30b, mmu-miR-221, and mmu-miR-19b) were downregulated in the DIO mice. [score:7]
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[+] score: 13
Other miRNAs from this paper: hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3, hsa-let-7b, hsa-let-7c, hsa-let-7d, hsa-let-7e, hsa-let-7f-1, hsa-let-7f-2, hsa-mir-16-1, hsa-mir-21, hsa-mir-16-2, mmu-let-7g, mmu-let-7i, mmu-mir-9-2, mmu-mir-151, mmu-mir-10b, hsa-mir-192, mmu-mir-194-1, mmu-mir-199a-1, hsa-mir-199a-1, mmu-mir-122, hsa-mir-10a, hsa-mir-10b, hsa-mir-199a-2, hsa-mir-199b, hsa-mir-210, hsa-mir-214, mmu-let-7d, hsa-let-7g, hsa-let-7i, hsa-mir-122, hsa-mir-9-1, hsa-mir-9-2, hsa-mir-9-3, hsa-mir-194-1, mmu-mir-192, mmu-let-7a-1, mmu-let-7a-2, mmu-let-7b, mmu-let-7c-1, mmu-let-7c-2, mmu-let-7e, mmu-let-7f-1, mmu-let-7f-2, mmu-mir-16-2, mmu-mir-21a, mmu-mir-10a, mmu-mir-210, mmu-mir-214, mmu-mir-199a-2, mmu-mir-199b, mmu-mir-9-1, mmu-mir-9-3, hsa-mir-194-2, mmu-mir-194-2, hsa-mir-365a, mmu-mir-365-1, hsa-mir-365b, hsa-mir-151a, gga-let-7i, gga-let-7a-3, gga-let-7b, gga-let-7c, gga-mir-16-1, gga-mir-194, gga-mir-10b, gga-mir-199-2, gga-mir-16-2, gga-let-7g, gga-let-7d, gga-let-7f, gga-let-7a-1, gga-mir-199-1, gga-let-7a-2, gga-let-7j, gga-let-7k, gga-mir-122-1, gga-mir-122-2, gga-mir-9-2, mmu-mir-365-2, gga-mir-9-1, gga-mir-365-1, gga-mir-365-2, hsa-mir-151b, mmu-mir-744, gga-mir-21, hsa-mir-744, gga-mir-199b, gga-mir-122b, gga-mir-10a, gga-mir-16c, gga-mir-214, sma-let-7, sma-mir-71a, sma-bantam, sma-mir-10, sma-mir-2a, sma-mir-3479, sma-mir-71b, mmu-mir-21b, mmu-let-7j, mmu-mir-21c, mmu-let-7k, gga-mir-365b, sma-mir-8437, sma-mir-2162, gga-mir-9-3, gga-mir-210a, gga-mir-9-4, mmu-mir-9b-2, mmu-mir-9b-1, mmu-mir-9b-3, gga-mir-9b-1, gga-mir-10c, gga-mir-210b, gga-let-7l-1, gga-let-7l-2, gga-mir-122b-1, gga-mir-9b-2, gga-mir-122b-2
For validation of the microarray results, the miRNAs that displayed the largest fold change were quantified by qRT-PCR and normalized to miR-16 (a total of 6 up-regulated miRNAs and 6 down-regulated miRNAs were examined). [score:7]
Figure S2Expression of miR-16 in liver and serum over the time course of S. mansoni infection. [score:3]
To account for differences in RNA extraction or qRT-PCR efficiency, the data were normalised to miR-16, which displayed stable expression in the liver during infection (Fig. S2). [score:3]
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[+] score: 13
To assess whether the depletion protocols could affect miRNA expression values, we determined the abundance levels of mmu-miR-16-5p (MIMAT0000527, miRBase v18), a miRNA that is reported to be highly and stably expressed in mouse serum, in depleted and control samples 22. [score:5]
Both depletion protocols were found to have an impact on miR-16 expression, with a 2-fold and 20-fold reduction in miR-16 expression levels for the beads -based depletion and RNase H -based depletion, respectively (Supplementary Figure S2). [score:5]
Hsa-miR-15a-5p, hsa-miR-16-5p, hsa-miR-30c, hsa-miR-451a, hsa-miR-191-5p, hsa-miR-486-5p and hsa-miR-335-3p for instance have been described to be aberrantly expressed in metastatic neuroblastoma tumors 23. [score:3]
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64
[+] score: 13
We found a significant up-regulation of oncogenic miRNAs and a significant down-regulation of tumor-suppressing miRNAs, which included let-7, miR-17-92, miR-10b, miR-15, miR-16, miR-26, and miR-181. [score:9]
Fifteen miRNAs were highly expressed in both liver and brain: miR-709, let-7a, let-7f, let-7c, let-7d, miR-26a, let-7b, let-7g, miR-26b, miR-29a, miR-126-3p, miR-23b, miR-30c, miR-16, and miR-23a. [score:3]
In this study, we found that many cancer-related miRNAs, such as let-7, miR-17-92, miR-10b, 125b, miR-146, miR-15, miR-200, and miR-16, were significantly affected by RDX exposure (Table 4). [score:1]
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65
[+] score: 12
Downstream molecules of the insulin signaling pathway are predicted targets of miR-15a and miR-16 (targetscan. [score:5]
Our previous study showed altered expression of miR-15 family members, miR-15b and miR-16, in retinal endothelial cells (REC) cultured in high glucose conditions [9]. [score:3]
REC were transfected with mimics (30 nM of final concentration) of miR-15a and/or miR-16 in high glucose conditions. [score:1]
REC were transfected with miRNA mimic (hsa-miR-15a-5p and hsa-miR-16-5p) (Invitrogen, Carlsbad, CA) using Oligofectamine (Invitrogen) following manufacturer instructions. [score:1]
Transfection was performed on REC in high glucose with miRNA mimic (hsa-miR-15a-5p, hsa-miR-16-5p). [score:1]
A final concentration of 30 nM was used when transfected separately (miR-15a and miR-16), and 15 nM was used in combination (miR-15a + miR-16). [score:1]
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66
[+] score: 12
Other miRNAs from this paper: hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3, hsa-let-7b, hsa-let-7c, hsa-let-7d, hsa-let-7e, hsa-let-7f-1, hsa-let-7f-2, hsa-mir-15a, hsa-mir-16-1, hsa-mir-17, hsa-mir-18a, hsa-mir-19a, hsa-mir-19b-1, hsa-mir-19b-2, hsa-mir-21, hsa-mir-23a, hsa-mir-30a, hsa-mir-98, hsa-mir-16-2, mmu-let-7g, mmu-let-7i, mmu-mir-15b, mmu-mir-30a, mmu-mir-30b, mmu-mir-101a, mmu-mir-125a, mmu-mir-125b-2, mmu-mir-9-2, mmu-mir-132, mmu-mir-133a-1, mmu-mir-135a-1, mmu-mir-150, mmu-mir-155, mmu-mir-204, mmu-mir-205, hsa-mir-30c-2, hsa-mir-30d, mmu-mir-30e, hsa-mir-34a, hsa-mir-204, hsa-mir-205, hsa-mir-217, mmu-mir-34c, mmu-mir-34b, mmu-let-7d, hsa-let-7g, hsa-let-7i, hsa-mir-15b, hsa-mir-30b, hsa-mir-125b-1, hsa-mir-132, hsa-mir-133a-1, hsa-mir-133a-2, hsa-mir-135a-1, hsa-mir-135a-2, hsa-mir-9-1, hsa-mir-9-2, hsa-mir-9-3, hsa-mir-125a, hsa-mir-125b-2, hsa-mir-150, mmu-mir-19b-2, mmu-mir-30c-1, mmu-mir-30c-2, mmu-mir-30d, mmu-let-7a-1, mmu-let-7a-2, mmu-let-7b, mmu-let-7c-1, mmu-let-7c-2, mmu-let-7e, mmu-let-7f-1, mmu-let-7f-2, mmu-mir-15a, mmu-mir-16-2, mmu-mir-18a, mmu-mir-21a, mmu-mir-23a, mmu-mir-34a, mmu-mir-98, mmu-mir-322, mmu-mir-338, hsa-mir-155, mmu-mir-17, mmu-mir-19a, mmu-mir-135a-2, mmu-mir-19b-1, mmu-mir-9-1, mmu-mir-9-3, mmu-mir-125b-1, mmu-mir-217, hsa-mir-30c-1, hsa-mir-34b, hsa-mir-34c, hsa-mir-30e, hsa-mir-338, mmu-mir-133a-2, mmu-mir-133b, hsa-mir-133b, hsa-mir-18b, hsa-mir-503, mmu-mir-541, mmu-mir-503, mmu-mir-744, mmu-mir-18b, hsa-mir-541, hsa-mir-744, mmu-mir-133c, mmu-mir-21b, mmu-let-7j, mmu-mir-21c, mmu-mir-30f, mmu-let-7k, mmu-mir-9b-2, mmu-mir-9b-1, mmu-mir-9b-3
The cohort of miRNA, which was upregulated during osteoblast maturation, including miR-30d, miR-155, miR-21 and miR-16, constitutes a marker of osteocytic differentiation and these miRNA may possibly repress stemness maintenance in osteoblasts. [score:4]
*miR-16 and miR-322/424 share targets. [score:3]
The expression level of miR-16 was decreased by the single stimulation (Fig. 4A, D). [score:3]
Both miR-34c and miR-16, which increased at 2w+, the stage of osteocytes, are possibly osteocyte markers and repressors of osteoblast-maintaining genes. [score:1]
miR-16 was also repressed by osteogenic stimulus as quantified by either miRNA array or qRT-PCR (Fig. 5G, H). [score:1]
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67
[+] score: 12
Other miRNAs from this paper: hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3, hsa-let-7b, hsa-let-7c, hsa-let-7d, hsa-let-7e, hsa-let-7f-1, hsa-let-7f-2, hsa-mir-15a, hsa-mir-16-1, hsa-mir-17, hsa-mir-18a, hsa-mir-19a, hsa-mir-19b-1, hsa-mir-20a, hsa-mir-22, hsa-mir-26a-1, hsa-mir-26b, hsa-mir-98, hsa-mir-101-1, hsa-mir-16-2, mmu-let-7g, mmu-let-7i, mmu-mir-1a-1, mmu-mir-15b, mmu-mir-101a, mmu-mir-126a, mmu-mir-130a, mmu-mir-133a-1, mmu-mir-142a, mmu-mir-181a-2, mmu-mir-194-1, hsa-mir-208a, hsa-mir-30c-2, mmu-mir-122, mmu-mir-143, hsa-mir-181a-2, hsa-mir-181b-1, hsa-mir-181c, hsa-mir-181a-1, mmu-let-7d, hsa-let-7g, hsa-let-7i, hsa-mir-1-2, hsa-mir-15b, hsa-mir-122, hsa-mir-130a, hsa-mir-133a-1, hsa-mir-133a-2, hsa-mir-142, hsa-mir-143, hsa-mir-126, hsa-mir-194-1, mmu-mir-30c-1, mmu-mir-30c-2, mmu-mir-208a, mmu-let-7a-1, mmu-let-7a-2, mmu-let-7b, mmu-let-7c-1, mmu-let-7c-2, mmu-let-7e, mmu-let-7f-1, mmu-let-7f-2, mmu-mir-15a, mmu-mir-16-2, mmu-mir-18a, mmu-mir-20a, mmu-mir-22, mmu-mir-26a-1, mmu-mir-26b, mmu-mir-29c, mmu-mir-98, mmu-mir-326, rno-mir-326, rno-let-7d, rno-mir-20a, rno-mir-101b, mmu-mir-101b, hsa-mir-1-1, mmu-mir-1a-2, hsa-mir-181b-2, mmu-mir-17, mmu-mir-19a, mmu-mir-181a-1, mmu-mir-26a-2, mmu-mir-19b-1, mmu-mir-181b-1, mmu-mir-181c, hsa-mir-194-2, mmu-mir-194-2, hsa-mir-29c, hsa-mir-30c-1, hsa-mir-101-2, hsa-mir-26a-2, hsa-mir-378a, mmu-mir-378a, hsa-mir-326, mmu-mir-133a-2, mmu-mir-133b, hsa-mir-133b, mmu-mir-181b-2, rno-let-7a-1, rno-let-7a-2, rno-let-7b, rno-let-7c-1, rno-let-7c-2, rno-let-7e, rno-let-7f-1, rno-let-7f-2, rno-let-7i, rno-mir-15b, rno-mir-16, rno-mir-17-1, rno-mir-18a, rno-mir-19b-1, rno-mir-19a, rno-mir-22, rno-mir-26a, rno-mir-26b, rno-mir-29c-1, rno-mir-30c-1, rno-mir-30c-2, rno-mir-98, rno-mir-101a, rno-mir-122, rno-mir-126a, rno-mir-130a, rno-mir-133a, rno-mir-142, rno-mir-143, rno-mir-181c, rno-mir-181a-2, rno-mir-181b-1, rno-mir-181b-2, rno-mir-194-1, rno-mir-194-2, rno-mir-208a, rno-mir-181a-1, hsa-mir-423, hsa-mir-18b, hsa-mir-20b, hsa-mir-451a, mmu-mir-451a, rno-mir-451, ssc-mir-122, ssc-mir-15b, ssc-mir-181b-2, ssc-mir-19a, ssc-mir-20a, ssc-mir-26a, ssc-mir-326, ssc-mir-181c, ssc-let-7c, ssc-let-7f-1, ssc-let-7i, ssc-mir-18a, ssc-mir-29c, ssc-mir-30c-2, hsa-mir-484, hsa-mir-181d, hsa-mir-499a, rno-mir-1, rno-mir-133b, mmu-mir-484, mmu-mir-20b, rno-mir-20b, rno-mir-378a, rno-mir-499, hsa-mir-378d-2, mmu-mir-423, mmu-mir-499, mmu-mir-181d, mmu-mir-18b, mmu-mir-208b, hsa-mir-208b, rno-mir-17-2, rno-mir-181d, rno-mir-423, rno-mir-484, mmu-mir-1b, ssc-mir-15a, ssc-mir-16-2, ssc-mir-16-1, ssc-mir-17, ssc-mir-130a, ssc-mir-101-1, ssc-mir-101-2, ssc-mir-133a-1, ssc-mir-1, ssc-mir-181a-1, ssc-let-7a-1, ssc-let-7e, ssc-let-7g, ssc-mir-378-1, ssc-mir-133b, ssc-mir-499, ssc-mir-143, ssc-mir-423, ssc-mir-181a-2, ssc-mir-181b-1, ssc-mir-181d, ssc-mir-98, ssc-mir-208b, ssc-mir-142, ssc-mir-19b-1, hsa-mir-378b, ssc-mir-22, rno-mir-126b, rno-mir-208b, rno-mir-133c, hsa-mir-378c, ssc-mir-194b, ssc-mir-133a-2, ssc-mir-484, ssc-mir-30c-1, ssc-mir-126, ssc-mir-378-2, ssc-mir-451, hsa-mir-378d-1, hsa-mir-378e, hsa-mir-378f, hsa-mir-378g, hsa-mir-378h, hsa-mir-378i, mmu-mir-378b, mmu-mir-101c, hsa-mir-451b, hsa-mir-499b, ssc-let-7a-2, ssc-mir-18b, hsa-mir-378j, rno-mir-378b, mmu-mir-133c, mmu-let-7j, mmu-mir-378c, mmu-mir-378d, mmu-mir-451b, ssc-let-7d, ssc-let-7f-2, ssc-mir-20b-1, ssc-mir-20b-2, ssc-mir-194a, mmu-let-7k, mmu-mir-126b, mmu-mir-142b, rno-let-7g, rno-mir-15a, ssc-mir-378b, rno-mir-29c-2, rno-mir-1b, ssc-mir-26b
let-7, miR-98, miR-130a and miR-16 showed uniform levels of expression in 13 different tissues but were hardly detected in pancreas (Figure 3A). [score:3]
Additionally, many other miRNAs, such as let-7, miR-98, miR-16, miR22, miR-26b, miR-29c, miR-30c and miR126, were also expressed abundantly in thymus (Figure 3). [score:3]
Similarly, let-7, miR-98, miR-16 and miR-130a are abundantly expressed in 13 of the 14 tissues (except in pancreas) (Figure 3A). [score:3]
Similarly, we found all members of the miR-15, miR-16, miR-18 and miR-133 families in our sequences, suggesting that all members belonging to these miRNA families are expressed in these three (heart, liver and thymus) tissues. [score:3]
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68
[+] score: 12
Murchison et al. first investigated the expression of miRNAs in mouse oocytes, and they demonstrated that the miR-30, miR-16 and let-7 family was overexpressed in mouse germinal vesicle (GV) oocytes, speculating, as a result, that miRNAs might play important regulatory roles in the expression of mRNAs during the process of follicular maturity [23]. [score:6]
The expression of miR-103, miR-16, miR-30b, miR-30c and let-7d played an important role in maintaining the stability of the spindle structure [10]. [score:3]
Furthermore, Tang et al reported that the miR-30, miR-16, let-7 and miR-17-92 family, which was detected in mature mouse oocytes, dynamically regulated oogenesis and early embryonic development. [score:3]
[1 to 20 of 3 sentences]
69
[+] score: 12
The top miRNAs (those with the highest number of targets in each time point) were miR-149-5p, miR-138-5p and miR-16-5p for 15, 30 and 45 dpi, with 14, 22 and 21 targets, respectively (targets described in Supplemental Tables  8– 10). [score:7]
miRNAs target several Nrf2-modulated genes (like the major effector of the antioxidant response heme oxygenase 1 HMOX1, targeted by miR-16-5p). [score:5]
[1 to 20 of 2 sentences]
70
[+] score: 12
miR-24 and miR-16, surrogate markers for total miRNA expression, were detectable in all samples. [score:3]
The expression profiles of miR-24 and miR-16 have been reported to be stable in several bodily fluids and tissues [24– 27], and were therefore used for normalization purposes. [score:3]
Amplification was performed using a qPCR Thermal Cycler (Applied Biosystems, Nieuwerkerk aan den IJssel, The Netherlands) with a denaturation step at 95 °C for 10 min, followed by 50 cycles of 95 °C for 15 s and 60 °C for 60 s. Data of these qPCR experiments were normalized using the geometric mean [26, 28] of the two uniformly expressed miRNAs, i. e., miR-16 and miR-24. [score:3]
Levels were normalized using the geometric mean of miR-24 and miR-16. [score:1]
Relative expression levels (REL) were calculated using the formula REL = 2 [− ∆Ct], where Ct is cycle threshold, and ∆Ct = Ct (miRNA) –  Ct (geometric mean of miR-16 and miR-24). [score:1]
Again, miR-24 and miR-16 were detected in all samples, while the proportion of individuals with undetectable miR-219 was higher in the MS patients’ groups (Fig. 1c). [score:1]
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71
[+] score: 11
Other miRNAs from this paper: mmu-mir-16-2, mmu-mir-210
Additionally, Wing et al found that EGCG could promote the apoptosis of human liver cancer cells by up -regulating the expression levels of miR-16 and down -regulating the expression levels of Bcl-2 [19]. [score:7]
Epigallocatechin gallate up-regulation of miR-16 and induction of apoptosis in human cancer cells. [score:4]
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72
[+] score: 11
Altered expression of many miRNAs is seen in several tumor types: e. g. B-cell lymphomas (clustered miR-17) [2], [3], malignant lymphomas (miR-15a, miR-16-1; targeting BCL2) [4], glioblastoma tumors (miR-21up-regulation) [5], colorectal neoplasia (miR-143, miR-145 down-regulated) [6], lung cancer (miR-29) [7], and breast cancer (miR-10b) [8], with several more tumor types under analysis. [score:11]
[1 to 20 of 1 sentences]
73
[+] score: 11
Previously, we had identified a specific whole blood–derived miRNA signature in mice exposed to LPS as there was a dose- and time -dependent upregulated expression of the miRNA targets (let-7d, miR-15b, miR-16, miR-25, miR-92a, miR-103, miR-107 and miR-451) follo-wing in vivo LPS injection [14]. [score:8]
In previous study, we had demonstrated that expression of multiple miRNAs (let-7d, miR-15b, miR-16, miR-25, miR-92a, miR-103, miR-107, and miR-451) is significantly altered in the whole blood of mice after exposure to LPS in a dose- and time -dependent fashion [14]. [score:3]
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74
[+] score: 11
Quantitative PCR analyses revealed that miR-375 and the broadly expressed miR-16 are both readily detected in the circulation of C57BL/6 mice (20.8 ± 0.3 Ct, n = 3, Fig.   5a). [score:3]
We observed that circulating miR-375 levels were increased by ≈2-fold in STZ -treated diabetic mice as compared to controls (Fig.   6c), while those of miR-16, a ubiquitously expressed miRNA was unaffected by STZ treatment (Fig.   6d). [score:2]
Absolute miRNA quantification was performed by reverse transcription of serial dilutions of synthetic oligonucleotides with sequence to mature mmu-miR-375 (5′-UUUGUUCGUUCGGCUCGCGUGA-3′), mmu-miR-16 (5′-UAGCAGCACGUAAAUAUUGGCG-3′), and mmu-miR-194 (5′-UGUAACAGCAACUCCAUGUGGA-3′). [score:1]
e Blood glucose and f circulating miR-375 and g miR-16 levels in WT and db/db (BKS-background) male mice at 8 weeks of age (n = 4–5). [score:1]
In contrast, plasma miR-16 levels were not different between WT, miR-375 KO, and β-Rescue animals (Fig.   5d). [score:1]
h Blood glucose, i pancreatic insulin content, and j circulating miR-375 and k miR-16 in C57BL/6 (WT) mice fed a normal or high-fat diet (HFD, for 25 weeks) and ob/ob (C57BL/6 background) mice (23-week-old) (n = 5). [score:1]
b Copy number of circulating miR-375 and miR-16 in C57BL/6 mice at 7 weeks. [score:1]
c Circulating miR-375 and d miR-16 copy number in plasma of 6-h fasted C57BL/6 WT mice (10-week-old) after being injected with STZ (+, 1 × 150 mg/kg) or PBS as control (−) for 3 days (n = 7–8). [score:1]
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75
[+] score: 11
Anti-miR chemistries suppressing miR-15 in mice were reported to reduce myocardial infarct size [15], while inhibition of either miR-15a or miR-16 enhanced post-ischemic neovascularization [19]. [score:5]
MiR-15 and miR-16 were reported to induce apoptosis by inhibiting Bcl-2 [18]. [score:3]
The miR-15 family members including miR-15a, miR-15b, miR-16, miR-195, miR-424, and miR-497, show 5′-end sequence similarity and many common targets [16, 17]. [score:3]
[1 to 20 of 3 sentences]
76
[+] score: 11
In many tumors, there is either overexpression of so-called oncogenic miRNAs (e. g., miR-155, miR-17−5p and miR-21) [15, 16] or downregulation of tumor suppressor miRNAs (e. g., miR-34, miR-15a, miR-16−1 and let- 7) [17– 20]. [score:8]
Consequently miRNAs have been demonstrated to act either as oncogenes (e. g., miR-155, miR-17−5p and miR-21) [15, 16] or tumor suppressors (e. g., miR-34, miR-15a, miR-16−1 and let- 7) [17– 20]. [score:3]
[1 to 20 of 2 sentences]
77
[+] score: 11
Interestingly, fluoxetine was shown previously to regulate miRNA expression, namely to alter the expression of miR-16 in serotonergic raphe nuclei (71). [score:6]
However, in the here presented study which analyzed miRNA expression levels in another brain region, i. e., the PFC, in a different experimental context, miR-16 expression was not significantly altered by fluoxetine treatment. [score:5]
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78
[+] score: 11
It should be noted that other microRNAs potentially regulating CCNE1 protein expression were also significantly changed, including down-regulation of miR-141, miR-16, miR-15a, miR-352, miR-15b and up-regulation of miR-518e, miR-29a, miR-192, and miR-29b, implicating a regulatory network fine-tuning the cell cycle checkpoints. [score:11]
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79
[+] score: 11
It was already demonstrated that p53 directly regulated the expression of tumor-suppressor miRNAs as the miR-34 family members [34], or mir-16 and mir-145, through a Drosha -mediated mechanism [35]. [score:7]
Calin G. A. Dumitru C. D. Shimizu M. Bichi R. Zupo S. Noch E. Aldler H. Rattan S. Keating M. Rai K. Frequent deletions and down-regulation of micro -RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia Proc. [score:4]
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80
[+] score: 10
Linsley et al. reported that, in a colon carcinoma cell line, the microRNA-16 family could regulate different targets in a coordinated fashion, including CDK6, CARD10, CDC27 and C10orf46, which act in cell cycle progression [49]. [score:4]
The miR-15a and miR-16 cluster was also demonstrated as having more than one target [9, 10, 49]. [score:3]
Bandi et al. documented that cell cycle arrest induced by miR-15a and miR-16 depended on the expression of Rb [9]. [score:3]
[1 to 20 of 3 sentences]
81
[+] score: 10
E. g. for miR-16, the peak1 in panel (b) (arrow) represents similar concentration as the peak1 in panel (d) (arrow). [score:1]
If a protein complex protects MIR2911 then proteinase K treatment would render MIR2911, like miR-16, sensitive to degradation by plasma RNases 14. [score:1]
In the context of either of the two exosome isolation protocols, the majority (over 90%) of the circulating miR-16 and MIR2911 were not found in the exosome vesicles (Fig. 7a). [score:1]
The copies of MIR2911, miR-16, and let-7a were quantified in each fraction by qRT-PCR and normalized to spiked-in 161 miRNAs. [score:1]
Levels of miR-16 and MIR2911 were quantified by qRT-PCR in the exosome pellets, or the supernatant fractions. [score:1]
Meanwhile, although MIR2911 did fractionate with miR-16, an AGO2 associated miRNA, MIR2911 was not proteinase K sensitive (Fig. 6c,d). [score:1]
The fractionation of let-7 and miR-16 were consistent with previous work in human plasma 14 while MIR2911 fractionated in the vicinity of the protein associated miRNA miR-16 (Fig. 7b). [score:1]
Given the large size of exosome associated miRNAs, they should elute from SEC columns early relative to protein associated miRNAs such as miR-16. [score:1]
The results showed that let-7a eluted earlier than both MIR2911 and miR-16. [score:1]
The copies of MIR2911, miR-16, and let-7a (an exosome -associated miRNA), were quantified in each fraction by qRT-PCR. [score:1]
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82
[+] score: 10
An RNA pool from 20 different tissues was used as a positive control and the results were normalized to the expression of miR-16 and miR-103. [score:3]
Expression was normalized to miR-16 and miR-103. [score:3]
Expression in the MeWo cells, although at rather low levels (average Cp value 34.7 compared to the controls (miR-16 and 103) at 25.9 and 27.8 respectively), indicates possible involvement in regulating the endogenous levels of MITF in this cell type. [score:3]
ND (1/9) ND (0/9) 34.69 (7/9) 37.39 (1/3) miR-124 34.37 (9/9) 34.73 (5/9) 35.09 (2/9) 32.68 (3/3) miR-506 35.19 (2/9) 34.73 (3/9) 32.87 (9/9) ND (0/9) miR-148a 27.90 (9/9) 28.29 (9/9) 28.82 (9/9) 33.12 (3/3) miR-148b 28.37 (9/9) 28.65 (9/9) 29.37 (9/9) 35.19 (1/3) miR-152 34.08 (9/9) 35.12 (9/9) 34.26 (9/9) 35.05 (2/3) miR-16 contr. [score:1]
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83
[+] score: 10
miRNA-16 and let-7a were used as endogenous controls to standardize miRNA expression for blood and tissue respectively [29]. [score:3]
MiR-16 and let-7a expression was stable across all tissue samples, and the average of both values was used as an endogenous control (C [T] range: 21–25 across all samples, Figure S3A). [score:3]
MiR-16 expression in the circulation was stable across all 60 samples (C [T] range: 27–30 across all samples, Figure S3B) and was used as an endogenous control. [score:2]
MiR-195 and miR-497 have been shown to both originate from the miR-16 super family [54]. [score:1]
The average of miR-16 and let-7a was used as endogenous controls for tissue samples and found to have a C [T] range of a 21–25 across all 38 tissue samples included in this study. [score:1]
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84
[+] score: 9
Indeed, using our conditional NPM/ALK lymphoma transgenic mouse mo del [46, 47], we observed that ALK works alongside HIF1α to boost VEGF (Vascular endothelial growth factor) expression by down -regulating miR-16, an inhibitor of VEGF mRNA [38]. [score:6]
2.3.2. miR-181a and miR-16 as Regulators of Tumor Microenvironment. [score:2]
Moreover, injection of miR-16 into the tumors of nude mice was found to decrease tumor growth in vivo [38]. [score:1]
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85
[+] score: 9
Moreover, it has been reported that PF inhibited proliferation and induced apoptosis of human glioma cells via upregulating microRNA-16 and downregulating matrix metalloproteinase-9 (MMP9) [19]. [score:9]
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86
[+] score: 9
Other miRNAs from this paper: hsa-mir-16-1, hsa-mir-17, hsa-mir-20a, hsa-mir-21, hsa-mir-23a, hsa-mir-100, hsa-mir-103a-2, hsa-mir-103a-1, hsa-mir-107, hsa-mir-16-2, mmu-mir-1a-1, mmu-mir-23b, mmu-mir-125b-2, mmu-mir-130a, mmu-mir-9-2, mmu-mir-145a, mmu-mir-181a-2, mmu-mir-184, mmu-mir-199a-1, hsa-mir-199a-1, mmu-mir-205, mmu-mir-206, hsa-mir-181a-2, hsa-mir-181b-1, hsa-mir-199a-2, hsa-mir-205, hsa-mir-181a-1, hsa-mir-214, hsa-mir-219a-1, hsa-mir-223, mmu-mir-302a, hsa-mir-1-2, hsa-mir-23b, hsa-mir-125b-1, hsa-mir-130a, hsa-mir-145, hsa-mir-9-1, hsa-mir-9-2, hsa-mir-9-3, hsa-mir-125b-2, hsa-mir-184, hsa-mir-206, mmu-mir-16-2, mmu-mir-20a, mmu-mir-21a, mmu-mir-23a, mmu-mir-103-1, mmu-mir-103-2, rno-mir-338, mmu-mir-338, rno-mir-20a, hsa-mir-1-1, mmu-mir-1a-2, hsa-mir-181b-2, mmu-mir-107, mmu-mir-17, mmu-mir-100, mmu-mir-181a-1, mmu-mir-214, mmu-mir-219a-1, mmu-mir-223, mmu-mir-199a-2, mmu-mir-9-1, mmu-mir-9-3, mmu-mir-181b-1, mmu-mir-125b-1, hsa-mir-302a, hsa-mir-219a-2, mmu-mir-219a-2, hsa-mir-302b, hsa-mir-302c, hsa-mir-302d, hsa-mir-367, hsa-mir-372, hsa-mir-338, mmu-mir-181b-2, rno-mir-9a-1, rno-mir-9a-3, rno-mir-9a-2, rno-mir-16, rno-mir-17-1, rno-mir-21, rno-mir-23a, rno-mir-23b, rno-mir-100, rno-mir-103-2, rno-mir-103-1, rno-mir-107, rno-mir-125b-1, rno-mir-125b-2, rno-mir-130a, rno-mir-145, rno-mir-181a-2, rno-mir-181b-1, rno-mir-181b-2, rno-mir-184, rno-mir-199a, rno-mir-205, rno-mir-206, rno-mir-181a-1, rno-mir-214, rno-mir-219a-1, rno-mir-219a-2, rno-mir-223, hsa-mir-512-1, hsa-mir-512-2, rno-mir-1, mmu-mir-367, mmu-mir-302b, mmu-mir-302c, mmu-mir-302d, rno-mir-17-2, hsa-mir-1183, mmu-mir-1b, hsa-mir-302e, hsa-mir-302f, hsa-mir-103b-1, hsa-mir-103b-2, rno-mir-9b-3, rno-mir-9b-1, rno-mir-9b-2, rno-mir-219b, hsa-mir-23c, hsa-mir-219b, mmu-mir-145b, mmu-mir-21b, mmu-mir-21c, mmu-mir-219b, mmu-mir-219c, mmu-mir-9b-2, mmu-mir-9b-1, mmu-mir-9b-3
From the top twenty miRNAs showing highest expression in A2B5+ GalC− cells, miR-130a, miR-16, miR-17, and miR-20a were also in the top twenty expressed miRNAs from our GPs. [score:5]
Similarly, miR-17, miR-20a, miR-21, miR-16, miR-103, and miR-107 identified in A2B5-GalC+ cells showed overlapping expression with our OPs. [score:3]
Interestingly, miR-16 was found to have an evolutionarily conserved 8mer target site with a high prediction score to GFAP, one of the most wi dely used markers for characterizing astrocytes. [score:1]
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87
[+] score: 9
Down regulated expression of has-mir-16, and has-mir-92 and increased levels of has-mir-765 correlated with the severe TBI, however, their utility in diagnosing mTBI was limited [24]. [score:4]
Therefore, miR-16 (Mean Ct  = 15.54±0.91) along with another miRNAs, miR-1937b (Mean Ct  = 14.91±0.90) were taken for use as endogenous controls in the miRNA expression validation experiments. [score:3]
One of the miRNAs (miR-16) has been described as stable serum miRNA in other studies [50]. [score:1]
Similar results were obtained with either of the selected stable miRNAs (data not shown) and data presented here used miR-16 as the stable endogenous miRNA. [score:1]
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88
[+] score: 9
miR-16, miR-31, miR-33a, miR-146a, miR-155, and miR-301a can suppress or promote IL-8 expression and secretion by inhibiting the expression of different proteins [31– 36]. [score:9]
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89
[+] score: 9
The combined use of B2M/18S/HPRT1 and miR-16/sno234 for normalization was validated as the optimal RGs for mRNA and microRNA expression data from liver, respectively. [score:3]
In agreement with previous data [35], [36], significant increase in both miRNAs was observed in MSG-obese mice in comparison to lean ones, when the two best RGs (miR-16 and sno234) were used as normalizer. [score:1]
The BestKeeper analysis of miRNA candidate genes ranked mir-16 as the best RG, followed by sno234 and miR-186 for liver tissue and sno234 followed by sno202 and miR-186 for SI (Table 4, Data S3). [score:1]
In the case of miRNA normalization, the best RGs according to geNorm analysis are miR-16 and sno234 for liver tissue and miR-186 and miR-200a for SI. [score:1]
According to our expectations, the increase in both miRNAs was observed, when the two best RGs (miR-16 and sno234) were used as normalizer both in combination (geometric mean) and as single RGs (Fig. 5). [score:1]
The most suitable combination of RGs in the liver tissue seems to be B2M, HPRT1, 18S for mRNA normalization and miR-16 and sno234 for miRNA normalization. [score:1]
When various grouping was applied, the best combination for liver tissue was miR-122 and miR-16. [score:1]
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90
[+] score: 9
We observed moderate elevation of miR-15a, miR-15b and miR-16 expression levels, and great increase in miR-497∼195 expression in CD31 [hi]Emcn [hi] endothelial cells compared to CD31 [lo]Emcn [lo] endothelial cells (Supplementary Fig. 1a). [score:4]
The expression of miR-15a, miR-15b and miR-16 showed no significant difference between miR-497∼195 [−/−] and miR-497∼195 [lox/lox] controls indicating that genetic manipulation of miR-497∼195 in ECs has little effect on other members of the miR-15 family (Supplementary Fig. 2b). [score:3]
qRT-PCR revealed five folds higher of miR-497∼195 expression in Tg mice as compared with that in controls (Supplementary Fig. 4a) and no alterations in the transcription of miR-15a, miR-15b and miR-16 (Supplementary Fig. 4b). [score:2]
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91
[+] score: 8
miR-15a, miR-16, and miR-503 were reported to inhibit tumor angiogenesis by targeting VEGFA [15],[16]. [score:5]
FEBS J. 15 Sun CY, She XM, Qin Y, Chu ZB, Chen L et al. (2013) miR-15a and miR-16 affect the angiogenesis of multiple myeloma by targeting VEGF. [score:3]
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92
[+] score: 8
However, the slightly up-regulated miR-16, miR-34c-3p and let-7i* miRNAs in this study have been demonstrated to be down-regulated in other cancer settings [56], [57], [58]. [score:7]
Int J Cancer In press 70 Aqeilan RI Calin GA Croce CM 2010 miR-15a and miR-16-1 in cancer: discovery, function and future perspectives. [score:1]
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93
[+] score: 8
Other miRNAs from this paper: mmu-mir-15a
Recent study suggested that one of the mechanisms was through up-regulation of MCL-1 expression via suppression of microRNA-15a/miR-16-1 [10]. [score:8]
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94
[+] score: 8
For instance, miR-16, acting as an oncogene, led to the inhibition of the cell adhesion capability and the promotion of cell migration in LSCC through directly suppressing Zyxin expression [6]. [score:8]
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95
[+] score: 8
Other miRNAs from this paper: mmu-mir-16-2
The miR16 expression in the mouse hippocampus regulates depression-like behavior via neurogenesis -dependent mechanisms (Launay et al., 2011). [score:4]
miR-16 targets the serotonin transporter: a new facet for adaptive responses to antidepressants. [score:3]
Raphe -mediated signals control the hippocampal response to SRI antidepressants via miR-16. [score:1]
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96
[+] score: 8
The most prominent LIS1 targeting miRNA family contained two downregulated miRNAs, miR-15a and miR-16, with three predicted targets in the LIS1 3′UTR (Table 1). [score:8]
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97
[+] score: 8
These upregulated miRNAs include certain known miRNAs (mmu-miR-148b-5p, mmu-miR-879-5p, mmu-miR-144-3p, mmu-miR-540-5p, mmu-miR-582-5p, mmu-miR-15b-5p, mmu-miR-210-5p, mmu-miR-871-3p, mmu-miR-3103-5p, mmu-miR-16-1-3p, mmu-miR-470-5p, mmu-miR-190b-5p, mmu-miR-384-5p and mmu-miR-490-5p), as well as some novel miRNAs (novel_mir_46, novel_mir_214 and novel_mir_213) with their stem loop structures by Miredp (S3 Fig). [score:4]
Through sequencing miRNAs for their quantifications, we show that some known miRNAs (miR-148b-5p, miR-879-5p, miR-144-3p, miR-540-5p, miR-582-5p, miR-15b-5p, miR-210-5p, miR-871-3p, miR-3103-5p, miR-16-1-3p, miR-470-5p, miR-190b-5p, miR-384-5p and miR-490-5p) are upregulated in CUMS -induced depression mice (Table 3), which degrade mRNAs listed in Table 1. In other words, the analysis from miRNA sequencing is consistent to the analysis from mRNA sequencing. [score:4]
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98
[+] score: 8
Other miRNAs from this paper: mmu-mir-16-2
In addition, condition medium derived from MSCs down-regulated the expression of vascular endothelial growth factor partially by miR-16 and suppressed angiogenesis 39. [score:8]
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99
[+] score: 7
For instance, miR-15b induced cell cycle arrest at G0/G1 phase by targeting cyclin E in glioma cells [23]; miR-16 induced G1 arrest partially by targeting cyclin D1 [24]; miR-122a can modulate cyclin G1 expression in human hepatocellular carcinoma-derived cell lines [25]. [score:7]
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100
[+] score: 7
The loss of the miRNAs miR-15a and miR-16-1 in patients with the 13q deletion contributes to the pathogenesis of the disease [7], [8], and altered miR expression is associated with disease progression and poor prognosis [9]. [score:7]
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