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321 publications mentioning mmu-mir-17 (showing top 100)

Open access articles that are associated with the species Mus musculus and mention the gene name mir-17. Click the [+] symbols to view sentences that include the gene name, or the word cloud on the right for a summary.

1
[+] score: 611
We compared the list of target genes de-repressed by miR-17~92 family miRNA deletion (ribo -upregulated T KO targets) with those suppressed by transgenic miR-17~92 overexpression (ribo -downregulated TG targets). [score:16]
Taken together, these results demonstrated that the global impact of miR-17~92 on its target gene mRNA levels is subtle, that only a subset of functionally relevant target genes are suppressed by transgenic miR-17~92 expression, that miR-17~92 -mediated suppression occurs predominantly at the protein level, and that this suppression is not caused by an altered translational environment in TG B cells. [score:15]
miR-17~92 regulates functional target gene expression mainly through translational repression in activated B cells and 5’UTR plays an important role in regulating target gene sensitivity to miRNA suppression. [score:13]
As our results showed that miR-17~92 suppresses target gene expression mainly through translational repression, we then focused on molecular features implicated in translational regulation [93]. [score:12]
Moreover, miR-17~92 controls key target gene expression mainly through translational repression and 5’UTR plays an important role in regulating target gene sensitivity to miRNA suppression. [score:12]
Therefore, only a small fraction of target genes respond to changes in miR-17~92 expression levels and miR-17~92 regulates its target gene expression mainly at the translational level. [score:12]
Ten targets (termed group 1 targets) were suppressed when miR-17~92 expression increased from T KO to WT levels, but showed little suppression in TG B cells. [score:11]
This is quite similar to our polysome profiling analysis of miR-17~92 target genes in WT and TG B cells, which showed that transgenic miR-17~92 expression shifted only a fraction of its target mRNAs from rapid translation states into slow translation states. [score:11]
Taken together, these results suggest that target mRNAs are compartmentalized: target mRNAs in the first peak are associated with miR-17~92 family miRNAs and undergo slow translation, while target mRNAs in the second peak are largely free of miR-17~92 family miRNAs and undergo more active translation. [score:11]
In this study, we showed that most functional target genes of miR-17~92 are suppressed at the translational level, but some target genes are suppressed by mRNA degradation, either completely or partially. [score:11]
When ribosome profiling data of T KO, WT, and TG B cells were analyzed together, it became clear that ribo -upregulated T KO targets as a group showed significant reduction in ribosome density when miR-17~92 family miRNA expression increased from almost zero in T KO B cells to WT levels, but did not show further reduction when miRNA expression increased from WT to TG levels (Fig 2B and S6 Table). [score:10]
Consistently, ribosome footprint abundance in 5’UTRs of ribo -downregulated TG targets was increased when miR-17~92 expression increased from WT to TG levels (Fig 5B), while other non-responsive target genes did not show significant changes in ribosome footprint abundance in their 5’UTRs in T KO, WT or TG B cells (Fig 5C). [score:10]
When miR-17~92 expression is further increased to the TG levels, less sensitive targets (such as ribo -downregulated TG targets) that do not respond to WT levels of miR-17~92 become responsive at this higher level. [score:10]
The other six targets (termed group 2 targets) showed suppression when miR-17~92 expression increased from WT to TG levels, and were de-repressed in T KO B cells (Fig 2E). [score:9]
Seven targets (termed group 3 targets) were suppressed when miR-17~92 expression increased from WT to TG levels, but showed only marginal de-repression in T KO B cells. [score:9]
As shown in S12 Fig, mutation of miR-17~92 binding sites led to increased activity of a luciferase gene fused to target gene 3’UTRs, therefore demonstrating direct regulation of these target genes by miR-17~92 in B cells. [score:8]
Only 13 of the 47 target genes showed significant reduction in protein levels in TG B cells (S7A Fig), including several inhibitors of the PI3K (Pten and Phlpp2) and NF-κB (Tnfaip3/A20, Itch, Rnf11, Tax1bp1, Cyld, and Traf3) pathways previously implicated in miR-17~92 -driven B cell lymphoma development [40, 65], and five additional tumor suppressor genes (Hbp1, Stk38, Arid4b, Rbbp8 and Ikzf1) [66– 69]. [score:8]
In the same analysis, the ribosome density of ribo -downregulated TG targets did not exhibit any significant changes between T KO and WT B cells, but showed significant reduction when miR-17~92 expression increased from WT to TG levels (Fig 2C and S6 Table). [score:8]
Moreover, our reporter assay experiments confirmed direct regulation of group 1 targets by miR-17~92 in wild type B cells, but the degree of de-repression in reporter activity caused by binding site mutation was often less than the degree of de-repression in target gene protein levels in T KO B cells (Fig 2E and S12 Fig). [score:7]
The differences in threshold and saturation levels underlie the different sensitivity of group 1, 2, 3 target genes to changes in miR-17~92 expression levels, while the differences in amplitude explain the various degrees of suppression or de-repression in TG and T KO B cells, respectively (Fig 2E). [score:7]
During B cell activation, there was indeed an inverse correlation between the expression levels of miR-17~92 and these target gene mRNAs at all time points examined, but the average change in target mRNA levels was only 3.7% in TG and 6.6% in T KO B cells (S5C and S5D Fig). [score:7]
This conditional transgene and knock-out strategy bypasses developmental defects caused by dysregulated miR-17~92 expression during the early stages of B cell development [50, 51]. [score:7]
Consistent with CD69 expression in B cells of these three genotypes (Fig 2E), the expression of renilla luciferase was more sensitive to miR-17~92 depletion than overexpression (S20A Fig). [score:7]
When the cutoff is set at 1.4 fold change in ribosome footprint abundance, only 80 of them are suppressed by the WT levels of miR-17~92 and qualify as responsive targets, amounting to 9% of experimentally identified targets. [score:7]
In summary, we conducted an integrated analysis of miR-17~92 family miRNAs, their target genes, and the functional consequences of these miRNA-target gene interactions in primary B cells expressing miR-17~92 family miRNAs at three different physiological levels. [score:7]
Transgenic miR-17~92 expression shifted a fraction of target mRNAs from the second peak into the first peak (Fig 4C), thereby reducing the overall translation rate and protein output (S7A Fig). [score:7]
When the relative protein levels of these 23 targets in T KO, WT, and TG B cells were plotted together, it became obvious that different targets exhibit different sensitivity to changes in miR-17~92 expression levels (Fig 2E). [score:7]
The differential responses of target genes to three different levels of miR-17~92 expression in T KO, WT and TG B cells were confirmed by immunoblot analysis of individual target genes (S11 Fig). [score:7]
Indeed, polysome profiling analysis confirmed that miR-17~92 represses CD69 expression at the translation level (Fig 6C), and deletion of the miR-17~92 family miRNAs led to a 4.5-fold increase in cell surface expression of CD69 in T KO B cells, with only marginal effect on its mRNA level (Fig 6D). [score:7]
Among the 780 transcribed miR-17~92 targets, 641 were detected by significant numbers of ribosome footprints (termed translated targets) (S10A Fig). [score:7]
Upon activation, both miR-17~92 miRNAs and their target mRNAs are up-regulated (Fig 3C and 3D), but the fold increase of the latter outpaces the former, thereby increasing the ratios between conserved binding sites and miRNA molecules to 2.8 (miR-92 family) and 8.7 (miR-18 family) in 25.5h activated B cells (Fig 3E). [score:6]
Here we take advantage of recent technical advances to globally examine the mRNA and protein levels of 868 target genes regulated by miR-17~92, the first oncogenic miRNA, in mutant mice with transgenic overexpression or deletion of this miRNA gene. [score:6]
We show that miR-17~92 regulates target gene expression mainly at the protein level, with little effect on mRNA. [score:6]
The relative contribution of translational repression and mRNA degradation to miR-17~92 regulation of these 13 target genes was approximately 4:1. CD19 and Actb were used as positive and negative control, respectively. [score:6]
miR-17~92 regulates functional target gene expression predominantly at the protein level. [score:6]
The transcribed genes included 85% (743 in naïve B cells) to 90% (780 in 25.5h activated B cells) of miR-17~92 target genes (termed transcribed targets). [score:5]
This analysis produced results consistent with polysome profiling analysis, showing that miR-17~92 target mRNAs were enriched in fractions 10–11 while depleted in fractions 14–16 in TG B cells, and miR-155 target mRNAs were depleted in fraction 10–11 while enriched in fractions 14–16 in miR-155 KO B cells (S16B and S16C Fig). [score:5]
Further studies show that the sensitivity of target genes to miR-17~92 is determined by a non-coding region of target mRNA. [score:5]
We next examined the effect of miR-17~92 on predicted target genes with the highest context++ scores based on the most recent TargetScan 7.0 algorithm (S3 Table) [55]. [score:5]
This suggested that miR-17~92 represses translation initiation of these target genes through their 5’UTRs (See discussion). [score:5]
Target gene mRNA copy number and the number of conserved miR-17~92 binding sites on each target mRNA. [score:5]
Predicted miR-17~92 target genes with the highest context++ scores based on the most recent TargetScan 7.0 algorithm. [score:5]
Target genes exhibit different sensitivity to changes in miR-17~92 expression. [score:5]
Target genes exhibit different sensitivity to miR-17~92 expression level changes. [score:5]
Global analysis of the impact of miR-17~92 on target gene expression. [score:5]
Ribosome footprint distribution in translated miR-17~92 targets in T KO, WT, and TG B cells. [score:5]
Mir-17-5p regulates breast cancer cell proliferation by inhibiting translation of AIB1 mRNA. [score:5]
Poly -RNA-seq analysis miR-17~92 targets in TG B cells and miR-155 targets in miR-155 KO B cells. [score:5]
Transgenic miR-17~92 expression shifts target mRNAs from heavy to light polysomes. [score:5]
When miR-17~92 target genes were analyzed, neither transgenic miR-17~92 expression nor deletion of miR-17~92 family caused significant global changes in their mRNA levels throughout B cell activation (Fig 1B and 1C and S2 Table). [score:5]
Among the 868 experimentally identified targets with conserved miR-17~92 binding sites [40], 780 are significantly transcribed and 641 are significantly translated. [score:5]
Interestingly, the ribosome footprint overlaps with the two sub-optimal start codons and the 5’ arm of the putative hairpin, while its abundance shows positive correlation with miR-17~92 expression levels and negative correlation with CD69 expression (Figs 6B and 2E). [score:5]
Surprisingly, only a small fraction of target genes respond to miR-17~92 expression changes. [score:5]
This suggests that miR-17~92 miRNAs are predominantly associated with target mRNAs undergoing slow translation. [score:5]
We have previously identified 868 target genes harboring a total of 1139 miR-17~92 binding sites conserved in human and mouse (termed miR-17~92 targets) by PAR-CLIP analysis of B cells [40]. [score:5]
We next assessed the impact of miR-17~92 expression on target genes using ribosome profiling (S2C Fig). [score:5]
Direct regulation of group 1 target genes by miR-17~92 in wild type B cells. [score:5]
Translated genes lacking miR-17~92 binding sites were used as negative control, whose ribosome density showed no significant alterations in B cells expressing miR-17~92 at three different levels (Fig 2D). [score:5]
Our quantification of miR-17~92 family miRNA molecules and their potential binding sites on target mRNAs in B cells suggested that only a fraction of target mRNA molecules are occupied by these miRNAs (Fig 3E). [score:5]
Deletion of miR-155 shifted miR-155 target mRNAs from fractions 10–11 to fractions 14–16, but had almost no significant effect on the distribution Actb and miR-17~92 target mRNAs. [score:5]
Immunoblot analysis of 16 translation regulators that lack miR-17~92 binding sites. [score:4]
Notably, the majority of targets investigated has been previously validated as direct miR-17~92 targets in various cellular contexts (S4 Table). [score:4]
Moreover, most of the small number of target genes that show greater than 1.4-fold changes in mRNA levels have not been previously implicated in lymphoma development, cell survival and proliferation, and are unlikely to mediate the functions of miR-17~92 in B cells (S2 Table). [score:4]
Analysis of target genes regulated by individual members of the miR-17~92 cluster came to the same conclusion (S5A and S5B Fig). [score:4]
in which these genes were implicated as direct targets for miR-17~92 miRNAs were included [40, 45, 49, 56, 75– 77, 80, 126, 129– 197]. [score:4]
Therefore, we investigated the possibility that miR-17~92 regulates the expression of functionally relevant target genes mainly at the protein level. [score:4]
We compiled a list of 63 miR-17~92 target genes, which were either validated in previous studies [59– 64], or are novel but functionally relevant to B cell lymphoma development or B cell immune responses (S4 Table). [score:4]
This list contains most of miR-17~92 target genes validated in previous studies. [score:3]
The impact of miR-17~92 on target gene mRNA and protein levels. [score:3]
Microarray analysis of T KO, WT and TG B cells with target genes subsetted according to individual subfamily of miR-17~92. [score:3]
This rather modest global effect of miR-17~92 on the mRNA levels of its target genes is consistent with the results from previous studies performing transcriptome analysis of T cells, B lymphoma cells, and embryonic heart and tail bud with genetic ablation of either the whole miR-17~92 cluster or its individual members [51, 56– 58]. [score:3]
miR-17~92 expression levels and activities of major signaling pathways during B cell activation. [score:3]
When multiple miR-17~92 binding sites (BS) are present in a target gene 3’UTR and are far away from each other, multiple reporter constructs were generated, with each construct harboring one binding site. [score:3]
Microarray analysis of miR-17~92 target gene mRNA levels in T KO, WT, and TG B cells. [score:3]
Molecular dissection of cis-elements of CD69 5’UTR in determining its sensitivity to suppression by miR-17~92. [score:3]
While the β-Actin mRNA (Actb) was enriched in heavy polysome fractions, mRNAs of all validated miR-17~92 target genes exhibited a bimodal distribution (Fig 4C). [score:3]
1, Jarid2 and Peli1) and miR-17~92 target mRNAs in the sucrose gradient [88– 91]. [score:3]
A summary of immunoblot analysis of miR-17~92 target genes in TG B cells. [score:3]
S20 FigMolecular dissection of cis-elements of CD69 5’UTR in determining its sensitivity to suppression by miR-17~92. [score:3]
128 top target genes were selected for each miRNA miR-17~92 subfamily, and the ones transcribed at greater than 0.5 copy per cell were analyzed. [score:3]
In the presence of WT levels of miR-17~92 family miRNAs, RISC complexes are recruited to these target mRNAs through their cognate binding sites in the 3’UTRs, and dissociate RNA helicases from the 5’UTRs. [score:3]
Our global analysis of miR-17~92 target genes in primary B cells provide insights into this question. [score:3]
The impact of miR-17~92 family miRNA deletion on the target gene protein levels. [score:3]
Ribosome footprint distribution analysis showed that there were ribosome footprints in 5’UTRs of miR-17~92 target genes, though their abundance was lower than ribosome footprint abundance in CDS (S18 Fig). [score:3]
miR-19, a component of the oncogenic miR-17~92 cluster, targets the DNA-end resection factor CtIP. [score:3]
microRNA-17~92 is a powerful cancer driver and a therapeutic target. [score:3]
In the absence of miR-17~92 family miRNAs (in T KO B cells), eIF4As or other RNA helicases facilitate the unwinding of these secondary structures, allowing PIC to scan through and to initiate translation. [score:3]
Consistent with the endogenous CD69 gene (Fig 2E), the reporter gene was more sensitive to miR-17~92 depletion than to transgenic miR-17~92 expression. [score:3]
The experimentally identified miR-17~92 binding sites by human B cell PAR-CLIP [40], Burkitt’s lymphoma cell HITS-CLIP [79], HEK293 PAR-CLIP [35] and TargetScan 7.0 [55] were indicated. [score:3]
We investigated the effect of transgenic miR-17~92 expression and complete deletion of the miR-17~92 family on the mRNA levels of these target genes during B cell activation. [score:3]
As discussed above, it is likely that only a fraction of these 80 target genes are relevant for the function of miR-17~92 in B cells. [score:3]
Deletion of the left arm of the hairpin reduced the sensitivity of renilla luciferase to miR-17~92 suppression, but no significant effect was observed for mutating the two sub-optimal start codons (S20B Fig). [score:3]
S5 Fig (A-B) PAR-CLIP identified miR-17~92 targets [40] were subsetted according to individual subfamily of miR-17~92. [score:3]
Replacing CD69 5’UTR with β- Actin 5’UTR significantly reduced the sensitivity of the renilla luciferase reporter gene to miR-17~92 suppression (Fig 7C). [score:3]
A comparison of the renilla/firefly luciferase activity ratio (hRluc/Fluc) between psiCheck-2-pd containing wt and mut CD69 3’UTR should reveal the sensitivity of the renilla luciferase mRNA to miR-17~92 -mediated suppression. [score:3]
We confirmed that miR-17~92 expression in TG B cells was 3 fold higher than in WT B cells, and was completely absent in T KO B cells (S4B and S4C Fig). [score:3]
Translated genes lacking miR-17~92 binding sites were used as control (D). [score:3]
A comparison of renilla luciferase activity normalized to firefly luciferase activity (hRluc/Fluc) between psiCheck-2-pd containing mut and wt CD69 3’UTR reveals the sensitivity of the renilla luciferase mRNA (hRluc) to miR-17~92 -mediated suppression. [score:3]
We found mRNAs with miR-17~92 binding sites tend to have longer 5’UTR, CDS, and 3’UTR, but their length did not predict target gene sensitivity. [score:3]
S17 Fig(A) The distribution of length of 5’UTR, CDS, and 3’UTR among miR-17~92 targets. [score:3]
miR-17-5p promotes human breast cancer cell migration and invasion through suppression of HBP1. [score:3]
We have now performed a comprehensive molecular analysis of primary B cells expressing miR-17~92 miRNAs at three different levels (T KO, WT and TG). [score:3]
Numbers in parenthesis indicate the numbers of transcribed genes and transcribed miR-17~92 targets analyzed by microarray. [score:3]
In addition, the distribution of miR-17~92 family miRNAs largely overlapped with the first peak of their target mRNAs (Fig 4B and 4C). [score:3]
In this study, we investigated miRNA mechanism of action in lymphocytes by conducting an integrated analysis of the transcriptomes and translatomes of primary B cells from miR-17~92 transgenic and knockout mice. [score:2]
Germline knockout of miR-17~92 family in mice is incompatible with life [49]. [score:2]
We next performed reporter assays in wild type B cells to investigate whether miR-17~92 exerts its effect on these target genes through its cognate binding sites on target mRNAs. [score:2]
Consistent with the previous observations that miR-17~92 regulation of Hbp1 occurs mainly at the mRNA level (Fig 1D and S9E Fig), the distribution of the Hbp1 mRNA in the sucrose gradient showed little change (Fig 4C). [score:2]
To understand the functional contribution of the putative hairpin and two sub-optimal start codons in CD69 5’UTR to the sensitivity of CD69 mRNA to miR-17~92 suppression, we deleted the left arm of the hairpin (ΔHP) or mutated these two sub-optimal start codons (Mut-uORF), and performed reporter assays in WT B cells. [score:2]
We introduced 3nt mutations into these binding sites to abolish their interactions with miR-17~92 miRNAs to generate a mutated form of CD69 3’UTR (mut). [score:2]
They fall into four miRNA subfamilies (miR-17, miR-18, miR-19, and miR-92 subfamilies), with members in each subfamily sharing the same seed sequence. [score:1]
Our calculation showed that each naïve B cell expresses 900–1,800 molecules of miR-17, miR-19, and miR-92 subfamily miRNAs, and 80 molecules of miR-18 subfamily miRNAs (Fig 3B and 3C and S7 Table). [score:1]
n. d., miR-17~92 family miRNA binding sites not detected in the CLIP dataset. [score:1]
miR-17~92 [fl/fl] mice were crossed with miR-106a~363 [-/-] mice, miR-106b~25 [-/-] mice and CD19-Cre mice to generate miR-17~92 [fl/fl];miR-106a~363 [-/-];miR-106b~25 [-/-];CD19-Cre (T KO) mice. [score:1]
The renilla luciferase reporter containing CD69 5’UTR showed a 4.4 fold de-repression when miR-17~92 binding sites in its 3’UTR were mutated, very similar to the fold de-repression of the endogenous CD69 gene in T KO B cells (Figs 6D and 7C). [score:1]
The wild type CD69 3’UTR (wt) contains three binding sites for miR-17~92 miRNAs (one for miR-17 subfamily and two for miR-92 subfamily). [score:1]
miR-17~92 target genes investigated in this study. [score:1]
Quantification of miR-17~92 miRNAs and binding sites in primary B cells. [score:1]
The miR-17~92 family consists of three miRNA clusters: miR-17~92, miR-106a~363, and miR-106b~25 (S1 Fig). [score:1]
S4 TableA list of miR-17~92 target genes investigated by immunoblot analysis. [score:1]
The conserved miR-17~92 binding sites were identified by PAR-CLIP analysis of human B cells [40]. [score:1]
Therefore, we conclude that only a fraction of potential binding sites are occupied by miR-17~92 miRNAs at any given time. [score:1]
MiR-17~92 Tg mice were crossed with CD19-Cre mice to generate miR-17~92 Tg/Tg;CD19Cre (TG) mice [40]. [score:1]
In contrast to miR-21, miR-17~92 miRNAs were mainly associated with light polysomes (Fig 4A and 4B). [score:1]
We also measured the protein levels of 16 genes that control translation initiation and elongation in a global manner and completely lack miR-17~92 binding sites in their mRNAs [70]. [score:1]
B cell-specific deletion of the miR-17~92 family (CD19-Cre;miR-17~92 [fl/fl];miR-106a~363 [-/-];miR-106b~25 [-/-], termed T KO mice) severely impaired antibody responses, while B cell-specific miR-17~92 transgenic (TG) mice develop lymphomas with high penetrance [40]. [score:1]
PIC contains 40S ribosome subunit, Met-tRNAi, and eIFs 1, 1A, 2, 3, and 5. (PDF) (XLSX) (XLSX) (XLSX) A list of miR-17~92 target genes investigated by immunoblot analysis. [score:1]
The generation of miR-17~92 Tg (Jax stock 008517), miR-17~92 [fl/fl] (Jax stock 008458), miR-106a~363 [-/-] mice (Jax stock 008461), miR-106b~25 [-/-] mice (Jax stock 008460), CD19-Cre (Jax stock 006785) was previously reported [49, 126, 127]. [score:1]
Genomic organization of the miR-17~92 family miRNAs in mice. [score:1]
The ratios between conserved miR-17~92 binding sites and miRNA molecules range from 0.5 (miR-92 subfamily) to 4.6 (miR-18 subfamily) in naïve B cells (Fig 3E). [score:1]
Taking non-conserved binding sites into account, potential miR-17~92 binding sites outnumber miRNA molecules even further, by as much as 20-fold. [score:1]
To test this, we determined the copy numbers of miR-17~92 miRNA molecules and miR-17~92 binding sites present in B cells during activation. [score:1]
Indicated amounts of synthetic miR-17, miR-18a, miR-19b and miR-92 were added to naïve and activated T KO B cells before RNA extraction. [score:1]
The miRNA molecule numbers in WT B cells were determined by quantitative Northern blot analysis of WT B cells and T KO B cells spiked with graded amounts of chemically synthesized mature miR-17~92 family miRNAs (Fig 3A–3C and S7 Table). [score:1]
1006623.g003 Fig 3(A,B) Quantitative Northern blot to determine miR-17~92 miRNA copy numbers. [score:1]
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[+] score: 514
De Summa et al. [74] show that overexpression of miR-17 in both mesenchymal-like BRCA1-proficient and in BRCA1- and BRCA2-mutated BC cell lines in addition to the significant overexpression of miR-17 in sporadic patients seems to suggest that downregulation of BRCA1, a presumed target of miR-17-5p mimics a ‘BRCAness’ phenotype, that is, a phenotype that some sporadic cancers share with BRCA1- or BRCA2-mutation carriers. [score:11]
In addition, both miR-17-5p and miR-17-3p are abundantly processed from precursor miR-17 and have synergetic effects on developing HCC by binding different targets on different signaling pathways: miR-17-5p targets PTEN, one of the most frequently lost tumor suppressor in human cancers, while miR-17-3p represses expression of vimentin, an intermediate filament with the ability to modulate metabolism, and GalNT7, an enzyme that regulates metabolism of liver toxin galactosamine. [score:10]
On the other hand, downregulation of miR-17-5p upregulates its target, the autophagy regulator beclin-1, which leads to apoptosis resistance of cancer cells upon paclitaxel treatment [81]. [score:10]
According to Matsubara et al. [80], inhibition of miR-17-5p and miR-20a with antisense oligonucleotides (ONs) can induce apoptosis selectively in lung cancer cells overexpressing miR-17-92, suggesting the possibility of targeting an ‘oncomiR addiction’ to expression of these miRNAs in a subset of lung cancers. [score:9]
Assuming that miR-17-5p inhibition would restore protein expression of tumor suppressive miR-17-5p targets Programmed cell death 4 (PDCD4) and Phosphatase and tensin homolog (PTEN), human TNBC cells were transfected with antisense oligonucleotides against miR-17-5p. [score:9]
To justify miR-17-5p acts as tumor suppressor, a study shows that low expression levels of miR-17 results in cisplatin resistance of NSCLC by high expression of CDKN1A (cyclin -dependent kinase inhibitor 1A) and RAD21 (Rad21 homolog (Schizosaccharomyces pombe)) [83]. [score:9]
Resveratrol and Pterostilbene decrease the levels of endogenous as well as exogenously expressed miR-17, miR-20a and miR-106b thereby upregulating their target PTEN [122] and eventually leading to reduced tumor growth in vivo. [score:8]
Inhibitors of miR-17 could potentially serve as adjuvants in chemotherapy as oncogenic miRNAs like miR-17 are upregulated in rapamycin-resistant cells and inhibition of miR-17 restored rapamycin sensitivity. [score:8]
A recent study from Liao XH et al. also established that miR-17-5p acts as a tumor suppressor by directly targeting STAT3 and inducing apoptosis in breast cancer cells by inhibiting STAT3/p53 pathway [70]. [score:8]
They found that miR-17-5p was highly expressed in strongly invasive, but not in weakly invasive BC cells, and that miR-17-5p overexpression enhanced migratory and invasive abilities of BC cells, while its downregulation had the opposite effect. [score:8]
As p21 and STAT 3 are direct targets of miR-17-5p and miR-20a, downregulation of miR-17-5p and miR-20a induces myeloid differentiation and growth arrest in AML cells in vitro and in vivo [112]. [score:7]
High levels of miR-17-5p, which further downregulate CDKN1A (Cyclin Dependent Kinase Inhibitor 1A), p21 and E2F1 tumor suppressor genes in imatinib sensitive and resistant chronic myeloid leukemia (CML) cells compared to peripheral blood mononuclear cells (PBMCs), have also been observed [111]. [score:7]
It acts as a tumor suppressor in normal growth conditions by inhibiting PTEN through miR-17-5p and at unfavorable conditions miR-17-3p promotes tumor cell survival by inhibiting MDM2 [124]. [score:7]
A follow-up study from the same group investigated the cellular mechanisms involving miR-17-5p in gastric cancer and found that miR-17-5p/20a promote gastric cancer by directly targeting the tumor suppressors p21 and p53 -induced nuclear protein 1 (TP53INP1), which results in unrestrained proliferation and apoptosis inhibition, respectively, and involve a positive regulatory circuit between miR-17-5p/20a and MDM2 (murine double minute 2). [score:7]
However, miR-17-5p was found downregulated in chronic lymphocytic leukemia both with normal p53 and with mutated/ deleted p53, but downregulation was more pronounced in the latter patient group [114, 115]. [score:7]
This correlates with the observation that serum levels of circulating miR-17-5p were upregulated in a relapse group of patients and downregulated in the post-operative group. [score:7]
miR-17-5p expression levels might be used as predictive factor for chemotherapy response and a prognostic factor for overall survival in CRC, since patients with high miR-17-5p expression in tumor tissue have shorter overall survival rates [97, 100] and respond better to adjuvant chemotherapy than patients with low miRNA expression [97]. [score:7]
On the one hand, miR-17-5p inhibits mTOR by inducing MKP7 (Mitogen-Activated Protein Kinase Phosphatase 7) via targeting ADCY5 (Adenylate Cyclase 5): Upon dephosphorylation of mTOR by MKP7, mTOR dimerizes with PRAS40 (40-kDa proline-rich AKT substrate) and gets inhibited [21, 47]. [score:7]
Expression of CCND1 was inhibited by overexpression of miR-17-5p [72]. [score:7]
The effect of miR-17-5p is highly dependent on many factors like type of cancer, mo del systems used and constructs used in mo del systems for knockdown or overexpression, as well as on the relative expression levels of miR-17-3p and miR-17-5p which was discussed in few cancer types where miR-17-3p did have synergistic or rescue effect. [score:6]
Another study shows that high levels of miR-17-5p decreased expression of its direct target TGFBR2 (transforming growth factor-β receptor 2), further promoting gastric cancer cell proliferation and migration [89]. [score:6]
The tumor suppressor BRCC2, which is thought to induce apoptosis in a caspase -dependent manner, is a direct target of miR-17-5p [107]. [score:6]
Downregulation of miR-17-5p by curcumin and its synthetic analogs inhibits CRC cell proliferation and induces apoptosis, and could provide the basis for future therapeutic approaches [103]. [score:6]
Even though correlating miR-17-5p expression levels with various tumor properties might be very useful in the development of biomarkers, it does not give evidence about its tumorigenic or tumour-suppressive potential. [score:6]
HIF-1α (Hypoxia Inducible Factor 1 Alpha Subunit) downregulates the expressions of miR-17-5p and miR-20a through a mechanism that is dependent of c-Myc but independent of its transcription partner HIF-1ß. [score:6]
When overexpressed, PTENP1 sequesters miR-17, which would otherwise target PTEN and the negative Akt-regulator PHLPP (PH Domain And Leucine Rich Repeat Protein Phosphatase). [score:6]
According to Li et al. [67], miR-17-5p promotes human breast cancer cell migration and invasion through suppression of HMG box-containing protein 1 (HBP1), which they confirmed as a direct target of miR-17-5p. [score:6]
Out of 4000 genes linked to BC progression, miR-17-5p was confirmed in vitro and in vivo as regulator of multiple pro-metastatic genes, hence had an anti-metastatic effect, while miR-17-5p inhibition in BC cells enhanced expression of pro-metastatic genes and accelerated lung metastasis from orthotopic xenografts. [score:6]
In addition, miR-17-5p directly targets RND3, a Rho Family GTPase that acts as tumor suppressor by promoting adhesion [98]. [score:6]
What causes miR-17-5p overexpression leading up to CRC pathogenesis and by what targets does it regulate proliferation? [score:6]
The therapeutic potential of miR-17-5p inhibition in triple negative BC (TNBC), one of the most aggressive breast cancer forms, was also assessed as a therapeutic target [71]. [score:5]
Expression of miR-17-3p is approximately half of the level of miR-17-5p except for PBMCs, where expression was below detection limits [32]. [score:5]
Conflicting results on tumor suppressor versus promoter function exist for prostate cancer (PC): Both mature miR-17-5p and passenger strand miR-17-3p target TIMP3 which has synergetic effect on enhancing prostate tumor growth and invasion [118]. [score:5]
Expression of miR-17-5p is also high in osteosarcoma, whereby PTEN seems to be an important target contributing to progression and metastasis [105]. [score:5]
By targeting SMAD7, miR-17-5p promotes nuclear translocation of β-catenin, enhances expression of COL1A1 (Collagen Type I Alpha 1 Chain) and finally facilitates the proliferation and differentiation of femoral head mesenchymal stem (HMS) cells promoting osteonecrosis [106]. [score:5]
Downregulation of p21 by miR-17-5p in turn promotes PCNA (proliferating cell nuclear antigen) activity, where p21 is a negative regulator of PCNA and thus ERα promotes breast cancer cell cycle progression and proliferation in p21/PCNA/E2F1 -dependent pathway [68]. [score:5]
It should be mentioned though, that although miR-17-5p expression levels allowed distinction between NSCLC and healthy control, it was not useful as diagnostic marker for discriminating between NSCLC and chronic obstructive pulmonary disease (COPD) [77]. [score:5]
Supporting in vitro and tissue level high expression of miR-17-5p, a clinical study proves serum levels of miR-17 along with miR-19a, miR-20a and miR-223 were significantly upregulated in CRC patients compared to controls [104]. [score:5]
Likewise, miR-17-5p increased the proliferation and growth of gastric cancer cells in vitro and in vivo, by targeting SOCS6, a cytokine -induced STAT inhibitor [88]. [score:5]
Expression profiling of acute myeloid leukemia (AML) identified a set of seven miRNAs comprising miR-17-5p that allows discrimination of three common AML-causing chromosomal translocations with a diagnostic accuracy of > 94%, and is significantly overexpressed in MLL (mixed lineage leukemia) rearrangements, which causes particularly aggressive leukemia with poor prognosis [108]. [score:5]
The results showed that miR-17-3p seems to act as a back-up mechanism of miR-17-5p for these targets, and therefore, due to the high sequence homology between the antisense molecules and miR-17-3p, as well as to excess binding sites for miR-17-3p on the 3′UTR of PDCD4 and PTEN mRNAs, the antisense oligo acted as a miR-17-3p mimic and reduced PDCD4 and PTEN expression instead of restoring it. [score:5]
Downregulation of E2F1 by miR-17-5p is of importance for proliferation both during embryonic colon development and colon carcinogenesis [95]. [score:5]
Other studies concluded that miR-17-5p expression levels did not have sufficient informative values to serve as diagnostic tool, at least using sputum miRNA profiling [78], This study confirms previous results of the same group [79], where miR-17-5p was not found either over-or under-expressed in human lung cancer. [score:5]
Apart from promoting breast cancer cell migration and invasion by miR-17-5p, Liao XH et al. showed that miR-17-5p also promotes cell proliferation by down -regulating p21 which is a direct target of miR-17-5p in ERα (Estrogen receptor α) -positive breast cancer cells. [score:5]
This is in accordance with the notion that miR-17-5p overexpression reduces cyto-protective autophagy by targeting Beclin-1 in paclitaxel resistant lung cancer cells [82]. [score:5]
However, high levels of miR-17-3p have also been reported to suppress tumorigenicity of PC cells through inhibition of mitochondrial antioxidant enzymes [119]. [score:5]
Accordingly, miR-17-5p targets P130 (Retinoblastoma-Like 2, a presumed tumor suppressor, present in a complex that represses cell cycle -dependent genes) and subsequently activates the WNT/β-catenin pathway [97]. [score:5]
Among all the miRNAs of the miR-17-92 cluster, miR-17-5p showed highest expression in epithelial colon cells and expression levels increased in the transitional zone from normal to adenoma to adenocarcinoma (N-A-AC), suggesting a role in sequential evolution of early colon cancer [91]. [score:5]
qPCR -based miRNA expression profiling revealed that miR-17-5p, miR-18a-5p and miR-20a-5p exhibit enhanced expression in tissue samples derived from triple -negative as compared to luminal A breast tumors, which are less aggressive and have much better prognosis as well as lower recurrence rate [64]. [score:4]
This is in contrast to studies that found miR-17 (no distinction between 5p and 3p) downregulated in lung adenocarcinoma initiating cells [76] and in non-small cell lung cancer (NSCLC). [score:4]
Transcriptional regulation and target mRNAs of miRNA-17-92 cluster and miR-17-5p. [score:4]
This further supports that upregulation of miR-17-5p is at least associated to myeloid leukemia. [score:4]
A potential anti-prostate cancer drug, glucosinolate-derived phenethyl isothiocyanate (PEITC), results in miR-17-5p -mediated suppression of PCAF and again AR-regulated transcriptional activity and cell growth of prostate cancer cells, suggesting a new mechanism by which PEITC modulates prostate cancer cell growth [121]. [score:4]
Validated gene targets of miR-17 and pathways affected by their regulation in cancers. [score:4]
Downregulation of AIB1 (“Amplified in breast cancer 1”) by miR-17-5p decreased proliferation and abrogated insulin-like growth factor 1 -mediated, anchorage-independent growth of breast cancer cells. [score:4]
Not only in solid tumors, but also in tumors of hematopoietic origin miR-17-5p is upregulated, like in both acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). [score:4]
In addition to PTEN, SMAD7 and thus Wnt signalling is a direct target for miR-17-5p in this context. [score:4]
The long noncoding RNA CCAT2, a WNT downstream target, induces miR-17-5p and MYC through TCF7L2 (Transcription Factor 7 Like 2) -mediated transcriptional regulation (Figure 2) [96]. [score:4]
Large-scale miRnome analysis on 540 samples including lung, breast, stomach, prostate, colon and pancreatic tumors identified miR-17-5p as upregulated in all solid tumors [50]. [score:4]
Cumulative data clearly point to a role of miR-17-5p in the development and progression of breast cancer, and is currently being explored as biomarker for diagnosis, prognosis and therapeutic target. [score:4]
miR-17-5p and miR-20a in turn negatively regulate E2F1 expression [28]. [score:4]
MicroRNA expression and sequence analysis database (mESAdb) [33], which integrates data from several databases like e. g. the one by Basekerville and Bartel [34] substantiates these findings and emphasize the importance of miR-17-5p in all tissues. [score:3]
In summary, in the context of gastric cancer, miR-17-5p clearly acts as oncogene and targets the components of many pathways involved in cell proliferation and migration. [score:3]
Elevated miR-17-5p expression is also observed in early embryonic colon epithelium, and is sustained only in the proliferative crypt progenitor compartment. [score:3]
Nonetheless, results derived from a SCID mouse mo del suggests the suitability of miR-17 as a therapeutic target for CLL treatment. [score:3]
In contrast, miR-17-5p was described as tumor suppressor [69]. [score:3]
Hence, miR-17-5p may be used as diagnostic and prognostic marker, but also as a potential target for molecular therapy of osteosarcoma. [score:3]
ERα plays an important role in cell-cycle progression by promoting the expression of PCNA and Ki-67 along with miR-17-5p. [score:3]
Among these, the miRNA-17-92 cluster seems of special interest as it has been the first oncomiR to be described, but one of the cluster members, miR-17-5p, has also been found to decrease with aging and might even prolong the life span of mice upon overexpression. [score:3]
What prognostic and therapeutic implications can be derived from miR-17-5p expression data? [score:3]
In addition to exploring its diagnostic potential, miR-17-5p might also serve as therapeutic target in lung cancer treatment. [score:3]
Hence miR-17-5p plays a tumor suppressor role in this setting. [score:3]
HCC cell lines overexpressing miR-17-5p injected either subcutanously or into the livers of nude mice generating an orthotopic intrahepatic tumor mo del, miR-17-5p supported tumor growth and intrahepatic metastasis [63]. [score:3]
While we here focus on the role of the single miR-17-5p in formation and progression of distinct cancer types, Xiang and Wu [57] have reviewed the tumor-suppressive and tumorigenic properties of the miR-17-92 cluster as a whole. [score:3]
MiR-17 along with miR-106a/b and miR-20a/b targets GABBR1(gamma-amino-butyric acid type B receptor 1) thus promoting colorectal cancer cell proliferation and invasion [99]. [score:3]
Therefore, the authors suggest miR-17-5p as a potential therapeutic target for treatment of basal-like breast cancer. [score:3]
By now a more differentiated view has emerged, as miR-17-5p alone, by stimulating T cells can suppress cancer growth [52], while still able to drive hepatocellular carcinoma in a transgenic mouse mo del. [score:3]
This effect seems mediated by p300/CBP -associated factor (PCAF) as a target of miR-17-5p modulating the androgen receptor transcriptional activity [120]. [score:3]
Several studies confirm miR-17-5p overexpression in CRC (colorectal cancer) tissue samples [92, 93, 94]. [score:3]
In support of miR-17-5p’s tumor-suppressive role, recent bioinformatics and in vitro analysis revealed that levels of miR-17-5p are decreased in triple negative breast cancer cells resulting increase in CCND1 (cyclin D1) levels which is reason for uncontrolled proliferation. [score:3]
Hence, miR17 might represent a biomarkers of ‘BRCAness’ phenotype, indicating which patients who could most benefit from PARP inhibitor therapies. [score:3]
The role of miR-17-5p as an oncomiR is supported by many studies, while also the opposite, a tumor suppressive role has been found in some studies. [score:3]
MiR-17 might be largely responsible for the effect of the cluster, since overexpression of pre-miR-17 in a transgenic mouse mo del results in hepatocellular carcinoma (HCC). [score:3]
Similarly, miR17-5p was identified as metastatic suppressor of basal-like breast cancer [53]. [score:3]
Targets of miR-17-92 cluster and miR-17-5p. [score:3]
On the other hand overexpression of miR-17 promotes the cancer cell migration by reducing cell adhesion and promoting cell detachment in immortalized rat prostate endothelial cells [54]. [score:3]
This assumption has been verified by Wang et al. [55], they not only found that concentrations of miR-17-5p/20a were significantly associated with the differentiation status and tumor progression, but also revealed that high expression levels of miR-17-5p/20a were significantly correlated with poor overall survival. [score:3]
In terms of ubiquitous transcription of miR-17-5p, it was found to be expressed in all 40 different normal human tissues tested including brain, muscle, circulatory, respiratory, lymphoid, gastrointestinal, urinary, reproductive and endocrine systems [32]. [score:3]
On the other hand miR-17-5p targets IRS1 thus activating AMPK (AMP-activated protein kinase) which stops phosphorylation of ULK1 (Unc-51 like autophagy activating kinase 1) by mTOR and promotes formation of ULK1-ATG13-FIP200 (ATG13, autophagy related 13; FIP200, focal adhesion kinase family kinase-interacting protein of 200 kDa) complex required for the initiation of autophagy, a major complex involved in the formation of autophagosome [21]. [score:3]
Indeed, this was shown to happen via inhibition of miR-17-5p. [score:3]
Thereby, miR-17-5p also targets the long non-coding RNA PTENP1, a pseudogene of PTEN. [score:3]
Summarized, the tumorigenic or tumor-suppressive functions of miR-17-5p might depend on the cellular context, that is, on the mo del system used, cell type, cancer stage and many other factors, like for example “BRCA-ness”. [score:3]
After all, elevated miR-17-5p expression could either contribute to tumor formation and progression, or could represent a defense mechanism that is intended to limit carcinogenesis. [score:3]
Expression of miR-17 as well as its seed region is strongly conserved in higher animals. [score:3]
A very similar observation was made in another tumor entity, pancreatic cancer, where an overexpressed nerve growth factor receptor (GFRα2) led to PTEN inactivation mediated by induction of miR-17-5p [102]. [score:3]
On the other hand, chemotherapy was found to further increase the expression levels of miR-17-5p in CRC cells in vitro, thereby repressing the pro-apoptotic factor PTEN and promoting chemoresistance [101]. [score:3]
For example, elevated miR-17-5p expression levels are present in tumor tissue and serum of lung cancer patients–including adenocarcinoma, squamous cell and adenosquamous carcinoma- compared to healthy controls. [score:2]
Circulatory/serum miR-17-5p levels are deregulated which also reflects the differential biology of breast cancer subtypes [73]. [score:2]
Further studies on miR-17-3p are required to establish a firm regulation between miR-17-5p and miR-17-3p. [score:2]
MiR-17-5p expression levels were associated with clinical stage, positive distant metastasis and poor response to neo-adjuvant chemotherapy. [score:2]
This shows how miR-17-5p tightly regulates the genes involved in cell proliferation and cell apoptosis. [score:2]
A very recent article explores the effects of miR-17-5p in osteosarcoma tumorigenesis and development. [score:2]
MiRNA-17-5p expression is highly elevated in patient-derived HCC tissues, especially in metastasis derived tissues when compared to controls [61]. [score:1]
Most interestingly, upon resistance to therapy of multiple myeloma with bortezomib, the exosomal transfer of several microRNAs seems to be altered, among them miR-17-5p, which was significantly reduced [110]. [score:1]
In addition, therapeutic potential for antagomirs against miR-17-5p/20a was suggested, which was applied as chemotherapeutics in a mouse tumor mo del. [score:1]
Thus, miR-17-5p can either promote or curb apoptosis of lung cancer cells. [score:1]
Hence PTENP1 functions as miR-17 antagonist, representing an appealing approach for HCC treatment based on miR-17 function in tumorigenesis [60]. [score:1]
Summarized, miR-17-5p possesses oncogenic activity in the context of hepatocellular carcinoma. [score:1]
Emerging evidence indicates that the miR-17-92 cluster and specifically miR-17-5p play an important role in carcinogenesis in the liver. [score:1]
We here have summarized studies that indicate that elevated levels of miR-17-5p might be an alarm signal for cancer, that might be sensitive, albeit not specific for a single type of cancer. [score:1]
miR-17-5p: a link between proliferation, cancer and aging. [score:1]
In addition, serum levels of miR-17-5p were associated with metastasis status and staging, suggesting that the miRNA in the serum indeed is tumor cell derived [62]. [score:1]
Indeed, levels of serum miR-17-5p/20a were notably reduced in post -treated mice with tumor volume regression. [score:1]
However, a follow-up study failed to assign a prognostic value to miR-17-5p plasma levels, since there was a slight, but not significant difference in the survival rates of patient groups exhibiting low or high miR-17-5p plasma levels, although the trend might turn significant when based on larger sample size (n = 31 vs. [score:1]
Still, circulating miRNAs as biomarkers or alarmiRs still lack sufficient studies to be able to define the range of inter-individual variation in the general healthy population and consequently define thresholds for e. g. miR-17-5p in serum or plasma that would lead to the decision of careful follow up clinical testing for the presence of a tumor. [score:1]
In cancers like glioblastomas, under stress conditions miR-17 plays a dual role depending on the conditions. [score:1]
The p38 MAPK-HSP27 pathway mediates miR-17-5p’s effect on migration, but, however, is not involved in its effect on proliferation. [score:1]
Several studies have investigated the relationship between miR-17-5p and lung cancer, mainly in view to its potential clinical application of miRNA expression profiles as diagnostic and prognostic marker. [score:1]
Overview of pathways affected by miR-17-5p in different cancer phenotypes leading to cell proliferation and migration. [score:1]
It was found that patients suffering from several different types of cancer have high circulating miR-17-5p levels in serum [55, 56], implying that increased serum levels of miR-17-5p could be an alarm signal for different types of cancers. [score:1]
Thus, in the context of the bone, miR-17-5p seems to have tumorigenic activity. [score:1]
Their findings in gastric cancer cells were backed-up by administering antagomiRs against miR-17-5p/20a to reduce tumor formation in a xenograft mouse mo del [87]. [score:1]
miR-17-5p and its role in cancer. [score:1]
We thereby surprisingly found that it is elevated in the serum or plasma of a large variety of solid and hematologic tumor types, which prompts us to here postulate a function of circulating miR-17-5p as an alarm signal that is sensitive for tumors in general, albeit not specific for a defined tumor type. [score:1]
These results state that miR-17-3p also plays an important role in different cancers either in synergetic way or as rescue for miR-17-5p. [score:1]
A study in multiple myeloma (MM) patients showed that high levels of miR-17-5p, miR-20a and miR-92-1 of miR-17-92 cluster are associated with shorter progression-free survival, suggesting poor prognosis [109]. [score:1]
Again, the final effect of miR-17-5p seems to be highly context -dependent. [score:1]
According to a recent report, circulating exosomes from prostate cancer cells carry long non-coding RNAs which are themselves enriched with miRNA seed regions that can bind to let-7 and miR-17 families like a miRNA sponge [123]. [score:1]
In Figure 2, we summarize the pathways effected by miR-17-5p in different cancer types. [score:1]
For a comprehensive review on the use of miRNAs as biomarkers for prognosis, diagnosis, therapeutic prediction and therapeutic tool in breast cancer, please refer to Bertoli et al. [66], who also discuss the potential of miR-17-5p as potential diagnostic biomarker. [score:1]
Briefly, miR17-5p plays a key role in colorectal cancer pathogenesis and progression. [score:1]
With this in mind, we set out to summarize the current knowledge of miR-17-5p in the context of cancer. [score:1]
For details on miR-17-5p’s role in aging, please refer to a recent review [21]. [score:1]
Hence, there exists a positive WNT signaling feedback loop involving miR-17-5p. [score:1]
Here in this review we focus on one of its member, miRNA-17-5p, and present current state of knowledge in the context of cancer, plasma or serum levels for specific type of tumors making it an ‘alarm signal’ for early detection of tumors. [score:1]
Henceforth miR-17-5p could be used as a diagnostic biomarker for colorectal cancer. [score:1]
In addition, serum miR-17-5p levels were inversely related to the survival of patients with lung cancer, that is, high levels correlated with shorter survival times [75]. [score:1]
The miR-17-92 cluster transcript comprises six miRNAs - miR-17-5p, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR-92a-1 - and is highly conserved among vertebrates [19, 20]. [score:1]
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As TIMP3 is a tumor suppressor frequently found downregulated in prostate cancer, our work is of clinical relevance, which may lead to a new approach in interrupting prostate cancer development and invasion by targeting miR-17 expression. [score:11]
As expression of some miRNAs in the miR-17∼92 cluster and its paralogs is upregulated in prostate cancer patients (47), miR-17 targeting TIMP3, p21 and PTEN appears to be a crucial event in prostate cancer development and invasion. [score:9]
Expression of TIMP3 was promoted by transfection of either miR-17-5p inhibitor or miR-17-3p inhibitor (Figure 7c, left). [score:7]
Similarly, when the vector -transfected cells were transfected with either a miR-17-5p inhibitor or miR-17-3p inhibitor, expression of TIMP3 was enhanced (Figure 7c, right). [score:7]
Although the miR-17-3p inhibitor (17-3pI) did not show any effects, the miR-17-5p inhibitor (17-5pI) enhanced p21 and PTEN expression (Figure 10d, middle for vector cells and right for miR-17 cells). [score:7]
In one of the extreme cases, we found that ectopic expression of miR-17 elevated tumor growth by >10-fold, as it repressed expression of these tumor suppressors, enhancing tumor cell proliferation, survival and invasion. [score:7]
Mutation of the miR-17-5p target site abolished the inhibitory effects of miR-17-5p (Figure 6e). [score:6]
Our finding that expression of miR-17 enhanced cell survival correlated with p21 and PTEN downregulation was consistent with previously reported results. [score:6]
One important function of miR-17 in tumor proliferation is the inhibition of p21, which is a target of miR-17-5p. [score:5]
We also tested the individual and combinatorial effects of miR-17-5p and miR-17-3p mimics, as well as miR-17-5p and miR-17-3p inhibitors on p21 and PTEN expression. [score:5]
We found that the promotion of TIMP3 expression by the inhibitors of miR-17-5p and miR-17-3p decreased the capacity of invasion in both miR-17 -transfected cells (Figure 7d, left) and the vector -transfected cells (Figure 7d, right, Supplementary Figure S2b). [score:5]
Although the miR-17-3p mimic (17-3pM) showed weak activity in repressing p21 and PTEN expression, the miR-17-5p mimic (17-5pM) showed a clear repressive effect on p21 and PTEN expression (Figure 10d, left). [score:5]
In addition, miR-20a, which is part of the mir-17–92 cluster, is overexpressed in prostate cancer, and its inhibition induces cell death and apoptosis in PC3 cells (5). [score:5]
Our results highlight the importance that both mature miR-17-5p and the passenger strand miR-17-3p targeted the same target TIMP3, which would enhance the power of a miRNA. [score:5]
We stably transfected human prostate cancer cell lines DU145 with our previously generated miR-17 expression construct (22) and confirmed that the levels of miR-17-5p and miR-17-3p expressions were significantly higher in the transfected cells than the control cells (Figure 1a). [score:5]
To further confirm the targeting, we silenced ectopic expressed miR-17-5p and miR-17-3p or endogenous miR-17-5p and miR-17-3p. [score:5]
Combining both inhibitors exerted a greater effect on enhancing TIMP3 expression, confirming a function of endogenous miR-17-5p and miR-17-3p. [score:5]
It was shown that the miR-17-5p targets the E2F1 pathway and the cell-cycle inhibitor, p21/WAF1 (41, 43). [score:5]
Similarly, when the cells were co -transfected with luc-TIMP-3p and miR-17-3p, but not miR-17-5p, we detected a significant inhibitory effect of miR-17-3p on luc-TIMP-3p activity, which was abolished when the target site was mutated (Figure 6e). [score:5]
We then examined the effects of TIMP3 repression on cell invasion and found that inhibition of TIMP3 expression by the miR-17-5p mimic and the miR-17-3p mimic significantly promoted cell invasion (Figure 7b, Supplementary Figure S2a). [score:5]
To validate the targeting results, we analyzed TIMP3 expression in the tumors formed by cells transfected with miR-17 or the control vector. [score:5]
In this study, we reported that miR-17-5p and miR-17-3p were not only both abundantly expressed from one single precursor but also acted in coordination to enhance the power of a miRNA precursor by repressing the same target. [score:5]
DU145 cells transfected with miR-17 were transiently transfected with a miR-17-5p inhibitor and/or a miR-17-3p inhibitor. [score:5]
Expression of anti-miR-17 inhibited tumor growth. [score:5]
The pre-miR-17 was ligated into a mammalian expression vector, BluGFP, which contains a Bluescript backbone, a CMV promoter driving expression of green fluorescent protein and a H1 promoter driving miR-17 as described previously (22). [score:5]
The miR-17-5p inhibitor (17-5pI) but not 17-3pI enhanced p21 and PTEN expression. [score:5]
When the cells were grown in serum-free medium, the reintroduction of TIMP3 into the miR-17 -expressing cells reversed the effect of miR-17 on cell survival: re -expression of TIMP3 was sufficient to cause cell death (Figure 8f, Supplementary Figure S3c). [score:5]
To study the function of TIMP3 in miR-17-regulated cell activities, we generated TIMP3 expression construct. [score:4]
As a target of miR-17-5p, PTEN can regulate the PI3K/AKT pathway, a major cell survival pathway, which plays an important role in prostate cancer (44–46). [score:4]
Although abnormal miR-17 expression is frequently observed in various types of cancers (37–39), the role of miR-17 in regulation of prostate cancer remains unclear. [score:4]
Our study showed that ability of mature miR-17-5p and the passenger strand miR-17-3p to synergistically enhance prostate tumor growth and invasion by repressing the same target TIMP3 may be a mode of miRNA -mediated gene regulation. [score:4]
Both miR-17-5p and miR-17-3p directly target TIMP3. [score:4]
To obtain direct evidence that TIMP3 was a target of both miR-17-5p and miR-17-3p, we generated two TIMP3 luciferase reporter constructs (luc-TIMP-5p and luc-TIMP-3p), one harboring the binding site for miR-17-5p and the other for miR-17-3p, and two mutant constructs in which the binding sites were mutated (Figure 6d, Supplementary Figure S1 for detail information of the constructs). [score:4]
Another important function of miR-17-5p in tumor formation and development is the inhibition of PTEN, a well-characterized tumor suppressor. [score:4]
Wang and colleagues have shown that miR-17-5p and miR-17-3p are differentially expressed during different time point in cell culture (49). [score:3]
It was found that DU145 cells transfected with miR-17-5p mimics or miR-17-3p mimics significantly decreased the expression of TIMP3 protein levels (Figure 7a). [score:3]
We sought to identify the target(s) of both miR-17-5p and miR-17-3p in mediating the observed effects, focusing on proteins that were computationally predicted to be bound by miR-17-5p and miR-17-3p. [score:3]
The miR-17- and vector -transfected cells were also treated with the miRNA inhibitors. [score:3]
It had also been reported that p21 was a potential target of miR-17 (31). [score:3]
DU145 cells stably transfected with miR-17 were transiently transfected with the TIMP3 expression construct or a control vector. [score:3]
We found that both miR-17-5p and miR-17-3p could target TIMP3 and coordinately function as an oncogene in prostate cancer. [score:3]
Expression of miR-17 decreased the number of cells in the G1 phase. [score:3]
Targeting the same mRNA (e. g. TIMP3) by both strands of a miRNA (e. g. miR-17-5p and miR-17-3p) can effectively and efficiently exert the biological functions of a miRNA. [score:3]
To confirm the targeting of TIMP3 by both miR-17-5p and miR-17-3p, we transiently transfected DU145 cells with miR-17-5p and/or miR-17-3p RNA mimics. [score:3]
Histological analysis of tumor sections showed that expression of miR-17 decreased tumor cell death in both PC3 cells (Figure 4c) and LNCaP cells (Figure 4d). [score:3]
Figure 6. Both miR-17-5p and miR-17-3p represses TIMP3 expression. [score:3]
As expression of miR-17 decreased DU145 cell death, we examined tumor sections stained with H&E and detected extensive cell death in the control group (stained as red, arrows) relative to the miR-17 group (Figure 3b). [score:3]
We have also reported that both miR-17-5p and miR-17-3p are highly expressed in miR-17 transgenic mice (22). [score:3]
It had been previously reported that PTEN was targeted by miR-17 (30). [score:3]
It was previously reported that p21 and PTEN were targeted by miR-17 (40–42). [score:3]
Figure 7. Confirmation of miR-17-5p and miR-17-3p targeting TIMP3. [score:3]
DU145 cells transfected with a control (middle) or miR-17 vector (right) were treated with miRNA inhibitors. [score:3]
Expression of miR-17 increased tumor volume. [score:3]
Cells transfected with RNA mimics (both miR-17-5p and/or miR-17-3p) repressed TIMP3 expression. [score:3]
Cell cycle analysis indicated that expression of miR-17 decreased G1 populations in both PC3 (Figure 1c) and LNCaP (Figure 1d) cells. [score:3]
Previously, we founded that transgenic mice expressing the miR-17 precursor produced comparable levels of mature miR-17-5p and passenger miR-17-3p (22). [score:3]
Thus, TIMP3, p21 and PTEN were collectively targeted by miR-17 in prostate cancer. [score:3]
In this study, we also found that both p21 and PTEN were targets of miR-17 in prostate cancer cells and tumors. [score:3]
To confirm the role of TIMP3 in mediating miR-17 functions, we delivered siRNA targeting TIMP3 into DU145 cells. [score:3]
Right, lysate prepared from tumors formed by the miR-17 or control -transfected cells was also analyzed for TIMP3 expression. [score:3]
This may explain why ectopic expression of miR-17 significantly promoted prostate tumor growth and invasion. [score:3]
To further validate our system, we analyzed PTEN and p21 expression in the tumors formed by cells transfected with miR-17 or the control vector. [score:3]
We found that expression of miR-17 significantly enhanced cell invasion through Matrigel. [score:3]
Repression of TIMP3 expression was seen in cells transfected with miR-17. [score:3]
To test the functions of endogenous miR-17, we stably transfected DU145 cells with an anti-miR-17 construct or a control vector, as DU145 were found to express high endogenous levels of miR-17 (Figure 1e). [score:3]
Herein we showed that repression of TIMP3 expression could be achieved by a miRNA miR-17. [score:3]
Repression of p21 and PTEN expression was seen in cells transfected with miR-17. [score:3]
These results suggest that the effect of miR-17 on enhanced invasion and survival was at least partly occurring through repression of TIMP3 expression. [score:3]
Figure 10. miR-17 represses TIMP3, PTEN and p21 expression in tumor sections. [score:3]
Expression of miR-17 increased tumor growth. [score:3]
We examined p21 levels and confirmed that p21 expression was repressed in the miR-17 -transfected cells (Figure 10a). [score:3]
To understand how expression of miR-17 might have induced tumor cell invasion, we conducted cell invasion assays in the DU145 (Figure 5d), PC3 (Figure 5e) and LNCaP (Figure 5f) cells transfected with miR-17 and the control vector placed in Matrigel-coated transwell chambers. [score:2]
We detected repression of PTEN expression in the miR-17 -transfected cells as compared with the controls (Figure 10a). [score:2]
Analysis of endogenous miRNA showed that DU145 cells expressed significantly higher levels of miR-17-5p and miR-17-3p, as compared to PC3 and LNCaP cells (Figure 1e). [score:2]
Expression of miR-17 produced an increased rate of tumor growth as compared to the control cells (Figure 3a, left). [score:2]
Mutation of the binding site reversed miR-17-5p’s effect. [score:2]
To test whether TIMP3 was repressed by miR-17 expression, we analyzed cell lysates prepared from the miR-17- and vector -transfected cells on western blot probed with anti-TIMP3 antibody and confirmed repression of TIMP3 in the miR-17 -transfected cells as compared with the control (Figure 6b, left). [score:2]
Our study has demonstrated that miR-17 played an important role in the development and invasion of prostate cancer. [score:2]
Expression of miR-17 in DU145, PC3 and LNCaP cells displayed enhanced cell survival compared with the control cells (Figure 2a). [score:2]
Cell lysates were prepared from DU145 cells stably transfected with miR-17 or the control vector and analyzed by western blotting probed with anti-PTEN antibody. [score:1]
Evidence of extensive cell death (decreased cell population) could be seen in the control but not in the miR-17 tumors. [score:1]
We examined whether miR-17 played a role in prostate cancer cell activities. [score:1]
Transfection with TIMP3 reversed the effect of miR-17 resulting in decreased cell invasion. [score:1]
Luciferase activity was significantly repressed when luc-TIMP-5p was co -transfected with miR-17-5p, but not with miR-17-3p. [score:1]
When the cells were transfected with a combination of miR-17-5p and miR-17-3p, repression of TIMP3 was further increased. [score:1]
To confirm these results, we reintroduced a miR-17 mimic into the anti-miR-17 -transfected cells. [score:1]
Five-week-old strain CD1 nude mice were injected with the miR-17- or vector -transfected cells at the cell number of 5 × 10 [5] cells or indicated in the legends in 50 µl of phosphate buffered saline with 50 µl of Matrigel per mouse. [score:1]
Cells transfected with miR-17 formed more and larger colonies than those transfected with the control vector (Figure 2c). [score:1]
In general, tumor sections from the miR-17 transfected cells exhibited much lower levels of TIMP3 than the control. [score:1]
We tested whether miR-17 played a role in tumor growth. [score:1]
Western blot analysis indicated that TIMP3 level was much lower in the miR-17 tumors than in the control tumors (Figure 4b, right). [score:1]
CD-1 nude mice were subcutaneously injected with DU145 cells transfected with miR-17 or the control vector. [score:1]
In this function, miR-17-5p promotes cell-cycle progression and hyperproliferation, which is consistent with our observation that miR-17 transfected prostate cancer cell lines tend to have increased proliferation rates. [score:1]
Figure 8. Confirmation of TIMP3 in mediating miR-17 functions. [score:1]
The effect of miR-17 on cell cycle progression was examined, and we found that miR-17 transfected DU145 cells had significantly less cells detected in the G1 phase than the control cells (Figure 1b). [score:1]
miR-17 promotes tumor cell proliferation, survival and colony formation. [score:1]
Two other prostate cancer cell lines PC3 and LNCaP were also stably transfected with miR-17 and the control vector. [score:1]
This occurred in the miR-17 group but not in the control group (Figure 5a). [score:1]
Bioinformatics analysis indicated that there was one potential binding sites for miR-17-5p and one for miR-17-3p in the 3′UTR of TIMP3 (Figure 6a). [score:1]
Evidence of extensive cell death (pink) could be seen in the control but not in the miR-17 tumors. [score:1]
These results suggest that TIMP3 was involved in a pathway essential for miR-17-enhanced cell invasion and survival. [score:1]
Western blot analysis indicated that PTEN and p21 levels were much lower in the miR-17 tumors than in the control tumors (Figure 10b). [score:1]
Figure 3. miR-17 enhances tumor growth in DU145 cells. [score:1]
The miR-17- and vector -transfected cells were also cultured in soft agarose. [score:1]
Figure 2. miR-17 promotes cell survival and colony formation but decreased apoptosis. [score:1]
The miR-17 cells were significantly more invasive than the control cells (left, ** P < 0.01, n = 5). [score:1]
The miR-17 tumor cells had fewer apoptotic cells than the control. [score:1]
Previous studies demonstrated that miR-17 played roles in the growth of a number of cancer types (26–29). [score:1]
Figure 1. miR-17 increased cell-cycle progression and cell proliferation. [score:1]
Evidence of invasive tissues could be seen in the miR-17 DU145 tumors, but it was absent in the control tumors. [score:1]
In general, tumor sections from the miR-17 transfected cells exhibited much lower levels of PTEN and p21 than the control. [score:1]
Although no sign of tumor invasion was seen, the anti-miR-17 tumor did not interact with the muscle tissue. [score:1]
As well, a combination of miR-17-5p and miR-17-3p exerted a greater effect on cell invasion than either one could when transfected alone. [score:1]
More apoptotic cells, Annexin V positive, were detected in the vector -transfected cells than in the miR-17 -transfected cells. [score:1]
The activity of luc-TIMP-5p decreased when co -transfected with miR-17-5p. [score:1]
Figure 5. miR-17 affects tumor cell invasion. [score:1]
Transfection with miR-17 rescued anti-miR-17’s effects on cell apoptosis (e) and invasion (f). [score:1]
Co-transfection with miR-17-3p mimic decreased luc-TIMP-3 activity but had no effect on the mutant. [score:1]
Our study and others suggest that miR-17 functions as a powerful oncogenic miRNA in prostate cancer. [score:1]
miR-17 enhances tumorigenesis and invasion. [score:1]
Figure 4. miR-17 enhances tumor growth in PC3 and LNCaP cells. [score:1]
Extensive cell death was detected in the smaller anti-miR-17 tumors, which displayed linking capacity to the muscle tissue (Figure 9h). [score:1]
Figure 9. Confirmation of miR-17 functions. [score:1]
DU145 cells were co -transfected with miR-17-5p and luc-TIMP-5p or luc-TIMP-5pmut. [score:1]
Transfection with miR-17 enhanced cell survival. [score:1]
There were many more apoptotic cells in the control group than in the miR-17 group (Figure 3c). [score:1]
These tests revealed a rescue effect of the miR-17 mimic on cell apoptosis (Figure 9e) and invasion (Figure 9f, Supplementary Figure S4c). [score:1]
In an extreme case, cells transfected with miR-17 formed tumors with >10-fold than the control cells (Figure 3a, right). [score:1]
Confirmation of TIMP3 in mediating miR-17 functions. [score:1]
In this report, we studied the role of miR-17 in prostate cancer in vivo and in vitro. [score:1]
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Q-PCR analysis revealed that compared with control kidneys, Pparα expression was downregulated by ∼80% in Pkd1 [F/RC] KO and Pkd2- KO kidneys, whereas its expression was upregulated in Pkd1 [F/RC]D KO and Pkd2-miR-17∼92 KO kidneys (Fig. 7d). [score:10]
miR-17 expression was significantly reduced in cyst epithelia of Pkd2-miR-17∼92 KO mice (negative control), whereas its expression was induced in renal tubules of kidney-specific miR-17∼92 -overexpressing transgenic mice (positive control), indicating that the in situ probe specifically detects miR-17. [score:7]
Finally, since miR-17 is upregulated, we determined whether PPARα is downregulated in human ADPKD cysts. [score:7]
Expression of PPARα targets was reduced in ADPKD mo dels, whereas their expression was increased after miR-17∼92 deletion. [score:7]
Anti-miR-17 inhibits cysts in in vitro human mo dels of PKDTo assess the translational potential of our findings, we studied the effects of miR-17 family inhibition in primary cell cultures derived from human ADPKD cysts. [score:7]
Our results have shown that miR-17 and miR-19 directly inhibit Pparα expression in cystic kidneys, but whether reducing Pparα gene dosage is sufficient to promote cyst growth is not known. [score:6]
Quantitative real-time PCR (Q-PCR) validated the microarray data and additionally demonstrated that miR-17 upregulation correlates with disease progression in both mo dels (Fig. 1d,e). [score:6]
Interestingly, Etfa, Etfb, Etfdh, Cpt2 and the various peroxisome genes were all downregulated in both Pkd1 and Pkd2 mutant kidneys, whereas their expression was increased after miR-17∼92 deletion. [score:6]
Deletion of c-Myc resulted in a 53.7% downregulation of pri-miR-17 expression, suggesting that c-Myc also promotes miR-17∼92 transcription in the context of ADPKD (Fig. 6f). [score:6]
Within the miR-17∼92 cluster, we decided to target the miR-17 family based on our observation that multiple members of this family were upregulated in ADPKD mo dels. [score:6]
RNA-Seq (Supplementary Fig. 9,10) and subsequent Q-PCR analyses (Fig. 9a,b) showed that a large network of OXPHOS/FAO-related PPAR α target genes were upregulated after miR-17∼92 deletion in both ADPKD mo dels. [score:6]
miR-17 expression was abolished in cysts of Pkd2-miR-17∼92 KO mice, whereas its expression was induced in renal tubules of miR-17∼92Tg mice, indicating that the in situ probe specifically detects miR-17. [score:5]
To assess the translational potential of our findings, we studied the effects of miR-17 family inhibition in primary cell cultures derived from human ADPKD cysts. [score:5]
To gain insights into the signalling events linked to dysregulated miR-17∼92 expression, we began by identifying PKD-relevant upstream regulators of miR-17∼92. [score:5]
Since miR-17 inhibition slows cyst growth in ciliopathy mo dels (Kif3a- KO and Nphp3 [pcy/pcy]), the beneficial effects of anti-miR-17 treatment may also be observed in other forms of cystic kidney disease besides ADPKD. [score:5]
We observed attenuated cyst growth, lower blood urea nitrogen (BUN) levels, downregulation of Kim1 and Ngal expression, reduced renal fibrosis and lower cyst epithelial proliferation in Pkd1 [RC/RC]; Ksp/Cre;miR-17∼92 [F/F] compared with Pkd1 [RC/RC]; Ksp/Cre mice (Fig. 3e–h and Supplementary Fig. 4). [score:5]
For PPPARα expression plasmid studies, mIMCD3 cells were grown in six-well dishes (2 × 10 [5] cells per well), and transfected with 0.6 μg of a Pparα expression plasmid (pPparα) or Control (pCMX), along with 5 nM of miR-17 or scrambled mimic (Scr). [score:5]
Thus, deletion of miR-17∼92 inhibited cyst proliferation and disease progression in both Pkd1- KO and Pkd2- KO mo dels. [score:5]
In contrast, compared with control kidneys, miR-17 expression was increased by 50.3% in Pkd1- KO kidneys, whereas its expression was reduced by 51.9% in Pkd1-miR-17∼92 KO kidneys (Supplementary Fig. 1B). [score:4]
Among the upregulated miRNAs, we focused on the miR-17 family because it contributed most substantially (>2.7%) to the total miRNA pool in both ADPKD mo dels (Fig. 1a,b). [score:4]
To study whether c-Myc regulates miR-17∼92 expression in ADPKD mo dels, Ksp/Cre; Pkd1 [F/F]; c-Myc [F/F] (Pkd1/ c-Myc- KO) mice were generated. [score:4]
Intriguingly, this analysis identified Pparα as one of 25 common direct targets of miR-17∼92 in the context of ADPKD (Fig. 7c and Supplementary Tables 4,5). [score:4]
miR-17 miRNA family is upregulated in ADPKD. [score:4]
To understand the biological relevance of miR-17 upregulation, the miR-17∼92 cluster was deleted in orthologous ADPKD mo dels. [score:4]
Next, we intersected the RNA-Seq data from both ADPKD mo dels with a list of high-probability direct mRNA targets of miR-17∼92. [score:4]
To identify the precise location of dysregulated miR-17 expression, we performed in situ hybridization using a locked nucleic acid (LNA) -modified probe against the mature miR-17 transcript. [score:4]
Pkd1 expression was unchanged whereas miR-17∼92 expression was reduced by 85.4% in Pkd1 [F/RC]D KO compared with Pkd1 [F/RC]S KO kidneys (Supplementary Fig. 3A,B). [score:4]
miR-17 is upregulated in kidney cysts of mouse and human ADPKD. [score:4]
In support of this conclusion, we show that genetic deletion of miR-17∼92 attenuates disease progression in ADPKD mouse mo dels irrespective of the mutated gene (Pkd1 or Pkd2), the type of mutation (null or hypomorphic) or the dynamics of cyst growth (rapidly fatal, aggressive but long-lived or slowly progressing). [score:4]
In contrast, compared with control kidneys, miR-17 expression was increased by 45.4% in Pkd2- KO kidneys, whereas its expression was reduced by 46.5% in Pkd2-miR-17∼92 KO kidneys (Supplementary Fig. 2C). [score:4]
We have recently developed a chemically modified anti-miR oligonucleotide (anti-miR-17) that sterically inhibits the activity of all miR-17 family members in cultured cells via complementary base pairing 16. [score:3]
miR-17 expression was increased in cyst epithelia of both Pkd1-mutant and Pkd2- KO kidneys (Fig. 1f). [score:3]
This analysis identified Pparα and 24 other common putative miR-17∼92 targets in the context of ADPKD. [score:3]
miR-17 expression was not detected by ISH in NHK kidney sections. [score:3]
Our work suggests that an important additional component of this metabolic re-wiring is the inhibition of FAO and OXPHOS mediated, at least in part, by the miR-17- Pparα axis (Fig. 10). [score:3]
Anti-miR-17 inhibits cysts in in vitro human mo dels of PKD. [score:3]
First, we validated that Pparα is inhibited by miR-17∼92 in ADPKD mo dels. [score:3]
We have previously used Pkd2- KO mice to analyse miR-17 expression by Q-PCR 12. [score:3]
miR-17 and miR-19 binding to Pparα 3′-UTR lead to reduced Pparα expression, which in turn affects mitochondrial metabolism in kidney epithelial cells. [score:3]
miR-17∼92 deletion attenuates disease progression in long-lived and slow cyst growth mo dels of ADPKD. [score:3]
In contrast, deletion of miR-17∼92 did not affect c-Myc expression in Pkd1- KO or Pkd2- KO kidneys, indicating that c-Myc functions upstream of miR-17∼92 in ADPKD (Fig. 6g,h). [score:3]
Therefore, we used this in situ probe to examine miR-17 expression in kidney samples from normal humans (NHK) and patients with ADPKD. [score:3]
Pkd2 [−/−] cells were transfected with 0.4 μg of a PPARα expression plasmid (pPpara) or Control (pCMX), along with 1 nM of miR-17 or scrambled mimic (Scr). [score:3]
The pre-clinical studies presented here indicate that miR-17∼92 is a novel drug target for ADPKD. [score:3]
In conclusion, miR-17∼92 promotes ADPKD progression through a new mechanism involving the inhibition of mitochondrial function. [score:3]
miR-17 modulates mitochondrial function by inhibiting Ppara. [score:3]
How to cite this article: Hajarnis, S. et al. microRNA-17 family promotes polycystic kidney disease progression through modulation of mitochondrial metabolism. [score:3]
c-Myc deletion reduced pri-miR-17 expression in Pkd1- KO kidneys. [score:3]
c-Myc promotes miR-17∼92 expression in PKD. [score:3]
miR-17∼92 deletion results in improved expression of mitochondrial and metabolism-related gene networks. [score:3]
We used the to assess whether this compound inhibits endogenous miR-17 in kidneys following systemic administration. [score:3]
c-Myc promotes miR-17∼92 expression in cystic kidneys. [score:3]
To explore the downstream mechanisms, we performed RNA sequencing (RNA-Seq) analysis to compare mRNA expression profiles between kidneys of 21-day-old Pkd1 [F/RC]S KO and Pkd1 [F/RC]D KO mice (n=3 biological samples), and 21-day-old Pkd2- KO and Pkd2-miR-17∼92 KO mice (n=5 biological samples). [score:3]
Q-PCR analysis revealed that primary miR-17 transcript (pri-miR-17) was upregulated by 9.3-fold in SBM kidneys compared with control kidneys, suggesting that c-Myc drives miR-17∼92 transcription (Fig. 6d). [score:3]
These findings are likely to be relevant to human ADPKD pathogenesis because inhibiting miR-17 also attenuated proliferation and cyst growth of primary human ADPKD cultures. [score:3]
While our data demonstrated that administration of anti-miRs up to 6 months is feasible, additional pre-clinical studies are needed to fully address the long-term safety profile of anti-miR-17 therapy and to further explore miR-17 as a drug target for PKD. [score:3]
Moreover, re -expression of Pparα normalized ATP -dependent OCR of miR-17 mimic -treated mIMCD3 and Pkd2 [−/−] cells (Fig. 9g–i and Supplementary Fig. 12). [score:3]
Importantly, miR-17 is a feasible and novel drug target for ADPKD. [score:3]
Finally, we examined the role of miR-17∼92 in a slow cyst growth mo del of ADPKD (Pkd1 [RC/RC]) that harbours homozygous germline Pkd1 RC mutations. [score:2]
Compared with Pkd2- KO mice, we observed a 149% increase in median survival, 31.8% improvement in serum creatinine levels, 15.8% reduction in cyst index, reduced Kim1 (down by 37.9%) and Ngal (down by 39.6%) expression, and a 58.9% decrease in the number of proliferating cyst epithelial cells in Pkd2-miR-17∼92 KO mice (Fig. 2e–h and Supplementary Fig. 2). [score:2]
Furthermore, upstream regulatory analysis revealed that gene networks controlled by key metabolism-related transcription factors Pparα, Pparg and Ppargc1a were activated upon miR-17∼92 deletion (Fig. 7b). [score:2]
Pparα expression was also increased in anti-miR-17 -treated compared with vehicle -treated Pkd2- KO kidneys. [score:2]
Expression of miR-17 (blue) was increased in cysts (cy) of Pkd1 [F/RC]S KO and Pkd2- KO compared with renal tubules of wild-type mice. [score:2]
Kidney-weight-to-body-weight ratios, BUN levels and Kim1 and Ngal expression were reduced in anti-miR-17 -treated compared with vehicle -treated Pkd2- KO mice (Fig. 4b–e). [score:2]
Q-PCR was performed by using the TaqMan Gene Expression Master Mix (Life Technologies) and pre-designed pri-miR-17∼92 primers from Life Technologies. [score:2]
Based on the unbiased analysis of our RNA-Seq data, we reasoned that miR-17∼92 -mediated direct repression of Pparα might provide one potential explanation for this phenomenon. [score:2]
PPARα expression was decreased in mIMCD3 cells treated with miR-17 mimic compared with scramble mimic. [score:2]
Collectively, these observations suggest that miR-17 promotes proliferation in cystic kidneys, at least in part, by reprogramming metabolism through direct repression of Pparα. [score:2]
Conversely, Pparα mRNA and protein expression was decreased in miR-17 mimic -treated compared with scramble mimic -treated mIMCD3 as well as Pkd2 [−/−] kidney epithelial cells (Fig. 7d,f). [score:2]
Compared with renal tubules in NHK, miR-17 expression was increased in kidney cysts from patients with ADPKD (Fig. 1g). [score:2]
We observed a 50% improvement in median survival, 22% reduction in serum creatinine levels, 26.8% reduction in kidney-weight-to-body-weight ratio and decreased expression of kidney injury markers Kim1 (down by 38.7%) and Ngal (down by 43.9%) in Pkd1-miR-17∼92 KO compared with Pkd1- KO mice (Fig. 2a,b,d and Supplementary Fig. 1D,E). [score:2]
miR-17 aggravates cyst growth through direct repression of Pparα. [score:2]
Q-PCR analysis showed that compared with control kidneys, Pkd1 expression was equally reduced in both Pkd1- KO and Pkd1-miR-17∼92 KO kidneys indicating a similar level of Cre/ loxP recombination (Supplementary Fig. 1A). [score:2]
Similarly, compared with mock or control oligonucleotide transfection, anti-miR-17 inhibited in vitro cyst growth of primary ADPKD cells in a dose -dependent manner (Fig. 5b,c and Supplementary Fig. 6B). [score:2]
Q-PCR and western blot analysis showed that compared with control kidneys, Pkd2 expression was equally reduced in both Pkd2- KO and Pkd1-miR-17∼92 KO kidneys indicating a similar level of Cre/ loxP recombination (Supplementary Fig. 2A,B). [score:2]
Treatment with anti-miR-17 produced a dose -dependent reduction in the proliferation of cyst epithelia from five human donors (Fig. 5a and Supplementary Fig. 6A). [score:1]
involving Pkhd1/Cre; Pkd2 [F/F] (Pkd2- KO) mice were utilized to determine the delivery and efficacy of the anti-miR-17 compound. [score:1]
The proposed mechanism by which miR-17∼92 promotes ADPKD progression. [score:1]
Thus, c-Myc functions upstream of miR-17∼92 in ADPKD mo dels. [score:1]
Anti-PS staining was observed in collecting duct-derived cysts (arrowheads) of Pkd2- KO mice injected with anti-miR-17 indicating that the compound was delivered to collecting duct cysts. [score:1]
Treatment with miR-17 mimics reduced basal and ATP -dependent OCR in mIMCD3 and Pkd2 [−/−] cells. [score:1]
Deleting the miR-17 binding site prevented miR-17 -mediated, but not miR-19 -mediated, repression. [score:1]
Primary cyst epithelial cultures derived from kidneys of ADPKD patients were transfected with anti-miR-17 (dose: 3 nM, 10 nM or 30 nM) or three different control oligonucleotides (dose: 30 nM, control oligo1, 2 and 3). [score:1]
The following miRNA mimics were purchased from Dharmacon, Inc (Thermo Fischer Scientific Inc) - miR-17 (catalogue # C-310561-07-0005), miR-19a (catalogue # C-310563-05-0005) and negative control or Scrambled (catalogue # CN-001000-01-05). [score:1]
The tissues were incubated with miR-17 (cat # 38461-01, Exiqon) or scramble (cat # 99004-01, Exiqon) probes. [score:1]
The percentage of miR-17 -positive cysts per kidney section from four human ADPKD patients (#6–9) is shown in the graph. [score:1]
To assess therapeutic efficacy, we injected Pkd2- KO mice with 20 mg kg [−1] of anti-miR-17 or vehicle at postnatal days (P) 10, 11, 12 and 19 and killed them at P28. [score:1]
In the cytoplasm, the mature miRNAs (miR-17 and miR-19) bind to Pparα 3′-UTR. [score:1]
To test whether the binding sites are functional, we co -transfected mIMCD3 cells with a luciferase reporter plasmid containing Pparα 3′-UTR and miR-17, miR-19, or scramble mimics (Fig. 8b). [score:1]
For WY14643 studies, mIMCD3 or Pkd2 [−/−] cells were grown at 37 °C, plated in six-well dishes (2 × 10 [5] cells per well), and transfected with 5 nM of miR-17 or scrambled mimic. [score:1]
The miR-17 miRNA family aggravates cyst growth in the Kif3a- KO ciliopathy mo del of PKD 12. [score:1]
Anti-miR-17 treatment reduces proliferation and cyst growth in in vitro mo dels of human ADPKD. [score:1]
Next, we evaluated whether anti-miR-17 treatment demonstrates therapeutic efficacy in a second, more long-term mo del of cystic kidney disease. [score:1]
Quantification of the Sirius red staining (g) and the number of proliferating cells (h) revealed that both interstitial fibrosis and tubular proliferation were reduced after miR-17∼92 deletion in Pkd1 [RC/RC] mice. [score:1]
To assess the therapeutic efficacy of this compound, Pkd2- KO mice were injected with anti-miR-17 or PBS at P10, 11, 12 and 19, and kidneys were harvested on P28. [score:1]
In situ hybridization (ISH) was performed using an LNA -modified anti-miR-17 probe. [score:1]
Pparα 3′-UTR harbours an evolutionarily conserved binding site for miR-17 and miR-19 families (Fig. 8a). [score:1]
Next, we studied the role of miR-17∼92 in Pkhd1/Cre; Pkd2 [F/F] (Pkd2- KO) mice, which with a median survival of ∼70-days exhibit a relatively less aggressive PKD. [score:1]
Kidney sections were co-stained with DBA (green, a marker of collecting ducts) and anti-PS antibody (red, antibody labels anti-miR-17 compound). [score:1]
Chromatin immunoprecipitation analysis showed that c-Myc binds to the miR-17∼92 promoter in cultured renal epithelial cells and mouse kidneys (Fig. 6a). [score:1]
Semi-qPCR analysis of the immunoprecipitated DNA revealed that c-Myc specifically binds to the miR-17∼92 promoter in mIMCD3 cells and mouse kidney tissue. [score:1]
Staining with the anti-PS antibody revealed that anti-miR-17 was delivered to collecting duct cysts even when administered after numerous cysts had already formed (Fig. 4a). [score:1]
Moreover, a 300 mg kg [−1] dose of anti-miR-17 did not produce acute liver or kidney toxicity in mice (Supplementary Fig. 5C). [score:1]
The first and second generation progeny were intercrossed to generate Ksp/Cre; Pkd1 [F/F] (Pkd1- KO) and Ksp/Cre; Pkd1 [F/F]; miR-17∼92 [F/F] (Pkd1-miR-17∼92 KO) mice. [score:1]
miR-17∼92 promotes cyst growth in early-onset ADPKD mo dels. [score:1]
Anti-miR-17 studies involving Pkhd1/Cre; Pkd2 [F/F] (Pkd2- KO) mice were utilized to determine the delivery and efficacy of the anti-miR-17 compound. [score:1]
Large interconnected gene networks controlled by URs PPARα, PPARg and PPARGC1a were predicted to be activated after miR-17∼92 deletion in both ADPKD mo dels. [score:1]
Therefore, we tested whether miR-17 affected these functions of PPARα. [score:1]
The generation of the following mouse lines is discussed in the results section: Pkd1-miR-17∼92 KO, Pkd2-miR-17∼92 KO, Ksp/Cre; Pkd1 [F/RC] (Pkd1 [F/RC]S KO), Ksp/Cre; Pkd1 [F/RC]; miR-17∼92  [F/F] (Pkd1 [F/RC]D KO), Pkd1 [RC/RC]; Ksp/Cre, Pkd1 [RC/RC];Ksp/Cre;miR-17∼92 [F/F] and Pkd1/c-Myc- KO. [score:1]
Similar miR-17 displacement was not observed in mice treated with a control oligonucleotide (Supplementary Fig. 5B and Supplementary Table 3). [score:1]
In a complementary pharmaceutical approach, we demonstrate that anti-miR-17 also slowed cyst growth in two orthologous mouse mo dels, including a long-lived, slow cyst growth mo del. [score:1]
Anti-miR-17 reduced proliferation and cyst count in a dose -dependent manner. [score:1]
Anti-miR-17 demonstrates therapeutic efficacy in short-term and long-term PKD mouse mo dels. [score:1]
mIMCD3 cells were transfected with 0.6 μg of pPpara or pCMX, along with 5 nM of miR-17 or Scr mimic. [score:1]
Moreover, similar to genetic deletion, treatment with anti-miR-17 also slowed the proliferation of cyst epithelial cells (Fig. 4f). [score:1]
The miR-17∼92 primary transcript is processed to yield the individual mature miRNAs. [score:1]
To examine whether miR-17 plays a similar pathogenic role in ADPKD, miR-17∼92 was genetically deleted in various orthologous ADPKD mouse mo dels. [score:1]
C57BL/6 mice (Jackson Laboratories) were injected with a single subcutaneous dose of 0.3, 3 or 30 mg kg [−1] of anti-miR-17, a control oligonucleotide or PBS. [score:1]
Ksp/Cre 42, Pkhd1/Cre 43, Pkd1 [F/F] 15, Pkd2 [F/F] 15, miR-17∼92 [F/F] (ref. [score:1]
The displacement values are reported in log [2] scale where the positive values reflect the loss of miR-17 from the high molecular weight polysome fractions. [score:1]
S KO indicates either Pkd1 [F/RC]S KO or Pkd2- KO, whereas D KO indicates either Pkd1 [F/RC]D KO or Pkd2-miR-17∼92 KO kidneys. [score:1]
44), miR-17 transgenic mice 45, Pkd1 [RC/RC] (ref. [score:1]
miR-17∼92 promotes cyst growth in long-lived ADPKD mo dels. [score:1]
Our studies point to pro-proliferative metabolic reprogramming induced by the c-Myc-miR-17- Pparα signalling axis as a potential new mechanism for PKD pathogenesis (Fig. 10). [score:1]
miR-17 modulates metabolic functions of PPARα. [score:1]
c-Myc binds to miR-17∼92 promoter and enhances its transcription in cystic kidneys. [score:1]
Pathway analysis suggested that the primary cellular consequence of miR-17∼92 deletion in both ADPKD mo dels was improved mitochondrial metabolism (Fig. 7a and Supplementary Figs 7 and 8). [score:1]
First, we deleted miR-17∼92 in Pkd1- KO mice, which develop an early-onset and rapidly fatal form of PKD. [score:1]
Luciferase reporter assays revealed that compared with scramble, both miR-17 and miR-19 mimics suppressed wild-type Pparα 3′-UTR. [score:1]
Similarly, deleting the miR-19 binding site abolished miR-19 -mediated, but not miR-17 -mediated, repression. [score:1]
miR-17∼92 [F/F] mice were bred with Ksp/Cre; Pkd1 [F/+] transgenic mice. [score:1]
We developed an antibody (anti-PS) that specifically binds to the chemically modified phosphate backbone of anti-miR-17 to determine its cellular distribution. [score:1]
We tested whether miR-17 affected these functions of PPARα. [score:1]
Watson-Crick base-pairing between miR-17/ PPARΑ 3′-UTR and miR-19/ PPARΑ 3′-UTR is shown. [score:1]
These networks were also differentially regulated in Pkd2-miR-17∼92 KO compared with Pkd2- KO kidneys. [score:1]
These cells were transfected with anti-miR-17 and control oligos using RNAiMAX (catalogue # 13778-150, Life Technologies) following the manufacturer's protocol at 2,500 cells per well density in a 96-well plate. [score:1]
The seed sequences for the miR-17 and the miR-19 binding sites were mutated in the WT-Pparα 3′-UTR construct to produce the Pparα 3′-UTR (Δ17) and Pparα 3′-UTR (Δ19) constructs. [score:1]
Both miR-17 and miR-19 repressed Pparα 3′-UTR. [score:1]
Therefore, we evaluated whether miR-17∼92 also influences disease progression in long-lived and slow cyst growth mo dels of ADPKD. [score:1]
Anti-miR-17 attenuates cyst growth in two PKD mo dels. [score:1]
mIMCD3 cells were co -transfected with this plasmid and scramble (scr, black), miR-17 mimic (red) or miR-19 mimic (blue) (n=3). [score:1]
The miR-17 family is highlighted in red. [score:1]
mIMCD3 cells were plated in six-well dishes (2 × 10 [5] cells per well) and transfected with 0.4 μg of pLS-Renilla-3′-UTR plasmids, and 10 nM of miR-17 or miR-19a mimic. [score:1]
Moreover, we did not observe any major adverse effects of long-term anti-miR-17 therapy such as weight loss, failure to thrive or death. [score:1]
The experimental approach for anti-miR-17 studies is shown in Supplementary Fig. 5. Littermate pairs of Pkhd1/Cre; Pkd2 [F/F] (Pkd2- KO) mice were administered either standard moist chow or standard moist chow supplemented with fenofibrate at a dose of 800 mg per day per kg body-weight for 10 days starting at postnatal day 18. [score:1]
The circles indicate predicted binding sites for the various miRNA families derived from the miR-17∼92 cluster. [score:1]
Anti-miR-17 studies. [score:1]
The human mature miR-17 sequence is identical to the mouse miR-17. [score:1]
Pkd2- KO and Pkhd1/Cre; Pkd2 [F/F]; miR-17∼92 [F/F] (Pkd2-miR-17∼92 KO) mice were generated using the strategy described earlier. [score:1]
miR-17∼92 deletion attenuates cyst growth in early-onset ADPKD mo dels. [score:1]
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b Flow cytometry analysis of the expression of α [4]-integrin, α [5]-integrin, CD44 and CXCR4 on miR-17 overexpressing CB CD34 [+] cells (green and blue lines) and CTRL CD34 [+] cells (red line) Ectopic expression of miR-17 alters CB CD34 [+] cell adhesion to hematopoietic niche componentsThrough adhesion to the corresponding components from niche in vivo, the adhesive moleculars expressed by hematopoietic cells regulated the interaction between hematopoietic cells and their niche. [score:10]
The increased expression of major adhesion molecules in miR-17 overexpressed CB CD34 [+] cells suggests that the adhesion between miR-17 overexpressed CB CD34 [+] cells and their niche in vivo is regulated abnormally, which may further lead to the reduced hematopoietic reconstitution capability of 17/OE cells in engrafted mice. [score:8]
b Flow cytometry analysis of the expression of α [4]-integrin, α [5]-integrin, CD44 and CXCR4 on miR-17 overexpressing CB CD34 [+] cells (green and blue lines) and CTRL CD34 [+] cells (red line) Through adhesion to the corresponding components from niche in vivo, the adhesive moleculars expressed by hematopoietic cells regulated the interaction between hematopoietic cells and their niche. [score:8]
Conversely, downregulation of miR-17 inhibited the expansion of CB CD34 [+] cells. [score:6]
The improper expression of N-cadherin and β [1]-integrin on CD34 [+] cells upon miR-17 overexpression raised the possibility that the adhesion between 17/OE cells and their niche in vivo is regulated abnormally, which further leads to the reduced hematopoietic reconstitution capability of 17/OE cells in vivo. [score:6]
By overexpression and knockdown studies, we showed that ectopic expression of miR-17 promotes long-term expansion and colony forming of CB CD34 [+] cells and CD34 [+]CD38 [−] cells in vitro. [score:6]
By overexpression and knockdown studies, we demonstrated that miR-17 regulates the growth of CB CD34 [+] cells and CD34 [+]CD38 [−] cells in vitro. [score:5]
The expression levels of miR-17 in CD34 [+]CD38 [−] cells are higher than those in CD34 [+]CD38 [+] cellsTo determine the expression level of miR-17 in human hematopoietic cells, we first obtained two populations from human CB MNCs through the analyses of human lineage-specific CD markers. [score:5]
In contrast to the more committed CB CD34 [+]CD38 [+] cells, CB CD34 [+]CD38 [−] populations express significantly higher levels of miR-17, suggesting that miR-17 may be a key regulator during the development of HSCs. [score:5]
miR-17 is abundantly expressed in murine hematopoietic progenitors and increased expression of AAAGUGC-seed containing miRNA in lineage negative bone marrow cells promotes replating capacity and expansion of myeloid progenitors [21]. [score:5]
The levels of miR-17 expression in GFP -positive cells from engrafted mice at 20 weeks were still up- or downmodulated (Fig.   3c), which indicated that this inconsistency between the in vivo and in vitro data was not due to the change of miR-17 expression in vivo. [score:5]
To confirm whether this inconsistency resulted from the change of miR-17 expression in vivo, we checked the levels of miR-17 expression in GFP -positive cells from engrafted mice at 20 weeks. [score:5]
We further found that the expression of selected major adhesion molecules on CB CD34 [+] cells was increased and the specific adhesion of these cells to N-cadherin and vascular cell adhesion molecule-1 (VCAM1) were also enhanced upon miR-17 overexpression in vitro. [score:5]
Fig. 4The adhesion molecule expression on CB CD34 [+] cells after miR-17 overexpression. [score:5]
Collectively, miR-17 levels are downregulated during the differentiation of human hematopoietic cells, which suggests that miR-17 may play a role in regulating HSC function. [score:5]
, ectopic expression of miR-17 in peripheral blood cells may inhibit both myeloid and erythroid colony growth [22]. [score:5]
The expression levels of miR-17 were up- or downregulated in 17/OE and 17/KD, respectively (Fig.   1b) compared to those in CTRL. [score:5]
All of these results indicated that interaction of miR-17 overexpressed CB CD34 [+] cells with their niche may be abnormal in vivo and further prevent the efficient and continuous production of blood cells, which may be, at least in part, responsible for the reduced hematopoietic reconstitution potential of miR-17 overexpressed CB CD34 [+] cells in vivo. [score:5]
a The expression of N-cadherin and β [1]-integrin on CB CD34 [+] cells after miR-17 overexpressing (17/OE) or control (CTRL) cells was analyzed by flow cytometry (left panels). [score:5]
To analyze the function of miR-17 on primitive human hematopoietic cells, the miR-17 overexpression (17/OE) and knockdown (17/KD or 17/KD1) mo dels were created using primary CB CD34 [+] cells. [score:4]
After knockdown of miR-17, there was a trend towards a decrease in the expression of β [1]-integrin and N-cadherin (Additional file 2: Figure S2), although statistical analyses of the cohort indicated that it did not meet statistical significance (p > 0.05). [score:4]
It seemed that the significantly reduced hematopoietic reconstitution potential of miR-17 overexpressed CB CD34 [+] cells in vivo is not a result of the defect of transplanted 17/OE cell migration between bones because the migration of miR-17 overexpressed CB CD34 [+] cells in vitro towards SDF1α was only slightly increased compared to that of CTRL cells. [score:4]
Our data imply that the adhesion between miR-17 -overexpressed CB CD34 [+] cells and their niche in vivo is regulated abnormally, which may further lead to the reduced hematopoietic reconstitution capability of 17/OE cells in engrafted mice. [score:4]
This study establishes that miR-17 is differentially expressed in human CB hematopoietic cells (Fig.   1a), which indicates that miR-17 may play distinct roles in hematopoietic cells at different developmental stages, although more CD markers are needed to further identify the subpopulations from CB CD34 [+] cells. [score:4]
Our data showed that miR-17 is significantly expressed in human CB CD34 [+]CD38 [−] cells compared to the levels expressed in the CD34 [+]CD38 [+] cells or mononuclear cells (MNCs). [score:4]
b Real-time PCR was performed to evaluate the expression level of miR-17 in CB CD34 [+] cells after transfection with vectors for miR-17 overexpression (17/OE), miR-17 knockdown (17/KD or 17/KD1), or control (CTRL). [score:4]
CB CD34 [+] cells were transfected with vectors for miR-17 overexpression (17/OE), miR-17 knockdown (17/KD), or control (CTRL) and cultured in cytokine -driven serum-free medium. [score:4]
As shown in Fig.   3c, the expression levels of miR-17 were still up- or downmodulated in GFP -positive cells from NPG recipients transplanted with CB 17/OE CD34 [+] cells or 17/KD CD34 [+] cells, respectively, although the expression levels of miR-17 became somewhat lower compared to that of corresponding initial cells. [score:4]
The adhesion potential of 17/OE CB CD34 [+] cells to VCAM1 was significantly reduced following β [1]-integrin knockdown, which suggested that β [1]-integrin expressed on 17/OE CD34 [+] cells mediated, at least in part, the increase in interaction between 17/OE CD34 [+] cells and VCAM1 caused by ectopic miR-17. [score:4]
The adhesion potential of 17/OE CD34 [+] cells to VCAM1 was significantly blocked upon β [1]-integrin knockdown (Fig.   5b), which suggested that β [1]-integrin expressed on 17/OE CD34 [+] cells mediated, at least in part, the increase in interaction between 17/OE CD34 [+] cells and VCAM1 caused by ectopic miR-17. [score:4]
The miR-17 overexpression and knockdown mo dels were created using primary CB CD34 [+] cells transfected by the indicated vectors. [score:4]
The expression of N-cadherin and β [1]-integrin on CB CD34 [+] cells after miR-17 knockdown (17/KD) or control cells (CTRL) was analyzed by flow cytometry (left panels). [score:4]
4.0 × 10 [4] miR-17 overexpression (17/OE), miR-17 knockdown (17/KD), or control (CTRL) CB CD34 [+] cells were injected intravenously into the sublethally irradiated NPG mice (n = 6 per group). [score:4]
We conclude that the proper expression of miR-17 is required, at least partly, for normal hematopoietic stem cell–niche interaction and for the regulation of adult hematopoiesis. [score:4]
c The levels of miR-17 expression in GFP -positive cells from engrafted mice at 20 weeks were tested by real-time PCR. [score:3]
The mechanisms underlying the enhanced expression of adhesive molecules in CB CD34 [+] cells upon ectopic miR-17 are largely unclear and will be explored further in our laboratory. [score:3]
Ectopic expression of miR-17 alters CB CD34 [+] cell adhesion to hematopoietic niche components. [score:3]
Fig. 1The expression of miR-17 in CB hematopoietic CD34 [+]CD38 [−]/CD38 [+] cells. [score:3]
The more precise subpopulations of human hematopoietic progenitors were established recently [29– 31] so further studies are still needed to get a more detailed expression profile of miR-17 in human CB HSCs based on the more precise hierarchy mo del. [score:3]
The CD34 [+]CD38 [−] populations expressed significantly higher levels of miR-17 in comparison to those of the CD34 [+]CD38 [+] populations or MNCs (Fig.   1a). [score:3]
Meenhuis A van Veelen PA de Looper H van Boxtel N van den Berge IJ Sun SM MiR-17/20/93/106 promotes hematopoietic cell expansion by targeting sequestosome1-regulated pathways in miceBlood. [score:3]
To examine whether the reduced hematopoietic reconstitution capability of CB CD34 [+] cells upon miR-17 modulation in vivo is a result of improper attachment or migration in engrafted mice, we examined the expression patterns of selected adhesion and homing molecules known to be important for the hematopoietic reconstitution of human HSCs in the engrafted mice. [score:3]
The expression pattern of miR-17 in human hematopoietic cells is consistent with that in 32D-CSF3R cells [21]. [score:3]
In support of this finding in vitro, a significant increase was observed in the adhesion of CB CD34 [+] cells to N-cadherin and VCAM1 following miR-17 overexpression (Fig.   5a). [score:3]
Although miR-17 contains a highly conserved seed sequence between species and is expressed in hematopoietic cells at different stages from both mouse and human origin, there still exist different opinions concerning the function of miR-17 on hematopoiesis [16, 21– 23]. [score:3]
Ectopic expression of miR-17 resulted in the promoted expansion of the phenotypic and functional CD34 [+]CD38 [−] compartment in vitro. [score:3]
Moreover, Li et al. showed that the miR-92a -induced erythroleukemia cell line, when overexpressing miR-17, displayed a significantly reduced proliferation rate, exhibited morphological features of apoptosis, and ultimately died 2 weeks post-transduction [16]. [score:3]
The expression levels of CD44 and CXCR4 were almost unchanged upon ectopic miR-17 (Fig.   4b). [score:3]
The number of CFU-Mix and BFU-E did not change significantly after miR-17 overexpression. [score:3]
The human pre-miR-17 gene was amplified by polymerase chain reaction (PCR) and subcloned into the vector pCMV-GFP to generate the expression constructs pCMV-GFP -pre-miR-17 (17/OE). [score:3]
miR-17 has been recognized either as an onco-miRNA or as a tumor suppressor depending on the cell type. [score:3]
Lane 1, one mouse without transplants; lane 2, one mouse receiving transplants of 17/OE CD34 [+] cells; lane 3, one mouse receiving transplants of 17/KD CD34 [+] cells; lane 4, one mouse receiving transplants of CTRL CD34 [+] cells; lane 5, one mouse receiving transplants of fresh CD34 [+] cells The expression of adhesion molecules on CB CD34 [+] cells upon miR-17 modulationEctopic miR-17 promotes the expansion of CB CD34 [+] cells in vitro but the hematopoietic reconstitution capability of 17/OE cells is reduced in vivo, which displays inconsistency. [score:3]
b CB CD34 [+] cells, transfected with ectopic miR-17 vector (17/OE or 17/KD), were further separated through the analysis of CD38 expression. [score:3]
The expression levels of miR-17 in CD34 [+]CD38 [−] cells are higher than those in CD34 [+]CD38 [+] cells. [score:3]
The expression of adhesion molecules on CB CD34 [+] cells upon miR-17 modulation. [score:3]
Based on shRNA influence on miR-17 expression in CB CD34 [+] cells, 17/KD was chosen for further studies. [score:3]
However, statistical analyses of this cohort indicated that it did not meet statistical significance (Fig.   5d), suggesting that the reduced hematopoietic reconstitution capability of miR-17 overexpressed CB CD34 [+] cells in vivo does not result from the migration defect of transplanted 17/OE cells. [score:3]
Fig. 5Effects of miR-17 overexpression on adhesion and migration of CB CD34 [+] cells. [score:3]
In vitro assays revealed that ectopic expression of miR-17 promoted long-term expansion, especially in the colony-forming of CB CD34 [+] cells and CD34 [+]CD38 [−] cells. [score:2]
Knockdown of miR-17 in CD34 [+] cells, on the other hand, resulted in reduced hematopoietic multipotential, which was followed by significantly diminishing CFC and CFU-GM output (Fig.   2c). [score:2]
d The migration of 17/OE CB CD34 [+] cells towards SDF1α was slightly increased compared with that of CTRL cells, but statistical analyses indicated it did not meet statistical significance (p > 0.3, Student’s t-test) To determine the expression level of miR-17 in human hematopoietic cells, we first obtained two populations from human CB MNCs through the analyses of human lineage-specific CD markers. [score:2]
After knockdown of miR-17, there was a trend toward a decrease in the total number of CD34 [+] cells, although statistical analyses of the cohort indicated that it did not meet statistical significance (p > 0.05). [score:2]
However, it is to be noted that the hematopoietic reconstitution potential of miR-17 -overexpressed CB CD34 [+] cells in vivo were significantly reduced in contrast with that of control CB CD34 [+] cells in repopulating assays with NPG mice. [score:2]
The percentage of CD45 [+]CD34 [+] cells in the 17/KD group whose transplants of CD34 [+] cells with miR-17 knockdown showed a tendency, although insignificant, to be lower than that of mice receiving transplants of CTRL CD34 [+] cells. [score:2]
However, the overexpression of miR-17 in vivo reduced the hematopoietic reconstitution potential of CB CD34 [+] cells compared to that of control cells. [score:2]
Compared with that from CD34 [+] cells after culturing for 7 days, there was a significant increase in the number of CFU-Mix from CD34 [+]CD38 [−] cells upon miR-17 overexpression (Fig.   2c). [score:2]
Although these studies all indicate that miR-17 is an important regulator of hematopoiesis, the function of miR-17 on hematopoiesis remains controversial. [score:2]
The function of miR-17 on human CB CD34 [+] cells is reinforced by knockdown studies on miR-17. [score:2]
After knockdown of miR-17, there was a trend toward a decrease in the colony forming capacity of CD34 [+]CD38 [−]/CD38 [+] cells, although statistical analyses of the cohort indicated that it did not meet statistical significance (p > 0.05) outside of the CFU-GM forming capacity from CD34 [+]CD38 [−] cells. [score:2]
It seems that miR-17 has almost no effect on the erythroid lineage development based on the lack of a significant difference between the number of BFU-E from 17/OE cells and that from the CTRL cells. [score:2]
It is of note that the migration of miR-17 overexpressed CB CD34 [+] cells towards SDF1α was slightly decreased compared to that of control cells. [score:2]
However, when the input cell numbers were similar, the 17/OE CD34 [+] cells contributed significantly less to hematopoietic reconstitution in recipient mice as opposed to the CTRL CD34 [+] cells, which was different from the in vitro expansion and colony forming assay, suggesting that the hematopoietic reconstitution capability of miR-17 -overexpressed CB CD34 [+] cells were reduced in vivo. [score:2]
After knockdown of miR-17, there was a trend toward a decrease in the expansion capacity of CD34 [+]CD38 [−]/CD38 [+] cells, although statistical analyses of the cohort indicated that it did not meet statistical significance (p > 0.05) outside of the first 5 days from CD34 [+]CD38 [−] cells. [score:2]
Knockdown of miR-17, on the other hand, resulted in decreased CB CD34 [+] cell expansion, which consequently diminished the cell number. [score:2]
a The adhesion of miR-17 overexpressing (17/OE) CB CD34 [+] cells to N-cadherin or vascular cell adhesion molecule-1 (VCAM1) was significantly increased compared with that of control (CRTL) cells. [score:2]
Taken together, these data indicate that the ectopic miR-17 in CD34 [+] cells preferentially supports a specific expansion of the CD34 [+]CD38 [−] populations in vitro. [score:1]
Effects of miR-17 modulation on the hematopoietic reconstitution capability of CB CD34 [+] cells in NPG miceTo further support our in vitro expansion results, we examined the hematopoietic reconstitution potential of CB CD34 [+] cells after miR-17 modulation in NPG mice. [score:1]
It is of great interest to note that the hematopoietic reconstitution potential of miR-17 overexpressed CB CD34 [+] cells in vivo is reduced compared to that of CTRL CB CD34 [+] cells according to SCID repopulating cells assays (Fig.   3). [score:1]
Moreover, the expanded CB CD34 [+] cells by ectopic miR-17 are capable of normal maturation ex vivo because they could differentiate into all of the lineages tested. [score:1]
miR-17 promotes expansion and colony forming of CB CD34 [+] cells. [score:1]
The miR-17 levels of the CD34 [+]CD38 [+] populations showed a tendency, albeit insignificant, to be higher than those of the MNCs. [score:1]
Although the percentage of human CD45 [+] cells gradually increased in the PB from all of the mice transplanted with miR-17-modulated CB CD34 [+] cells, the PB from 17/KD recipients displayed a significantly higher percentage of human CD45 [+] cells at 4 weeks than CTRL recipients, indicating that the hematopoietic reconstitution potential of 17/KD CD34 [+] cells is higher than that of CTRL CD34 [+] cells during the first 4 weeks (Fig.   3a left panel). [score:1]
The two miR-17-specific small hairpin RNAs (17/KD and 17/KD1) [24] and β1-integrin-specific shRNA (β1/KD) oligomers [26] were tested. [score:1]
The expanded hematopoietic cells upon ectopic miR-17 can promote myeloid lineage fate (Fig.   2c), which is consistent with the results from Meenhuis group [19], demonstrating the conservative function of miR-17 between mouse and human HSC to a certain extent. [score:1]
Effects of miR-17 modulation on the hematopoietic reconstitution capability of CB CD34 [+] cells in NPG mice. [score:1]
We temporally monitored the PB of NPG recipients transplanted with miR-17-modulated CB CD34 [+] cells for 20 weeks by analyzing the percentage of human CD45 [+] cells using flow cytometry every 4 weeks. [score:1]
a The expression level of miR-17 in CB CD34 [+]CD38 [−]/CD38 [+] cells was evaluated by real-time PCR. [score:1]
c The expression level of miR-17 in 17/OE CD34 [+] cells or 17/KD CD34 [+] cells 5 days after sorting was evaluated by real-time PCR. [score:1]
Moreover, most of the data about miR-17 to date were obtained from murine studies while the relevance to human HSC still needs to be substantiated. [score:1]
a Effect of miR-17 modulation on repopulation of CB CD34 [+] cells in NOD prkdc [scid] Il2rg [null] (NPG™) mice. [score:1]
Here, we reported that miR-17 is also necessary in the cell-intrinsic control of governing the biological properties of human cord blood (CB) CD34 [+] cells in vitro and in vivo. [score:1]
We further examined the multipotent differentiation of CD34 [+]CD38 [−]/CD38 [+] cells upon miR-17 modulation after cultured for 14 days in vitro. [score:1]
Recently, we have found that miR-17 is necessary in the cell-extrinsic control of HSC and HPC function, which is, at least in part, through the augmented HIF-1α signal pathways in osteoblasts [24]. [score:1]
miR-17 (also called miR-17-5p), an important member of the miR-17-92 cluster, contains the AAAGUGC-seed sequence [20]. [score:1]
After incubation with the coated ligands, CB CD34 [+] cells showed a significant increase in the adhesion to N-cadherin or VCAM1 upon ectopic miR-17 according to the CFC output (Fig.   5a). [score:1]
We have recently found that miR-17 is necessary in the cell-extrinsic control of cord blood (CB) CD34 [+] cell function. [score:1]
Here, we demonstrated that the proper level of miR-17 is also necessary in the cell-intrinsic control of the hematopoietic properties of CB CD34 [+] cells. [score:1]
Fig. 3Effect of miR-17 modulation on the hematopoietic reconstitution potential of CB CD34 [+] cells in NPG mice. [score:1]
The data are presented as the ratio of miR-17 levels (relative to U6) in 17/OE, 17/KD or 17/KD1 to that in CTRL. [score:1]
This idea is evidenced by the fact that ectopic miR-17 promotes the proliferation of CB CD34 [+]CD38 [−] cells, especially within the first 15 days of culture when the cells are the least differentiated (Fig.   2b). [score:1]
Lane 1, one mouse without transplants; lane 2, one mouse receiving transplants of 17/OE CD34 [+] cells; lane 3, one mouse receiving transplants of 17/KD CD34 [+] cells; lane 4, one mouse receiving transplants of CTRL CD34 [+] cells; lane 5, one mouse receiving transplants of fresh CD34 [+] cells Ectopic miR-17 promotes the expansion of CB CD34 [+] cells in vitro but the hematopoietic reconstitution capability of 17/OE cells is reduced in vivo, which displays inconsistency. [score:1]
Fig. 2The effect of miR-17 modulation on the expansion of CB CD34 [+]CD38 [−]/CD38 [+] cells. [score:1]
Our studies demonstrated that miR-17 may specifically affect hematopoietic stem and early progenitor cells. [score:1]
Together, our data suggest that miR-17 in CD34 [+] cells preferentially promotes a specific expansion of the CB CD34 [+]CD38 [−] populations in vitro and the expanded CD34 [+]CD38 [−] cells could differentiate into all of the lineages tested. [score:1]
a The expansion of CB CD34 [+] cells upon miR-17 modulation after culturing for 20 days. [score:1]
In summary, our data suggest the potential contribution of miR-17 in the in vitro and in vivo function on human HSCs and HPCs. [score:1]
b Flow cytometry analysis of the human CB CD34 [+] cell repopulation in a representative NPG mouse after miR-17 modulation. [score:1]
As shown in Fig.   2a, CD34 [+] cells were expanded significantly upon ectopic miR-17. [score:1]
These data add adhesive molecules to the signaling network affected by miR-17 and suggest a miR-17–N-cadherin (β [1]-integrin) pathway in human CB HSCs and HPCs. [score:1]
Therefore, further investigations of miR-17 on hematopoiesis in vivo raises the possibility that miR-17 may play a wider role in regulating hematopoietic development. [score:1]
To further support our in vitro expansion results, we examined the hematopoietic reconstitution potential of CB CD34 [+] cells after miR-17 modulation in NPG mice. [score:1]
As shown in Fig.   2b, although both CD34 [+]CD38 [−] and CD34 [+]CD38 [+] cells were expanded to different extents upon miR-17 modulation, the ectopic miR-17 tends to expand CD34 [+]CD38 [−] cells more, rather than CD34 [+]CD38 [+] cells, especially during the first 15 days. [score:1]
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In conclusion, the results of molecular, cellular and in vivo experiments demonstrated that the circadian-regulated miR-17-5p inhibits the translation of Clock by targeting the 3′UTR of Clock mRNA and participates in the regulation of free-running period in mice. [score:9]
One of the major findings of the present study was that miR-17-5p and Clock exhibited reciprocal regulation: miR-17-5p bound to two sites on the 3′UTR of Clock mRNA and strongly suppressed the expression of Clock, whereas CLOCK protein directly bound to the promoter of miR-17 and enhanced the expression of miR-17-5p. [score:9]
MiR-17-5p represses the translation of Clock by targeting its 3′UTRWe used three computational prediction algorithms, miRanda, PicTar and TargetScan, to screen miRNAs that target the 3′UTR of Clock mRNA. [score:9]
However, overexpression of miR-17-5p decreased the protein expression of CLOCK and its paralog, NPAS2 (Fig. 6F), whereas the inhibition of miR-17-5p increased the expression of CLOCK and several clock proteins, including BMAL1, PER1 and CRY1 (Fig. 6F). [score:9]
Thus, we demonstrated that miR-17-5p inhibited Clock expression by targeting its 3′UTR. [score:7]
Overexpression of miR-17-5p inhibited the expression of CLOCK and NPAS2, this effect may endow miR-17-5p the rationality to shorten the period length, as Clock- or Npas2 -null mice display shortened free running period 34. [score:7]
The double mutation of Clock 3′UTR significantly attenuated the inhibitory effect of miR-17-5p on Luc expression (Fig. 1B). [score:6]
In addition, we packed the lentivirus that carried miR-17-5p sponges and infected N2a cells, aiming to use this tool to compete with the binding sites of the corresponding miRNAs and effectively attenuate the inhibitory effect of miR-17-5p on its target genes. [score:5]
Note that the overexpression or subtraction of miR-17-5p increased the CRY1 expression in N2a cells. [score:5]
These experiments suggest that miR-17-5p inhibits the translation, rather than promotes the mRNA degradation, of Clock. [score:5]
As shown above, i. c. v. injection of antagomir-17-5p suppressed the expression/action of miR-17-5p in the SCN and led to an acceleration of the circadian locomotor rhythm and a shortening of the period length. [score:5]
As shown above, miR-17-5p inhibited the expression of Clock and CLOCK promoted the transcription of miR-17-5p in vitro. [score:5]
We also focused on the potential output molecule(s) of the circadian clock by which miR-17-5p affected the period length, and demonstrated that a consistent increase or decrease of miR-17-5p lead to a unidirectional change of a clock gene, i. e., the promotion of Cry1 expression, whereas Clock exhibited bidirectional changes in response to increase or decrease of miR-17-5p. [score:5]
We observed that the application of inhibitor or antagomir sharply repressed the endogenous expression of miR-17-5p (Fig. 1C). [score:5]
Western blot results indicated that the overexpression of miR-17-5p substantially decreased the level of CLOCK protein, whereas a miR-17-5p inhibitor or antagomir promoted it (Fig. 1D). [score:5]
The rhythmic expression patterns of miR-17-5p in synchronised cells and SCN implicate that miR-17-5p expression may be under the control of circadian clock. [score:5]
These findings demonstrated that miR-17-5p inhibited the expression of Clock. [score:5]
Direct regulation of miR-17-5p expression by CLOCK. [score:5]
The introduction of antagomir-17-5p weakened the inhibition of miR-17-5p on CLOCK expression, and thus promoted the circadian rhythm process. [score:5]
Thus, we used cycloheximide (CHX), an inhibitor of protein synthesis in eukaryotic cells, to inhibit the synthesis of CLOCK and to determine whether miR-17-5p would affect the degradation speed of CLOCK. [score:5]
In addition, as indicated in Fig. 6F, the interference of miR-17-5p indirectly boosted the expression of several important clock genes on the protein level, which may potentially result in a circadian process impetus. [score:4]
In the present study, miR-17-5p had two binding sites in the 3′UTR of Clock mRNA and functioned as a stronger inhibitor of Clock compared with its other targets. [score:4]
Taken together, these results indicate that CLOCK binds to the promoter of miR-17 and regulates the expression of miR-17-5p. [score:4]
In addition to the finding that miR-17-5p affected the CLOCK level in cell lines, we also demonstrated that miR-17-5p was rhythmically expressed in the SCN compared with its expression pattern in the cortex. [score:4]
As shown in Fig. 6F, both overexpression and knockdown of miR-17-5p increased CRY1 protein level. [score:4]
Western blot results indicated that CLOCK was rhythmically expressed with a shifted peak value compared with miR-17-5p (Fig. 2B), whereas miR-17-5p and CLOCK had similar oscillatory periods of approximately 24 hours in expression. [score:4]
MiR-17-5p represses the translation of Clock by targeting its 3′UTR. [score:4]
CRY1 was not the direct target of miR-17-5p. [score:4]
The knockdown of miR-17-5p also did not affect the mRNA expression of most clock genes (Fig. S3). [score:4]
We transfected Neuro-2a (N2a) cells with miR-17-5p mimics or a pMIRNA1 overexpression construct with the insertion of pre-miR-17. [score:3]
The injection of antagomir-17-5p contributed to the inhibition of miR-17-5p in the SCN of the mice even after 14 days (Fig. 5F). [score:3]
After screening for several generations with G418, a cell line stably overexpressing miR-17-5p was constructed. [score:3]
To further clarify the action mechanism of miR-17-5p on Clock, we performed experiments at the translation process. [score:3]
At the cellular level (shown in Figs S4 and 6F), miR-17-5p also targeted NPAS2, a paralog of CLOCK. [score:3]
To further validate the impact of miR-17-5p on Clock, we performed overexpression and knockdown assays in cell lines. [score:3]
Rhythmic expression of miR-17-5p and CLOCK in the SCN of C57BL/6J mice. [score:3]
To confirm the interaction sites between miR-17-5p and the Clock 3′UTR, we identified two putative recognition sites in the Clock 3′UTR by utilising TargetScan. [score:3]
The transcription of miR-17 is directly regulated by CLOCK. [score:3]
This finding suggests that miR-17-5p represses the translation of Clock. [score:3]
Thus, a smaller quantity of miR-17-5p may be sufficient to exert a moderate repression of CLOCK expression. [score:3]
The overexpression vector had two products, miR-17-5p and miR-17-3p. [score:3]
We selected CT20 as the injection time because this time point preceded the time of miR-17-5p expression peak. [score:3]
Nevertheless, these two sites were primarily responsible for the binding and inhibition of miR-17-5p on Clock mRNA. [score:3]
MiR-17-5p may target additional regulatory genes of the free-running period. [score:3]
The expression of miR-17-5p was decreased (Fig. 4C), suggesting that the level of miR-17-5p is under the control of Clock. [score:3]
We further identified that miR-17-5p decreased the CLOCK protein level not by promoting Clock mRNA or CLOCK protein degradation, but by repressing the translation of Clock. [score:3]
The antagomirs of miR-17-5p used in this study were similar with the inhibitors of miR-17-5p in action but with different modifications. [score:3]
The overexpression of miR-17-5p did not significantly affect the mRNA levels of most clock genes, with the exception of a slight increase in the mRNA levels of Cry1, Cry2 and Per2 (Fig. S3). [score:3]
It is possible that the inhibitory effect of miR-17-5p on the 3′UTR of Clock mRNA is relieved by antagomir-17-5p. [score:3]
Rhythmic expression of CLOCK and miR-17-5p in synchronised NIH/3T3 cells. [score:3]
A representative example of Western blots (upper of B) and the grey values (lower of B) of each corresponding time point of three independent experiments are shown in panel B. Each n = 3 in A or B. (A) RT-qPCR results indicate the circadian expression pattern of miR-17-5p in mouse SCN or cortex (CTX) in LD 12:12 (h) or DD conditions. [score:3]
As shown in Fig. 3A, the miR-17-5p expression exhibited similar rhythmic patterns in both LD and DD. [score:3]
The N2a cells that expressed miR-17-5p were obtained by screening with 800 μg/ml G418. [score:3]
We further demonstrated that miR-17-5p and CLOCK were expressed in different phases in synchronised cells and in opposite phases in the SCN. [score:3]
We demonstrated that miR-17-5p was expressed in a circadian oscillatory pattern in the synchronised cells (Fig. 2A). [score:3]
Plasmids for the overexpression of miR-17-5p were constructed and transfected to N2a cells. [score:3]
However, whether miR-17-5p plays a role in the regulation of circadian rhythm has not been reported. [score:2]
To confirm the importance of miR-17-5p in circadian rhythm regulation, we applied a specific antagomir in vivo to reduce the level of mature miR-17-5p in the mouse SCN. [score:2]
As shown in the present study, miR-17-5p was involved in the regulation of molecular clock. [score:2]
To further confirm whether CLOCK directly regulates miR-17, we performed a chromatin immunoprecipitation (ChIP) assay. [score:2]
Overall, miR-17-5p may be a novel factor in the modulation of the circadian molecular feedback loops and an intermediate molecule that provides circadian regulation to many other physiological and pathological pathways. [score:2]
The present study demonstrated an interesting function of miR-17-5p in the regulation of circadian clock period. [score:2]
MiR-17-5p expresses in the mouse SCN in a circadian manner. [score:2]
Although the present study shows relevance between CRY1 and the period length, we do not intend to suggest a causal and exclusive output role of CRY1 in the regulation of circadian period by miR-17-5p. [score:2]
Thus, these findings may basically endow miR-17-5p as a regulator of circadian rhythm. [score:2]
Among these miRNAs, we focused on miR-17-5p because the influence of miR-17-5p transfection on Clock expression in NIH/3T3 cells (Fig. S1C) was similar with the dual luciferase reporter assay (Fig. S1B). [score:2]
Thus, we speculated that CLOCK may mediate the regulation of miR-17-5p on CRY1. [score:2]
As shown in Fig. 1G,H, when protein degradation was blocked by MG-132, an inhibitor of major proteasomes, miR-17-5p could still decrease the CLOCK protein level compared with the nonsense sequence. [score:2]
The increase of CRY1 protein level in response to bidirectional changes of miR-17-5p were also demonstrated in vivo (Fig. 6H). [score:2]
MiR-17-5p that were sponge packed with adenovirus, a competitive inhibitor of small RNAs, were designed as reported 39 and ordered from Hanbio Company (Shanghai, China). [score:2]
We determined that the oscillatory amplitude of the miR-17-5p level was not as large as Per1 and other well-known clock genes, this result suggests a fine-tuning role of this miRNA in circadian clock regulation. [score:2]
MiR-17-5p is rhythmically expressed in synchronised NIH/3T3 cells. [score:2]
As indicated in Fig. 4F, compared to the vector control, co -expression of Clock and Bmal1 promoted the transcription of miR-17-Luc. [score:2]
Our results revealed that blocking transcription via actinomycin significantly decreased the Clock mRNA level and introducing miR-17-5p into cells did not exert a further influence on the Clock mRNA (Fig. 1E). [score:1]
The upstream 5-kb and downstream 2-kb sequences of miR-17 transcription start site were examined for putative CLOCK -binding sites. [score:1]
Assessment of the CLOCK protein half-life demonstrated that miR-17-5p did not affect the stability of CLOCK (Fig. 1I,J). [score:1]
The promoters of miR-17 (1.8 kb), Per1 (1.8 kb) and Per2 (1.7 kb) were cloned from the genome of the NIH/3T3 cell line and ligated to pGL3-basic 37. [score:1]
However, the possibility that miR-17-5p promotes the degradation of CLOCK cannot be excluded in this condition. [score:1]
Moreover, miR-17-5p was under the control of CLOCK: CLOCK bound to the proximal promoter region of miR-17 and promoted its transcription, and a stable decrease of CLOCK led to reduction of miR-17-5p. [score:1]
We examined the miR-17-5p levels in the SCN during a 24-h period in mice in LD (light:dark) 12:12 (h) or DD (constant dark) circumstances. [score:1]
These findings indicate that miR-17-5p acts as a modulator of the circadian period length. [score:1]
The sequences of pre-miR-17 and its adjacent region were cloned from the genome of the mice and ligated to pcDNA3.1 or pMIRNA1. [score:1]
Cry1 was not the only changeable gene in response to the increase or decrease of mir-17-5p and Clock in the present study. [score:1]
These unexpected results suggest that there may be an equilibrium mechanism to stabilise the miR-17-5p level in the SCN, and changes in the miR-17-5p level would accelerate the circadian clock process and lead to period shortening, potentially via specific output signalling molecule(s). [score:1]
The transfection of the pMIRNA-miR-17 construct increased the abundance of mature miR-17-5p by more than 10 fold (Fig. 1C). [score:1]
As this attenuation did not reach 100%, we speculated that there might be other interacting regions on the Clock 3′UTR for miR-17-5p. [score:1]
As shown in Fig. 4E, CLOCK bound to the promoter region of miR-17, approximately from the upstream 1.5 kb to the transcription start site. [score:1]
These results obtained at the cellular and integral levels were in accordance with the behavioural results that both increase and decrease of miR-17-5p in the SCN lead to an accelerated process of the circadian clock (period shortening). [score:1]
These findings suggest that miR-17-5p may be under the circadian control. [score:1]
We delivered agomir-17-5p to the lateral ventricle of the mouse brains, a performance equal to an increase of miR-17-5p level in the SCN. [score:1]
In addition, we cloned the promoter region of miR-17 and validated the transactivation of CLOCK and BMAL1 on miR-17-Luc. [score:1]
Because period shortening is associated with the output signalling of the circadian clock, we speculated that miR-17-5p may exert an effect on the output genes of the circadian clock, for example, Per1 and Pk2 (prokineticin 2). [score:1]
CRY1 may be an important output signaling molecule of Clock in mediating the period shortening elicited by miR-17-5p, although we cannot exclude other signaling molecules involved in the output process of rhythm. [score:1]
Identification of two major binding sites for miR-17-5p on the 3′UTR of Clock mRNA. [score:1]
Taken together, these findings suggest that miR-17-5p is a distinct molecule in equilibrating the period length of the circadian rhythm, potentially via the modulation of CRY1 level through CLOCK in the SCN. [score:1]
Moreover, miR-17-5p reduced the CLOCK protein level regardless of transcription blocking (Fig. 1F), suggesting that miR-17-5p may potentially affect the renewal of CLOCK protein, rather than the degradation of Clock mRNA. [score:1]
In addition, Npas2 takes a similar role as Clock in the function of miR-17-5p. [score:1]
To investigate the expression patterns of miR-17-5p and CLOCK, we synchronised NIH/3T3 cells with 50% horse serum. [score:1]
The relative miR-17-5p levels were higher at late night and early morning but were lower in the middle of the day. [score:1]
We speculated that there was a specific association between miR-17-5p and CLOCK. [score:1]
Thus, the miR-17-5p level may be relevant to the circadian clock in the SCN. [score:1]
It is notable that the miR-17-5p level increased to a zenith value when the cytoplasmic CLOCK decreased to a nadir value. [score:1]
We are curious regarding how miR-17-5p affected the CRY1 protein level. [score:1]
Constructs of 80 ng pGL3-basic, miR-17-Luc, Per1-Luc or Per2-Luc were mixed with 8 ng Renilla-luciferase vector, respectively, and transfected into 293T cells. [score:1]
Cells were transfected with miR-NC or miR-17-5p mimics followed by treatment with 50 nM MG-132 or DMSO for 8 hours. [score:1]
These experiments suggest that the circuit between miR-17-5p and CLOCK may comprise a mechanism that stabilises the molecular feedback loops of the circadian clock. [score:1]
MiR-17-5p significantly decreased the expression of Clock in both assays. [score:1]
The decrease in miR-17-5p led to a shortening of the free-running period. [score:1]
This result further suggests a role of miR-17-5p in the function of the master clock. [score:1]
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[+] score: 289
In this paper, using two independent cancer cell mo dels, we unveiled a novel regulatory mechanism where inhibition of miR-17-5p and/or miR-106-5p leads to recover TRIM8 -mediated p53 tumour suppressor activity and strong inhibition of N-MYC -dependent cell proliferation by p53 -dependent N-MYC destabilization through miR-34a up-regulation. [score:11]
To demonstrate that miR-106b-5p as well as miR-17-5p directly inhibit the expression of TRIM8, we first used an in silico approach to identify the miR-106b-5p and miR-17-5p -binding sequence in the TRIM8 3’UTR region by using Target Scan (Release7.0, August 2015) [25], the database of conserved 3’UTR miRNA targets. [score:10]
Here, to elucidate the molecular mechanisms leading to this gene expression alteration, we tested the possible correlation with the expression levels of two miRNA, miR-17-5p and miR-106b-5p, since they are up-regulated in chemo/radio-resistant cancers and the miR-17-92 cluster targets TRIM8 mRNA [8, 9, 13– 15]. [score:10]
As we observed in vitro, molecular analyses of the excised masses confirmed p53 stabilization following exogenous HA-TRIM8/RING expression, miR-17-5p down-regulation and, at the same time, the up-regulation of miR-34a (Fig.   9c-e - Additional file 11: Figure S10b). [score:9]
Considering the variation of miR-17-5p expression in each tissue pair and Fuhrman grade, no relevant variation was detected suggesting that the up-regulation of miR-17-5p expression might be independent from the severity of this type of tumour (Fig.   1a). [score:8]
These results clearly depict a novel pathway through which the overexpression of miR-17-5p and its paralogue miR-106b-5p inhibits TRIM8, leading, on one hand, to the de-stabilization of the p53 tumour suppressor protein and, on the other hand, to the activation of the N-MYC oncogene, turning on cancer cells proliferation (Fig.   5a). [score:7]
A feed-forward loop is thus created, whereby N-MYC fosters its own protein expression via the inhibition of TRIM8 mRNA by promoting miR-17-5p and miR-106b-5p expression (Fig.   5a). [score:7]
c, d, e The stabilization of p53, miR-34a and down-regulation of miR-17-5p in xenografts treated with HA-TRIM8, HA-RING (T1-T5) or LacZ (C1-C3) was demonstrated by western blotting analysis of p53 (Actin was used as loading control) and by RT-qPCR on miR-17-5p (* p-value < 0.05) and miR-34a expression (* p-value < 0.05). [score:6]
When miR-17-5p and miR-106b-5p expression levels were compared to those of TRIM8 in the same sample, only miR-17-5p showed a strong negative linear relationship with TRIM8 (r = -0.7878; p-value < 0.0001), while miR-106b-5p was not linear correlated to its target gene expression (r = -0.02684; p-value = 0.8694) (Additional file 2: Figure S1c and d). [score:6]
miR-17-5p and miR-106b-5p expression levels are up-regulated in ccRCC patients. [score:6]
Data are shown as the average with standard deviation of at least 3 independent experiments (** p-value < 0.005; *** p-value < 0.001) Altogether, our experiments demonstrated that TRIM8 is a direct target of miR-106b-5p as well as of miR-17-5p and that their co -expression synergizes in decreasing TRIM8 mRNA, eventually resulting in increased cell proliferation (Fig.   3a-d). [score:6]
Data are shown as the average with standard deviation of at least 3 independent experiments (** p-value < 0.005; *** p-value < 0.001) Altogether, our experiments demonstrated that TRIM8 is a direct target of miR-106b-5p as well as of miR-17-5p and that their co -expression synergizes in decreasing TRIM8 mRNA, eventually resulting in increased cell proliferation (Fig.   3a-d). [score:6]
In UOK-257 cells, upon anti-miR-17-5p overexpression, p21 protein expression increased 2.3 fold compared to the control cells coherently with mRNA expression (Fig.   4b and e - Additional file 6: Figure S5a). [score:6]
Relative expression ratios were measured respect to the sample transfected with the Negative Control miRNA Mimic and normalized by the expression levels of RPL13 for TRIM8 and p21, and by the expression level of U6 snRNA for miR-34a and miR-17-5p. [score:5]
We showed that TRIM8 is a target of miR-17-5p and miR-106b-5p, whose expression is promoted by N-MYC, and that alterations of their levels affect cell proliferation, acting on the TRIM8 transcripts stability, as confirmed in ccRCC patients and cell lines. [score:5]
Intriguingly, the UOK-257 carcinoma cell line (harbouring a mutant p53) showed miR-17-5p expression levels comparable to cancer RCC-Shaw and HCT116 cells, while miR-106b-5p expression levels were comparable to non-cancer HK-2 cells (Fig.   1c and d), a condition similar to that observed in G1-G2 Fuhrman grade patients (where p53 was wild-type though). [score:5]
Conversely, by inhibiting the action of miR-17-5p and miR-106b-5p on TRIM8, p53 becomes stable and activates miR-34a expression, which in turn quenches the N-MYC protein (Fig.   5b). [score:5]
On the contrary, the suppression of miR-106b-5p or miR-17-5p by specific anti-miRNAs increased TRIM8 expression levels with a reduction of cell proliferation in all cell lines (Fig.   3c and d). [score:5]
Fig. 3Effects of overexpression/silencing of miR-17-5p and miR-106b-5p on TRIM8 and p21 expression and cell proliferation. [score:5]
This increase in p21 expression seems to be p53-independent since we did not observe any increase of p53 protein upon anti-miR-17-5p overexpression (Adiitional file 6: Figure S5a). [score:5]
As stated before, colorectal HCT116 cells show higher expression level of TRIM8 and coherently lower miR-17-5p and miR-106b-5p expression levels than RCC-Shaw cells (Fig.   1c and d - Additional file 2: Figure S1b). [score:5]
Interestingly, we did not observe alterations of miR-106a-5p, miR-17-5p and miR-106b-5p expression in benign renal oncocytoma samples compared to non-tumour matched epithelial tissues (Additional file 3: Figure S2b), suggesting that only miR-17-5p and miR-106b-5p up-regulation is a signature of a malignant phenotype. [score:5]
Surprisingly, RT-qPCR demonstrated that RCC cells treated with chemotherapeutic drugs showed an inexplicable increase of miR-17-5p and miR-106b-5p expression levels (Additional file 7: Figure S6b and e) that in addition to keeping low TRIM8 protein levels, also keeps low the levels of crucial cell cycle inhibitors such as p21 and PTEN [14, 15]. [score:5]
Nevertheless, among all the targets regulated by miR-17-5p, we demonstrated that TRIM8 is pivotal to trigger cell sensitivity to chemotherapy. [score:4]
Even though chemotherapeutic drugs are able to reduce cell proliferation rate, this reduction became more pronounced when we increased TRIM8 and p53 proteins levels by transfecting anti-miR-17-5p or anti-miR-106b-5p (Fig.   7a-b), leading to miR-34a up-regulation and N-MYC decrease (Fig.   7b - Additional file 9: Figure S8a-f). [score:4]
The successful neutralization of miR-17-5p by anti-miR-17-5p was demonstrated by the stabilization of one of its targets, i. e. PPP2R2B (Protein Phosphatase 2 Regulatory subunit B, beta) (Fig.   8b) [37]. [score:4]
Moreover, we demonstrate that a TRIM8 deficit, due to miR-17-5p/miR-106b-5p up-regulation, contributes to oncogenesis and chemo-resistance. [score:4]
Here we showed that the up-regulation of miR-17-5p and miR-106b-5p leads to TRIM8 deficit, which in turn leads to failure of p53 protein activation, preventing the cells response to chemotherapy. [score:4]
In this scenario, TRIM8 recovery via miR-17-5p and miR-106b-5p silencing seems to be a winning move as it renders effective the tumour suppressor activity of p53, promoting the transcription of miR-34a that knocks out the oncogenic potential of N-MYC. [score:4]
The luciferase reporter assays demonstrated that both miR-106b-5p and miR-17-5p significantly suppressed the firefly luciferase activity of pMIR-3’UTR-TRIM8-wt (2.63- and 2.44-fold in HK-2, 1.82- and 2.6-fold in HCT116, respectively), whereas they failed to work when the target site was mutated (Fig.   2b and c). [score:4]
Next, we tested if TRIM8, among all the targets regulated by miR-17-5p, was pivotal to trigger cell sensitivity to chemotherapy. [score:4]
We first measured miR-17-5p and miR-106b-5p expression levels in the same ccRCC samples in which we found TRIM8 down-regulation (Fig.   1a and b – Additional file 2: Figure S1a; [7]). [score:4]
An increasing number of papers reported that miR-106b-5p and miR-17-5p, above all the microRNAs of the miR-17-92 cluster, are overexpressed in many different chemo/radio-resistant cancers, including ccRCC, glioma and Colorectal Cancers (CRC). [score:3]
In patients affected by colorectal cancer, neuroblastoma, breast and pancreatic cancer, it has been extensively reported that miR-17-5p and miR-106b-5p are overexpressed and are capable to confer chemo-resistance [14, 15, 42, 43]. [score:3]
Western blot of Actin was conducted as control Noteworthy, RT-qPCR demonstrated that in RCC-Shaw cells all the drugs induced a great increase of miR-17-5p and weaker of miR-106b-5p expression levels (Additional file 7: Figure S6b and e). [score:3]
The bars represent the Standard Error of the Mean These results led to the conclusion that TRIM8, among all miR-17-5p targets, is pivotal in controlling cell sensitivity to chemotherapy. [score:3]
The bars represent the Standard Error of the Mean These results led to the conclusion that TRIM8, among all miR-17-5p targets, is pivotal in controlling cell sensitivity to chemotherapy. [score:3]
Consistently with the cell proliferation decrease upon anti-miR-17-5p and anti-miR-106b-5p overexpression, only in p53wt background, but not in p53-mutated UOK-257 cells, TRIM8 protein levels raised, p53 became stabilized and promoted p21 and miR-34a transcription, in turn decreasing N-MYC protein levels (Fig.   6d-f and Additional file 8: Figure S7a-i). [score:3]
We did not treat tumours with anti-miR-17-5p because its inhibitory effects on tumour growth have been reported [26], while our aim here was to point out the critical role of TRIM8 on tumour growth. [score:3]
To assess miRNA/target interaction, the TRIM8 3’UTR fragment containing miR-17-5p/miR-106b-5p binding site wild type or mutant (wt or mut), and the p21 3’UTR fragment containing miR-17-5p/miR-106b-5p binding site wild type (wt) were cloned into pMIR Luciferase reporter vector (Life Technologies) downstream of the reporter luciferase gene. [score:3]
Western blot of Actin was conducted as control Noteworthy, RT-qPCR demonstrated that in RCC-Shaw cells all the drugs induced a great increase of miR-17-5p and weaker of miR-106b-5p expression levels (Additional file 7: Figure S6b and e). [score:3]
TRIM8 3’UTR is a target of both miR-17-5p and miR-106b-5p. [score:3]
On the other side, the inhibition of both endogenous miR-106b-5p and miR-17-5p by anti-miR-106b-5p and anti-miR-17-5p resulted in increasing firefly luciferase activity of pMIR-3’UTR-TRIM8-wt, unlike the mutant construct (Fig.   2d and e). [score:3]
Here we report an inverse correlation between miR-17-5p and TRIM8 expression in vivo. [score:3]
Correlation analysis of TRIM8 and miR-17-5p/miR-106b-5p expression levels was performed using GraphPad Prism 7 (GraphPad Software, San Diego, CA). [score:3]
Significantly, ccRCC tissues and their non-tumour counterpart showed no differences at all in the expression levels of miR-106a-5p, belonging to the same miR-17 “seed” family and whose “seed” region matched with TRIM8-3’UTR (Additional file 3: Figure S2a). [score:3]
To further confirm our hypothesis, we analysed the effect of miR-106b-5p or miR-17-5p overexpression or repression by transient transfection of the specific anti-miR-106b-5p and anti-miR-17-5p in HK-2, clear cell Renal Carcinoma (RCC-Shaw) and colorectal HCT116 cell lines. [score:3]
As shown in Fig.   8a-b and in Additional file 10: Figure  9a-d, anti-miR-17-5p plus control shRNA blocked cell proliferation because TRIM8 and p53 expression levels were increased, p21 and miR-34a were transactivated by p53, and N-MYC was destabilized. [score:3]
a, b miR-17-5p and miR-106b-5p expression in ccRCC samples and their paired non-tumour tissues. [score:3]
Altogether, these results clearly indicate a strong correlation between TRIM8 mRNA expression and miR-17-5p and miR-106b-5p levels, suggesting that these miRNAs could mediate TRIM8 mRNA degradation. [score:3]
In this paper we provided evidence that TRIM8 and its regulators miR-17-5p and miR-106b-5 participate to a feedback loop controlling cell proliferation through the reciprocal modulation of p53, miR-34a and N-MYC. [score:2]
TRIM8 Drug resistance N-MYC p53 miR-17 family The efficacy of current cancer treatments is often limited by the development of therapeutic resistance whose mechanisms still remain not fully elucidated. [score:2]
The increase of miR-17-5p and of miR-106b-5p (such as that we observed in G3 stage renal carcinomas, Fig.   1a and b) might account for a TRIM8 deficit, possibly responsible for p53 inactivation even in the absence of p53 mutations. [score:2]
Potential therapeutic applications that target miR-17-5p/miR-106b-5p using specific anti-miRNAs should be considered more closely for chemo-resistant tumours. [score:2]
The expression levels of miR-17-5p were significantly higher in tumours (T in Fig.   1a), compared to their corresponding non-tumour tissue (NT in Fig.   1a) (3.64-fold; p-value < 0.005), which held true for all the sample pairs analysed. [score:2]
miR-17-5p, miR-106b-5p, miR-106a-5p, and miR-34a expression levels were measured in triplicate by TaqMan MicroRNA Assay (Life Technologies™) using the ABI PRISM 7900HT platform (Applied Biosystems [®], Life Technologies™) and normalized to snU6 expression (Life Technologies™). [score:2]
We cloned the putative binding sites (wild-type or suitably mutated) of miR-106b-5p and miR-17-5p downstream of the luc2 firefly luciferase gene, under the control of the human PhosphoGlycerateKinase (PGK) promoter (pMIR-3’UTR-TRIM8-wt or pMIR-3’UTR-TRIM8-mut) and transfected them in the HK-2 and HCT116 cell lines with Negative Control miRNA Mimic (Ambion), miR-106b-5p, miR-17-5p, anti-miR-106b-5p, anti-miR-17-5p, both miRNAs or both anti-miRNAs (Fig.   2b-e). [score:1]
The HK-2 (normal kidney cells), the RCC-Shaw (ccRCC-derived cell line with wt-p53), the UOK-257 cells (RCC cell line with mutated p53) and HCT116 (colorectal cancer with wt-p53) were transfected with anti-miR-17-5p and anti-miR-106b-5p. [score:1]
To address this critical question, renal HK-2, RCC-Shaw and UOK-257 cells were transfected with control miRNA, anti-miR-17-5p or anti-miR-106b-5p and treated with Nutlin-3 (N) and Cisplatin (C), which induce p53 -dependent cell cycle arrest in tumours with wild type p53. [score:1]
Cells transfected with anti-miR-17-5p or anti-miR-106b-5p, or with control miRNA and treated with the different drugs have been normalized with respect to this calibrator. [score:1]
Data are shown as the average with standard deviation of at least 3 independent experiments (** p-value < 0.005; *** p-value < 0.001) As a final important result of miR-17-5p and miR-106b-5p knocking-down, RCC and HCT116 cell proliferation rate decreased as demonstrated by MTT and colony-forming assays (Fig.   3d - Additional file 5: Figure S4c). [score:1]
These evidences confirmed in vivo the pathway we identified in vitro, with TRIM8 emerging as key actor of the activation in the p53-(miR-34a)-(N-MYC)-(miR-17) family. [score:1]
d, e, f Western Blotting of the indicated proteins in HK-2 (control), RCC-Shaw and UOK-257 after transfection with Negative Control miRNA Mimic, anti-miR-17-5p, anti-miR-106b-5p without drug (-) or after chemotherapeutic drug treatment with Nutlin-3 (N)(10 μM), Cisplatin (C)(7.5 μM), Sorafenib (S)(10 μM) or Axitinib (A)(10 μM). [score:1]
This cluster is located on chromosome 13q31.3 in the third intron of the C13orf25 gene, and contains 6 miRNAs (miR-17, miR-18, miR-19a, miR-20a, miR-19b-1, miR-92-1). [score:1]
Both miR-17-5p and miR-106b-5p link p53 to the N-MYC pathway. [score:1]
Anti-miR-17-5p and anti-miR-106b-5p render chemotherapy effective. [score:1]
In patients with a more aggressive tumour behaviour (G3 Fuhrman grade) we observed also a significant increase of miR-106b-5p, but not miR-106a-5p, belonging both to the same miR-17 seed -family. [score:1]
Data are shown as the average with standard deviation of at least 3 independent experiments (** p-value < 0.005; *** p-value < 0.001) As a final important result of miR-17-5p and miR-106b-5p knocking-down, RCC and HCT116 cell proliferation rate decreased as demonstrated by MTT and colony-forming assays (Fig.   3d - Additional file 5: Figure S4c). [score:1]
To tackle this issue, RCC-Shaw cells were transfected with Negative Control miRNA Mimic (Ambion), anti-miR-17-5p plus pRS control vector or anti-miR-17-5p plus specific TRIM8 short hairpins (pRS-shRNA-TRIM8) and treated with chemotherapeutics. [score:1]
b Western blotting analysis of the indicated proteins in RCC-Shaw transfected with Negative Control miRNA Mimic or anti-miR-17-5p plus control short hairpin -RNA or specific short hairpin against TRIM8 (shRNA-TRIM8). [score:1]
On the x-axis the Ct of TRIM8 minus the arithmetic mean of Cts calculated for ACTB and RPL13 (used as housekeeping genes in) is reported, while the y-axis shows the Ct of miR-17-5p/miR-106b-5p minus the Ct of U6 snRNP (used to normalize miRNAs expression levels). [score:1]
In addition, reducing the levels of miR-17-5p/miR-106b-5p, we increased the chemo-sensitivity of RCC/CRC-derived cells to anti-tumour drugs used in the clinic. [score:1]
Fig. 5Schematic representation of the opposite p53-TRIM8 and N-MYC-miR17-5p/miR106b-5p networks in cellular response to treatments. [score:1]
a, c, e, f Expression levels of TRIM8 and p21 were measured by RT-qPCR in HK-2, RCC-Shaw and HCT116 cells transfected with Negative Control miRNA Mimic, miR-17-5p or miR-106b-5p (alone or together) anti-miR-17-5p or anti-miR-106b-5p (alone or together). [score:1]
The HK-2 and the HCT116 cells were transfected with Negative Control miRNA Mimic, miR-17-5p, miR-106b-5p, anti-miR-17-5p or anti-miR-106b-5p, along with pMIR luciferase reporter construct containing p21 3’UTR. [score:1]
Cells transfected with anti-miR-17-5p plus control shRNA or shRNA-TRIM8, or with control miRNA and treated with the different drugs have been normalized with respect to this calibrator. [score:1]
Together these three miRNA clusters contain a total of 15 miRNAs constituting four “seed” families: the miR-17, the miR-18, the miR-19 and the miR-92 family. [score:1]
2.5 × 10 [5] human HK-2 or HCT116 cells were plated in six-well plates (60–80% confluency) and transfected with 50 pmol of miR-17-5p -mimic, miR-106b-5p -mimic, anti-miR-17-5p, anti-miR-106b-5p or Negative Control miRNA Mimic (Ambion), using SiPORT NeoFX Transfection Agent (Ambion). [score:1]
The HK-2 and HCT116 cells were transfected with Negative Control miRNA Mimic, miR-17-5p or miR-106b-5p (alone or together), anti-miR-17-5p or anti-miR-106b-5p (alone or together), along with pMIR luciferase reporter construct containing TRIM8 3’UTR (wt or mut). [score:1]
Fig. 2Structure and functional characterization of the putative miR-17-5p/miR-106b-5p target identified in the TRIM8 3’UTR sequence. [score:1]
Therefore we demonstrated that, by counteracting the action of miR-17-5p and miR-106b-5p by specific anti-miRs, the cells regained sensitivity to chemotherapeutic treatments. [score:1]
Conversely, both anti-miR-17-5p and anti-miR-106b-5p induced a significant reduction in proliferation rate in RCC-Shaw and in HK-2 cells, but not in UOK-257 cells, which became more pronounced when cells were treated with chemotherapeutics (Fig.   6a-c). [score:1]
Western blot of Actin was conducted as control Altogether, these experiments demonstrated that anti-miR-17-5p and anti-miR-106b-5p increase the efficacy of chemotherapy treatments in resistant cancer cell lines. [score:1]
The co-transfection of both miR-106b-5p and miR-17-5p further decreased the luciferase activity (4.2-fold in HK-2 and 3.56-fold in HCT116 cells) (Fig.   2b and c), indicating they may act synergistically. [score:1]
a, d, e Expression levels of TRIM8, p21 and miR-34a were measured by RT-qPCR in HK-2, RCC-Shaw, UOK-257 and HCT116 transfected with Negative Control miRNA Mimic, anti-miR-17-5p or anti-miR-106b-5p. [score:1]
On the contrary, anti-miR-17-5p plus specific TRIM8 short hairpin failed to stall cell proliferation because p53 was not stabilized by TRIM8, and was therefore no longer able to transactivate p21 and miR-34a (Fig.   8b - Additional file 10: Figure S9a-d). [score:1]
2.0 × 10 [5] HK-2, RCC-Shaw, UOK-257 or HCT116 cells were plated in six-well plates (50–60% confluency) and transfected with 50 pmol of anti-miR-17-5p, anti-miR-106b-5p or Negative Control miRNA Mimic (Ambion) using SiPORT NeoFX Transfection Agent (Ambion). [score:1]
Fig. 7Anti-miR-17-5p and anti-miR-106b-5p render chemotherapy treatments effective in CRC. [score:1]
Fig. 6Anti-miR-17-5p and anti-miR-106b-5p render chemotherapy treatments effective in ccRCC. [score:1]
Western blot of Actin was conducted as control Altogether, these experiments demonstrated that anti-miR-17-5p and anti-miR-106b-5p increase the efficacy of chemotherapy treatments in resistant cancer cell lines. [score:1]
Therefore, the effectiveness of anti-miR-17-5p and anti-miR-106b-5p in rendering the cells sensitive to chemotherapeutic drugs may be explained by their capability to increase TRIM8 mRNA steady state levels. [score:1]
2.5 × 10 [5] human HK-2, RCC-Shaw, UOK-257 or HCT116 cells were plated in 6-well plates (60–80% confluency) and transfected with 50 pmol of miR-17-5p -mimic, miR-106b-5p -mimic, anti-miR-17-5p, anti-miR-106b-5p or Negative Control miRNA Mimic (Ambion) using SiPORT NeoFX Transfection Agent (Ambion). [score:1]
Below it is shown the sequence alignment between the miR-17-5p/miR-106b-5p “seed” sequence and the TRIM8 3’UTR, as well as the evolutionary conservation across species. [score:1]
b Western Blotting of the indicated proteins in HCT116 cells after transfection with Negative Control miRNA Mimic, anti-miR-17-5p, anti-miR-106b-5p without drug (-) or after chemotherapeutic drug treatment with Nutlin-3 (N)(10 μM), Cisplatin (C)(7.5 μM), Sorafenib (S)(10 μM) or Axitinib (A)(10 μM). [score:1]
In this paper, we describe for the first time how TRIM8 plays a crucial role in p53 activation and in N-MYC quenching in a complex signalling involving the miR17 family. [score:1]
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Other miRNAs from this paper: mmu-mir-18a, mmu-mir-20a, mmu-mir-19a, mmu-mir-19b-1
Our data demonstrated that uPAR upregulated miR-17 and miR-20a expression via c-myc, and uPAR reduced cell apoptosis by increasing miR-17-5p/ 20a expression, which caused inhibition of TRAIL -induced apoptosis. [score:10]
We found that uPAR induces miR-17-5p and miR-20a expression by upregulating the transcription factor c-myc, whereas miR-17-5p/20a inhibit breast cancer apoptosis by suppressing DR4 and DR5. [score:10]
To further determine whether miR-17-5p/20a inhibits apoptosis and cell growth by suppressing DR4 and DR5, miR-17-5p -expressing or miR-20a -expressing cells were co -transfected with DR4 and DR5 siRNA. [score:9]
However, in our mo del system, downregulation of uPAR increased TRAIL -induced apoptosis through induction of the expression of miR-17-5p and miR-20a and inhibition of DR4 and DR5. [score:8]
Treatment with miR-17-5p/20a mimics caused a decrease in DR4 or DR5 mRNA, whereas miR-17-5p/20a inhibitors caused an increase in DR4 or DR5 mRNA (Figure 2D and 2E) and protein (Figure 2D and 2E) expression levels in these two cells, indicating that both DR4 and DR5 are inhibited by miR-17-5p/20a. [score:7]
Inhibition of miR-17-5p and miR-20a expression suppresses tumor growth in nude mice. [score:7]
We assessed whether inhibiting uPAR -induced miR-17-5p/20a could suppress growth of triple negative MDA-MB-231 tumor xenografts that highly express uPAR. [score:7]
Given that miR-17-5p/20a are expressed predominantly in malignant cells but not in normal cells, our study supports the notion that the inhibition of miR-17-5p/20a activity might provide a novel therapeutic approach for uPAR -overexpressing breast cancer. [score:7]
uPAR upregulates c-myc -induced miR-17-5p/20a expression. [score:6]
uPAR upregulates miR-17-5p/20a expression via c-myc. [score:6]
Inhibition of miR-17-5p and miR-20a levels by antagomir-17-5p and antagomir-20a delivery induces apoptosis and suppresses breast tumor growth in nude mice. [score:5]
Mimics and inhibitors of miR-17-5p or miR-20a, cholesterol-conjugated miR-17-5p and miR-20a inhibitors, c-myc, uPAR, and DR5-specific small interfering RNA (siRNA) were chemically synthesized by RiboBio Co. [score:5]
Besides inhibition of TRAIL -induced apoptosis, miR-17/20a enhances cell proliferation (see Figure 3D), which might be the result of suppression of other factors. [score:5]
Figure 4 (A–C) MDA MB 231 cells were co -transfected with DR4/DR5 siRNA and miR-17-5p or miR-20a inhibitor or a randomized oligonucleotide as an inhibitor control, and MCF-7 cells were co -transfected with DR4/DR5 siRNA and miR-17-5p or miR-20a mimic or a randomized oligonucleotide as a mimic control. [score:5]
Conversely, overexpression of uPAR in the poorly uPAR -expressing MCF-7 cells resulted in an increase in miR-17-5p/20a levels (Figure 5C). [score:5]
Figure 3 (A–D) MDA MB 231 cells were transfected with miR-17-5p or miR-20a inhibitor or a randomized oligonucleotide as an inhibitor control, and MCF-7 cells were transfected with miR-17-5p or miR-20a mimic or a randomized oligonucleotide as a mimic control. [score:5]
Because a previous study demonstrates that c-myc binds to the miR-17-cluster locus and enhances miR-17 and miR-20a expression [27], we speculated that uPAR might induce miR-17-5p/20a expression via c-myc. [score:5]
Another important gene that is suppressed by miR-17-92 is PTEN [37, 49], and a recent study confirms that TGFBR2 is suppressed by miR-17-5p/20a [50]. [score:5]
This outcome might occur because the uPAR depletion -induced decrease (approximately 50%, see Figure 5B) of miR-17-5p and miR-20a preferentially reduces DR4/5 translation efficiency, because transfection of miR-17-5p/20a inhibitor caused more evident changes in protein levels of DR4/5 than in their mRNA levels (Figure 2D and 2E). [score:5]
These data suggest that the inhibition of cell apoptosis by miR-17-5p/20a is largely dependent on suppression of DR4 and DR5. [score:5]
Collectively, these data indicate that uPAR enhances miR-17-5p/20a expression by c-myc and thus inhibits cell apoptosis. [score:5]
Our data demonstrate that uPAR induces miR-17-5p/20a expression via c-myc in breast cancer and DR4 and DR5 are suppressed by miR-17-5p/20a. [score:5]
Conversely, transfection of their mimics in MCF-7 cells, which express relatively low levels of miR-17-5p and miR-20a, suppressed apoptosis (without TRAIL) and TRAIL -induced cell apoptosis (all P < 0.01) (Figure 3B and Supplementary Figure 2B). [score:5]
Transfection of MDA MB 231 cells, which express relatively high levels of miR-17-5p and miR-20a, with miR-17-5p or miR-20a inhibitors significantly induced apoptosis (without TRAIL) and increased TRAIL -induced cell apoptosis compared with controls (both P < 0.01) (Figure 3A and Supplementary Figure 2A). [score:4]
In this study, we found that uPAR knockdown increased the pro-apoptotic DR4 and DR5 protein levels in the MDA MB 231 TNBC cell line and confirmed that DR4 and DR5 are suppressed by miR-17-5p and miR-20a, which is similar to the results of Krishnamoorty et al. [34] showing that uPAR -depleted glioma cells have higher levels of DR4 and DR5. [score:4]
We examined the mechanism of uPAR -induced miR-17-5p/20a upregulation. [score:4]
Depletion of miR-20a and miR-17-5p (Figure 6E) and upregulation of DR4/DR5 mRNA and protein levels (Figure 6F and 6G) in tumors were verified by real-time PCR and Western blotting, respectively. [score:4]
MiR-17-5p/20a inhibits TRAIL -induced apoptosis by suppressing DR4 and DR5 in breast cancer cells. [score:4]
Given the multiple functions of the miR-17-92 cluster in the cell cycle, apoptosis, and tumorigenesis, the finding that uPAR induces miR-17-5p/20a expression might broaden our knowledge of the function of uPAR in cancer development, progression, and metastases. [score:4]
No differences in cell apoptosis were observed between uPAR siRNA -treated and control siRNA -treated cells under miR-17-5p/20a depletion by inhibitors (Figure 5G). [score:3]
Similarly, miR-17-5p and miR-20a inhibitors or mimics could not cause a change in activated caspase 8 and caspase 3 levels in DR4 and DR5 siRNA -treated cells (Figure 4C). [score:3]
Inhibition of TRAIL -induced apoptosis by miR-17-5p/miR-20a in breast cancer cells. [score:3]
We determined that DR4 and DR5 are suppressed by miR-17-5p/20a, which blocks cell apoptosis in breast cancer. [score:3]
The DR4 and DR5 3′untranslated region (UTR), which contains miR-17-5p or miR-20a binding sites, was amplified by PCR. [score:3]
This activity might explain the discrepancy between uPAR and miR-17-5p/20 expression in BT-474 cells. [score:3]
The miR-17 and miR-20a miRNAs are usually expressed as miR-17/20a because they share an identical sequence in both humans and mice [35, 36]. [score:3]
miR-17-5p/20a antagomirs inhibited the growth of triple -negative breast tumor xenografts in nude mice. [score:3]
We further examined the influence of miR-17-5p/20a on DR4 or DR5 expression in breast cancer cells. [score:3]
Additionally, increased cleaved caspase 3 was also observed in antagomir-17-5p/20a -treated tumors (Figure 6G) Figure 6 (A and B) MDA MB 231 cells were treated with cholesterol-conjugated miR-17-5p and miR-20a inhibitors or a cholesterylated randomized oligonucleotide as a control. [score:3]
Antagomir-17-5p and antagomir-20a are synthesized cholesterylated stable miR-17-5p and miR-20a inhibitors that have two oxygen methylation modifications and a sulfur -modified phosphate. [score:3]
The expression levels of miR-17-5p/20a are relatively high in MDA-MB-231 cells and low in MCF-7 cells (Figure 2C). [score:3]
DR4 and DR5 are suppressed by of miR-17-5p/20a. [score:3]
As shown in Figure 5A, a similar expression of uPAR and miR-17-5p/20a occurred in these cell lines except in the BT-474 cell lines, in which higher levels of miR-17-5p/20a occurred with higher uPAR levels and vice versa. [score:3]
Therefore, miR-17/20a antagomir -mediated tumor inhibition in mice might not be totally attributable to antagomir -induced cell apoptosis. [score:3]
Additionally, increased cleaved caspase 3 was also observed in antagomir-17-5p/20a -treated tumors (Figure 6G) Figure 6 (A and B) MDA MB 231 cells were treated with cholesterol-conjugated miR-17-5p and miR-20a inhibitors or a cholesterylated randomized oligonucleotide as a control. [score:3]
Inhibition of TRAIL -induced apoptosis by miR-17-5p/20a through DR4 and DR5 in breast cancer cells. [score:3]
The uPAR -inhibited cell apoptosis was largely blocked by depletion of miR-17-5p/20a or by DR4 and DR5 in breast cancer cells (see Figure 4 and Figure 5). [score:3]
Cholesterol-conjugated miR-17-5p and miR-20a inhibitor (10 nmol/mouse) or cholesterylated randomized oligonucleotide were intratumor- delivered five times every 3 days. [score:3]
Treatment with miR-17-5p or miR-20a inhibitor abruptly increased the protein levels of activated caspase 8 and caspase 3, whereas treatment with miR-17-5p or miR-20a mimics decreased the levels of caspase 8 and caspase 3 (Figure 3C). [score:3]
The effects of miR-17-5p and miR-20a inhibitors or mimics on cell apoptosis (Figure 4A and Supplementary Figure 1) and cell growth (Figure 4B) were largely attenuated by DR4 and DR5 depletion. [score:3]
At 48 hours after transfection with miR-17-5p/20a, miR-17-5p/20a inhibitor, or a randomized oligonucleotide as a control, cells were treated with 50 ng/mL TRAIL for additional 8 hours, and cellular apoptosis was detected. [score:3]
Overexpression of c-myc in MCF-7 cells resulted in an increase in miR-17-5p by 7.0-fold and miR-20a levels by 6.9-fold (Figure 5F). [score:3]
We observed that uPAR RNAi in MDA MB 231 cells led to decreased protein levels of DR4 and DR5 but not decreased mRNA levels (Figure 1C and 1D), whereas miR17-5p/20a inhibited both the mRNA levels and the protein levels in transfection experiments (see Figure 2). [score:3]
This report is the first showing that uPAR induces miR-17 and miR-20a expression. [score:3]
Transfection with uPAR siRNA in MDA MB 231 cells with uPAR overexpression led to a decrease in miR-17-5p/20a levels (Figure 5B). [score:3]
CCK-8 assays revealed that treatment with miR-17-5p or miR-20a inhibitor resulted in decreased cell growth, whereas an miR-17-5p or miR-20a mimic caused an increase in cell growth (all P < 0.05) (Figure 3D). [score:2]
Mutations in the seed region of the miR-17-5p (up) or miR-20a (lower) binding sites in the DR4 (nucleotides 1630-1647) or DR5 UTR (nucleotides 1892-1909) are marked in the box. [score:2]
These in vitro and in vivo experiments suggested that uPAR contributes to resistance to tumor apoptosis and that directing therapy at uPAR -induced miR-17-5p/20a is a potential therapeutic option in breast cancer. [score:2]
The PCR products were cloned into the pGL3 vector, and the recombinant plasmids (DR4-UTR-wt and DR5-UTR-wt) were mutated in the seed sequences of miR-17-5p or miR-20a by site-directed mutagenesis. [score:2]
Mutations were made in the seed region of the miR-17-5p/20a binding site as a control. [score:2]
We focused on miR-17-5p and miR-20a in this study because they belong to the same miRNA cluster (miR-17-92 cluster), which has strong oncogenic potential [23, 24, 25]. [score:1]
As seen in Figure 2A, perfect matches of the seed sequence are shown by vertical lines between the DR4 3′-UTR (nucleotides 1630–1647) or DR5 3′-UTR (nucleotides 1892–1909) and miR-17-5p or miR-20a. [score:1]
miR-17-5p or miR-20a levels were detected by real-time PCR, and c-myc protein levels were determined by Western blotting at 48 hours after transfection. [score:1]
At 48 hours after transfection, miR-17-5p or miR-20a levels were detected by real-time PCR. [score:1]
Our study reveals a new underlying miR-17-5p/20a- mediated pathway by which uPAR induces cell apoptosis in breast cancer. [score:1]
Thus, uPAR acted through the miR-17/5p/20a- DR4/DR5 pathway to block cell apoptosis. [score:1]
These results suggest that disrupting uPAR -induced miR-17/20a is a potential therapeutic option for TNBC cancer. [score:1]
Blocking uPAR -induced miR-17-5p/20a by antagomir treatment significantly attenuated TNBC tumor growth in mice. [score:1]
The miR-17-5p/20a miRNAs reduced the activity of a firefly luciferase reporter by binding to the wild-type (DR4-wt or DR5-wt) but not the mutant (DR4-mut or DR5-mut) DR4 or DR5 3′UTR (Figure 2B), confirming that miR-17-5p/20a interacts with this binding site. [score:1]
The miR-17-92 cluster includes six miRNAs (miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR-92-1) that are located within an 800-bp region of human chromosome 13. [score:1]
The miR-17-5p and miR-20a miRNAs were selected from the top 40 miRNAs according to context score percentiles, because they belong to the miR-17-92 cluster that is involved in cell proliferation and apoptosis [23, 24, 25] and they bind to both DR4 and DR5. [score:1]
The 293T cells were co -transfected with DR4-UTR-wt (DR5-UTR-wt) or DR4-UTR-mu (DR5-UTR-mu), pRL-TK plasmid, and miR-17-5p/miR-20a or a randomized oligonucleotide as a control. [score:1]
Five breast cancer cell lines were selected to detect uPAR mRNA and miR-17-5p/20a levels by real-time PCR. [score:1]
Our results underscore the potential of miR-17-5p/20a as an option for tailored therapy of breast cancers, including TNBC. [score:1]
Figure 5 (A) uPAR, miR-20a, and miR-17-5p levels in MDA MB 231, MCF-7, SK-BR3, BT-4T4, and ZR-751 cells were detected by real-time PCR. [score:1]
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Targets of the miR-17∼92 family clusterWe recently reported that the miR-17∼92 cluster down-regulates bone morphogenic protein (Bmp) receptor type 2 (Bmpr2), the receptor for Bmp-2, -4 and -7 and that higher levels of Bmpr2 are detected in cerebella of P7 miR-17∼92c KO; miR-106b∼25 KO mice compared to those of controls (Murphy et al., 2013). [score:5]
We found no significant up-regulation of microRNAs encoded by this cluster in cerebella lacking both miR-17∼92 and miR-106b∼25 clusters (data not shown). [score:4]
The miR-17/92 polycistron is up-regulated in sonic hedgehog -driven medulloblastomas and induced by N-myc in sonic hedgehog -treated cerebellar neural precursors. [score:4]
Treatment of SHH-MB with tiny LNAs directed against miR-17/20a/106b/93 and miR-19a/b-1 from the miR-17∼92 cluster family inhibits proliferation of SHH-MB in vitro and in vivo (Murphy et al., 2013). [score:4]
Analysis of previously published targets (Brock et al., 2009; Concepcion et al., 2012; Mogilyansky and Rigoutsos, 2013; Sun et al., 2013) revealed that Bmpr2, but not PTEN and p21 [Cip1], is regulated by the miR-17∼92 cluster (Murphy et al., 2013). [score:4]
The miR-17∼92 cluster is absolutely required for SHH-MB development MiRs encoded by the miR-17∼92 cluster are overexpressed in both mouse and human tumors (Uziel et al., 2009; Northcott et al., 2009). [score:4]
It has been previously reported that the miR-17∼92 cluster down-regulates multiple components of the TGF-β pathway (Tagawa et al., 2007; Petrocca et al., 2008; Dews et al., 2010; Mestdagh et al., 2010; Concepion et al., 2012; Li et al., 2012; Mogilyansky and Rigoutsos, 2013). [score:4]
The miR-17∼92 and miR106b∼25 clusters control the number of GNPs during post-natal cerebellar developmentWe previously reported that microRNAs from the miR-106a∼363 cluster are not expressed in wild-type cerebella (Uziel et al., 2009). [score:4]
We found that Bmpr2 was upregulated in the cerebella of miR-17∼92c KO; miR-106b∼25 KO mice, which might be responsible, in part, for the cerebellar anomalies. [score:4]
Our results are in agreement with a study suggesting that miR-17∼92 cluster downregulates two different signaling pathways, Bmp and TGF-β pathways (Brock et al., 2009). [score:4]
The miR-17∼92 and miR-106b∼25 clusters are direct targets of Myc and Mycn (O'Donnell et al., 2005; Northcott et al., 2009; de Pontual et al., 2011). [score:4]
The miR-17/92 cluster is targeted by STAT5 but dispensable for mammary development. [score:4]
To identify potential targets of the miR-17∼92 cluster family, we compared the gene expression profile of cerebella from control and miR-17∼92c KO; miR-106b∼25 KO mice at P4, and P7, times at which difference in cerebellar size was detectable. [score:4]
In agreement with these reports, we found, by GSEA analysis, that the TGF-β pathway was upregulated in the cerebella of miR-17∼92c KO; miR-106b∼25 KO mice. [score:4]
Silencing of the miR-17∼92 cluster family inhibits medulloblastoma progression. [score:3]
The miR-17/92 cluster: a comprehensive update on its genomics, genetics, functions and increasingly important and numerous roles in health and disease. [score:3]
Loss of the miR-17∼92 but not the miR-106b∼25 cluster inhibits Ptch1 [+/−] -induced SHH-MBs. [score:3]
SHH signaling induces the transcription of Mycn, which, in turn, induces the expression of the miR-17∼92 and miR-106b∼25 clusters that encode 6 and 3 microRNAs, respectively. [score:3]
Expression of miR-19a and miR-106b encoded by the miR-17∼92 cluster family in cerebella during embryogenesis. [score:3]
We previously found that enforced expression of the miR-17∼92 cluster collaborates with SHH signaling to induce SHH-MB (Uziel et al., 2009). [score:3]
The miR-17∼92 cluster is expressed in proliferative GNPs but not in post-mitotic granule neurons. [score:3]
Taken together, our findings demonstrate that the miR-17∼92 cluster is dispensable for cerebellar development, but required for SHH-MB development. [score:3]
In contrast, none of the 12 mice transplanted with GNPs purified from the cerebella of P7 miR-17∼92c KO; Ptch1 [+/−]; Cdkn2c [+/−] mice and expressing Mycn developed medulloblastoma, 180 days post-implantation. [score:3]
As expected, 100% (9/9) of the animals transplanted with GNPs purified from the cerebella of P7 miR-17∼92 [+/+]; Ptch1 [+/−]; Cdkn2c [+/−] mice and overexpressing Mycn developed SHH-MBs (Fig.  8A) (Zindy et al., 2007; Kawauchi et al., 2012). [score:3]
However, in contrast, the miR-17∼92 cluster alone or in combination with miR-106b∼25 is dispensable during retinal development (Conkrite et al., 2011; Nittner et al., 2012), mammary development (Feuermann et al., 2012) and Langerhans cells (Zhou et al., 2014). [score:3]
Fig. 9. The miR-17∼92 cluster suppresses TGF-β signaling. [score:3]
Fig. 1. Expression of miR-19a and miR-106b encoded by the miR-17∼92 cluster family in cerebella during embryogenesis. [score:3]
However, unlike Mycn, enforced expression of the miR-17∼92 cluster in GNPs purified from the cerebella of Trp53 [−/−]; Cdkn2c [−/−] mice failed to induce SHH-MBs after orthotopic transplantation demonstrating that activation of the Patched signaling pathway was required for miR-17∼92 induction of SHH-MBs (Uziel et al., 2009). [score:3]
Analysis of the microRNAs expressed by the two clusters revealed that miR-19a and miR-19b-1 that share the same seed sequence are encoded only by the miR-17∼92 but not the miR-106b∼25 cluster (Fig.  10). [score:3]
The miR-17∼92 cluster is required for medulloblastoma formationWe previously reported that GNPs purified from Ptch1 [+/−]; Cdkn2c [−/−] mice and overexpressing the miR-17∼92 cluster induced SHH-MB after transplant in the cortex of naïve CD-1 nu/nu recipient mice (Uziel et al., 2009). [score:3]
Further analysis using Clip-Seq approaches will be required to identify the bona fide miR-17∼92 targets in mouse and human MBs (Darnell, 2010). [score:3]
The miR-17∼92 cluster is a downstream target of Myc (c-Myc) (O'Donnell et al., 2005) and Mycn (Northcott et al., 2009; de Pontual et al., 2011). [score:3]
We previously reported that GNPs purified from Ptch1 [+/−]; Cdkn2c [−/−] mice and overexpressing the miR-17∼92 cluster induced SHH-MB after transplant in the cortex of naïve CD-1 nu/nu recipient mice (Uziel et al., 2009). [score:3]
Targeted deletion reveals essential and overlapping functions of the miR-17 through 92 family of miRNA clusters. [score:3]
Co-inactivation of the miR-17∼92 and miR-106b∼25 clusters reduced cerebellar size and foliation by limiting proliferationWe previously found that microRNAs encoded by the miR-17∼92 and miR-106b∼25, but not the miR-106a∼363, clusters are expressed in proliferating GNPs in the postnatal cerebellum (Uziel et al., 2009). [score:3]
miR-17∼92 miRNA cluster promotes kidney cyst growth in polycystic kidney disease. [score:3]
Fig. 8. Enforced Mycn expression in GNPs from Ptch1 [+/−]; Cdkn2c [+/−] but not from miR-17∼92c KO; Ptch1 [+/−]; Cdkn2c [+/−] mice induces SHH-MBs. [score:3]
Similarly when overexpressed in wild-type GNPs, the miR-17∼92 cluster collaborates with SHH signaling to provide GNPs with a proliferative advantage (Northcott et al., 2009). [score:3]
We previously found that microRNAs encoded by the miR-17∼92 and miR-106b∼25, but not the miR-106a∼363, clusters are expressed in proliferating GNPs in the postnatal cerebellum (Uziel et al., 2009). [score:3]
We recently reported that the miR-17∼92 cluster down-regulates bone morphogenic protein (Bmp) receptor type 2 (Bmpr2), the receptor for Bmp-2, -4 and -7 and that higher levels of Bmpr2 are detected in cerebella of P7 miR-17∼92c KO; miR-106b∼25 KO mice compared to those of controls (Murphy et al., 2013). [score:3]
In contrast, GNPs purified from the cerebella of P7 Ptch1 [+/−]; miR-17∼92 [floxed/ floxed]; Nestin-Cre [+] animals and overexpressing Mycn failed to induce tumors in recipient animals. [score:3]
Given the absolute requirement of the miR-17∼92 cluster for Ptch1 [+/−] induced SHH-MB formation, the identification of bone fide miR-17∼92 targets is clearly warranted. [score:3]
Overexpression of the miR-17∼92 cluster in GNPs heterozygous for the SHH receptor, Patched 1 (Ptch1 [+/−]), induces early onset of SHH-MB formation after orthotopic transplant in the cortices of naïve recipient animals (Uziel et al., 2009). [score:3]
Enforced Mycn expression in GNPs from Ptch1 [+/−]; Cdkn2c [+/−] but not from miR-17∼92c KO; Ptch1 [+/−]; Cdkn2c [+/−] mice induces SHH-MBs. [score:3]
MiRs encoded by the miR-17∼92 cluster are overexpressed in both mouse and human tumors (Uziel et al., 2009; Northcott et al., 2009). [score:3]
Deletion of the miR-17∼92 cluster was sufficient to completely abolish tumor development, pointing to the absolute requirement for the miR-17∼92 cluster, and to the lack of compensation by the miR-106b∼25 cluster, for MB development in collaboration with constitutively activated SHH signaling. [score:3]
This suggests that these two microRNAs might be sufficient for tumor development and that they might recapitulate the oncogenic function of the miR-17∼92 cluster, as reported previously for the development of Eμ-Myc B cell lymphoma (Mu et al., 2009; Olive et al., 2009). [score:3]
Targets of the miR-17∼92 family cluster. [score:3]
MicroRNAs encoded by the miR-17∼92 cluster, also called oncomiR-1, are overexpressed in various cancers (Concepcion et al., 2012; Mogilyansky and Rigoutsos, 2013) including mouse and human medulloblastomas with constitutively activated SHH signaling (Uziel et al., 2009; Northcott et al., 2009). [score:3]
The miR-17∼92 cluster suppresses TGF-β signaling. [score:3]
The miR-17∼92 and miR106b∼25 clusters control the number of GNPs during post-natal cerebellar development. [score:2]
miR-17∼92 cooperates with RB pathway mutations to promote retinoblastoma. [score:2]
The miR-17∼92 cluster is absolutely required for SHH-MB development. [score:2]
These results suggest that the miR-17∼92 cluster is required for SHH -induced PNL formation and for SHH-MB development. [score:2]
To assess their role in cerebellar and medulloblastoma (MB) development, we deleted the miR-17∼92 cluster family in Nestin -positive neural progenitors and in mice heterozygous for the Sonic Hedgehog (SHH) receptor Patched 1 (Ptch1 [+/−]). [score:2]
We here show that the miR-17∼92 cluster and its paralog the miR-106b∼25 cluster, are differently required for cerebellar and medulloblastoma development. [score:2]
In summary, while the miR-17∼92 and miR-106b∼25 clusters are essential for cerebellum development and homeostasis, the miR-17∼92, but not the miR-106b∼25, cluster is required for tumor initiation in Ptch1 [+/−] mice. [score:2]
Cerebellar development of miR-17∼92c KO; miR-106b∼25 KO mice appeared normal during embryogenesis until birth, a time when GNPs respond to SHH to rapidly proliferate with maximal proliferation between P5 and P7. [score:2]
Remarkably, deletion of the miR-17∼92 cluster abolished the development of SHH-MB in Ptch1 [+/−] mice. [score:2]
Schematic representation of the miR-17∼92 cluster family and its regulation by SHH signaling. [score:2]
These results suggested that decreased proliferation of GNPs from cerebella of miR-17∼92c KO; miR-106b∼25 KO mice might account for the diminished pool of GNPs during post-natal development. [score:2]
Strikingly, deletion of the miR-17∼92 cluster in Nestin -positive cells from Ptch1 [+/−]; Cdkn2c [+/−] mice completely abolished SHH-MB development. [score:2]
Synergistic action of the microRNA-17 polycistron and Myc in aggressive cancer development. [score:2]
These results suggested that, besides its role in SHH-MBs, the miR-17∼92 cluster might play a role in cerebellar development. [score:2]
The miR-17∼92 cluster not only regulates proliferation but also apoptosis (Concepcion et al., 2012; Mogilyansky and Rigoutsos, 2013). [score:2]
lacking one copy of miR-17∼92 show skeletal and growth defects recapitulating the Feingold syndrome observed in patients harboring MYCN mutations or hemizygous deletion of MIR-17∼92 (de Pontual et al., 2011). [score:2]
Here we analyzed the role of the miR-17∼92 cluster family during cerebellar development by conditional deletion the miR-17∼92 cluster alone or together with the miR-106b∼25 cluster (miR-17∼92c KO; miR-106b∼25 KO) in neural progenitors. [score:2]
Mid-sagittal sections through the cerebellum of control (D), miR-17∼92c KO (E), miR-106b∼25 KO (F) and miR-17∼92c KO; miR-106b∼25 KO (G) mouse at 1 month of age stained by H&E. [score:1]
Fig. 5. Premature termination of EGL proliferation in mice lacking the miR-17∼92 and miR-106b∼25 clusters. [score:1]
Using the Biocarta gene sets, we found that the Transforming Growth Factor (TGF)-β responsive signaling pathway was enriched in cerebella from P7 miR-17∼92c KO; miR-106b∼25 KO mice relatively to controls (Fig.  9B). [score:1]
Since miR-17∼92 -null mice die shortly after birth (Ventura et al., 2008), the miR-17∼92 cluster was conditionally deleted in Nestin -positive neural progenitors using a Nestin-Cre transgenic mouse (miR-17∼92 [floxed/ floxed]; Nestin-Cre [+]) in a wild-type miR-106b∼25 (referred as miR-17∼92c KO) or in a miR-106b∼25 -null (referred as miR-17∼92c KO; miR-106b∼25 KO) background. [score:1]
To assess whether the miR-17∼92 cluster was required for MB formation, we bred miR-17∼92 [floxed/+]; Nestin-Cre [+] with Ptch1 [+/−]; Cdkn2c [−/−] mice. [score:1]
Mid-sagittal sections through the cerebellum of a control (A,E), miR-17∼92c KO (B,F), miR-106b∼25 KO (C,G) and miR-17∼92c KO; miR-106b∼25 KO (D,H) mouse at E18.5 (A–D) and P7 (E–H) of age were stained by H&E. [score:1]
MiR-17∼92c KO (blue line, n = 20) and miR-17∼92 [+/+] (red line, n = 22) mice. [score:1]
The cerebellar folia from miR-17∼92c KO; miR-106b∼25 KO and miR-17∼92c KO mice were misshapen with shallow fissures. [score:1]
Premature termination of EGL proliferation in mice lacking the miR-17∼92 and miR-106b∼25 clusters. [score:1]
This was associated with a significant decrease in the number of 3 week old Ptch1 [+/−]; Cdkn2c [+/−] mice lacking the miR-17∼92 cluster that exhibit PNLs on the surface of their cerebella. [score:1]
Our data also showed that the miR-106b∼25 did not compensate for the loss of the miR-17∼92 cluster in tumor initiation. [score:1]
MiR-17∼92, miR-106a∼363 and miR-106b∼25 clusters encode 6, 6 and 3 mature microRNAs (colored boxes), respectively. [score:1]
At E18.5, cerebella of all 4 genotypes were indistinguishable displaying the 5 cardinal lobes separated by four principal fissures (Fig.  2A–D) despite complete Cre -mediated recombination of the two miR-17∼92 alleles (supplementary material Fig. S1A) and reduced levels of miR-19a in cerebella of miR-17∼92c KO mice (supplementary material Fig. S1B, lane 2). [score:1]
Fig. 4. Reduced proliferation in cerebella of mice lacking the miR-17∼92 and miR-106b∼25 clusters. [score:1]
This demonstrated that, despite the small brain size of the miR-17∼92c KO; miR-106b∼25 KO mice, their cerebella was significantly smaller than expected. [score:1]
Scattered ectopic clusters of mature granule neurons were found on the surface of the molecular layer in miR-17∼92c KO; miR-106b∼25 KO mice (Fig.  3G–I). [score:1]
However, only 33% (2/6) of cerebella from miR-17∼92c KO; Ptch1 [+/−]; Cdkn2c [+/−] mice showed PNLs (p = 0.0248) (Fig.  6F). [score:1]
The myc-miR-17∼92 axis blunts TGFbeta signaling and production of multiple TGFbeta -dependent antiangiogenic factors. [score:1]
Scattered foci of non-proliferating, differentiated neurons (negative for Ki67 but positive for NeuN and GABA (A) receptor α6 subunit) were observed on the surface of the cerebellar ML of miR-17∼92c KO; Ptch1 [+/−]; Cdkn2c [+/−] mice (Fig.  6D,E, Fig.  7H–J, arrows). [score:1]
Fig. 7. Foci of differentiated neurons at the cerebellar surface of 3 week old miR-17∼92c KO; Ptch1 [+/−]; Cdkn2c [+/−] mice. [score:1]
Reduced proliferation in cerebella of mice lacking the miR-17∼92 and miR-106b∼25 clusters. [score:1]
While lack of the miR-106b∼25 cluster had no obvious phenotype, loss of the miR-17∼92 cluster induced a reduction in cerebellar size and foliation. [score:1]
lacking the miR-17∼92 cluster die shortly after birth from lung and heart defects while mice lacking each of its two paralogs do not show any obvious phenotypes (Ventura et al., 2008). [score:1]
Consistent with this possibility, we observed premature exhaustion of proliferative GNPs at P14 in miR-17∼92c KO; miR-106b∼25 KO mice. [score:1]
Foci of differentiated neurons at the cerebellar surface of 3 week old miR-17∼92c KO; Ptch1 [+/−]; Cdkn2c [+/−] mice. [score:1]
To visualize PNLs, we stained the entire brains of 3 week old miR-17∼92c KO; Ptch1 [+/−], Cdkn2c [+/−] and miR-17∼92 [+/+]; Ptch1 [+/−], Cdkn2c [+/−] mice with X-gal, since a portion of the wild-type allele of Patched is replaced by LacZ in the Ptch1 [+/−] mice (Goodrich et al., 1997). [score:1]
Co-inactivation of the miR-17∼92 and miR-106b∼25 clusters reduced cerebellar size and foliation by limiting proliferation. [score:1]
Therefore, the miR-17∼92 cluster plays a critical role in GNPs during SHH-MB initiation as was shown in retinoblastoma (Nittner et al., 2012). [score:1]
GNPs purified from the cerebella of P7 miR-17∼92c KO; Ptch1 [+/−]; Cdkn2c [+/−] and miR-17∼92 [+/+]; Ptch1 [+/−]; Cdkn2c [+/−] mice were infected with retroviruses encoding Mycn and the red fluorescence protein (RFP), and stereotactically implanted 2 days later into the cortices of naïve recipient animals. [score:1]
While, as expected, 45.5% (10/22) of miR-17∼92 [+/+]; Ptch1 [+/]; Cdkn2c [+/−] mice succumbed to SHH-MBs, none (0/20) of the miR-17∼92c KO; Ptch1 [+/−], Cdkn2c [+/−] mice developed tumors over a period of 300 days (Fig.  6A). [score:1]
Fig. 3. Mice lacking the miR-17∼92 and miR-106b∼25 clusters have small brains and small cerebella. [score:1]
Principal component analysis revealed that cerebella from control mice clustered together but independently from those of miR-17∼92c KO; miR-106b∼25 KO mice for each time point (Fig.  9A). [score:1]
The miR-17∼92 cluster collaborates with the Sonic Hedgehog pathway in medulloblastoma. [score:1]
Mid-sagittal sections through the cerebellum of P14 control (A–F) and miR-17∼92c KO; miR-106b∼25 KO (G–L) mice stained with DAPI (A,C,G,I), or antibodies raised against BrdU (B,D,H,J), p27 [Kip1] (E,K) and cyclin D2 (F,L). [score:1]
Thus, the requirement for microRNAs encoded by the miR-17∼92 cluster is cell context specific. [score:1]
Fig. 2. Progressive foliation defects in cerebella of mice lacking the miR-17∼92 and miR-106b∼25 clusters. [score:1]
Mice lacking the miR-17∼92 and miR-106b∼25 clusters have small brains and small cerebella. [score:1]
Fig. 6. The mir-17∼92 cluster is required for medulloblastoma formation. [score:1]
Mouse lines carrying conditional alleles of miR-17∼92 (miR-17∼92 [floxed/ floxed]) (Ventura et al., 2008), or lacking miR-106b∼25 (miR-106b∼25 [−/−]) (Ventura et al., 2008) were generously provided by Dr Tyler Jacks (Boston, MA, USA). [score:1]
The miR-17∼92 cluster controls the number of oligodendroglial cells (Budde et al., 2010), renal tubular cells (Patel et al., 2013), neural stem cells (Bian et al., 2013) and cardiomyocytes (Chen et al., 2013). [score:1]
We also generated miR-17∼92 [floxed/ floxed]; Nestin-Cre [−]; miR-106b∼25 [−/−] (referred as miR-106b∼25 KO) and miR-17∼92 [floxed/ floxed]; Nestin-Cre [−]; miR-106b∼25 [+/+] (referred as control) littermates. [score:1]
Co- deletion of the two clusters miR-17∼92 and miR-106b∼25 led to the exhaustion of progenitor neurons in the cerebellum resulting in reduction in cerebellar size and the number of folia. [score:1]
Role of miR-17 family in the negative feedback loop of bone morphogenetic protein signaling in neuron. [score:1]
Arrows indicate 2 clusters of granule neurons out of cycle, on the surface of the cerebellum, in miR-17∼92c KO; miR-106b∼25 KO mice. [score:1]
The miR-17∼92 cluster family is composed of three members encoding microRNAs that share seed sequences. [score:1]
Germline deletion of the miR-17∼92 cluster causes skeletal and growth defects in humans. [score:1]
Cerebella from 3 week old Ptch1 [+/−]; Cdkn2c [+/−] mice wild type (A–E) or null (F–J) for the miR-17∼92 cluster were stained with H&E (A,B,F,G), an antibody against Ki67 (C,H), NeuN (D,I), and GABA(A) receptor α6 subunit (GABARa6) (E,J). [score:1]
The fact that the loss of the miR-17∼92 and miR-106b∼25 clusters attenuated proliferation of GNPs but still caused folia formation suggests that both clusters are required but not sufficient to induce the cerebellar phenotype of Mycn; Myc double -null mice. [score:1]
These results point to an absolute requirement for the miR-17∼92, but not for the miR-106b∼25, cluster in SHH-MB initiation. [score:1]
This suggested that the miR-106a∼363 cluster is unlikely to contribute to the phenotype seen in miR-17∼92c KO; miR-106b∼25 KO mice. [score:1]
Progressive foliation defects in cerebella of mice lacking the miR-17∼92 and miR-106b∼25 clusters. [score:1]
We detected PNLs in 84.5% (11/13) miR-17∼92 [+/+]; Ptch1 [+/−], Cdkn2c [+/−] mice (Fig.  6B,C,F). [score:1]
The miR-17∼92 cluster encoded by chromosome 14 in the mouse (13 in humans) has two paralogs, the miR-106b∼25 and miR-106a∼363 clusters, each of which is located on different chromosomes (Fig.  1A). [score:1]
We show that mice in which we conditionally deleted the miR-17∼92 cluster (miR-17∼92 [floxed/ floxed]; Nestin-Cre [+]) alone or together with the complete loss of the miR-106b∼25 cluster (miR-106b∼25 [−/−]) were born alive but with small brains and reduced cerebellar foliation. [score:1]
However, the miR-17∼92 and miR-106b∼25 clusters share overlapping functions since mice with combined deletion exhibit a more profound phenotype than those lacking miR-17∼92 alone (Ventura et al., 2008). [score:1]
However, cerebella of miR-17∼92c KO; miR-106b∼25 KO mice and, to a lesser extent, cerebella of miR-17∼92c KO mice, demonstrated foliation defects mainly in folia VI–VII and folia I–V (compare Fig.  2H and Fig.  2F with Fig.  2E, respectively, see arrows), while the folia in the cerebella of miR-106b∼25 KO mice appeared similar to those of control mice (compare Fig.  2G with Fig.  2E). [score:1]
Genetic dissection of the miR-17∼92 cluster of microRNAs in Myc -induced B-cell lymphomas. [score:1]
The mir-17∼92 cluster is required for medulloblastoma formation. [score:1]
To gain insights into the pathways affected by the deletion of miR-17∼92 and miR-106b∼25 clusters we performed GSEA. [score:1]
The miR-17∼92 cluster is required for medulloblastoma formation. [score:1]
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Quantitative proteome-wide expression analysis and alignment of miR-17∼19b seed regions identified multiple apoptosis regulators as being downregulated by miR-17∼19b-overexpression. [score:9]
[14] Conversely, moderate overexpression of miR-17∼92 causes a reduction in Bim and Pten expression, resulting in lymphoproliferation and autoimmune disease. [score:7]
As miR-92a expression was unchanged between BCR-ABL -positive and -negative ALL cells (Figure 1), we transduced TonB cells to overexpress miR-17∼19b, a derivative of miR-17∼92 suitable for transgenic expression. [score:7]
[18] In normal lymphopoiesis, loss of miR-17∼92 results in upregulation of Bim (Bcl2l11) and increased apoptosis, inhibiting the pro-B to pre-B transition. [score:6]
Having validated the anti-apoptotic protein BCL2 as a miR-17∼92 target, we have demonstrated the direct control of BCL2 expression by miR∼17 and miR∼18 family members by the canonical RNAi effector protein AGO2. [score:6]
To verify that Bcl2 is a direct target of miR-17∼19b miRNAs, we transfected luciferase reporter constructs containing murine Bcl2 sequences into miR-17∼19b overexpressing NIH3T3 cells. [score:6]
Efficient downregulation of BCL2 protein in SupB15 cells by lentivirally mediated overexpression of both miR-17∼19b and anti-BCL2 shRNA was further confirmed by fluorescence microscopy demonstrating co-localisation of BCL2 (red) and the mitochondrial protein COX4 (green) (Figure 5e). [score:6]
From these, BCL2 was validated as a direct target of the miR-17 and miR-18a, and BCL2 knockdown resulted in strong induction of apoptosis in BCR-ABL -positive, but not BCR-ABL -negative ALL cells. [score:5]
Inhibition of BCL2 and BCR-ABL in BCR-ABL -positive ALL cells resulted in different kinetics of cell death, turn-over of BCL2 protein and induction of miR-17∼92 expression. [score:5]
Overexpression of the miR-17∼92 derivative miR-17∼19b resulted in reduced proliferation and notably a substantial pro-apoptotic effect, with a significantly enriched subG1 population and concomitant cleavage of caspase 3. This effect is surprising, as previous studies of miR-17∼92 have shown direct regulation of ‘pro-apoptotic' molecules Bim and Pten in normal lymphopoiesis, [14] MYC -driven lymphomas 18, 20 and immunodeficiency or lymphoproliferative states. [score:5]
Anti-Bcl2 shRNA reduced Bcl2 protein expression by ∼70% – a similar level to overexpression of miR-17∼19b (∼80%) (Figure 5a). [score:5]
Having confirmed murine Bcl2 as a target of miR-17∼19b, we next analysed specific targeting of the human BCL2 transcript by the cluster. [score:5]
We next over-expressed the miR-17∼92 derivative miR-17∼19b in an inducible mo del of BCR-ABL -positive ALL, thereby identifying impaired apoptosis as a key determinant of reduced miR-17∼92 function in this disease setting. [score:5]
Expression of miR-17∼19b reduced BCL2 protein expression in BCR-ABL -positive ALL cell lines by 30–70% (Supplementary Figure 4A). [score:5]
This shows that miR-17∼19b overexpression results in increased binding of BCL2 mRNA to AGO2 demonstrating specific targeting of BCL2 mRNA by miR-17∼19b miRNAs. [score:5]
These data demonstrate that the expression of murine Bcl2 is directly regulated by miR-17∼19b through miRNA binding within the 5′-UTR. [score:5]
We found that ABT-737 treatment led to a decrease in BCL2 protein levels and an increase in miR-17∼92 miRNA expression in a BCR-ABL -dependent manner, suggesting that BCL2 inhibition results in the perturbation of a complex signalling network. [score:5]
They also suggest that inhibition of BCL2 by ABT-737 results in perturbations in wider signalling networks that leads to expression changes in both miR-17∼92 and BCL2 protein. [score:5]
[25] In this setting, expression of BCR-ABL was associated with a 2.3–3.3-fold reduction in expression of miR-17, -18a and -19a, in agreement with our findings in primary ALL samples (Supplementary Figure 1). [score:5]
Furthermore, the TonB mo del of inducible BCR-ABL expression on a murine B-lymphoid precursor background demonstrated a significant reduction in mature miR-17∼92 expression following induction of BCR-ABL, confirming the specificity of this finding for BCR-ABL -positive ALL. [score:5]
Overexpression of miR-17∼19b also led to a further increase in caspase 3 cleavage in BCR-ABL expressing cells (Figure 2c, lanes 3 and 5), but not in cells grown in the presence of IL-3 (Figure 2c, lanes 2 and 4). [score:5]
Repression of murine and human Bcl2 expression mimics miR-17∼19b overexpression. [score:5]
Given that miR-17∼19b overexpression was a driver of cell death in TonB cells in a BCR-ABL-specific manner, we hypothesised that identifying key targets of the cluster could provide novel therapeutic opportunities in BCR-ABL -positive ALL. [score:5]
miR-17∼19b suppresses expression of Bcl2. [score:5]
miR-17∼92 is downregulated in BCR-ABL -positive human ALL samples. [score:4]
miR-17∼19b targets regulators of apoptosis. [score:4]
[36] Our data suggest that downregulation of miR-17∼92 may be an important mediator of this effect. [score:4]
miRNAs predominantly affect protein expression, so we initially used a stable isotope labelling in cell culture (SILAC) -based approach to identify miR-17∼19b- and miR-20a-regulated proteins. [score:4]
To investigate whether differential miR-17∼92 expression is controlled by BCR-ABL, we used an inducible murine mo del of BCR-ABL expression. [score:3]
Furthermore, after ABT-737 but not imatinib treatment, a delayed increase in expression of miR-17∼92 encoded miRNAs (starting at approximately 24 h after addition of ABT-737, Figure 7f, Supplementary Figure 5B) has been observed. [score:3]
These data point to a direct role for miR-17∼19b encoded miRNAs in the regulation of apoptosis in BCR-ABL -positive ALL. [score:3]
miR-20a overexpression showed similar, but weaker, effects to miR-17∼19b in preliminary experiments (data not shown). [score:3]
As shown in Figure 3b, protein expression of Adseverin (Scin), Bcl2, Sialophorin (Spn), Aifm-1 (Aif), Sequestosome-1 (Sqstm1) and Shp-1 (Ptpn6) was reduced in the presence of miR-17∼19b (from 0.2- to 0.65-fold), whereas protein levels of Granzyme B (Gzmb) and DnaJB6 remained unchanged. [score:3]
To study the functional contribution of Bcl2 to the miR-17∼19b -induced phenotype, we lentivirally transduced TonB cells to express either anti-Bcl2- or control shRNA. [score:3]
To our surprise, patient samples showed significantly lower expression of mature miR-17∼92 elements in BCR-ABL -positive ALL than in either BCR-ABL -negative ALL or normal CD34+ cells. [score:3]
We further confirmed this by a complementary approach using lentiviral overexpression of antagomirs against miR-17, miR-18 and miR-20a in the human BCR-ABL -positive BV173 cell line. [score:3]
We next analysed the functional effects of miR-17∼19b overexpression in human BCR-ABL -positive ALL cell lines. [score:3]
[14] Moreover, we observed a high expression of miR-17∼92 in adult heart and in postnatal cardiomyocytes. [score:3]
Assessment of miR-17∼19b levels following transduction confirmed overexpression and AGO2 association of all three miRNAs (Figure 4b). [score:3]
Together, these results demonstrate a specific role for BCL2 in the proliferation of human BCR-ABL -positive ALL cells and suggest that the miR-17∼92 cluster suppresses BCL2 in BCR-ABL -positive cells. [score:3]
In contrast, transduction of the BCR-ABL -negative ALL cell lines REH, Nalm-6, and 697 with miR-17∼19b had no, or only minor, inhibitory effects on cell proliferation (Figure 5c, right). [score:3]
Lentiviral supernatants expressing miR-17∼19b and control vector SIEW were used to transduce ∼1 × 10 [6] 293 cells with an MOI of ∼2. [score:3]
Whereas cell cycling was only marginally affected, apoptosis was markedly enhanced by overexpression of miR-17∼19b following induction of BCR-ABL. [score:3]
29, 30, 31, 32 As shown in Figure 3a, all targets analysed have at least two miRNA -binding sites for the miR-17∼19b cluster. [score:3]
In keeping with the phenotype described for the TonB mo del, an unbiased global proteomic approach identified several apoptosis-related proteins as miR-17∼19b targets. [score:3]
18, 20 Based on our previous work in chronic myeloid leukaemia, [21] we first analysed miR-17∼92 expression in ALL and observed a significantly lower expression in ALL as compared to normal CD34+ cells with further reduction in BCR-ABL -positive as compared to -negative ALL cells. [score:3]
In contrast, BCR-ABL -mediated cell proliferation was strongly inhibited by miR-17∼19b (Figure 2a, right). [score:3]
21, 26 miRNA expression was increased between 5- and 16-fold upon transduction (miR-17 5.2-fold, miR-18a 2.1-fold, miR-19a 9-fold, miR-19b 10.6-fold, and miR-20a 15.8-fold). [score:3]
Our data indicate a selective advantage for low miR-17∼92 expression in primary BCR-ABL -positive ALL cells. [score:3]
We next analysed the presence of putative miR-17∼92 -binding sites (seed matches) within the target mRNAs. [score:3]
[14] Based on this, we analysed expression of miR-17∼92 encoded miRNAs in 14 BCR-ABL -negative and 13 BCR-ABL -positive ALL samples, as well as normal CD34+ cells, using miR-qRT-PCR. [score:3]
The polycistronic microRNA cluster miR-17∼92 encodes miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR-92-1. [13] Notably, miR-17∼92 -deficient mice suffer significant developmental cardiac defects and lung hypoplasia though interrogation of haematopoiesis identified isolated defects in B-lineage development. [score:3]
Transgenic expression of miR-17∼19b increased the subG1 fraction from 33 to 67% compared to vector controls (Figure 2b, right), whereas in BCR-ABL -negative cells it remained almost unchanged (7 and 11%, respectively, Figure 2b, left). [score:2]
To study the impact of miR-17∼19b on cell cycle regulation and apoptosis, transgenic TonB cells were analysed for DNA content in the presence and absence of BCR-ABL. [score:2]
We previously demonstrated an increased miR-17∼92 expression in chronic phase chronic myeloid leukaemia CD34+ cells, compared to normal CD34+ cells from healthy donors. [score:2]
Proliferation of TonB cells grown in the presence of IL-3 was only slightly reduced by transgenic miR-17∼19b expression as compared to controls (SIEW) (Figure 2a, left). [score:2]
[17] Conditional knockout of the cluster-revealed modulation of apoptosis as the predominant mechanism of action of miR-17∼92. [score:2]
Whereas levels of BCL2 mRNA in the input fraction were unchanged following miR-17∼19b overexpression, anti-AGO2 immunoprecipitates specifically pulled down 3.2-fold more BCL2 mRNA as compared to controls (Figure 4c). [score:2]
As we have shown upregulation of the oncomir miR-17∼92 in chronic phase chronic myeloid leukaemia, [21] we investigated the role of this cluster in BCR-ABL -positive ALL. [score:2]
In total, 84 proteins were regulated more than 1.7-fold by miR-17∼19b, with 31 exhibiting higher abundance and 53 exhibiting lower abundance in miR-17∼19b transgenic TonB cells. [score:2]
Upon lentiviral transduction, miR-17∼19b miRNA expression was increased between 3- and 12-fold as compared to controls (Supplementary Figure 2A). [score:2]
As shown in Figure 5c, lentiviral transduction of miR-17∼19b into the human BCR-ABL -positive cell lines Tom-1, BV173 and SupB15 inhibited cell proliferation by 40–55% as compared to controls (Figure 5c, left). [score:2]
Together, these data demonstrate direct and functional miRNA binding of miR-17∼19b members namely miR-17/miR-20a and miR-18a to human BCL2 mRNA. [score:2]
As shown in Figure 4a, miR-17∼19b significantly repressed luciferase activity for the wildtype but not for mutated miR-17 and miR-18a -binding sites in the murine Bcl2 5′UTR. [score:1]
Notably, six binding sites for miR-17∼19b miRNAs (three sites for miR-18a, two sites for miR-17 and one site for miR-20a) are located within the 5′UTR and CDS of murine Bcl2 (Supplementary Figure 3A). [score:1]
In human BCL2, we identified 13 binding sites for miR-17∼19b miRNAs (five sites for miR-17, six sites for miR-18a and two sites for miR-20a) located within the CDS and 3′UTR (Supplementary Figure 3B). [score:1]
TonB cells are dependent on interleukin-3 (IL-3) for survival and growth; induction of BCR-ABL allows cytokine-independent proliferation, making them an ideal system to study miR-17∼92 function in the context of BCR-ABL. [score:1]
We used qRT-PCR to investigate the association of BCL2 mRNA with AGO2, the catalytic component of RISC in miR-17∼19b overexpressing human 293 cells. [score:1]
They also strongly suggested that miR-17∼19b mediated repression of Bcl2 could contribute to the pro-apoptotic effects of the cluster in the context of BCR-ABL. [score:1]
[19] It is interesting to note that while this effect, in MYC -driven lymphoma at least, is primarily mediated by miR-19 family members (miR-19a/b), we have identified principally a miR-17 family- (miR-17, miR-20a/b, miR-106a/b and miR-93) and miR-18 family(miR-18a/b) -driven effect in BCR-ABL -positive ALL on BCL2, indicating differences in pro- and anti-apoptotic functions of miR-17∼92 between the various cellular contexts. [score:1]
TonB cells metabolically labelled with heavy, medium, or light isotope lysine and arginine versions were lentivirally transduced with miR-17∼19b, miR-20a, or a control vector (SIEW). [score:1]
These results suggested that miR-17∼92 miRNAs could have previously undiscovered anti-oncogenic functions under certain circumstances. [score:1]
miR-17∼92 has also been strongly implicated in both solid and haematopoietic malignancies. [score:1]
These data demonstrate different mechanisms of action for ABT-737 and imatinib in BCR-ABL -positive ALL cells and suggest a role for miR-17∼92 encoded miRNAs in BCL2 -mediated apoptotic pathways in these cells. [score:1]
Together, these data demonstrate that miR-17∼19b decreases cell proliferation and markedly increases apoptosis in a BCR-ABL-specific manner. [score:1]
[19] Dissection of the miR-17∼92 cluster has demonstrated that miR-19 is both necessary and sufficient to abrogate apoptosis, at least in Myc -mediated lymphomagenesis most likely by repression of PTEN and BIM. [score:1]
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[+] score: 242
Other miRNAs from this paper: hsa-mir-17
The expression of HIF-1α was up-regulated after miR-17 over-expressed whereas the expression of SDF-1, KL and EPO did not changed obviously. [score:10]
We also confirmed the up-regulation of HIF-1α in primary BMSCs after ectopic expression of miR-17 upon interaction with CB CD34 [+] cells. [score:6]
MiR-17 (also called miR- 17- 5p), an important member of the miR-17-92 cluster [22], is expressed abundantly in hematopoietic progenitors and promotes hematopoietic cell expansion by targeting sequestosome 1 (sqstm1) regulated pathways in mice [23]. [score:6]
Consistent with this data, expression of miR-17 is detected in human CD34 [+] cells and is shown to be significantly down-regulated during in vitro differentiation toward mature megakaryocytes, monocytes and monocytopoiesis [17], [24]. [score:6]
It seemed that this special environment is vital for the up-regulation of HIF-1a caused by miR-17, because HIF-1α was not changed without the existence of CB CD34 [+] cells regardless of the level of miR-17 expression (Figure 4C ). [score:6]
MiR-17 Up-regulates HIF-1α Expression upon Interaction with CB CD34 [+] CellsTo explore the mechanisms by which miR-17 promotes the function of FBMOB-hTERT in supporting hematopoiesis thus causing a specific expansion of the erythroid lineage, we examined the production of hematopoietic supporting growth factors including the hypoxia-inducible transcription factor (HIF-1α), stromal cell-derived factor (SDF-1), stem cellfactor/c-kit ligand (SCF/KL) and erythropoietin (EPO) by 17/OE and CTRL cells during interaction with CB CD34 [+] cells. [score:6]
Conversely, deficient expression of miR-17 partly inhibited the hematopoietic supporting ability of FBMOB-hTERT. [score:5]
Conversely, deficient expression of miR-17 in FBMOB-hTERT suppressed CD34 [+]CD38 [−] cell expansion (CD34 [+]CD38 [−] cells: 13.27- versus 18.45-folds). [score:5]
HIF-1α Knock Down Partially Abrogate the Hematopoietic Supporting Ability of Osteoblastic miR-17 Since ectopic miR-17 in FBMOB-hTERT cells can significantly up-regulate HIF-1α upon interaction with CB CD34 [+] cells, we examined whether or not the hematopoietic supporting ability of osteoblastic miR-17 is dependent on the augmented HIF-1α activity in FBMOB-hTERT cells. [score:5]
The full-length human pre-miR-17 expression clone was inserted into the retroviral vector, yielding the expression constructs, pCMV- pre-miR-17 (17/OE). [score:5]
MiR-17 up-regulates HIF-1α expression upon interaction with CB CD34 [+] cells. [score:5]
MiR-17 is Endogenously Expressed in FBMOB-hTERT and Primary BMSCsTo test our hypothesis that osteoblastic miR-17 may influence hematopoiesis, we first determined the miR-17 expression level in the FBMOB-hTERT and primary bone marrow stromal cells (BMSCs). [score:5]
0070232.g004 Figure 4 MiR-17 up-regulates HIF-1α expression upon interaction with CB CD34 [+] cells. [score:5]
MiR-17 Up-regulates HIF-1α Expression upon Interaction with CB CD34 [+] Cells. [score:5]
Overall, by changing the expression of hematopoietic supporting factors, ectopic expression of miR-17 in osteoblastic cells may create a suitable niche for HSC expansion, in particular the specific expansion of the erythroid lineage. [score:5]
The higher expression of miR-17 in FBMOB-hTERT cells was of interest given that such an expression was likely to have hematopoietic functional consequences. [score:5]
Conversely, miR-17 knockdown in FBMOB-hTERT suppressed the hematopoietic supporting ability of FBMOB-hTERT, in particular the mature erythroid cell growth. [score:4]
The Function of osteoblastic miR-17 on Expansion of CB CD34 [+] CellsTo analyze the function of osteoblastic miR-17 on the expansion of HSCs and HPCs, the miR-17 overexpressing and knockdown mo dels were created using FBMOB-hTERT cells by using retroviral vectors. [score:4]
Since ectopic miR-17 in FBMOB-hTERT cells can significantly up-regulate HIF-1α upon interaction with CB CD34 [+] cells, we examined whether or not the hematopoietic supporting ability of osteoblastic miR-17 is dependent on the augmented HIF-1α activity in FBMOB-hTERT cells. [score:4]
B: Real-time RT-PCR was performed to evaluate the expression level of miR-17 in FBMOB-hTERT cells after retrovirally transduced with vectors for miR-17 overexpression (17/OE), miR-17 knockdown (17/KD or 17/KD1), or control (CTRL). [score:4]
HIF-1α expression is markedly enhanced in miR-17 overexpressed FBMOB-hTERT upon interaction with CB CD34 [+] cells compared to other niche associated factors. [score:4]
To analyze the function of osteoblastic miR-17 on the expansion of HSCs and HPCs, the miR-17 overexpressing and knockdown mo dels were created using FBMOB-hTERT cells by using retroviral vectors. [score:4]
The expression of HIF-1α was significantly enhanced in miR-17 overexpressed FBMOB-hTERT upon interaction with CB CD34 [+] cells compared with other niche associated factors such as KL, SDF-1 and EPO. [score:4]
Taguchi et al [30] suggested that HIF-1α was repressed by miR-17–92 only under a normoxic condition, whereas HIF-1α was robustly induced under hypoxia regardless of the level of miR-17–92 expression [30]. [score:3]
Although only a limited number of secondary recipients have been analyzed, these data suggest that the ability of FBMOB-hTERT to maintain the multipotency of CB CD34 [+] cells in vitro was partly promoted through miR-17 over -expression. [score:3]
E: Luciferase reporter assays to check whether miR-17 directly target HIF-1α in FBMOB-hTERT. [score:3]
The expression of miR-17 in FBMOB-hTERT cells. [score:3]
To test our hypothesis that osteoblastic miR-17 may influence hematopoiesis, we first determined the miR-17 expression level in the FBMOB-hTERT and primary bone marrow stromal cells (BMSCs). [score:3]
Our study demonstrated that, in addition to regulating the cellular constituents of HSCs and HPCs, miR-17 may also participate in the regulation of hematopoietic microenvironment and be involved in intercellular communications between HSCs and their niche cells. [score:3]
1.0×10 [4] CD34 [+] CB cells were co-cultured with FBMOB-hTERT cells after transduced with vectors for miR-17 overexpression (17/OE), miR-17 knockdown (17/KD), or control (CTRL) for 5-8 weeks and then subject to colony-forming-unit (CFU) assay. [score:3]
These data suggested that the different expressions of HIF-1a in different culture environments were caused by miR-17. [score:3]
Based on shRNA influence on miR-17 expression, 17/KD was chosen for further studies. [score:3]
One of the mechanisms is likely mediated by a variety of HSC-supporting growth factors, such as HIF-1α, which are constitutively activated by overexpressed miR-17 upon interaction with CB CD34 [+] cells. [score:3]
These results suggest that ectopic miR-17 in FBMOB-hTERT augmented the expression of niche associated genes during co-cultured with CB CD34 [+] cells, which may be responsible for the hematopoietic supporting ability of osteoblastic miR-17. [score:3]
The ectopic expression of miR-17 partly promoted the ability of FBMOB-hTERT to support human CB CD34 [+] cell expansion and maintain their self-renewal and multipotency. [score:3]
Using these cells, we found that miR-17 was significantly overexpressed. [score:3]
D. The transcripts of the niche associated genes were analyzed by real-time RT-PCR in bone marrow stromal cells (BMSCs) with over-expressed miR-17 and the control cells upon interaction with CB CD34 [+] cells. [score:3]
0070232.g001 Figure 1The expression of miR-17 in FBMOB-hTERT cells. [score:3]
B: Effect of miR-17 modulation in FBMOB-hTERT cells on repopulation of CB CD34 [+] cells in non-obese diabetic/severe combined immunodeficient disease (NOD/SCID) mice. [score:3]
Compared with primary BMSCs, FBMOB-hTERT expressed a significantly higher level of miR-17 (Figure 1A ). [score:2]
In summary, our data suggested the potential contribution of miR-17 in bone marrow stem cell niches and an osteoblastic- miR-17- HIF-1α-HSC crosstalk in hematopoietic development. [score:2]
More interestingly, the specific erythroid lineage expansion of CB CD34 [+] cells caused by osteoblastic miR-17 was abrogated by HIF-1α knock down. [score:2]
HIF-1α knockdown partially abrogates the hematopoietic supporting ability of osteoblastic miR-17. [score:2]
Using the immortalized clone with the characteristics of osteoblasts, FBMOB-hTERT, in vitro expansion, long-term culture initiating cell (LTC-IC) and non-obese diabetic/severe combined immunodeficient disease (NOD/SCID) mice repopulating cell (SRC) assay revealed that the ectopic expression of miR-17 partly promoted the ability of FBMOB-hTERT to support human cord blood (CB) CD34 [+] cell expansion and maintain their multipotency. [score:2]
Real-time RT-PCR assay showed that miR-17 is endogenously expressed in these cells. [score:2]
Collectively, these examples illustrate a more general role for the autocrine production of miR-17 as a regulator of critical pathways determining normal hematopoietic cell fate and differentiation. [score:2]
Knockdown of miR-17 in FBMOB-hTERT cells, on the other hand, resulted in reduced hematopoietic support, which was followed by diminishing CFU output. [score:2]
Defining the role of miR-17 in osteoblasts on hematopoiesis raises the possibility that miR-17 may play a key part in regulating the hematopoietic niche. [score:2]
HIF-1α Knock Down Partially Abrogate the Hematopoietic Supporting Ability of Osteoblastic miR-17. [score:2]
More interestingly, selective expansion of the erythroid lineage of CB CD34 [+] cells through osteoblastic miR-17 was abrogated by HIF-1α knock down, demonstrating that HIF-1α was, at least partly, a mediator of miR-17 -induced CB CD34 [+] cell expansion in FBMOB-hTERT. [score:2]
MiR-17 is Endogenously Expressed in FBMOB-hTERT and Primary BMSCs. [score:2]
While evidence is accumulating for a crucial role of intrinsic miR-17 in regulating HSCs and HPCs, whether miR-17 signaling pathways within the hematopoietic niche, especially in osteoblasts, are also necessary in the cell-extrinsic control of hematopoiesis has not yet been examined. [score:2]
Although only a limited number of secondary recipients have been analyzed, these data suggest that the specific erythroid lineage expansion of CB CD34 [+] cells caused by osteoblastic miR-17 was abrogated by HIF-1α knock down. [score:2]
It is interesting that the number of mature erythroid (CFU-Es) from the cells co-cultured with the CTRL for 7 or 8 weeks was significantly higher than that of the cells co-cultured with HIF1α/KD, which further suggested that the specific erythroid lineage expansion of CB CD34 [+] cells caused by osteoblastic miR-17 was abrogated by HIF-1α knock down. [score:2]
While evidence is accumulating for an important role of intrinsic miR-17 in regulating HSCs and HPCs, whether miR-17 signaling pathways are also necessary in the cell-extrinsic control of hematopoiesis hereto remains poorly understood. [score:2]
These results confirmed our in vitro data and demonstrated that the specific erythroid lineage expansion of CB CD34 [+] cells caused by osteoblastic miR-17 was abrogated by HIF-1α knock down. [score:2]
0070232.g005 Figure 5HIF-1α knockdown partially abrogates the hematopoietic supporting ability of osteoblastic miR-17. [score:2]
MiR-17 is also endogenously expressed in cord blood (CB) CD34 [+] cells (data not shown), which is consistent with the previous reports [16], [23]. [score:2]
These suggested that the hematopoietic supporting ability of osteoblastic miR-17 was partially abrogated by HIF-1α knock down. [score:2]
Increased EPO by miR-17 may be a feasible explanation for the inclination of 17/OE cells to support the growth of mature erythroid cells. [score:1]
A: The expression level of miR-17 in FBMOB-hTERT cells was evaluated by real-time RT-PCR. [score:1]
In summary, in addition to intracellular miR-17, our data raised the possibility that miR-17 was also necessary in the cell-extrinsic control of HSCs and HPCs function, which is, at least in part, through the augmented HIF-1α signal pathways. [score:1]
The effect of miR-17 modulation in FBMOB-hTERT cells on CB CD34 [+] cells. [score:1]
To explore the mechanisms by which miR-17 promotes the function of FBMOB-hTERT in supporting hematopoiesis thus causing a specific expansion of the erythroid lineage, we examined the production of hematopoietic supporting growth factors including the hypoxia-inducible transcription factor (HIF-1α), stromal cell-derived factor (SDF-1), stem cellfactor/c-kit ligand (SCF/KL) and erythropoietin (EPO) by 17/OE and CTRL cells during interaction with CB CD34 [+] cells. [score:1]
We further identified that HIF-1α is responsible for, at least in part, the promoted hematopoietic supporting ability of FBMOB-hTERT caused by miR-17. [score:1]
C: Effect of miR-17 modulation in FBMOB-hTERT cells on repopulation of CB CD34 [+] cells in secondary NOD/SCID mice. [score:1]
A: The effect of miR-17 modulation in FBMOB-hTERT cells on long-term culture initiating cells activity of CB CD34 [+] cells. [score:1]
0070232.g002 Figure 2The effect of miR-17 modulation in FBMOB-hTERT cells on CB CD34 [+] cells. [score:1]
The Function of osteoblastic miR-17 on Expansion of CB CD34 [+] Cells. [score:1]
The miR-17 levels in the FBMOB-hTERT cells transduced with the indicated virus were determined using real-time RT-PCR. [score:1]
It is of interest to note that osteoblastic miR-17 seemed to be more prone to support erythroid lineage expansion because the number of mature erythroid (CFU-E) from the cells co-cultured with miR-17 modulated FBMOB-hTERT for 7 weeks was significantly changed in comparison to the cells co-cultured with CTRL cells. [score:1]
All these suggested that the ectopic miR-17 signal pathway in FBMOB-hTERT cells may create a niche which can partly promote HSC and HPC expansion and is more suitable for erythroid progenitor differentiation, which subsequently leads to more mature erythroid cells. [score:1]
The data are presented as the ratio of miR-17 levels (relative to U6) in 17/OE, FBMOB-hTERT, 17/KD or 17/KD1 to that in CTRL. [score:1]
Interestingly, the number of mature erythroid (CFU-E) from the cells co-cultured with 17/OE cells for 7 or 8 weeks was significantly higher than that of the cells co-cultured with the CTRL, whereas only after co-cultured for 8 weeks, the number of total CFU-Mix’s from the cells co-cultured with 17/OE cells was significantly higher than that of the cells co-cultured with the CTRL, suggesting that ectopic miR-17 in FBMOB-hTERT preferentially supports a specific expansion of the erythroid lineage. [score:1]
Cloning of Human pre-miR-17 Gene, the Derivative Construction and Retroviral Infection. [score:1]
The data are presented as the ratio of miR-17 levels (relative to U6) in FBMOB-hTERT to that in bone marrow stromal cells (BMSCs). [score:1]
It also seemed that osteoblastic miR-17 is prone to cause a specific expansion of the erythroid lineage. [score:1]
These data suggested that the function of osteoblastic miR-17 on HSCs and HPCs was through, at least in part, the HIF-1α signaling pathway. [score:1]
0070232.g003 Figure 3Effect of miR-17 modulation on repopulation of CD34 [+] cells in primary and secondary NOD/SCID mice. [score:1]
These results suggest that miR-17 in FBMOB-hTERT cells can promote CD34 [+]CD38 [−] cell expansion in vitro. [score:1]
It is noted that ectopic miR-17 in FBMOB-hTERT preferentially supports a specific expansion of the erythroid lineage. [score:1]
Of great interest, our experiments on ex vivo expansion, LTC-IC and SRC in vivo assay revealed that selective expansion of the erythroid lineage of CB CD34 [+] cells through osteoblastic miR-17 was abrogated by HIF-1α knock down (Figure 5 ). [score:1]
Except for the environment, the intricate and finely tuned relationship between HIF-1α and miR-17 is also likely dependent on cellular context and appears to be promoter-independent in FBMOB-hTERT. [score:1]
These results partly confirmed our in vitro data and suggest that osteoblastic miR-17 partly promotes the ability of FBMOB-hTERT to maintain the multipotency of CB CD34 [+] cells in vitro. [score:1]
The two miR-17-specific small hairpin RNAs (17/KD and 17/KD1) and HIF-1α-specific shRNA (HIF1α/KD) oligomers [29] were tested. [score:1]
On the basis of our results and previous reports, we put forth the idea that different microenvironments lead to different effects of miR-17 and that mechanism is the key point of our further research. [score:1]
We further identified that HIF-1α is responsible for, at least in part, the promoted function of ectopic miR-17 in FBMOB-hTERT on hematopoiesis. [score:1]
Although the relationship between miR-17 and HIF-1α is dependent on the environment and cellular context, our data showed a functional link between HIF-1α and miR-17, which has also been demonstrated by other research groups [30], [40]. [score:1]
All these suggested that HIF-1α is, at least partly, a mediator of CB CD34 [+] cell expansion caused by miR-17 in FBMOB-hTERT cells. [score:1]
In addition, Jin et al [34] found that miR-17 modulated the diverse effect of canonical Wnt signaling in different microenvironments [34]. [score:1]
It also seemed that osteoblastic miR-17 was prone to cause a specific expansion of the erythroid lineage. [score:1]
It seems that the ability of miR-17 in FBMOB-hTERT to promote CB CD34 [+] cell expansion requires a significant amount of time. [score:1]
Further characterization of miR-17 and other miRNAs on this field will be particularly important, not only for a better understanding of the detailed mechanisms behind HSC self-renewal and lineage commitment, but also for developing novel and efficient molecular targets to prevent and treat hematopoietic disorders. [score:1]
Our data demonstrated that CB CD34 [+] cell expansion can be partly promoted by osteoblastic miR-17, and in particular, ectopic miR-17 can cause a specific expansion of the erythroid lineage through augmenting HIF-1α in osteoblasts. [score:1]
Effect of miR-17 modulation on repopulation of CD34 [+] cells in primary and secondary NOD/SCID mice. [score:1]
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[+] score: 240
While transgenic miR-17~92 expression predominantly suppressed target gene expression at the translation level (Figure 5D), the effect of lentiviral miR-17~92 expression was target gene-specific: it suppressed the expression of Phlpp2 and Bim by translation repression, and Pten mainly through mRNA degradation (Figure 5F). [score:21]
Similar to transgenic miR-17~92 expression in B cells (Jin et al., 2013), a 2–5-fold increase in miR-17~92 expression in HeLa cells was sufficient to suppress the protein output of previously validated miR-17~92 target genes (Phlpp2, Pten, and Bim) (Figure 5E) (Ventura et al., 2008; Xiao et al., 2008; Rao et al., 2012; Jin et al., 2013; Kang et al., 2013; Xu et al., 2015). [score:9]
In stark contrast, the 4–20-fold increase in miR-17~92 expression brought about by transient transfection of low concentrations of miRNA mimics was not sufficient to suppress target gene expression (Figures 8A–C). [score:9]
Mature miR-17 expression level in naïve B cells was set as 1. Other commonly used methods to ectopically express miRNAs are through lentiviral infection, retroviral infection, plasmid transfection and transgene expression. [score:7]
On the other hand, stable transduction of HeLa cells with a miR-17~92 expressing lentivirus at MOI (multiplicity of infection) of 10–100 led to a 2–5-fold, while transient transfection of the same cells with a miR-17~92 expressing plasmid led to a 2-fold increase in the expression of these miRNAs. [score:7]
Similar to miR-17~92, miR-155 is frequently up-regulated in human B cell lymphomas (Eis et al., 2005; Rai et al., 2008), and transgenic expression of miR-155 is sufficient to drive B cell lymphoma development in mice (Costinean et al., 2006). [score:7]
Next we examined the effect of lentivirally expressed miR-17~92 on its target gene expression by. [score:7]
The percentage reduction in target gene protein levels was similar to, or even higher than, that achieved by transgenic miR-17~92 expression (Figures 5D–F), which is able to drive lymphoma development with high incidence (Jin et al., 2013). [score:6]
Figure 5 The effect of transgenic and lentiviral miR-17~92 expression on target genes. [score:5]
The maximal miR-17~92 expression level in these cell lines was about 10-fold of that found in naïve B cells (Figures 4B,C), consistent with the 2–36-fold overexpression found in biopsies from human Burkitt's lymphoma patients (Schmitz et al., 2012) (Figure 4C). [score:5]
In addition, transient transfection of both miR-17~92 and cel-mir-67 mimics led to reduction in protein levels of these target genes, though the kinetics differs for different target genes (Figures 6B,C). [score:5]
Mature miRNA expression levels in L1236 were set as 1. (C) Summary of miR-17 expression levels in samples used in this study. [score:5]
We also examined the effect of transgenic and lentiviral miR-17~92 expression on target gene mRNA levels. [score:5]
When transfected into HeLa cells at individual concentrations of 16.7 nM, they achieved a 10-20-fold (miR-92) or 200–400-fold (miR-17/20a, 18a, 19a/19b) overexpression (Figure 3), far exceeding the 3–5-fold increase that was sufficient to drive B cell lymphoma development in mice (Jin et al., 2013), as well as the 2–36-fold increase found in biopsies of human Burkitt's lymphomas, which consistently exhibit activation of the c-Myc-miR-17~92 axis (Schmitz et al., 2012). [score:4]
To directly compare the degree of overexpression achieved by this low transfection concentration with miR-17~92 overexpression in lymphomas and leukemias, we measured the endogenous miR-17~92 levels in nine human lymphoma and leukemia cell lines. [score:4]
The miR-17~92 -expressing-lentivirus (LV-miR-17~92), plasmid (Plasmid-miR-17~92), and transgene (TG) used in this study all contain the same insert, a 1007 bp fragment of human genomic DNA encoding miR-17~92 (human chr13: 91350568–91351574) (Xiao et al., 2008). [score:3]
We speculate that the mutated miR-17~92 miRNAs, together with antisense miR-17, may cause the non-specific alterations in gene expression in miRNA mimics -transfected HeLa cells (Figure 6). [score:3]
Importantly, this low concentration transfection achieved a 4–20-fold increase in miR-17~92 expression (Figure 4A). [score:3]
A biogenesis step upstream of microprocessor controls miR-17~92 expression. [score:3]
Numbers below blots indicate miR-17~92 expression normalized to U6. [score:3]
We examined miR-17~92 expression in primary B cells isolated from these mice in the presence and absence of stimulation. [score:3]
Note that non -transfected HeLa cells do not express antisense miRNAs or mutant miRNAs at levels higher than this cut off, while HeLa cells transfected with miR-17~92 mix contain many of these species. [score:3]
L1236, a human Hodgkin's lymphoma cell line that expresses 2–3-fold more miR-17~92 than HeLa cells, was used as a universal reference throughout the manuscript. [score:3]
Mature miRNA expression levels in non -transfected HeLa cells were set as 1. (B) analysis of endogenous miR-17~92 levels in nine human lymphoma or leukemia cell lines. [score:3]
To imitate miR-17~92 expression levels found in the biopsies of human Burkitt's lymphomas (2–36-fold) (Schmitz et al., 2012), we transfected an equal molar mix of miR-17, 19a, 20a, and 19b at 0.5nM for each miRNA. [score:3]
HeLa cells were seeded in 48-well plates at a density of 5 × 10 [3] cells/well on the afternoon prior to transduction with control or miR-17-92 -expressing lentiviruses (LV-Control and LV-miR-17~92, respectively). [score:3]
miR-17~92 is broadly and abundantly expressed in mammalian tissues and cell lines (Lu et al., 2005; Kuchen et al., 2010). [score:3]
We then examined the effect of transiently transfected miR-17~92 mimics on the same group of target genes. [score:3]
Targeted deletion reveals essential and overlapping functions of the miR-17~92 family of miRNA clusters. [score:3]
At 70% confluency, 0.4 μg of miR-17~92 -expressing pCNX2 plasmid (Plasmid-miR-17~92) or empty pCNX2 plasmid (Plasmid-Empty) were transfected into each well using FugeneHD (Promega #E2311) following manufacturer's instructions. [score:3]
Our previous study demonstrated that the miR-17~92 family miRNAs are expressed at similar levels in HeLa cells and in murine B cells (Xiao et al., 2008). [score:3]
As shown in Figure 5A, transgenic miR-17~92 expression led to a 2–6-fold increase in the amounts of these miRNAs. [score:3]
The following synthetic miRNA mimics were used in this study: Mimic Transfection Control with Dy547 (cel-mir-67 conjugated with Dy547), Dharmacon CP-004500-01-10 miRIDIAN microRNA Mimic Negative Control #1 (cel-mir-67), Dharmacon CN-001000-01-10 miRIDIAN Mimic hsa-miR-17, Dharmacon C-300485-05-0005 miRIDIAN Mimic hsa-miR-18a, Dharmacon C-300487-05-0005 miRIDIAN Mimic hsa-miR-19a, Dharmacon C-300488-03-0005 miRIDIAN Mimic hsa-miR-20a, Dharmacon C-300491-03-0005 miRIDIAN Mimic hsa-miR-19b, Dharmacon C-300489-03-0005 hsa-miR-92a, custom synthesized by Shanghai GenePharma miRIDIAN Mimic hsa-miR-155, Dharmacon C-300647-05-0010 Generation of miR-17~92 -expressing lentivirus was previously described (Hong et al., 2010). [score:3]
We have previously generated a conditional miR-17~92 transgene (termed miR-17~92 Tg), whose expression can be turned on in B cells by CD19-Cre, and showed that the resulting transgenic mice (TG) developed lymphomas with high penetrance (Jin et al., 2013). [score:3]
Therefore, these three methods achieved similar fold increases in miR-17~92 expression (Figures 5A–C), which were in the same range as that observed in human lymphoma and leukemia cell lines (Figure 4C). [score:3]
Importantly, transient transfection of cel-mir-67 did not alter the expression level and size of endogenous miRNAs, including miR-17, miR-18a, miR-19b, miR-92a, and miR-16 (Figure 2B). [score:3]
ML produced control and miR-17~92 -expressing lentiviruses. [score:3]
HeLa cells were transfected with empty plasmid or miR-17~92 -expressing plasmid (Plasmid-miR-17~92) and harvested at indicated time points (C). [score:3]
microRNA-17~92 is a powerful cancer driver and a therapeutic target. [score:3]
The microRNA cluster miR-17~92 promotes TFH cell differentiation and represses subset-inappropriate gene expression. [score:3]
for primary B cells, lentivirally transduced (LV-17~92) and plasmid transfected (Plasmid-17~92) HeLa cells were from Figure 5. Red and blue dashed lines indicate the maximal and mean miR-17 expression levels in human Burkitt's lymphoma patients, respectively (Schmitz et al., 2012). [score:3]
Upon transfection with 100 nM miR-17~92 mimics, the mRNA levels of all three target genes were reduced by 60~80% at 30 min post-transfection, and started to recover after 12 h (Figure 6A). [score:3]
HeLa cells were stably transduced with control or miR-17~92 expressing lentiviruses at indicated MOI (Multiplicity of infection) (B). [score:3]
miR-17~92 cooperates with RB pathway mutations to promote retinoblastoma. [score:2]
The total read counts of non -transfected HeLa cells (4.14 × 10 [6]) and miR-17~92 miRNA mimics -transfected HeLa cells (4.08 × 10 [6]) were almost identical, so that we can directly compare the read counts of individual RNA species in these two samples. [score:2]
MicroRNAs of the miR-17~92 family are critical regulators of T(FH) differentiation. [score:2]
HeLa cells were transfected with 100 nM unconjugated cel-mir-67 and analyzed by to detect cel-mir-67 (A) and endogenous miR-17, miR-18a, miR-19b, miR-92a, and miR-16 (B). [score:1]
miR-17~92 miRNAs are marked in green, and their mutant forms are marked in orange. [score:1]
Surprisingly, mutated forms of miR-17~92 miRNAs were present in high abundance in miRNA mimics -transfected HeLa cells, while completely absent in non -transfected HeLa cells. [score:1]
Interestingly, this led to accumulation of not only mature miR-17~92 miRNAs, but also high molecular weight RNA species similar to those observed for cel-mir-67 (Figures 2A, 3A), suggesting that the generation of high molecular weight RNAs is a general phenomenon associated with transient transfection of synthetic miRNA mimics. [score:1]
Figure 3 analysis of miR-17~92 miRNAs in HeLa cells transiently transfected with miR-17~92 mimics. [score:1]
SNORD29 contains sequence region highly homologous to miR-19b and is detected in both non -transfected and miR-17~92 mix -transfected HeLa cells. [score:1]
In addition, miRNA concatemers, hybrids between miR-17~92 miRNAs and other endogenous miRNAs, and polyadenylated miR-17~92 miRNAs were also identified, but their abundance was much lower than those with 5′- and 3′-end tailing (Figures 11B,C). [score:1]
1 μg total RNA from non -transfected HeLa cells and HeLa cells transfected with an equal molar mixture of miR-17, 18a, 19a, 20a, 19b, and 92a (16.7 nM each with a total transfection concentration of 100 nM) was used for Next Generation Sequencing following the standard Illumina protocol, TruSeq_SmallRNA_SamplePrep_Guide_15004197_C. [score:1]
The miRNA-17~92 cluster mediates chemoresistance and enhances tumor growth in mantle cell lymphoma via PI3K/AKT pathway activation. [score:1]
Primary B cells were purified from CD19-Cre (Control) and miR-17~92 Tg/Tg; CD19-Cre (TG) mice, stimulated in vitro for indicated amounts of time (A). [score:1]
Studies from us and other investigators estimated that miR-17~92 miRNAs are expressed at a few thousand copies per cell (Mukherji et al., 2011; Bosson et al., 2014). [score:1]
Figure 4 analysis of miRNAs in HeLa cells transiently transfected with low concentrations of miR-17~92. [score:1]
These antisense miR-17 sequences differ from miR-17 [*], the endogenous miR-17 passenger strand, by a few nucleotides (Figure 10A). [score:1]
To isolate RNA species containing a string of mature miR-17~92 or mutated miR-17~92 sequences, vcountPattern function with max. [score:1]
For this purpose, we transfected HeLa cells with a mixture of miR-17~92 miRNA mimics at 0.5 nM for each member (3 nM in total) and measured target gene mRNA and protein levels. [score:1]
Consistent with the results (Figure 3), there was a significant increase in the read counts for miR-17~92 miRNAs in miRNA mimics -transfected HeLa cells (Figure 10A). [score:1]
In experiments similar to those of cel-mir-67, we mixed the six miR-17~92 miRNAs at equal molar concentrations and transfected HeLa cells with a total concentration of 100 nM (16.7 nM for each miRNA). [score:1]
The purified B cells were cultured at 5 × 10 [6] cells/ml, and rested for 3 h before harvesting (naïve) or activated for indicated amounts of time with 2 μg/ml anti-IgM (for miR-17~92 transgenic B cells) or 25 μg/ml LPS and 5 ng/ml IL-4 (for miR-155 transgenic B cells) in 37°C cell culture chambers. [score:1]
For example, the probe mixture for the miR-17 subfamily contains probes for miR-17, miR-20a, miR-106a, miR-20b, miR-106b, and miR-93, the probe mixture for the miR-18 subfamily contains probes for miR-18a and miR-18b, the probe mixture for the miR-19 subfamily contains probes for miR-19a and miR-19b, and the probe mixture for the miR-92 subfamily contains probes for miR-92, miR-363, and miR-25. [score:1]
Taken together, transient transfection of a miR-155 mimic at 100 nM led to a few hundred fold increase in the mature miRNA level and accumulation of high molecular weight RNA species, which are similar to miR-17~92 and cel-mir-67 mimics. [score:1]
Transfection of miR-17~92-expresing plasmid was previously described (Xiao et al., 2008). [score:1]
miR-17 and miR-20a belong to the miR-17 family, while miR-19a and miR-19b belong to the miR-19 family. [score:1]
Therefore, transient transfection at individual concentrations of 16.7 nM brings the total amount of miR-17~92 miRNAs to a million copies per cell, which is similar to the estimated copy number of cel-mir-67 when transfected at 100 nM (Figure 2C). [score:1]
HeLa cells were transfected with an equal molar mixture of miR-17~92 miRNAs at 0.5 nM for each miRNA with a total concentration of 3 nM. [score:1]
On the afternoon of the following day, different amounts of LV-Control or LV-miR-17~92 lentiviruses corresponding to multiplicities of infection (MOI) 10, 20, 50, and 100 were added to cells and overnight infection was performed in the presence of 8 μg/ml polybrene. [score:1]
At this transfection concentration, the cellular levels of miR-17 and miR-19 family miRNAs reached 10-fold of endogenous levels in 30 min post-transfection, peaked around 23-fold at 3–6 h, and started to decline afterwards (Figure 4A). [score:1]
The following synthetic miRNA mimics were used in this study: Mimic Transfection Control with Dy547 (cel-mir-67 conjugated with Dy547), Dharmacon CP-004500-01-10 miRIDIAN microRNA Mimic Negative Control #1 (cel-mir-67), Dharmacon CN-001000-01-10 miRIDIAN Mimic hsa-miR-17, Dharmacon C-300485-05-0005 miRIDIAN Mimic hsa-miR-18a, Dharmacon C-300487-05-0005 miRIDIAN Mimic hsa-miR-19a, Dharmacon C-300488-03-0005 miRIDIAN Mimic hsa-miR-20a, Dharmacon C-300491-03-0005 miRIDIAN Mimic hsa-miR-19b, Dharmacon C-300489-03-0005 hsa-miR-92a, custom synthesized by Shanghai GenePharma miRIDIAN Mimic hsa-miR-155, Dharmacon C-300647-05-0010 Transfection of miR-17~92-expresing plasmid was previously described (Xiao et al., 2008). [score:1]
miRNA signals were detected with probes for individual miRNAs, or a mixture of probes detecting all members of a miRNA subfamily (i. e., miR-17 subfamily). [score:1]
The blot from Figure 3A Replicate 1 was hybridized with a probe specific for antisense miR-17. [score:1]
MiR-17~92 Tg mice were crossed with CD19-Cre mice to generate miR-17~92 Tg/Tg; CD19-Cre (TG) mice (Jin et al., 2013). [score:1]
Similar to miR-17~92 and cel-mir-67 mimic, transient transfection of miR-155 mimic also caused accumulation of high molecular weight RNA species (Figure 9A). [score:1]
Most of these long reads contain non-templated addition of nucleotides (tailing) to the 5′ or 3′ end of miR-17~92 miRNAs. [score:1]
The generation of miR-17~92 Tg (Jax stock 008517), CD19-Cre (CD19 [cre∕+]) (Jax stock 006785), and miR-155 transgenic mice was previously reported (Rickert et al., 1997; Costinean et al., 2006; Xiao et al., 2008). [score:1]
This is unexpected because cel-mir-67 bears no sequence similarity to any mammalian miRNAs including miR-17~92. [score:1]
Consistent with miR-17~92 results, there was no miR-155-specific high molecular weight RNA species in activated B cells, transgenic B cells, and lymphoma and leukemia cells (Figures 9A,B). [score:1]
miR-17~92 mix 100 nM contains an equal molar mixture of miR-17~92 miRNAs at 16.7 nM for each miRNA with a total concentration of 100 nM. [score:1]
In addition, sequences perfectly antisense to mature miR-17 (termed antisense miR-17) were found in high abundance, probably arising from the passenger strand of the miR-17 mimic. [score:1]
Antisense miR-17 sequences (marked in red) are perfectly complementary to miR-17, and differ from miR-17 [*], the endogenous form of miR-17 passenger strand, by a few nucleotides. [score:1]
MicroRNA-17~92 regulates effector and memory CD8 T-cell fates by modulating proliferation in response to infections. [score:1]
The presence of antisense miR-17 (unnatural miR-17 [*]) in transfected HeLa cells was confirmed by analysis (Figure 10B). [score:1]
Consistent with results, miR-17~92 miRNA mimics -transfected HeLa cells did contain high molecular weight RNA species harboring individual miR-17~92 miRNA sequences. [score:1]
Since miR-17~92 miRNA mimics were frequently mutated and trimmed in transfected cells, and probes used in our experiments can potentially hybridize with RNA species with imperfect complementarity, we searched the deep sequencing data for high molecular weight RNA species containing miR-17~92 miRNA sequences, allowing a few nucleotide mismatches. [score:1]
Germline deletion of the miR-17 ~ 92 cluster causes skeletal and growth defects in humans. [score:1]
Since cel-mir-67 is a C. elegans miRNA that has no homolog in mammalian species, we decided to perform the same experiments using microRNA-17~92 (miR-17~92), a miRNA cluster encoding six mature miRNAs (miR-17, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92). [score:1]
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In agreement with Wang et al. [14] we found that Rb2 is likely a target of miR-17-5p, as its expression can be stimulated by miR-17-5p inhibition but inhibited by miR-17 overexpression. [score:11]
Briefly, HFD consumption may upregulate adipogenic differentiation via increasing the expression of miR-17-5p, which downregulates Tcf7l2 expression and the Wnt signalling cascade. [score:11]
[14] We show here that Rb2 expression was also stimulated by miR-17-5p inhibition (Supplementary Figure 5A) and inhibited by miR-17 overexpression (Supplementary Figure 5B). [score:9]
As the repressive effect of exogenous TCF7L2 over -expression on can be reversed by miR-17 over -expression, it is likely that β-cat/Tcf7l2 is not the solo target of miR-17-5p in facilitating adipogenic differentiation. [score:7]
In mouse primary hepatocytes, miR-17 over -expression and miR-17-5p inhibition also generated repressive and stimulatory effect on Tcf7l2 mRNA expression, respectively (Supplementary Figure 3). [score:7]
With our experimental settings, we demonstrated the stimulatory effect of curcumin on Tcf7l2 mRNA and protein expression and β-cat S675 phosphorylation, associated with reduced miR-17-5p level and increased expression of the Wnt target Axin2. [score:7]
[34] PPRA α over -expression, however, stimulated miR-17-5p expression, indicating the existence of complicated reciprocal regulation. [score:6]
We then assessed whether the repressive effect of TCF7L2 over -expression on can be reversed by miR-17 over -expression. [score:5]
To test the effect of miR-17-5p over -expression, a stable cell selection procedure with puromycin was utilized for either control vector or the miR-17 precursor expressing vehicle was included; as the DNA transfection efficiency in this cell line was relatively low (Figure 1g). [score:5]
Figures 1k and l show that miR-17 inhibition (K) and over -expression (L) generated opposite effects on mRNA levels of five genes that encode adipogenic differentiation markers, including aP2 (adipocyte fatty acid -binding protein), C/EBP α (CCAAT/enhancer -binding protein α), C/EBP β (CCAAT/enhancer -binding protein β), Cidea (cell death-inducing DFFA-like effector a) and PPAR γ (peroxisome proliferator-activated receptor γ). [score:5]
This inhibition protocol significantly reduced the miR-17-5p expression level (Supplementary Figure 2A). [score:5]
[10] Due to a technical issue we cannot eliminate the involvement of miR-17-3p in our over -expression experiment; the inhibitor utilized in this study, however, was specific for miR-17-5p. [score:5]
Repressed miR-17-5p expression in response to curcumin treatment was associated with increased Tcf7l2 expression at mRNA and protein levels in 3T3-L1 cells (Figures 5c and d). [score:5]
It is worth to mention here that recently, Li et al. reported that in human adipocyte-derived mesenchymal stem cells, BMP2 is a direct target of miR-17-5p and miR-106a, while BMP2 knockdown repressed osteogenesis but increased adipogenesis. [score:5]
A paradoxical observation in this study is that although the repression of curcumin on 3T3-L1 differentiation was associated with repressed miR-17-5p expression and increased Tcf7l2 expression, basal Tcf7l2 level in the absence of curcumin treatment was not reduced after the differentiation. [score:5]
8, 22, 23, 24, 25 It should be emphasized here that curcumin can regulate many cellular events while miR-17-5p may have numerous downstream targets. [score:4]
We then directly tested the effect of miR-17-5p on Tcf7l2 expression in undifferentiated 3T3-L1 cells. [score:4]
Tcf7l2 is likely a direct downstream target of miR-17-5p. [score:4]
It is worth to mention that Rb2 is a previously identified target of the miR-17/92 cluster. [score:3]
Before doing so, we tested the effect of curcumin on miR-17-5p and Tcf7l2 expression in undifferentiated 3T3-L1 cells. [score:3]
Western blotting was then performed in undifferentiated 3T3-L1 cells with miR-17 over -expression or miR-17-5p repression. [score:3]
Furthermore, for each of the experiments with miR-17 over -expression, the elevation of miR-17-5p was confirmed by qRT-PCR. [score:3]
The generation of Ad-TCF7L2, Ad-TCF7L2DN and miR-17 over -expression plasmid has been previously presented. [score:3]
Repressed miR-17-5p (Figure 6a) or its precursor (Figure 6b) expression in response to curcumin treatment was appreciable at days 3 and 5 at both dosages. [score:3]
Figure 1e shows the result of our semi-quantitative analyses of ORO staining, and Figure 1f shows that miR-17 inhibition reduced the triglyceride level in differentiated 3T3-L1 cells. [score:3]
7, 8 Based on a recent study that miR-17-5p repressed another TCF member TCF7L1 in another cell lineage [13] and a previous investigation that miR-17/92 cluster over -expression stimulated, [14] we tested our hypotheses that miR-17-5p positively regulates adipogenesis via negatively regulating TCF7L2, and that curcumin can restore the Wnt activity and hence represses adipogenesis. [score:3]
How curcumin represses miR-17-5p expression remains to be explored. [score:3]
miR-17 inhibitor was the product of Shanghai GenePharma Co, Ltd (Shanghai, China). [score:3]
Figure 4g shows the correspondent alterations on adipogenic gene expression in 3T3-L1 cells infected with the control virus, or Ad-TCF7L2, or Ad-TCF7L2 and miR-17. [score:3]
In addition, the stimulatory effect of HFD on miR-17-5p expression was not observed in mouse liver (Supplementary Figures 1A–B). [score:3]
The stimulatory effect of miR-17 over -expression on miR-17-5p and its repressive effect on endogenous Tcf7l2 mRNA level were shown in Figures 4b and c, while Figure 4d shows the detection of exogenous TCF7L2 (80 kDa) and endogenous Tcf7L2 (78 and 58 kDa) in cells infected with the control virus, or Ad-TCF7L2, or Ad-TCF7L2 and miR-17 lentivirus. [score:3]
Figures 1h and i show that differentiated 3T3-L1 cells with miR-17 over -expression exhibited elevated ORO staining, comparing with cells transfected with the same amount of the control vector and underwent the same stable-cell selection procedure. [score:3]
A PPAR response element was located within the promoter region of the miR-17/92 cluster and we found previously that in the liver, PPAR α is a miR-17-5p target. [score:3]
We then tested the effect of miR-17-5p inhibition on (Figure 1c). [score:3]
These observations collectively indicate that miR-17-5p inhibition repressed. [score:3]
The repressive effect of Ad-TCF7L2 infection can be attenuated by miR-17 over -expression. [score:3]
Nevertheless, mouse visceral adipose tissue expression of miR-17-5p, but not the other cluster members, was shown to be stimulated by HFD feeding. [score:3]
miR-17 over -expression reduced the levels of the two major Tcf7l2 isoforms (78 and 58 kDa) (Figure 2g). [score:3]
miR-17 or its inhibitor transfection were performed with Lipofectamine. [score:3]
Figure 1j shows that miR-17 over -expression increased the cellular triglyceride level in differentiated 3T3-L1 cells. [score:3]
[41] It remains to be determined whether miR-17-5p plays a role in human adipogenesis, involving TCF7L2 expression and alternative splicing. [score:3]
[31] Wang et al. found that over -expression of the whole miR-17/92 cluster in 3T3-L1 cells accelerated their adipogenic differentiation, and their mechanistic exploration implicated Rb2 repression. [score:3]
As shown in Figures 1a and b, 12-week HFD feeding significantly increased the expression level of miR-17-5p, but not the other cluster members. [score:3]
Luciferase-reporter constructs were generated for testing whether this motif potentially affect miR-17-5p -mediated gene expression. [score:3]
HFD feeding also did not affect expression of other members of the miR-17/92 cluster in the mouse liver (Supplementary Figure 1C). [score:3]
miR-17-5p inhibition, however, generated the opposite effect (Figure 2h). [score:3]
The inhibition of miR-17-5p, however, resulted in a significant increase on Tcf7l2 mRNA level (Figures 2c and d). [score:3]
As shown in Figures 4e and f, the repressive effect of TCF7L2 on adipogenic differentiation was blocked with miR-17 over -expression. [score:3]
miR-17-5p positively regulates. [score:2]
We found that miR-17-5p promotes chemotherapeutic drug resistance and colon cancer metastasis, and that fatty liver development induced by dexamethasone injection in mice can be attenuated by reducing miR-17-5p levels. [score:2]
[34] miR-17 precursor can produce two functional regulatory microRNAs, miR-17-5p and miR-17-3p. [score:2]
We hence suggest that miR-17-5p positively regulates 3T3-L1 differentiation. [score:2]
Curcumin represses, associated with miR-17-5p repression and Tcf7l2 stimulation. [score:1]
A previous study demonstrated that the transfection of the whole miR-17/92 cluster accelerated. [score:1]
The miR-17 vehicle transfection increased miR-17-5p levels (Figure 2a), associated with reduced Tcf7l2 mRNA levels (Figure 2b). [score:1]
It is also necessary to point out that in response to HFD feeding, increased miR-17-5p level was observed in the white adipose tissue but not in the liver, although pathological effects of miR-17-5p in the liver have been documented. [score:1]
Further investigations are needed to determine how the repression of Tcf7l2, Rb2 and other miR-17-5p targets collectively contribute to the facilitation of adipogenesis in response to miR-17-5p elevation. [score:1]
[14] Here we located the stimulatory effect of this cluster to its first member miR-17, although we cannot eliminate the involvement of other members. [score:1]
34, 35 Recently, Jacovetti et al demonstrated that microRNAs including miR-17-5p and miR-181b play a central role in postnatal β-cell maturation. [score:1]
In addition, when rat mature adipocytes were treated with curcumin for 6 h, miR-17-5p level was also significantly repressed, associated with increased Tcf7l2 protein levels (Supplementary Figure 4). [score:1]
Curcumin, however, can reduce the miR-17-5p level, releasing its repression on Tcf7l2. [score:1]
A potential miR-17-5p binding motif was located within the mouse Tcf7l2 3'UTR (Figure 2i). [score:1]
Nevertheless, the above observations are consistent with our finding that miR-17-5p stimulates adipogenic differentiation. [score:1]
Supplementary Figure 2B shows the elevation of miR-17 level in 3T3-L1 cells after the miR-17 vehicle transfection. [score:1]
As shown in Figures 5a and b, curcumin treatment (2 μM or 10 μM for 6 h) repressed miR-17-5p precursor or mature miR-17-5p levels. [score:1]
For this purpose, 3T3-L1 cells were infected by either the control virus, or Ad-TCF7L2, or Ad-TCF7L2 and the miR-17 lentivirus (Figure 4a). [score:1]
16, 47, 48 miR-17 lentivirus construct was generated by inserting two copies of miR-17 precursor into the plv vector (Biosettia, CA, USA). [score:1]
In summary, our current study revealed the positive effect of miR-17-5p on white adipocyte differentiation. [score:1]
Here we focussed on miR-17-5p as its metabolic role has been demonstrated in several previous studies. [score:1]
34, 35 Thus, if miR-17-5p is a central switch in response to HFD consumption, adipose tissue could be a more sensitive organ. [score:1]
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Inhibition of miR-17 and miR-20a in cells overexpressing the miR-17-92 cluster can induce apoptosis, while inhibition of miR-18a and miR-19a did not have the same effect and inhibition of miR-92-1 resulted in only a modest reduction of cell growth [25]. [score:9]
CYP7A1 was confirmed as a target gene of miR-17, and the expression of CYP7A1 was found to be negatively regulated in both the transgenic mice liver cells and the miR-17 -expressing HepG2 cells. [score:8]
In our study, forced expression of mature miRNA-17 in HepG2 cells significantly reduced the expression of CYP7A1 through direct binding to the 3’UTR, which is vital for efficient activation of downstream pro-lipolytic gene expression. [score:8]
To further confirm that CYP7A1 was a target of miR-17 in humans, we generated a luciferase construct (psiCHECK-CYP7A1) harboring the binding sites of miR-17 and constructs in which miR-17 target sites were mutated (psiCHECK-CYP7A1 mut HepG2 cells were co -transfected with either CYP7A1 3’UTR luciferase constructs (psiCHECK-CYP7A1) or mutant constructs (psiCHECK-CYP7A1 mut), along with miR-17 mimics or inhibitors. [score:7]
c Upregulation of CYP7A1 by CYP7A1 expression construct transfection in miR-17 HepG2 cells. [score:6]
We developed both a miR-17 -expressing transgenic mouse mo del and a miR-17 -expressing HepG2 cell mo del, the latter was established via stable transfection. [score:5]
miR-17 also suppresses cell proliferation and invasion by targeting ETV1 in breast cancer [17]. [score:5]
When HepG2 cells were transfected with the CYP7A1 expression construct after miR-17 overexpression, a significant decrease in steatosis was observed compared with the control vector (Fig. 4d). [score:4]
miR-17 upregulates the migration and proliferation of HCC cells by activating the p38 mitogen-activated protein kinase (MAPK) pathway It also increases the phosphorylation of heat shock protein 27 (HSP27) [15]. [score:4]
CYP7A1 is a direct target of miR-17. [score:4]
Fig. 3CYP7A1 is a direct target of miR-17. [score:4]
d Luciferase activity changed in human psiCHECK-CYP7A1 and miR-17 co-transfection but not for psiCHECK-CYP7A1 mutant The targeting between CYP7A1 and miR-17 is conserved between mouse and human mo dels (Additional file 3: Figure S2). [score:3]
ating showed CYP7A1 expression was repressed in the liver of miR-17 transgenic mice. [score:3]
All data are shown as means ± S. D. Transgenic mice expressing miR-17 were generated as described previously [21]. [score:3]
miR-17 was significantly upregulated in hepatocellular carcinoma samples compared with its level in paracarcinomatous liver tissues. [score:3]
c Protein expression of CYP7A1 was decreased in miR-17 HepG2 cells. [score:3]
Expression of miR-17 was higher in transgenic mice than in wild-type mice (Additional file  1: Figure S1) and stable in the transgenic mouse mo del (Additional file  2: Figure S4). [score:3]
Transgenic mice expressing miR-17 were generated as described previously [21]. [score:3]
miR-17 expression level was stable in miR-17 transgenic mice. [score:3]
d Re-introduction of CYP7A1 into miR-17 HepG2 cells could partially alleviate steatosisTo further confirm that miR-17 targeting of CYP7A1 can affect cell steatosis, rescue experiments were performed. [score:3]
A cDNA sequence containing two pre-miR-17 units was inserted into the mammalian expression vector pEGFP-N1 in the restriction enzyme sites BglII and HindIII. [score:3]
To fully exploit how single members of the cluster can affect physiologic and pathologic processes, we produced a transgenic mouse mo del that exhibits stable overexpression of miR-17. [score:3]
and western blot were applied to measure the expression levels of miR-17 and the potential target gene CYP7A1. [score:3]
There was significant decrease in the mRNA (Fig. 3b) and protein expressions (Fig. 3c) in the miR-17 transfected cells. [score:3]
CYP7A1 expression level was stable in miR-17 transgenic mice. [score:3]
This plasmid was expected to simultaneously express miR-17 and green fluorescent protein (GFP). [score:3]
To examine how the expression of miR-17 can affect the metabolism of mouse tissue, organs from transgenic mice were examined and extensive steatotic changes were found in the liver. [score:3]
CYP7A1 was found to participate in miR-17 -induced steatosis, as its repressed expression in miR-17 HepG2 cells exacerbated steatotic change. [score:3]
B – CYP7A1 is predicted to be a target of miR-17 in mice. [score:3]
A – CYP7A1 is predicted to be a target of miR-17 in humans. [score:3]
b Expression of CYP7A1 mRNA decreased in miR-17 HepG2 cells. [score:3]
The miR-17 overexpression was confirmed using real time-PCR (Fig.   2a). [score:3]
“ctrl” represent cell groups without oleic acid treatment, while “oil” represent cell groups with oleic acid treatment Bioinformatic analysis indicated that miR-17 potentially targets many mRNAs. [score:3]
d Re-introduction of CYP7A1 into miR-17 HepG2 cells could partially alleviate steatosis To further confirm that miR-17 targeting of CYP7A1 can affect cell steatosis, rescue experiments were performed. [score:3]
a miR-17 overexpression in HepG2 cells stably transfected with miR-17. [score:3]
A fragment of cDNA harboring four copies of pre-miR-17 was generated to enhance miR-17 expression. [score:3]
We confirmed the effect of miR-17 on CYP7A1 expression in the HepG2 cell line. [score:3]
At the molecular level, the liver of transgenic mice revealed an overexpression of miR-17 that was accompanied by a strong repression of the lipid metabolism-related protein CYP7A1. [score:3]
“GFP” (green fluorescent protein) represent native control plasmid which didn’t express miR-17. [score:3]
d Luciferase activity changed in human psiCHECK-CYP7A1 and miR-17 co-transfection but not for psiCHECK-CYP7A1 mutantThe targeting between CYP7A1 and miR-17 is conserved between mouse and human mo dels (Additional file 3: Figure S2). [score:3]
b Quantitative analysis of liver triglyceride shows that the transgenic mouse liver exhibits significantly higher levels of triglyceride than the wild-type mouse liver (n = 10 for each group) To further analyze the role of miR-17 in inducing steatosis in liver, HepG2 cells were stably transfected with an miR-17 -expressing construct or GFP construct. [score:3]
Circulating miR-17 was found to be higher in patients with coronary artery disease and to be associated with the blood lipid levels in those patients [20]. [score:3]
Mechanistically, miR-17 acts as a novel inhibitor of CYP7A1 signaling in the hepatocyte and holds clinical promise as a therapeutic molecule for NAFLD. [score:3]
The cultures were maintained at 37 °C for 24 h, and then the luciferase reporter constructs psiCHECK-CYP7A1 or psiCHECK-CYP7A1 mut were co -transfected with miR-17 mimics, inhibitors or mock miRNAs (Shenggong) using Lipofectamine 2000. [score:3]
We observed that expression of miR-17 retards early-stage tissue growth in these transgenic mice. [score:3]
Mutation of the binding sites reversed this repressive effect of miR-17 (Fig. 3d). [score:2]
Expression of miR-17 increases in transgenic mice compared with wild-type mice. [score:2]
HepG2 cells stably transfected with miR-17 were transiently transfected with CYP7A1 expression constructs and a control vector for the steatosis assay. [score:2]
Development of fatty liver in miR-17 transgenic mice. [score:2]
MiRNA-17 Steatosis Fatty liver CYP7A1 Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of metabolic syndrome -associated liver pathologies progressing from simple steatosis through nonalcoholic steatohepatitis (NASH) and fibrosis to cirrhosis and hepatocellular carcinoma (HCC) [1]. [score:2]
It was found to be repressed in the miR-17 transgenic mice compared with its expression in wild-type mice (Fig.   3a). [score:2]
miR-17 is a novel regulator of CYP7A1 signaling in hepatic lipid metabolism, suggesting a potential therapeutic approach for fatty liver. [score:2]
However, those tumors did not arise on the cirrhotic background that is typical of most human HCCs, suggesting that miR-17 can directly cause hepatocellular carcinoma through pathways that are independent of NAFLD. [score:2]
Our results above indicate that miR-17 induced the development of fatty liver and these effects could partially be attributed to the mediation of CYP7A1. [score:2]
This further confirmed that the CYP7A1 -mediated pathway is essential for miR-17 to affect HepG2 cell steatosis. [score:1]
The resulting miR-17 construct was digested with ApaI and StuI to release the miR-17 transgene followed by microinjection into the oviducts of pseudo-pregnant recipient females using a standard protocol approved by the Animal Care and Use Committee of Fujian Medical University. [score:1]
MiR-17 regulates the steatosis level in HepG2 cell. [score:1]
Generation and genotyping of miR-17 transgenic mice. [score:1]
The luciferase assay was used to confirm direct binding of miR-17 and CYP7A1. [score:1]
Re-introduction of CYP7A1 into miR-17 HepG2 cell partially alleviated steatosis. [score:1]
This study provides the first evidence that miR-17 has a functional role in liver steatosis. [score:1]
b Transfection of miR-17 enhanced oleic acid (OA) uptake. [score:1]
Confirmation that CYP7A1 can mediate miR-17 -induced steatosis. [score:1]
In a previous study, an miRNA-17 transgenic mouse mo del was established [21]. [score:1]
Transfection with miR-17 enhanced oleic acid uptake (Fig. 2b and c). [score:1]
As observed in the liver of some miR-17 transgenic mice, steatosis was accompanied by HCC [21], indicated that steatosis may be a mechanism in the progression of miR-17 -mediated HCC. [score:1]
Besides its critical role in the progress of hepatic carcinoma, miR-17 is also involved in some other pathophysiologic processes. [score:1]
Then we treated miR-17- or GFP -transfected HepG2 cells with oleic acid followed by Oil-Red-O staining to reveal steatosis. [score:1]
Using western blot and real time-PCR, the protein and mRNA levels of CYP7A1 were analyzed in HepG2 cells stably transfected with miR-17 or GFP. [score:1]
This study aimed to explore whether miR-17, one of the most functional miRNAs in the miR-17-92 family, participates in the process of steatosis in hepatoma cells. [score:1]
Burton B. Yang of the Sunnybrook Science Center for generously donating the miR-17 transgenic mice. [score:1]
Luciferase activity changed in mouse psiCHECK-CYP7A1 and miR-17 co-transfection but not for psiCHECK-CYP7A1 mutant. [score:1]
We hypothesize that miR-17 may play an important role in energy homeostasis and lipid metabolism in hepatocytes and thus lead to steatosis and fatty liver. [score:1]
To serve as a negative control, we synthesized the 3’UTR of CYP7A1 with the miR-17 binding sites replaced by a random nucleotide sequence. [score:1]
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[+] score: 202
Stable ectopic expression of RUNX1-MTG8, CBFB-MYH11, or miR-17 in U937 cells (a representative U937 clone is shown for each construct) leads to downregulation of miR-193a, a RUNX1-regulated miRNA targeting KIT (left), and miR-27a, a RUNX1-regulated miRNA involved in myeloid differentiation (right). [score:10]
Additional miRNAs regulated by RUNX1 showing upregulation in non-CBF-AML (A) In addition to miR-17, Targetscan analysis identifies several other miRNAs targeting RUNX1-3′UTR. [score:9]
By examining published miRNA expression datasets from AML patients [24, 25], we observed that miR-17 is upregulated in approximately 60% of non-CBF-AML cases, while it is mostly downregulated in CBF-AML cases (Additional file 1: Figure S1B, top). [score:9]
For instance, in silico analysis of published miRNA expression datasets allowed us to highlight several miRNAs that could potentially target RUNX1-3′UTR and that, like miR-17, are upregulated in non-CBF-AML. [score:8]
Evidence of concomitant upregulation of KIT and miR-17 in non-CBF-AML (A) Scheme showing the putative miRNA-target sites in the RUNX1-3′UTR, as predicted by TargetScan analysis. [score:8]
In the first part of this study, we show that ectopic expression of miR-17, which controls RUNX1 level by targeting RUNX1-3′UTR [22], in human U937 cells leads to deregulation of a core RUNX1-regulated miRNA mechanism that is similarly affected by the t(8;21) RUNX1-MTG8 and inv(16) CBFB-MYH11 fusion proteins. [score:7]
In the first part of this study we show that KIT expression can be also upregulated by miR-17, a regulator of RUNX1, the gene encoding a CBF subunit. [score:7]
Stable expression of miR-17 downregulates RUNX1-regulated miRNAs of myeloid differentiation. [score:7]
Human (U937) or mouse (32D) myeloid clonal lines were used, respectively, to test: 1) the effect of RUNX1-MTG8 and CBFB-MYH11 fusion proteins, or upregulation of miR-17, on KIT -induced proliferation and myeloid differentiation, and 2) the effect of upregulation of KIT -induced proliferation per se on myeloid cell differentiation. [score:7]
Of note, both miR-17 and CBF-AML fusion proteins can affect other RUNX1-regulated miRNAs targeting KIT-3′UTR (see TargetScan analysis in Additional file 3: Table S2). [score:6]
In the case of miR-17 overexpression, which is mainly associated with the M5 FAB subtype, the phenotype could be due to deregulation of other miR-17 targets, besides RUNX1. [score:6]
Among these, we focused on miR-17-5p (hereafter simply referred to as miR-17), a miRNA that targets RUNX1-3′UTR, plays a key role in RUNX1 -mediated control of myeloid differentiation [22], and is often upregulated in leukemia [24]. [score:6]
Based on these preliminary observations, we set out to assess the effects of stable ectopic miR-17 expression in U937 cells on both miR-221 and KIT expression. [score:5]
Samples were analyzed for KIT (CD117) expression by flow cytometry and for miR-17 expression by qRT-PCR. [score:5]
Consistently, the U937 [miR-17], U937 [RUNX1-MTG8] and U937 [CBFB-MYH11] clones also displayed significant downregulation of RUNX1-regulated miRNAs involved in myeloid differentiation, such as miR-223 (Figure  3C) and miR-27a (Additional file 1: Figure S2, right) [13, 26]. [score:5]
Interestingly, miR-17 upregulation is mostly associated with the FAB M5 subtype (Additional file 1: Figure S1B, bottom), which is frequently characterized by KIT upregulation [18]. [score:5]
Additional miRNAs regulated by RUNX1 and deregulated by ectopic expression of CBF-AML fusion proteins or miR-17. [score:5]
DNA sequencing of RUNX1 exons excluded the presence of mutations in those samples showing upregulation of both KIT and miR-17. [score:5]
In this study we focused on miR-17, a miRNA that, by targeting RUNX1-3′UTR, plays a key role in the control of RUNX1 expression and myeloid differentiation [22]. [score:5]
Altogether, these findings show that ectopic miR-17 expression deregulates the same RUNX1-miR-221-KIT axis, which is also deregulated by CBF-AML fusion proteins (Figure  2G). [score:5]
Consistent with these observations, we found evidence of concomitant KIT (CD117) and miR-17 upregulation in three out of 10 non-CBF-AML patient samples analyzed in our laboratories (Additional file 1: Figure S1C). [score:4]
Figure 2 Stable expression of miR-17 deregulates the same core RUNX1-miR221-KIT axis affected by CBF-AML fusion proteins. [score:4]
Stable expression of miR-17 deregulates the same core RUNX1-miR-221-KIT axis affected by CBF-AML fusion proteins. [score:4]
For instance, as shown in Additional file 1: Figure S2, left, miR-193a is significantly downregulated in U937 [miR-17], U937 [RUNX1-MTG8], and U937 [CBFB-MYH11] clones. [score:4]
Notably, acute myeloid leukemia with t(8;21), inv(16), or upregulation of miR-17 fall into FAB subtypes with distinct phenotypic features. [score:4]
In the first part of this study we found that stable miR-17 upregulation affects, like the CBF-AML fusion proteins (RUNX1-MTG8 or CBFB-MYH11), a core RUNX1-miRNA mechanism leading to KIT -induced proliferation of differentiation-arrested U937 myeloid cells. [score:4]
The expression of miR-17, miR-18a, miR-20a, miR-93, and miR-181 in was evaluated from published gene expression datasets [24, 25]. [score:3]
Interestingly, both CBF leukemia fusion proteins and miR-17, which targets RUNX1-3′UTR, negatively affect a common core RUNX1-miRNA mechanism that forces myeloid cells into an undifferentiated, KIT -induced, proliferating state. [score:3]
Apparently, in the U937 cell context miR-17 ectopic expression significantly reduced miR-221 level, thus recapitulating the effect of RUNX1-MTG8 and CBFB-MYH11 (Figure  2D, left). [score:3]
Cytofluorimetric analysis shows GFP expression in representative U937 [miR-17] and U937 [Scram] clones (right). [score:3]
To develop U937 clones stably expressing ectopic miR-17 or cognate control clones, cells were transfected with pEZX-MR04 plasmid (GeneCopoeia, Rockville, MD) containing either the miR-17 precursor or a scrambled sequence, respectively. [score:3]
Shown here one representative clone out of 3 clones stably expressing RUNX1-MTG8, CBFB-MYH11, or miR-17. [score:3]
Clones with decreased luciferase expression (a prototypic clone out of three is shown in Figure  2B, bottom) were bona fide miR-17 -positive clones. [score:3]
We found that the effects of ectopic miR-17 expression mimic the biological effects induced by the RUNX1-MTG8 and CBFB-MYH11 fusion proteins by affecting the same core mechanism: the RUNX1-miR-221-KIT axis and miR-223. [score:3]
Next, we selected stable U937 [miR-17] and U937 [Scram] clones positive for GFP expression (Figure  2A, right) and transfected them with a Luc-RUNX1-3′UTR reporter carrying the luciferase sequence upstream of RUNX1-3′UTR (Figure  2B, top). [score:3]
To this end, we stably transfected U937 cells with a plasmid co -expressing a GFP tracking insert adjacent to either the miR-17 precursor or a scrambled control sequence (Figure  2A, left). [score:3]
Notably, miR-17 is only one of many miRNAs targeting RUNX1-3′UTR (Additional file 2: Table S1). [score:3]
Stable ectopic expression of wild type KIT in the U937 context (U937 [KIT]) at a level comparable (5-10%) to the one detected in U937 [miR-17], U937 [RUNX1-MTG8], and U937 [CBFB-MYH11] clones (Figure  2F, left) was per se sufficient to increase EdU-proliferation (Figure  2F, right). [score:3]
Scheme showing that miR-17 and the RUNX1-MTG8 and CBFB-MYH11 fusion proteins interfere with the same core RUNX1-miRNA mechanism that regulates KIT -mediated proliferation and myeloid differentiation. [score:2]
In addition to CBF-AML fusion proteins and miR-17, other factors could deregulate RUNX1 function or level. [score:2]
The overall findings of the first part of this study (schematically summarized in Figure  4) let us conclude that miR-17 deregulates a core RUNX1-miRNA mechanism of CBF-AML pathogenesis, since it recapitulates the same effects of both RUNX1-MTG8 and CBFB-MYH11 fusion proteins. [score:2]
Figure 4 MiR-17 deregulates a core RUNX1-miRNA mechanism of CBF-AML pathogenesis. [score:2]
Specifically, 52 non-CBF-AML and 31 CBF-AML were analyzed for miR-17, 31 non-CBF-AML and 18 CBF-AML were analyzed for miR-18a, 53 non-CBF-AML and 34 CBF-AML were analyzed for miR-20a, 34 non-CBF-AML and 18 CBF-AML were analyzed for miR-93 and miR-181. [score:1]
U937 [miR-17] clones (Figure  3A, right), similar to U937 [RUNX1-MTG8] and U937 [CBFB-MYH11] clones (Figure  3B, middle and right), displayed a decrease of CD11b -positive cells in response to PMA, thus indicating myeloid differentiation arrest. [score:1]
On the one hand, miRNAs (e. g. miR-17) can mimic the effects of CBF-AML fusion proteins by affecting a core RUNX1-miRNA mechanism of KIT -induced proliferation of undifferentiated myeloid cells. [score:1]
U937 [miR-17] clones showed a higher proportion of KIT (CD117) -positive cells (Figure  2C, left) as well as increased EdU-proliferation (Figure  2C, right) relative to control U937 [Scram] clones. [score:1]
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[+] score: 195
Fontana et al. reported that in therapy-resistant neuroblastoma, an increased miR-17-5p expression causes the downregulation of p21 and the tumor suppressor gene Bcl-2-interacting mediator (BIM) of cell death, which could be antagonized by miR-17-5p AS ODN, representing a novel oncogenic pathway to explain neuroblastoma progression and its resistance to therapy [25]. [score:8]
To confirm the association of c-Myc with the miR-17 family and p21, Wang et al. antagonized the expression of each miR-17 family member with a specific AS ODN in transfectants constitutively overexpressing c-Myc, in the presence of c-Myc, and showed that this treatment strongly restored p21 expression. [score:7]
Quantitative results of the apoptosis array revealed that miR-17-5p downregulated the expression of p21, p53, TNF RI, and FADD. [score:6]
In the present work, miR-17-5p downregulated the expression of p21, p-p53 (phosphorylated at S15, S46, and S392), TNF RI, and FADD, which might play roles in the radiosensitivity of irradiated tumor cells. [score:6]
However, miR-17-5p upregulated the expressions of cIAP-1, HIF-1α, and TRAIL R1. [score:6]
MicroRNA-17-5p -downregulated the apoptotic proteins of p21, p53, TNF RI, and FADD, and upregulated the apoptotic proteins of cIAP-1, HIF-1α, and TRAIL R1 in irradiated OC3 cells. [score:6]
Our study also revealed that miR-17-5p upregulates the expression of TRAIL R1, which might confer higher resistance to RT in betel nut chewing-related oral cancer. [score:6]
MiR-17-5p -downregulated (left panel) or -upregulated (right panel) proteins are indicated. [score:6]
We demonstrated that miR-17-5p decreased the expression of the cyclin -dependent kinase inhibitor p21 in the OC3 cells following irradiation. [score:5]
The results revealed that irradiation -induced miR-17-5p expression was significantly inhibited by miR-17-5p AS ODN but not by control ODN in irradiated OC3 cells. [score:5]
A. The OC3 cells without or with a p53 -overexpressing clone or p53 over -expression clone that treated with miR-17-5p AS ODN were irradiated with 5 Gy; after 48 h, the cell cycle was determined through propidium iodide staining and flow cytometry. [score:5]
Furthermore, the inhibition effect of miR-17-5p AS ODN on irradiation -induced miR-17-5p expression in the OC3 cells was verified through real-time PCR (Figure 1A). [score:5]
We showed that irradiation induced miR-17-5p expression and played a role in suppressing apoptosis. [score:5]
Our previous study revealed that miR-17-5p, a miR-17-92 polycistronic miR, is enhanced in the irradiated OC3 cancer cell line, and miR-17-5p was also observed to inhibit the downstream p21 expression and reduce radiosensitivity [16]. [score:5]
That study also revealed that translation inhibition mediated by the miR-17/Oncomir-1 miR polycistron is associated with an apoptotic response. [score:5]
Figure 4 A. The OC3 cells without or with a p53 -overexpressing clone or p53 over -expression clone that treated with miR-17-5p AS ODN were irradiated with 5 Gy; after 48 h, the cell cycle was determined through propidium iodide staining and flow cytometry. [score:5]
Furthermore, the stable overexpression of c-Myc elevated the expression of some members of the miR-17 family and of their primary transcripts. [score:5]
Wang et al. studied the mechanism underlying p21 suppression by the miR-17 family and observed that c-Myc expression was associated with miR-17 family members. [score:5]
Because miR-17-5p downregulated p-p53 (phosphorylated at S15, S46, and S392) in the OC3 cells, we further determined the effect of p53 on irradiated OC3 cells. [score:4]
The results revealed that the miR-17-5p -induced downregulation of p53 plays a critical role in OC3 radiosensitivity. [score:4]
The results revealed that the effect of miR-17-5p on the expression of p53 contributed to the modulation of the radiosensitivity of irradiated OC3 cells. [score:3]
MicroRNA-17-5p-regulated apoptosis-related protein expression in irradiated OC3 cells. [score:3]
These results suggest that c-Myc further repressed p21 expression at the post-transcriptional level in some members of the miR-17 family [45]. [score:3]
Moreover, we found that the effects of the miR-17-5p AS ODN on OC3 cells potentially reduced the expression of these radio-resistant proteins to overcome therapeutic resistance. [score:3]
Thus, we could use miR-17-5p AS ODN for multiple targets to enhance therapeutic effects. [score:3]
MiR-19, which is a component of the miR-17/Oncomir-1 miR polycistron, interferes with the expression of the antiapoptotic Ras homolog B (rhoB). [score:3]
A. Six hours later, the expression of miR-17-5p was determined through real-time PCR. [score:3]
MiR-17-5p-regulated protein expressions in OC3 cells. [score:3]
The expression level of miR-17-5p was 5.34 ± 0.51 folds in the control ODN group and 1.3 ± 0.2 folds in the miR-17-5p AS ODN group (P < 0.05). [score:3]
Our data might be the first to reveal that a high cIAP1 expression stimulated by miR-17-5p results in radiation resistance. [score:3]
Therefore, we examined radiation -induced changes in miR-17 expression and its function in oral carcinoma 3 (OC3) cells, an oral carcinoma cell line that was established from a 57-year-old Taiwanese patient having oral squamous cell carcinoma; this patient was a long-term betel nut chewer who did not smoke [15]. [score:3]
In conclusion, our results reveal that miR-17-5p plays a crucial role in the expression of apoptotic proteins, namely p21, p53, TNF RI, FADD, cIAP-1, HIF-1α, and TRAIL R1, in irradiated OC3 cells. [score:3]
Images of the apoptosis array (Figure 1) revealed that apoptosis-related proteins, namely p21, p53 (phosphorylated at S15, S46, or S392), TNF RI, FADD, cIAP-1, HIF-1α, and TRAIL R1, exhibited different expression levels in the irradiated OC3 cells pretreated with miR-17-5p AS ODN and the cells treated with control ODN (Figure 1B). [score:3]
While treated with mir-17-5p AS ODN to irradiated p53 expressing cells, the effect of p53 on cell cycle arrest was significantly enhanced (Figure 4). [score:3]
The IHC of p53 also revealed the p53 was abounding expressed in the miR-17-5p antisense ODN plus irradiation group (group 4) but not in other three groups (Figure 5B). [score:3]
Effects of miR-17-5p AS ODN on the expression of OC3 cell apoptosis-related proteins. [score:3]
In our study, we transfected the OC3 cells with miR-17-5p to verify its biological role; our results also showed G [2]/M arrest in response to radiation, which resulted in the inhibition of radiation -induced apoptosis. [score:3]
In our study, effects of the miR-17-5p AS ODN on OC3 cells potentially enhanced the expression of apoptosis-related proteins to achieve radiosensitivity. [score:3]
To determine the effects of miR-17-5p on OC3 cell apoptosis related-protein expression, the OC3 cells were pretreated with the miR-17-5p AS ODN or control ODN for 48 h before irradiation with 5 Gy. [score:3]
MiR-17-5p AS ODN therapy enhanced p53 expression and radiosensitivity of OC3 cell tumor growth in vivo. [score:2]
In this study, we used miR-17-5p AS ODN in vivo; AS ODN may be a powerful tool for clinical use. [score:1]
The results are presented as ratios of miR-17-5p AS ODN treatment versus the control ODN treatment; we defined ratios of >1.5 or <0.6 as a significant change. [score:1]
These results indicated that miR-17-5p increased or decreased apoptosis-related proteins, namely p21, p53, TNF RI, FADD, cIAP-1, HIF-1α, and TRAIL R1 in the OC3 cells. [score:1]
The role of the miR-17 polycistron in response to RT in oral cancer remains unclear. [score:1]
Figure 3 The OC3 cells were pretreated with the miR-17-5p AS ODN or control ODN for 48 h. After 24 h, the total cell lysates were collected for. [score:1]
Our previous study revealed that miR-17-5p could be induced in irradiated OC3 cells [16]. [score:1]
The mice were treated with miR-17-5p AS ODN or control ODN on day 7; on day 8, the mice in groups 2–4 were processed for tumor irradiation (10 Gy). [score:1]
After tumor formation on day 7, mice were divided into four groups, including sham group (group 1), irradiated alone group (group 2), control ODN plus irradiation group (group 3), and miR-17-5p antisense ODN plus irradiation group (group 4). [score:1]
Our finding from animal study revealed that microRNA-17-5p AS ODN therapy enhanced the radiosensitivity of OC-3 tumor growth, suggesting that miR-17-5p AS ODN therapy may be a strategy for betel nut chewing -associated oral cancer. [score:1]
In this study, we used microRNA-17-5p (miR-17-5p) antisense (AS) oligonucleotides (ODN) and a human apoptosis protein array to identify apoptosis-related proteins that are increased or decreased by miR-17-5p. [score:1]
These results also support that miR-17-5p AS ODN therapy may be a strategy for betel nut chewing -associated oral cancer. [score:1]
MiR-17 encodes seven mature miRs, and several studies have revealed their involvement in cancer [20, 21]. [score:1]
MicroRNA-17-5p antisense ODN therapy enhanced the radiosensitivity of OC3 tumor growth in vivoTo determine the in vivo therapeutic effect of miR-17-5p AS ODN, we used an OC3 xenograft mo del and SCID mice to determine the effect of miR-17-5p AS ODN on tumor irradiation. [score:1]
A 5′-ACUACCUGCACUGUAAGCACUUUG-3′ 2′-O-methyl oligonucleotide (Dharmacon, USA) was used for miR-17-5p antisense oligonucleotide. [score:1]
The sham group (group 1); irradiation-alone group (group 2); control ODN plus irradiation group (group 3), and miR-17-5p AS ODN plus irradiation group (group 4). [score:1]
Mice were treated with miR-17-5p antisense ODN or control ODN on day 7 and 8, mice in groups 2 to 4 were processed for tumor irradiation (10 Gy). [score:1]
Quantitative results of apoptosis protein arrays from miR-17-5p AS ODN -treated OC3 cells. [score:1]
Quantitative detection of miR-17-5p. [score:1]
The effect of mir-17-5p AS ODN on cell cycle arrest in irradiated p53 expressing cells was also evaluated. [score:1]
The OC3 cells were pretreated with the miR-17-5p AS ODN or control ODN for 48 h. After 24 h, the total cell lysates were collected for. [score:1]
Figure 1The OC3 cells were pretreated with miR-17-5p AS ODN or control ODN for 48 h, followed by irradiation with 5 Gy. [score:1]
After tumor formation on day 7, 10 mice each were divided into the sham group (group 1), irradiation-alone group (group 2), control ODN plus irradiation group (group 3), and miR-17-5p AS ODN plus irradiation group (group 4). [score:1]
The OC3 cells were pretreated with miR-17-5p AS ODN or control ODN for 48 h, followed by irradiation with 5 Gy. [score:1]
Quantitative results of apoptosis protein arrays, as described in Figure 1. The figure shows the ratios of the miR-17-5p AS ODN treatment versus the control ODN treatment (n = 3). [score:1]
To determine the in vivo therapeutic effect of miR-17-5p AS ODN, we used an OC3 xenograft mo del and SCID mice to determine the effect of miR-17-5p AS ODN on tumor irradiation. [score:1]
Figure 2Quantitative results of apoptosis protein arrays, as described in Figure 1. The figure shows the ratios of the miR-17-5p AS ODN treatment versus the control ODN treatment (n = 3). [score:1]
These findings would be useful for designing therapeutic strategies, in which RT and miR-17-5p -based gene therapy can be co-administered. [score:1]
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xlsx”): Column 1: predicted target gene list used for GSEA; Column 2: subset list of predicted target genes present on microaarray; Column 3: leading edge subset of genes that were found to be either up or downregulated by comparing −H−D vs +H+D (normalized p value indicated on the top of the column); Column 4: leading edge subset gene list that were found to be either up or downregulated by comparing −H−D vs +H+D (normalized p value indicated on the top of the column); Column 5: intersection between leading edge gene lists in columns 3 and 4. Lists of leading edge common targets for miR-17 and miR-20 (intersection of the four gene lists in columns 3 and 4 on sheets miR-17 and miR-20), miR-19a and miR19b (intersection of the four gene lists in columns 3 and 4 on sheets miR-19a and miR-19b) as well as for miR-451 (intersection of gene lists in columns 3 and 4 in sheet miR-451) that have been used to generate the histograms presented in Figure 3B, C and D are given in sheet named «Gene list profile». [score:13]
During this study, we found that several other known miR-17-92 targets such as PTEN (miR-19 and miR-17/20a target) [24], TxNIP/VDUP (miR-17/20a target) [44] E2F1 (miR-17/20a) [45], [46] and P21 (miR-17/20a target) [45], [47] were surprisingly not regulated in response to miR-17-92 expression variations in NN10#5 and 745A#44 cells. [score:12]
Up-regulation of miR-17-20a and 19a-19b predicted targets following Spi-1 and Fli-1 knockdown in 745A#44 cells. [score:7]
Clones NN10#17-92a and #17-92b expressing inducible exogenous miR-17-92 and clone NN10#17-20 expressing inducible exogenous miR-17 and miR-20a were derived from NN10#5 cells after transfection with expression vector pcDNA4/TO (Invitrogen, Cergy Pontoise, France) carrying the corresponding miRNA coding regions followed by zeocin selection. [score:7]
This analysis (Figure 3A) revealed that genes belonging to the predicted targets lists of miR-17, miR-20a, miR-19a and miR19b but not to those of miR-18a or miR-92a were significantly over-represented among genes up-regulated in response to HMBA and Dox (resulting in down regulation of miR-17-92 cluster). [score:7]
B: Mean relative expression profiles of the leading edge subsets of common predicted targets for miR-17/20a, miR-19a/19b and miR-451 identified by GSEA in response to progressive decrease of miR-17-92 expression or progressive increase of miR-451 induced by indicated combinations of HMBA and Dox treatment in 745A#44 cells. [score:7]
In summary, these analyses indicated that miR-17-92 cluster in 745A#44 cells is mainly involved in the down-regulation of predicted targets of miR-17/miR-20a and miR-19a/miR-19b rather than miR-18a or miR-92a. [score:6]
We further demonstrated that loss of proliferation induced by Fli-1 knockdown can be at least partially rescued by exogenous whole miR-17-92 cluster re -expression or by miR-17 and miR-20a only re -expression. [score:6]
We also compared mean expression levels of genes in the leading edge subsets of common miR-17/miR-20a, common miR-19a/miR-19b and miR-451 targets identified in response to HMBA in the other two conditions (+Dox only or +HMBA only) which are associated with intermediate pri-miR-17-92 expression levels (Figure 1). [score:6]
As expected from authentic target genes, variations in the levels of miR-17/miR-20a and of miR-19a/miR-19b targets were inversely correlated with the variations of pri-miR-17-92a levels (compare Figure 3B with pri-miR-17-92a and miRNAs profiles in Figure 1A). [score:5]
We then looked for other possible candidates in the transcriptome analysis presented in Figure 3. We noticed that HBP1, which has been identified as a miR-17 target in human breast cancer cells [37] was included in the leading edge genes subset whose expression increases in response to HMBA in 745A#44 cells. [score:5]
In contrast, in cells harboring functional P53 such as normal erythroid progenitors (B), increased Myc activity mediated by deregulated expression of miR17 and miR20a induces oncogenic stress thus explaining their anti-proliferative effect. [score:4]
Fli-1 directly activates miR-17-92 promoter and contributes to miR-17 and miR-20a expression in NN10#5 cells. [score:4]
Due to this property of HBP1, our finding that miR17 and miR20a downregulate HBP1 strongly suggests that these two miRNA contribute to increase Myc activity. [score:4]
We decided to focus first on miR-17 and miR-20a which are the most significantly downregulated miRNA of the cluster after Dox treatment. [score:4]
In fully transformed cells with no functional P53 such as established erythroleukemic cells lines (A) increased Myc activity induced by deregulated expression of miR17 and miR20a cannot induce oncogenic stress thus explaining their pro-proliferative effect. [score:4]
A: Parental NN10#5 cells and their derivative clones #17-92a, #17-92b or #17-20 harboring inducible exogenous whole miR-17-92 miRNA cluster or miR-17-20a sub-cluster were cultured for two days in the presence or absence of Dox and the relative levels of pri-miR-17-92, miR-17 and miR-20a levels were determined by qRT-PCR as in Figure 4. are expressed as +Dox/-Dox ratios (means and standard deviations from 3 independent experiments). [score:3]
One possibility could be that miR-17 levels in NN10#5 cells are insufficient to reduce proliferation as it is observed following strong overexpression in erythroid progenitors [29]. [score:3]
Having established miR-17 and miR-20a functionality in NN10#5 cells proliferation downstream of Fli-1, we tried to identify miR-17/20a targets able to explain this effect. [score:3]
Similarly, Fli-1 has been shown to contribute to the high malignancy of the breast cancer cell line MDA-MB231 [55] whereas another study independently showed the inhibition of Hbp1 by miR-17 in the same cells [37]. [score:3]
Except for miR-18, the oncogenic contributions of miR-17/20a, miR-19a/b and miR-92 have all been demonstrated and several functional targets identified, including E2F1, PTEN and BIM1 [26]. [score:3]
First, Li et al. achieved robust miR-17 overexpression by retroviral transduction. [score:3]
In that context, the identification of Hbp1 as a miR-17 target in NN10#5 and 745#44 cells is interesting. [score:3]
Using the same approach, we derived #17-20a cells allowing Dox-inducible expression of miR-17 and miR-20a only instead of the whole miR-17-92 cluster. [score:3]
Since it seems reasonable to think that miR-17 effect could be dose -dependent, it would be interesting to compare miR-17 levels between NN10#5 cells and cell lines overexpressing it from Li et al. study. [score:3]
In contrast, miR-17 and miR-20a anti-miRs transfection completely suppressed the proliferation rescue of #17-92a cells in the presence of Dox, thus confirming that exogenous miR-17-92 cluster contribution is strictly dependent on miR-17 and miR-20a function. [score:3]
#17-92a, #17-92b or #17-20 cell clones have been derived from parental NN10#5 cells following stable transfection of a Dox-inducible expression cassettes of the whole miR-17-92 cluster or only miR-17-20a sub-cluster, respectively. [score:3]
Surprisingly, Western blots analyses revealed that none of several miR-17/miR-20a targets already identified in other cell contexts (including p21, PTEN, E2F1 or TXNIP) displayed the expected increased level in response to Dox treatment (Figure 7A). [score:3]
In addition, miR-17 and miR-20a are two closely related miRNA which share a common seed sequence and have several well described targets. [score:3]
Identification of HBP1 as a miR-17/miR-20a target in NN10#5 and 745A#44 cells. [score:3]
We used a miR-17 and miR-20a anti-miRs mixture since both miRNA share a common seed sequence and have essentially identical targets. [score:3]
Interestingly, when compared to parental NN10#5 cells, #17-20a cells displayed the same proliferation rescue in Dox as did #17-92 a and #17-92b cells (Figure 6A), thus indicating that combined miR-17 and miR-20a re -expression is sufficient to reproduce the effect of the whole miR-17-92 cluster. [score:2]
We thus identified clones #17-92a and #17-92b that maintained stable levels of mature miR-17 and miR-20a in the presence or absence of Dox (Figure 5A) while still displaying Dox-inducible Fli-1 knock down (Figure 5B). [score:2]
Altogether these data lead us to propose a working mo del where the duality of miR-17 effect on cell proliferation could be explained by its dose -dependent and Hbp1 -mediated regulation of Myc activity and its strong dependence on the p53 status of the cells (see diagram in Figure S5). [score:2]
miR-17 and miR-20a are sufficient for partial rescue of Fli-1 knock-down induced cell proliferation arrest. [score:2]
miR-17 and miR-20a contribute to Hbp1 regulation in NN10#5 and 7451#44 cells. [score:2]
miR-17-92 functional implication in NN10#5 cells proliferation control downstream of fli-1. Restoration of initial levels of miR-17 and miR-20a following Dox -induced Fli-1 knockdown in NN10#5 cells. [score:2]
Moreover, we showed that restoration to their initial levels of only two members of this cluster, miR-17 and miR-20a, is sufficient to partially rescue the loss of proliferation induced by Fli-1 knock-down in erythroleukemic cells harboring activated fli-1 locus. [score:2]
From these results, we conclude that HBP1 can be considered as an authentic miR-17 and miR-20a target in both NN10#5 and 745A#44 cells. [score:2]
Like in #17-92a and #17-92b, miR-17 and miR-20a levels remained roughly unaffected by Dox treatment in #17-20 cells (Figure 5A) while Fli-1 remained strongly down regulated (Figure 5B). [score:2]
Our experimental results support the functional implication of miR-17 and miR-20a in erythroleukemic cells proliferation downstream of Fli-1 based on the rescue of proliferation loss induced by Fli-1 knock down following the restoration of these two miRNA to their initial levels. [score:2]
The absence of functional P53 in NN10 (F Moreau-Gachelin, personal communication) and 745A cell lines [39] might therefore be another cause of the lack of antiproliferative effect of miR-17 in these cell lines. [score:1]
pcDNA4/17-92 and pcDNA4/17-20 were obtained by cloning PCR-amplified genomic DNA corresponding to the whole mouse miR-17-92 cluster or to the miR-17 and miR-20a coding segments downstream to the Dox-inducible CMV promoter in pcDNA4/TO plasmid (Invitrogen, Cergy Pontoise, France). [score:1]
We also noticed that the antiproliferative effect of miR-17 was associated with increased levels of P53, thus suggesting [29] that P53 might be functionally involved. [score:1]
This conclusion is in apparent contradiction to the recent demonstration of the anti-proliferative effect of miR-17 in normal erythroid progenitors by Li et al [29]. [score:1]
This miR-17-92 cluster comprises six miRNAs that can be grouped into four sub-families based on their seed sequence (miR-17 and miR-20a, miR-18a, miR-19a and b and miR-92a) [19]. [score:1]
Moreover, transfection of miR-17 and miR-20a anti-miRs confirmed that this rescue is not due to clone effect and further supports our conclusion. [score:1]
Identification of miR-17/miR-20a and miR-19a/miR-19b signatures in the transcriptome of 745A#44 cells displaying decreased levels of miR-17-92 cluster. [score:1]
B: Equal numbers of NN10#5 or #17-92a cells were transfected with control, anti-miR-92a or a mixture of anti-miR-17 and anti-miR-20a and cultured in the presence or absence of Dox. [score:1]
Figure S4 Comparison of miR-17, miR18, miR-19a, miR19b and miR92 levels between NN10#5, 745A#44 and K16 erythroleukemic cells. [score:1]
However, interesting comparison between the two studies may help to understand the basis of these opposite effects of miR-17 on cell proliferation. [score:1]
Dox treatment of NN10#5 led to a strong reduction of Fli-1 levels associated with a 60% decrease of pri-miR-17-92 transcript level and a 40% decrease of mature miR-17 and miR-20a levels (Figure 4A). [score:1]
Given the very high similarity between miR-17 and miR-20a or miR-19a and miR-19b, this retrospective analysis was performed on the intersection of the leading edge subset of miR-17 and miR-20a or miR-19a and miR-19b, respectively. [score:1]
Figure S5 Diagram summarizing the inter-relationships established in our study and illustrating the pro-proliferative (A) or anti-proliferative (B) alternative contribution of miR-17-20a. [score:1]
Intriguingly however, miR-17/20a have also been reported to behave as an anti-oncogene [27]– [29] but the cell context differences that determine their oncogenic versus anti-oncogenic properties remain poorly understood. [score:1]
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[+] score: 177
Since both pathways are under regulation of miR-17, the global immune suppression we observed in mice with miR-17 overexpression could be the result of a vastly complex mechanism of interconnected regulatory networks. [score:7]
As a result of miR-17 overexpression, the expression levels of STAT3 were suppressed in the spleens of transgenic mice (Figure 4c). [score:7]
Moreover, inhibition of STAT3 expression can achieve the same effect as miR-17 over -expression. [score:7]
Interestingly, when melanoma cells were growing in the mice, effective immune response was observed in miR-17 overexpressing mice, leading to inhibited tumor development. [score:6]
Moreover, decreased expression of STAT3 was also detected in human T lymphocyte Jurkat cells transfected with the miR-17 overexpression plasmid (Figure 4c). [score:5]
In our study, we found that microRNA-17 was able to target STAT3 in tumor microenvironment, thus inhibited melanoma tumor growth by stimulating the tumor infiltrating CD8+ T cells response. [score:5]
CD8+ expression increased in the miR-17 -overexpressed Jurkat cells. [score:5]
Compared to the wild type mice, miR-17 overexpression in spleen was associated with reduced expression of MAP3K14 and STAT3 (Figure 3b). [score:4]
Compared to the wild type mice, miR-17 overexpression in spleen was associated with reduced expression of MAP3K14 and STAT3. [score:4]
We confirmed that mutation of the miR-17 binding site interfered with miR-17-target interaction, which led to restoration of luciferase activities (Figure 4b). [score:4]
To mimic the function of miR-17 in vitro, we further knocked down the expression of STAT3 by using siRNA against STAT3. [score:4]
By modulating STAT3 associated immune response in tumor microenvironment, the negative regulatory loop between miR-17 and STAT3 may be an important factor in tumor -associated immune tolerance and a potential immunotherapeutic target against cancer. [score:4]
When co-cultured with B16 cells, more cells overexpressing miR-17 were detected in S phase (34.22% vs. [score:3]
In line with what we have seen in non-tumor-bearing mice, the population of CD45+ cells was relatively lower in tumor-bearing mice with miR-17 overexpression (40.90% vs. [score:3]
Notably, the positive rate of CD8 was increased in these cells overexpressing miR-17 (Figure 4c). [score:3]
Our previous study showed that CD8+ cells differentiation was impaired in miR-17 overexpression mice [7]. [score:3]
Overall, provocative reactions were observed in the spleens of mice with miR-17 overexpression. [score:3]
Its 3′-untranslated region (3′-UTR) contains a base pairing sequence complementary to the seed region of miR-17 (Figure 4a). [score:3]
Tumor invasion was inhibited in miR-17 transgenic mice. [score:3]
Thus it is suggested that miR-17 promotes Jurkat cell differentiation in vitro, by targeting STAT3. [score:3]
Computational analysis showed that STAT3 is a candidate for miR-17 targeting. [score:3]
The melanoma tumors formed in mice overexpressing miR-17 were less than that in wild type mice. [score:3]
Figure 5 (A) Jurkat cells overexpressing miR-17 grew significantly faster when co-cultured with B16 cells. [score:3]
Our findings demonstrated that forced expression of miR-17 in Jurkat cells promoted cell proliferation and survival in the presence of B16 cells. [score:3]
B16 cells were co-cultured with Jurkat cells transfected with GFP mock control or miR-17 overexpression plasmid. [score:3]
The percentage of CD8+ T cells was suppressed in miR-17 transgenic mice before melanoma cell injection. [score:3]
The transgenic mice were developed by the microinjection of a miR-17 overexpression plasmid into C57BL/6 mice zygotes [7]. [score:3]
Decreased levels of STAT3 were associated with miR-17 over -expression. [score:3]
In summary, miR-17 targeted STAT3, promoted Jurkat-cell mitosis and proliferation during co-culture with melanoma B16 cells. [score:3]
When Jurkat cells were co-cultured with B16 cells, they benefited each other in proliferation: Jurkat cells overexpressing miR-17 grew significantly faster when co-cultured with B16 cells (Figure 5a). [score:3]
Stable overexpression of miR-17 could be observed in these cells for two weeks after transfection. [score:3]
and protein analysis indicated that STAT3 was the target of miR-17. [score:3]
Meanwhile, Jurkat cells transfected with mock-control or miR-17 overexpressing plasmid were co-cultured with B16 cells. [score:3]
B16 melanoma cells were injected into wild type and miR-17 overexpressing transgenic mice. [score:3]
However, when co-cultured with B16 cells, more cells overexpressing miR-17 were detected in S phase (34.22%). [score:3]
MiR-17 inhibits melanoma growth by stimulating CD8+ T cells mediated host immune response, which is due to its regulation of STAT3. [score:3]
Figure 4 (A) A potential miR-17 target site was found in the 3′UTR of STAT3. [score:3]
Consistently, miR-17 overexpressing cells in S phase also decreased to 13.89%, compared to 23.41% of control group (Figure 6a). [score:2]
Our finding also confirmed that the 3′-UTR of STAT3 harbors a miR-17 binding site and is subject to negative regulation of miR-17. [score:2]
Our studies show that STAT3 mediates the function of miR-17 in regulating T-cell activities, thus providing novel insight into the mechanisms that may underlie immune evasion in melanoma cells. [score:2]
Figure 6 (A) Left, Without co-culture, miR-17 overexpression increased cell number in G1 phase compared with the control cells (50.24% vs. [score:2]
Jurkat cells overexpressing miR-17 survived better in serum-free media compared to the controls. [score:2]
MiR-17 targets STAT3 in melanoma tumor microenvironment. [score:2]
When they grew independently, miR-17 overexpression in Jurkat cells increased the cell number in G1 phase (50.24%), compared to that in control group (42.84%) (Figure 6a). [score:2]
In summary, compared to the mice without tumor, a significant increase in CD8+ cells was observed in miR-17 overexpressing mice, but not in the wild type controls. [score:2]
MiR-17 targets STAT3 in tumor stromal cells. [score:2]
MiR-17 overexpressing cells in S phase also decreased (13.89% vs. [score:2]
In brief, U343 cells were seeded onto 12-well tissue culture dishes at a density of 1 × 10 [5] cells/well and co -transfected with the luciferase reporter constructs and miR-17-5p mimic with Lipofectamine 3000 (Life Technologies). [score:1]
We thereby designed a luciferase reporter construct which has a miR-17 binding site in the 3′-UTR of STAT3. [score:1]
As opposed to miR-17, knocking down STAT3 reduced the cell population in S phase (13.13%) and increased it in G1 phase (52.23%), compared to 21.46% in S phase and 38.04% in G1 phase of negative control oligos (Figure 6b). [score:1]
In the miR-17 transgenic mice, grafted tumors were still surrounded by an intact plasma membrane and less hemorrhagic necrosis can be found inside tumor (Figure 3a). [score:1]
Right, Typical distributions of CD45 positive cells in the miR-17 transgenic and wildtype mice. [score:1]
In the miR-17 transgenic mice, an increase of 10% after melanoma implantation was detected (30.80% vs. [score:1]
Right, Typical distributions of CD4, and CD8 subtypes in the miR-17 transgenic and wildtype mice. [score:1]
The CD8/CD4 ratio was close to 1 in both wild type and miR-17 transgenic mice (1.17 vs. [score:1]
Given the fact that miR-92a and miR-17 belong to a same microRNA cluster, their roles in immune mediation could be alike. [score:1]
When B16 cells were injected into the peritoneal cavities of miR-17 transgenic and wild type mice, they were capable of seeding on the surface of internal organs such as liver, bowels and omentum. [score:1]
In miR-17 transgenic mice, high levels of CD8+ T cells were detected in the spleen as well as peripheral blood. [score:1]
In addition, the miR-17 tumors were less invasive and less angiogenic. [score:1]
For example, there is a highly conserved STAT3 -binding site in the promoter of the miR-17 (C13orf25) [28]. [score:1]
Taken together, grafted melanoma cells were less invasive in the miR-17 transgenic mice than in the wild type mice. [score:1]
In contrast, both miR-17 and miR-92a promote immune cell mediated anti-tumor response. [score:1]
Meanwhile, intact plasma membrane and less hemorrhagic necrosis was seen in the miR-17 transgenic mice. [score:1]
We next injected mouse melanoma B16 cells intraperitoneally into wild type and miR-17 transgenic mice. [score:1]
We previously reported that decreased numbers and sizes of germinal centers were observed in non-tumor-bearing miR-17 transgenic mice [7]. [score:1]
CD8+ cells increased in tumor-bearing miR-17 transgenic mice. [score:1]
Previous work from our lab showed that miR-17 is essential for hematogenesis and differentiation [7]. [score:1]
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While the expression level of miR-17 was decreased from E12.5 to P0, p21 expression was continuously increased, suggesting a reciprocal expression between miR-17 and its target p21 during cortical development (Figures 3C–E). [score:10]
These results further suggest that higher expression of miR-17 in embryonic cortices is crucial for suppressing the p21 level and promoting NP proliferation, while low expression of miR-17 in postnatal cortices allows p21 expression to induce differentiation. [score:9]
miR-17 displays decreased expression in mouse cortices at embryonic and postnatal stages, which is opposite to that of p21, suggesting a reciprocal expression between miR-17 and its target p21. [score:7]
In E14.5 cortices, electroporated with the miR-17 precursor to overexpress miR-17 at E13.5, there was a significant increase in the percentage of BrdU [+]/GFP [+] cells, which suggests an up-regulation of proliferation of cortical NPs (Figures 4A,B). [score:6]
One of the mechanisms that control precise expression levels of these regulators is through the miRNA silencing regulation such as miR-17. [score:5]
In this study, we have demonstrated miR-17 targeting effect on p21 expression. [score:5]
miR-17 promotes proliferation of RGCs and IPs through suppressing p21 expression. [score:5]
We next examined whether the expression level of p21 is correlated with miR-17 expression in developing cortices by performing qRT-PCR. [score:5]
Our studies have revealed a mechanism of suppressing endogenous p21 expression by miR-17 in NPs. [score:5]
Furthermore, we have found the direct silencing action of miR-17 on p21 in vivo, since co -expression of p21 and miR-17, but not miR-17 mutations, can block negative effects of p21 on cortical NP proliferation. [score:5]
Figure 6. (A,B) Co -expression of miR-17 with p21 in E13.5 mouse cortices (n = 6), analyzed at E14.5, significantly rescued the reduced number of proliferating cells caused by p21 alone, while co -expression of a mutated miR-17 with p21 (n = 3) failed to do so. [score:5]
Our study here has shown that miR-17, a member of the miR-17–92 cluster, is an important regulator of p21 expression and expansion of the cortical neural progenitor pool. [score:4]
Since there are six miRNAs produced from the miR-17–92 cluster, and each miRNA usually has multiple targets, the molecular mechanisms of miR-17–92 function in cortical development remain an exciting research topic. [score:4]
Furthermore, miR-17 likely maintains the neural progenitor pool by regulating several targets. [score:4]
To directly test the silencing activity of miR-17 on p21 in vivo, we speculated that co -expression of miR-17 with p21 should be able to rescue the negative effect of p21 on proliferation of NPs. [score:4]
Our studies have identified a mechanism that controls p21 expression levels in NPs in vivo by miR-17 during cortical development. [score:4]
Here, we have not ruled out the likely possibility that the regulation of additional target genes by miR-17 contributes to its ability to promote NP proliferation. [score:4]
However, when a mutated miR-17 containing mutations in the seed sequence, which is responsible for binding to p21 3′UTR, was used, the targeting effect of miR-17 was abolished (Figure 3B). [score:4]
For instance, miR-17 has been shown to enhance NSC self-renewal by targeting Trp53inp1, a gene in the p53 pathway, to promote NP proliferation by silencing bone morphogenetic protein type II receptor (42, 43). [score:3]
These results indicate that the miR-17 sponge, which can block the endogenous miR-17 silencing activity, suppresses proliferation of cortical NPs. [score:3]
The targeting effects of miR-17 and p21 have been observed in oral carcinoma cells, acute myeloid leukemia cells, and Hodgkin’s lymphoma (39– 41). [score:3]
The percentages of BrdU [+]/GFP [+], Pax6 [+]/GFP [+], and Tbr2 [+]/GFP [+] cells versus total GFP [+] cells were all decreased significantly when the miR-17 sponge was expressed (Figures 5C–H). [score:3]
p21 is a putative target of miR-17. [score:3]
These results indicate that p21 is a specific target of miR-17. [score:3]
To further test miR-17 functions in expansion of cortical NPs, we applied a loss-of-function approach by expressing miR-17 sponges (36). [score:3]
These data indicate that the miR-17 sponge is able to block the silencing function of miR-17 to its target gene p21. [score:3]
Utilizing in utero electroporation and miRNA sponge, we have found that miR-17 specifically blocks p21 expression, thereby promoting the expansion of the cortical neural progenitor pool. [score:3]
In this study, we show that while p21 negatively regulates NP proliferation by reducing the numbers of both RGCs and IPs in the developing mouse cortex, miRNA miR-17 has an opposite effect on NP development. [score:3]
Figure 4. (A,B) Expression of miR-17 in E13.5 mouse cortices (n = 3), analyzed at E14.5, increased the number of proliferating cells co-labeled with GFP and BrdU. [score:3]
On the other hand, mutations of miR-17 in the seed sequence (miR-17 mut) had no effect on proliferation of RGCs and IPs, indicating a specific effect of miR-17 on NP development (Figures 4C–F). [score:3]
However, co -expression of p21 and the mutated miR-17 failed to rescue NP proliferation (Figures 6A–F). [score:3]
Figure 3. (A) A predicted targeting site of miR-17 on the 3′UTR of p21. [score:3]
Moreover, the percentages of Pax6 [+]/GFP [+] and Tbr2 [+]/GFP [+] cells were also increased, indicating an expansion of RGCs and IPs by miR-17 overexpression (Figures 4C–F). [score:3]
For co -expression of p21 and miR-17, the concentrations of p21 and miR-17 are 0.5 and 1.5 μg/μl, respectively, maintaining a total plasmid concentration of 2 μg/μl. [score:3]
Mutations of miR-17 in the seed sequence were generated using the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies) using the following primers: F-5′-gtcagaataatgtcgttgtgcttacagtgcaggtagtgatgtgtgcatctactgcagtgagggcacaagtagcattatgctgac-3′; R-5′-gtcagcataatgctacttgtgccctcactgcagtagatgcacacatcactacctgcactgtaagcacaacgacattattctgac-3′. [score:2]
miR-17 positively regulates proliferation of cortical neural progenitors. [score:2]
To prove the targeting effect of miR-17 on p21, we designed a luciferase assay in which the 3′UTR sequence of p21 was cloned into a luciferase vector and co -transfected with miR-17. [score:2]
Compared to p21 alone, co -expression of miR-17 and p21 significantly rescued NP proliferation, as demonstrated by increased percentages of BrdU [+]/GFP [+], Pax6 [+]/GFP [+], and Tbr2 [+]/GFP [+] cells, which were compatible to those electroporated with the control vector (Figures 6A–F). [score:2]
An important miRNA family is the miR-17–92 cluster, which produces miR-17, 18, 19, and 92, each with conserved seed sequences, and regulates proliferation and survival of various cells (29– 33). [score:2]
These results suggest that miR-17 silencing regulation of p21 likely controls NP population in embryonic cortices. [score:2]
miR-17 rescues the negative effect of p21 on proliferation of neural progenitors. [score:1]
The reduction of the relative luciferase activity, which is normally caused by the silencing effect of miR-17, was significantly rescued by the miR-17 sponge (Figure 5B). [score:1]
The miR-17 sponge was cloned in the 3′UTR of a coding gene iCre, and co -transfected with miR-17 and the construct containing the luciferase gene followed by the 3′UTR sequence of p21. [score:1]
A mutated miR-17 (n = 3) had no effect on the number of RGCs. [score:1]
A mutated miR-17 (miR-17 mut) (n = 3) had no effect on cell proliferation. [score:1]
Figure 5. (A) The design of the miR-17 sponge. [score:1]
Our results suggest that miR-17 promotes proliferation of cortical NPs. [score:1]
We designed the miR-17 sponge (miR-17 sp), which consists of three narrowly spaced, bulged binding sites for miR-17 (Figure 5A). [score:1]
A mutated miR-17 sponge (miR-17 sp-mut) (n = 3) failed to do so. [score:1]
It is likely that silencing p21 by miR-17 in proliferative cells is a general rule. [score:1]
org), we searched the 3′UTR of p21 and found that it contains one binding site for miR-17 (Figure 3A). [score:1]
To further test the effect of miR-17 sponge on expansion of cortical NPs in vivo, the miR-17 sponge was electroporated into the cortices of E13.5 mice, and analyzed at E14.5. [score:1]
Our own work and others have shown that miR-17–92 promotes proliferation of cortical NSCs and NPs (34, 35). [score:1]
A mutated miR-17 sponge (n = 3) failed to do so. [score:1]
While relative luciferase activities in constructs containing the 3′UTR of p21 were not affected by a control miRNA miR-9, they were significantly reduced by miR-17 (Figure 3B). [score:1]
However, a mutated miR-17 sponge at the seed sequence showed no such rescue effect. [score:1]
Blocking miR-17 using its sponge causes reduced proliferation of cortical neural progenitors. [score:1]
For luciferase assays, the miR-17 precursor and its mutation were subcloned into the pCDNA3.1 vector. [score:1]
A mutated miR-17 sponge (n = 4) failed to do so. [score:1]
The p21 full length cDNA with the 3′UTR was cloned into the pCAGIG vector, co-electroporated with miR-17 into E13.5 cortices, and analyzed at E14.5. [score:1]
A mutated miR-17 (n = 3) had no effect on the number of IPs. [score:1]
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When inhibit miR-17 in TNF-α-stimulated PDLSCs, protein expression level of osteogenic marker alkaline phosphatase (ALP) was downregulated (Fig.   3c). [score:8]
According to previous results in our lab, overexpression of miR-17 decreased expression levels of osteogenic markers and bone matrix formation, inhibition of miR-17 alone increased osteoblast marker genes, suggesting that miR-17 is a negative regulator of osteogenic differentiation in H-PDLSCs [3]. [score:8]
Furthermore, inhibition of miR-17 by anti-miR-17 oligonucleotides (si-miR-17) resulted in the upregulation of the protein expression level of HDAC9 (Fig.   3d). [score:8]
Interestingly, downregulation of HDAC9 by si-HDAC9 in P-PDLSCs restored the expression of pri-miR-17-92a as well as the mature miR17-92a, though, miR-18 was not affected, suggesting that HDAC9 inhibited miR17-92a (Fig.   3a, b). [score:8]
miR17-92a cluster is first described in 2001 [20] in mammalians, known as tissue-specific expressed onco-miR, forms signaling loop with myc protein, miR17-92a regulates more than a hundred targets involved in proliferation depending on different cellular context, their role in affecting the HDAC, which is responsible for the global proliferation inhibition remains unknown [21]. [score:8]
Consistent with these results, in the current study, Alizarin red staining showed that downregulation of miR-17 inhibited calcified nodule formation in TNF-α-stimulated H-PDLSCs (Fig.   3f). [score:6]
ChIP experiments revealed the promoter region of miR-17-92a HDAC9 enrichment in P-PDLSC samples, suggesting that HDAC9 inhibits the expression of miR17-92a by direct deacetylation (Fig.   3e). [score:6]
miR-17 in periodontal ligament stem cells targets the 3′ untranslated regions of a Smad ubiquitin regulatory factor one(Smurf1), which when activated under chronic inflammation, would lead to increased degradation of various osteoblast-specific factors [3]. [score:6]
We demonstrated that the inhibition of HDAC by NaB downregulated miR17-92a family and partially rescued inflammation impaired osteogenesis in vitro and in vivo. [score:6]
Comparison of the protein expression level of ALP (c) and HDAC9 (d) between P-PDLSCs and miR-17 inhibitor -treated P-PDLSCs. [score:5]
The miRNAs clusters, miR-17-92a, miR-106b-25, and miR-106a-363, have been found to control EZH2 expression, which is involved in H3K27me3 -mediated tumor suppressor genes in cancer [32]. [score:5]
Furthermore, simultaneous addition of si-miR-17 and NaB inhibited osteogenesis to a similar extent than using si-miR17 alone in TNF-α-stimulated H-PDLSCs, suggesting that the rescue of osteogenesis by NaB largely depended on the expression of miR-17 (Fig.   3f). [score:5]
When HDAC9 is inhibited by HDI, miR-17 has an inhibitory role in osteogenesis of PDLSC (data not shown). [score:5]
The expression of pri-miR-17~92a was downregulated in P-PDLSCs compared to H-PDLSCs (Fig.   3a). [score:5]
In our study, we showed that miR-17 is a new member of the epi-miRNA which inhibited the protein expression level of HDAC9. [score:5]
In the physiological conditions, miR-17 as well as HDAC9 forms an inhibitory balance to regulate the differentiation of PDLSCs and affect adjacent cells to regulate bone formation. [score:5]
Fig. 6 The mutual inhibition between HDAC9 and miR-17 in P-PDLSCs regulates the osteogenesis of PDLSCs and affects the bone regeneration in inflammatory condition a Schematic illustration of the LPS -induced periodontitis mo del in SD rats. [score:4]
miR-17 and HDAC9 negatively inhibit each other in regulation of osteogenic differentiation of PDLSCs in vitro. [score:4]
mir-17 and HDAC9 negatively inhibit each other in regulation of osteogenic differentiation of PDLSCs in vitro. [score:4]
Fig. 6 The mutual inhibition between HDAC9 and miR-17 in P-PDLSCs regulates the osteogenesis of PDLSCs and affects the bone regeneration in inflammatory condition Since adult stems cells have long been recognized as a critical population in restoring tissue function under inflammatory conditions, we asked if inflammation affects PDLSCs, the adult stem cells from periodontal ligament tissue and what specific effects has inflammation brought to PDLSCs. [score:4]
We further complement the story of HDAC9 and MSC by first identifying HDACs expression in PDLSC and finding that HDAC9 participates in osteogenic differentiation through interacting with miR-17 to repress RUNX2 transcription. [score:3]
According to our results, the overall role of miR-17 is likely to be closely associated with the expressional level of HDAC9. [score:3]
Fig. 3 a Comparison of the RNA expression level of pri-miR17-92a cluster between H-PDLSCs, P-PDLSCs, and si-HDAC9 -treated P-PDLSCs. [score:3]
miR-17 induced osteogenesis of inflamed periodontal adult stem cell through inhibition of HDAC9. [score:3]
f Analysis of calcified nodules by Alizarin red staining in NaB, si-miR-17, or NaB plus si-miR-17 -treated TNF-α-stimulated PDLSCs In order to find out if miR-17 plays important role in the inhibitory loop in osteogenesis of PDLSCs, we examined the effects of miR-17 on osteogenesis of PDLSCs. [score:3]
Finally, we revealed that the rescue of osteogenesis by HDAC inhibitor depended on miR-17. [score:3]
Taken together, the miR-17 and HDAC9 formed inhibitory loop under inflammatory conditions. [score:3]
Interestingly, the role of miR-17 in PDLSC differentiation can be shifted to either promotion of osteogenesis or inhibition of osteogenesis [16]. [score:3]
f Analysis of calcified nodules by Alizarin red staining in NaB, si-miR-17, or NaB plus si-miR-17 -treated TNF-α-stimulated PDLSCs a Comparison of the RNA expression level of pri-miR17-92a cluster between H-PDLSCs, P-PDLSCs, and si-HDAC9 -treated P-PDLSCs. [score:3]
b Comparison of the RNA expression of mature miR-17-92a between P-PDLSCs and si-HDAC9 -treated P-PDLSCs. [score:3]
This may explain why a co-inhibit relationship is needed between HDAC9 and miR-17. [score:3]
Inhibition of HDAC by NaB as well as si-miR-17 rescues osteogenesis of the human inflammatory PDLSCs. [score:3]
Statistical quantification of RUNX2 and OCN signal (right panel) The mutual inhibition between HDAC9 and miR-17 in P-PDLSCs regulates the osteogenesis of PDLSCs and affects the bone regeneration in inflammatory condition One of the key characteristics of chronic inflammation is its persistence. [score:2]
In previous studies, miR-17 promotes proliferation in the regulation of B cell lymphoma growth and retinoblastoma, and thus might delay differentiation 15, 26. [score:2]
Furthermore, miR-17 became a positive regulator of osteogenic differentiation in P-PDLSCs [3]. [score:2]
These evidences prompt us to verify that if miR-17-92a could regulate HDAC modulated dental tissue differentiation under inflammatory conditions. [score:2]
Furthermore, the transcription of miR-17 itself is regulated via deacetylation by HDAC9 in the promoter regions under inflammatory conditions. [score:2]
miR-17 is essential for NaB to rescue the osteo-differentiation of TNF-α-stimulated PDLSCs. [score:1]
We found a new epi-miRNA, the miR-17, which forms a reciprocal signaling loop with HDAC9 in PDLSCs under inflammation condition. [score:1]
However, the mechanisms underlying the shift of the role of miR-17 in osteogenesis needed further exploration. [score:1]
mir-17 and HDAC9 negatively affect each other under chronic inflammatory conditions in the adult stem cell in tooth tissue. [score:1]
e Analysis of the enrichment of HDAC9 protein at the pri-miR-17-92a cluster promoter region by Chromatin Immunoprecipitation (ChIP). [score:1]
We first examined the association of HDAC9 with miR-17~92a in PDLSCs of periodontitis patients. [score:1]
This is surprising since HDAC9 promotes proliferation and miR-17~92a belongs to the onco-miR family. [score:1]
Schematic illustration of the relationship between HDAC9, miR-17, and bone regeneration. [score:1]
We discovered that the onco-miR, miR-17 is a member of the epi-miRNAs. [score:1]
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[+] score: 166
mir-17~92 loss does not impact normal pancreas developmentPrior miRNA expression profiling studies of human PDAC specimens demonstrated elevated expression of components of the mir-17~92 cluster in PDAC. [score:6]
We utilized the TargetScan database [48] to identify known and putative mRNA targets of mir-17~92 that are implicated in the regulation of ERK phosphorylation. [score:6]
In particular, DUSP2, DUSP7 and DUSP10 suppress ERK activity and are demonstrated or predicted targets of mir-17~92 miRNAs [48, 58– 60]. [score:5]
Based on the partial residual expression of the miR-17 and miR-92 families (Figure 7B, 7C), and the generally very low expression of the miR-18 family (Figure 7D, note y axis units), we hypothesized that loss of the miR-19 family was responsible for the defective invasion of 17KPC cell lines. [score:5]
In contrast, miR-19 family miRNAs can only be expressed from the mir-17~92 and mir-106a~363 clusters, and 17KPC lines were found to completely lack expression of this miRNA family (Figure 7E). [score:5]
Prior miRNA expression profiling studies of human PDAC specimens demonstrated elevated expression of components of the mir-17~92 cluster in PDAC. [score:5]
We explored the possibility that mir-17~92 increases ERK activation via suppression of dual-specificity phosphatases (DUSPs), which are well-known regulators of numerous MAPK family proteins, including ERK [57]. [score:4]
Additional studies using inducible expression or repression of mir-17~92 miRNAs, coupled to mRNA and proteomic profiling approaches, will aid in elucidating the mechanisms by which this cluster regulates ERK phosphorylation in the early stages of pancreatic tumorigenesis. [score:4]
In addition, mir-17~92-encoded miRNAs are induced in precursor PanIN lesions, implicating them in early stages of PDAC development [24], and miR-17 overexpression has been associated with reduced pancreatic cancer patient survival [25]. [score:4]
Moreover, ectopic mir-17~92 expression in murine PanIN cell lines [46, 47] increased p-ERK levels under both serum replete and serum starved conditions (Figure 3A, 3B), confirming that mir-17~92-encoded miRNAs regulate this pathway. [score:4]
To ascertain whether mir-17~92 deletion differentially impacted primary versus metastatic disease processes, we stratified survival data based on the presence or absence of grossly visible metastases at euthanasia. [score:3]
ERK signaling is increased by mir-17~92 overexpression in PanIN cell lines. [score:3]
Together, these findings suggest that heterozygous deletion of Trp53 compensates for the loss of mir-17~92 and promotes disease progression in mir-17~92 deficient animals. [score:3]
Profiling of human pancreatic tumors and pancreatic cancer cell lines has shown that miRNAs encoded by the mir-17~92 cluster and its paralogs– mir-106b~25 and mir-106a~363–are upregulated in tumors compared to normal pancreatic tissue or chronic pancreatitis [22, 23]. [score:3]
mir-17~92 has been implicated in a variety of cancer contexts [19], and inhibition of members of this cluster has been shown to impair tumor growth and survival [20, 21]. [score:3]
Moreover, the enhanced ERK phosphorylation observed in PanIN cell lines following ectopic mir-17~92 expression was not associated with changes in DUSP7 or DUSP10 protein levels (Supplementary Figure 4). [score:3]
We find that mir-17~92 miRNAs are consistently overexpressed in PDAC cell lines (Supplementary Figure 1). [score:3]
Together, these in vivo findings suggest that loss of the mir-17~92 cluster may impact PDAC cell invasion, a feature associated with later stages of disease. [score:3]
In their work, the authors report that ectopic mir-17~92 expression promotes the proliferation of pancreatic cancer stem cells, resulting in their premature depletion and consequent reduced cell transformation and tumorigenicity. [score:3]
Several microRNAs, including those in the mir-17~92 cluster, display increased expression in PDAC as well as precursor PanIN lesions [22– 24], suggesting that these miRNAs may play a role in tumorigenesis. [score:3]
Future experiments in genetically engineered PDAC mouse mo dels with enhanced mir-17~92 expression may be required to resolve this discrepancy. [score:3]
Figure 3ERK signaling is increased by mir-17~92 overexpression in PanIN cell lines(A) Representative immunoblot for pERK and total ERK in the PanIN cell lines RP2294 and AH2375 stably infected with PIG- mir-17~92 or empty vector. [score:3]
The mir-17~92 cluster encodes six miRNAs encompassing four miRNA seed families (Figure 7A), implicating thousands of predicted mRNA targets as downstream effectors of the cluster's invasive program. [score:3]
In addition, we observe that mir-17~92 -null PanIN lesions have reduced ERK phosphorylation, and PanIN cell lines with ectopic mir-17~92 expression display elevated p-ERK levels. [score:3]
To ascertain whether mir-17~92 miRNAs have elevated expression in PDAC cells, we profiled a panel of PDAC cell lines as well as the immortalized pancreatic epithelial cell line HPNE. [score:3]
The observed residual mir-17~92 expression is likely from endocrine cells, many of which are derived from a PTF1A-independent lineage [40, 42], as well as a small population of acinar and ductal cells that have avoided recombination due to the fact that Cre drivers are not 100% efficient [43]. [score:3]
Quantitative RT-PCR demonstrated that 17KPC cell lines are indeed null for miRNAs from mir-17~92, however they retain robust expression from mir-106b~25 (Figure 7B, 7C). [score:3]
In fact, the mir-106b~25 locus is sufficient to drive expression of miRNAs for the miR-17 and miR-92 families to levels close to those observed in KPC lines, suggesting that loss of the miR-17 and -92 families may not be primarily responsible for the invasive defect of 17KPC cell lines. [score:3]
Thus, the detailed mechanisms regulating ERK phosphorylation in PanINs downstream of mir-17~92 remain unknown. [score:2]
In contrast, our studies demonstrate that ectopic mir-17~92 increases MEK/ERK signaling, a feature required for the maintenance of established PanIN lesions [6, 67], suggesting that elevated mir-17~92 levels should enhance pancreatic cancer development. [score:2]
mir-17~92 loss does not impact normal pancreas development. [score:2]
These findings demonstrate that mir-17~92 is not required for normal pancreas development. [score:2]
The mechanisms underlying the regulation of ERK activity by mir-17~92 are unknown. [score:2]
In addition, we find that mir-17~92 miRNAs, in particular miR-19 family miRNAs, promote PDAC cell invasion by regulating the formation of extracellular matrix-degrading invadopodia rosettes. [score:2]
mir-17~92 loss promotes PanIN loss and exocrine recoveryTo assess the impact of mir-17~92 deletion on the development and progression of precursor precancerous PanIN lesions, we crossed mir-17~92 [flox/ flox], Ptf1a-Cre mice onto the LSL-Kras [G12D] background [44]. [score:2]
Thus, the precise mechanisms through which mir-17~92 regulates ERK phosphorylation remain unknown. [score:2]
Taken together with the phosphorylation of MEK, these data suggest that the alterations in ERK phosphorylation do not reflect an overall reduction in KRAS signaling, and further suggest that mir-17~92 regulates PanIN maintenance by specifically influencing ERK pathway activity downstream of KRAS [G12D] and MEK. [score:2]
To determine the effect of mir-17~92 loss on pancreatic development, we induced pancreas-specific deletion of the mir-17~92 cluster using the conditional mir-17~92 [flox] allele and the recombination driver Ptf1a-Cre [40, 41]. [score:2]
Together, these findings illustrate important roles for mir-17~92 miRNAs during multiple phases of PDAC development and progression. [score:2]
To assess the impact of mir-17~92 deletion on the development and progression of precursor precancerous PanIN lesions, we crossed mir-17~92 [flox/ flox], Ptf1a-Cre mice onto the LSL-Kras [G12D] background [44]. [score:2]
To aid in our determination of which miRNA families may be most important in the invasive phenotype, we evaluated nine KPC and nine 17KPC cell lines for their expression of miR-17, -18, -19, and -92 family miRNAs across the three cluster paralogs: mir-17~92, mir-106b~25, and mir-106a~363. [score:1]
The mir-17~92 cluster was initially identified as oncogenic over a decade ago [18, 26]. [score:1]
of RNA from whole pancreata showed a strong reduction in the levels of mir-17~92 constituent miRNAs in compound mir-17~92 [flox/ flox], Ptf1a-Cre mice (Supplementary Figure 2A). [score:1]
Together, these findings suggest that mir-17~92 loss promotes the redifferentiation of PanINs into acinar cells. [score:1]
We observed similar overall survival, rates of metastasis, and histological prevalence of invasion at sacrifice between KPC and 17KPC mice, suggesting that Trp53 loss can compensate for mir-17~92 deletion. [score:1]
Here we report that deletion of the mir-17~92 miRNA cluster results in the regression of KRAS [G12D] -driven PanIN lesions and the expansion of normal acinar tissue in place of neoplastic cells over time. [score:1]
mir-17~92 -deficient cell lines are less invasive in vitroTo better understand the biology of mir-17~92 deficient pancreatic cancer cells, we generated a collection of cell lines from KPC and 17KPC tumors. [score:1]
These data suggest that mir-17~92 plays a role in PDAC invasiveness. [score:1]
mir-17~92 -deficient tumors display a delayed invasion phenotype. [score:1]
Loss of mir-17~92 prolongs survival in mice without metastases. [score:1]
However, mir-17~92 -null PanIN lesions display no changes in MEK phosphorylation or PI3K/AKT signaling, suggesting a specific impact of mir-17~92 on ERK activation. [score:1]
mir-17~92 -deficient cell lines are less invasive in vitro. [score:1]
mir-17~92 null PDAC cell lines have reduced invasive capacity in vitro. [score:1]
Figure 4Loss of mir-17~92 prolongs survival in mice without metastases(A) Kaplan-Meier survival plot for KPC and 17KPC mice. [score:1]
Despite efficient depletion of mir-17~92 miRNAs from the pancreas, organ size and exocrine and endocrine architecture and composition were unperturbed (Supplementary Figure 2B). [score:1]
We find that deletion of mir-17~92 impairs MEK/ERK signaling in PanIN lesions and this correlates with the presence of fewer PanINs, as well as their regression over time. [score:1]
These data suggest that loss of mir-17~92 decreases invasion, at least in part, as a result of reduced matrix-degrading capacity. [score:1]
mir-17~92 null PanINs regress with age. [score:1]
Together, our findings demonstrate important roles for mir-17~92-encoded miRNAs during early stages of pancreatic tumorigenesis, as well as tumor progression and invasion. [score:1]
Together, these data suggest that loss of mir-17~92 impairs the maintenance of PanIN lesions. [score:1]
Among the earliest described oncogenic miRNAs were members of the mir-17~92 cluster [17, 18]. [score:1]
Figure 6 mir-17~92 null PDAC cell lines form fewer invadopodia rosettesImmunofluorescence staining for the invadopodia constituent proteins cortactin (A, E), actin (B, F) and paxillin (C, G) in representative KPC and 17KPC cell lines. [score:1]
However, immunohistochemical staining of DUSP2, DUSP7 and DUSP10 failed to demonstrate any difference between KC and 17KC PanIN lesions, and modulation of mir-17~92 levels in PanIN cell lines also failed to change the levels of these phosphatases. [score:1]
However, we have not evaluated the self-renewal capacity of mir-17~92 expressing PanIN cell lines, nor have we tested their tumorigenic capacity upon implantation into recipient mice. [score:1]
Figure 7(A) Schematic representation of the mir-17~92 cluster and its paralogs mir-106b~25 and mir-106a~363. [score:1]
Importantly, apoptotic rates are not increased in mir-17~92 -null PanINs, suggesting that these lesions are not lost by apoptosis. [score:1]
Together, these data suggest that mir-17~92 contributes to the morbidity and mortality caused by primary KPC tumors and does not impact time to metastasis. [score:1]
Importantly, our experiments do not preclude significant roles for the other miRNAs encoded within the mir-17~92 cluster in PDAC invasion. [score:1]
Breeding pairs were designed to cross mir-17~92 [flox/wt], LSL-Kras [G12D] mice with mir-17~92 [flox/wt], Ptf1a-Cre mice in order to generate littermate mir-17~92 [wt/wt], LSL-Kras [G12D], Ptf1a-Cre and mir-17~92 [flox/ flox], LSL-Kras [G12D], Ptf1a-Cre mice (hereafter ‘KC’ and ‘17KC’). [score:1]
However, immunostaining of PanIN lesions with antibodies against these phosphatases did not show any differences between mir-17~92 wild type and deficient PanINs (data not shown). [score:1]
Therefore, we experimentally tested the requirement for mir-17~92 in a mouse mo del of pancreatic cancer. [score:1]
Figure 2 mir-17~92 null PanINs display reduced MEK/ERK pathway activationImmunostaining of PanIN lesions identified in 4- and 9-month old KC and 17KC mice for phosphorylated ERK (A– D), phosphorylated MEK (E– H), AKT phosphorylated at Thr [308] (I– L), and AKT phosphorylated at Ser [473] (M– P). [score:1]
mir-17~92 null PDAC cell lines form fewer invadopodia rosettes. [score:1]
To better understand the biology of mir-17~92 deficient pancreatic cancer cells, we generated a collection of cell lines from KPC and 17KPC tumors. [score:1]
mir-17~92 null PanINs display reduced MEK/ERK pathway activation. [score:1]
mir-17~92 loss promotes PanIN loss and exocrine recovery. [score:1]
The Ptf1a-Cre [40], mir-17~92 [flox] [41], LSL-Kras [G12D] [44], and Trp53 [flox] [68] mouse strains have been described previously. [score:1]
To assess whether mir-17~92 deletion impairs progression to carcinoma, we accelerated the KC mo del by including conditional loss of one copy of Trp53 (LSL-Kras [G12D], Trp53 [flox/wt], Ptf1a-Cre and mir-17~92 [flox/ flox], LSL-Kras [G12D], Trp53 [flox/wt], Ptf1a-Cre; hereafter “KPC” and “17KPC” mice). [score:1]
Studies in pancreatic cancer cell lines additionally demonstrated roles for the mir-17~92 cluster in PDAC cell proliferation, transformation and invasion [25, 38, 39]. [score:1]
mir-17~92 -deficient tumors display a delayed invasion phenotypeTo assess whether mir-17~92 deletion impairs progression to carcinoma, we accelerated the KC mo del by including conditional loss of one copy of Trp53 (LSL-Kras [G12D], Trp53 [flox/wt], Ptf1a-Cre and mir-17~92 [flox/ flox], LSL-Kras [G12D], Trp53 [flox/wt], Ptf1a-Cre; hereafter “KPC” and “17KPC” mice). [score:1]
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[+] score: 138
In terms of same “seed” sequence shared by miR-17 and miR-20a,1,220 conserved target genes for both miR-17 and miR-20a are predicted by TargetScan tool, in which quite a few genes are involved in cellular apoptosis regulation including caspase 7, caspase 2, BCL2-like 11, et al. Ingenuity Pathway Analysis (IPA) on the 1,220 predicted target genes revealed a cell death network (Supplemental Figure S5) and a cellular apoptosis pathway (Supplemental Figure S6). [score:8]
Furthermore, cell conditioned media from miR-17/20 overexpressing MCF-7 cells inhibited the invasiveness of MDA-MB-231 cells by inhibiting secretion of several cytokines and plasminogen activators. [score:7]
The miR-17/20 -mediated inhibition of breast cancer cellular proliferation via cyclin D1 repression, together with the finding that miR-17/20 inhibits breast cancer cell invasiveness (42), is consistent with a mo del in which miR-17/20 is a negative growth regulator in breast cancer cells. [score:6]
miR-17/20 overexpression induced the expression of p53, Bax, Cyto C and caspases which are key components of p53 -mediated apoptosis pathway (Figure 2A and Supplementary Figure S4). [score:5]
Western analysis showed that miR-17/20 overexpression promoted p53 expression in Akt1 [+/+] cells rather than Akt1 [−/−] cells (Fig. 4D). [score:5]
Consistent with a mo del in which miRNA-17 retards cellular growth in transgenic mice, these studies demonstrated miR-17/20 inhibits cyclin D1 gene expression via a 3' UTR binding site. [score:5]
In order to identify the mechanisms by which miR-17/20 regulates cellular apoptosis, analysis was conducted of apoptosis -regulating pathways and target genes (p53, p57, Cyto C, Caspase 3, Caspase 9, Bax, and PARP). [score:5]
The miR-17/20 cluster functions as a tumor suppressor in human breast cancer by decreasing AIB1 and cyclin D1 expression (33, 34). [score:5]
The target gene prediction of hsa-miR-17 and hsa-miR-20 was performed using TargetScan Human 6.2 version (released June 2012) (http://www. [score:5]
Consistent with the observation, transfection of MCF-7 cells with anti-miR-17 or anti-miR-20a decreased p53 expression (Fig. 1E). [score:3]
miR-17/20 overexpression decreased the IC [50] of MCF-7 cells to doxorubicin (48h treatment). [score:3]
p57 expression was unchanged by miR-17/20 treatment (Fig. 1C). [score:3]
miR-17/20 sensitizes doxorubicin -induced apoptosis, and increase p53 expression in breast cancer cells. [score:3]
D, Western blots showing the increased p53 expression in miR-17/20 transduced MCF-7 cells in the presence (lane 1 and 2) or absence (lane 3 and 4) of doxorubicin treatment. [score:3]
Our previous studies demonstrated the suppression of cellular proliferation in human breast cancer cells by miR-17/20. [score:3]
In transgenic mice, miR-17/20 overexpression reduced overall tissue growth resulting in small organs (41). [score:3]
Consistent with the role of the miR-17/20 in growth inhibition, the human miR-17/20 cluster, which is located on chromosome 13q31, undergoes loss of heterozygosity in a number of malignancies, including breast cancer. [score:3]
Deletion of Akt1 abrogated both the inhibition of cellular proliferation and the induction of apoptosis by miR-17/20. [score:3]
As shown in Figure 2B, miR-17/20 overexpression attenuated relative cell survival in the presence of tamoxifen (Fig. 2B, C). [score:3]
The expression of p53 and p27 [KIP1] increased in miR-17/20 transduced MCF-7 cells. [score:3]
A, Western blot showing the regulation of apoptosis pathway-related genes in miR-17/20 transduced MCF-7 cells. [score:2]
Apoptotic cells were increased in miR-17/20 overexpressing MCF-7 cells compared to control cells. [score:2]
Using Akt1 knockout mammary epithelial cells, and breast cancer derived cell lines from transgenic mice, we demonstrated the induction of p53 by miR-17/20 required Akt1. [score:2]
Figure 2 A, Western blot showing the regulation of apoptosis pathway-related genes in miR-17/20 transduced MCF-7 cells. [score:2]
In order to determine the potential role of miR-17/20 in regulating breast cancer cell apoptosis, MCF-7 cells were transduced with a retrovirus encoding the miR-17/20 cluster. [score:2]
Schematic representation of the molecular mechanisms by which miR-17/20 and Akt1 regulate p53 abundance and thereby apoptosis. [score:2]
In breast cancer cells, the cell cycle is controlled through a cyclin D1-miR-17/20 auto-regulatory feedback loop (33). [score:2]
Tamoxifen resistance occurs in ERα+ breast cancer cells including MCF-7. miR-17/20 transduced MCF-7 cells and control were treated with 15 uM tamoxifen up to 36 hours. [score:1]
In the current work we found the apoptosis induction by miR-17/20 in breast cancer cells. [score:1]
C, Western blots showing the increased p53 and p27KIP1 in miR-17/20 transduced MCF-7 cells. [score:1]
MCF-7 Cells were infected with the pMSCV [puro]-miR-17/20 cluster or the pMSCV [puro] empty vector. [score:1]
miR-17/20 sensitized the ErbB2 -induced NAFA cell line to apoptosis. [score:1]
We therefore derived breast tumor cell lines from MMTV-ErbB2/Akt1 [−/−] and MMTV-ErbB2/Akt1 [+/+] litter mate control mice and thereby assessed the role of Akt1 in miR-17/20 mediated apoptosis. [score:1]
The reduction in cell survival of miR-17/20 transduced cells was most pronounced (~20% vs. [score:1]
E, Western blot showing decreased p53 in anti-miR-17 and anti-miR-20a- transduced MCF-7 cells. [score:1]
The current studies demonstrated that miR-17/20 induces apoptosis in response to the DNA damaging agents doxorubicin and tamoxifen. [score:1]
miR-17/20 sensitized stress signal -induced apoptosis in breast cancer cells. [score:1]
In MCF-7 cells, doxorubicin -induced cellular apoptosis, assessed by TUNEL analysis, was abolished by anti-miR treatment in miR-17/20 transduced MCF-7 cells (Fig. 1B). [score:1]
Figure 5 miR-17/20 transduction was able to sensitize the Akt1 [+/+] cells to doxorubicin treatment, but not Akt1 [−/−] cells (Fig. 4B, C). [score:1]
D, Western blot of miR-17/20 transduced Akt1−/− and Akt+/+ murine ErbB2 breast tumor cells for p53. [score:1]
Akt1 is required for miR-17/20 sensitization of breast tumor cells to doxorubicin -induced apoptosis. [score:1]
B, Cell survival curves of miR-17/20 and control transduced MCF-7 treated with 0.5 μM doxorubicin for 0, 24, 48, and 72 hours. [score:1]
In the current studies, miR-17/20 enhanced tamoxifen -induced apoptosis. [score:1]
G, Western blot showing increased p27KIP1 BAX, p-γ/H2A, PARP and Caspase 9 in miR-17/20 transduced NAFA cells. [score:1]
F, miR-17/20 sensitizes NAFA cells to doxorubicin -induced apoptosis. [score:1]
B, Phase contrast images of miR-17/20 or control transduced MCF-7 cells after 36h treatment (15uM tamoxifen). [score:1]
The human miR-17/20 cluster's genomic location, chromosome 13q31, correlates with loss of heterozygosity in a number of different cancers including breast cancer (31, 32). [score:1]
In the current studies, Akt1 was required for miR-17/20 -mediated induction of p53 abundance. [score:1]
B, Annexin V staining as a marker of apoptosis in miR-17/20 transduced Akt1+/+ or Akt1−/− murine ErbB2 breast tumor cells, **p<0.01. [score:1]
The induction of apoptosis in miR-17/20 transduced NAFA cells was associated with induction of p27, Bax, p-γ-H2A, caspase 9 and cleaved PARP (Fig. 1G and Supplementary Figure S2). [score:1]
miR-17/20 transduction increased the sensitivity of NAFA cells to doxorubicin -induced apoptosis as assessed by TUNEL staining (Fig. 1F). [score:1]
At both 24h and 35h timepoints, control MCF-7 cells showed more resistance to tamoxifen than miR-17/20 transduced cells (Fig. 2C). [score:1]
The effect of miR-17/20 was next assessed in the NAFA cell line which was derived from an ErbB2 -induced mouse mammary tumor. [score:1]
Figure 3 A, Cell survival of miR-17/20 or control transduced MCF-7 cells after treatment with doxorubicin at indicated concentrations for 48h and 72h. [score:1]
Thus, miR-17/20 increased MCF-7 sensitivity to DNA damage inducing agents after doxorubicin or UV radiation (Fig. 1A and Supplemental Fig. S1). [score:1]
miR-17/20 increases the sensitivity of MCF-7 cells to doxorubicin. [score:1]
miR-17/20 increased MCF-7 cell sensitivity to tamoxifen. [score:1]
A, Cell survival of miR-17/20 or control transduced MCF-7 cells after treatment with doxorubicin at indicated concentrations for 48h and 72h. [score:1]
Doxorubicin treatment of MCF-7 cells enhanced miR-17/20 induction of p53 (Fig. 1D). [score:1]
miR-17/20Retroviral production and infection methods were described in detail before (40). [score:1]
In order to corroborate the effects of miR-17/20 on apoptosis, anti-miR-17 and anti-miR-20a were applied to block the function of endogenous miR-17 and miR-20a. [score:1]
miR-17/20 transduced cells and control were cultured in medium containing doxorubicin (0.05μM). [score:1]
miR-17/20 attenuated doxorubicin resistance in MCF-7 cell. [score:1]
In this regard, miR-17/20 levels were reduced in highly invasive breast cancer cell lines and node -positive breast cancer specimens (42). [score:1]
miR-17/20 increases tamoxifen sensitivity of MCF-7 cells. [score:1]
Similarly, miR-17/20 induced apoptosis in MCF-7 cells was associated with induction of Bax, Cyto C, Bcl-xs, caspase 3 and cleavage of caspase 9 and PARP (Fig. 2A). [score:1]
The mammary tumor cell lines were transduced with either miR-17/20 or control. [score:1]
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[+] score: 129
We grouped 22k mRNA (genes) into three groups, 20 K mRNA without miR-17 binding site (predicted with TargetScan), 2 K mRNA with miR-17 binding site that have context score larger than −0.3 (a score TargetScan used to measure how likely miR can inhibit target mRNA, the lower the score the more likely), another 277 mRNA with miR-17 binding site that have context score less than −0.3. [score:7]
To demonstrate that PMIS-miR-17-18 can also inhibit endogenous miR-17 from repressing BMPR2 expression, the BMPR2 3′UTR (untranslated region) was cloned into the luciferase vector and co -transfected with miR-17 or vector only and PMIS-miR-17-18 or vector only. [score:7]
Distributions of changes (0.1 unit bins) for mRNA UTRs containing no site (black line), site with a score >−0.30 (red line) and site with a score <−0.30 (blue line) shows mRNA targets of miR-17 are upregulated in PMIS-miR-17 transfected cells (Figures 8b and c). [score:6]
PTEN (phosphatase and tensin homolog) was identified in a screen for genes regulated by miR-17 and we show that inhibition of miR-17 by PMIS-miR-17-18 increased PTEN protein expression (Figure 2c). [score:6]
The miR-17 targets were separated into two groups based on TargetScan prediction score, one with a context+ score <−0.30 and the other with a context+ score >−0.30. [score:5]
The miR-17 inhibitor (PMIS-miR-17) reduced endogenous mature miR-17 levels to ~25% in 293 cells, whereas the miR-200c inhibitor (PMIS-miR-200c) does not change the level of miR-17 (Figure 2a). [score:5]
This miR inhibitor recovers over 90% of the inhibition of luciferase activity by endogenous miR-17 (Figure 1d). [score:5]
To construct the miR inhibitor clone vector, we replaced the miR-17 binding site with two BsmBI sites in the most effective inhibitor design. [score:5]
24, 29 Bim expression, a proapoptotic gene involved in B-cell development and a known target of miR-17, [38] was elevated in cells transfected with PMIS-miR-17 compared with cells with empty vector and PMIS-miR-200a (Figure 2c). [score:5]
These results demonstrate that PMIS-miR-17 specifically and effectively inhibit miR-17, which in turn results in the increase in transcripts targeted by miR-17. [score:5]
[27] miR-17 was used to test the inhibitor design, as miR-17 is one of highest expressed miRs. [score:5]
As expected, a single mutation of the inhibitor corresponding to the seed sequence (nts 2–8, mut-2 to 8) of miR-17 almost completely eliminated its effects (Figures 3a and b). [score:4]
Briefly, total RNA including miR from cells stably expressing PMIS vector, PMIS-miR-17 or PMIS-miR-17-18 was extracted. [score:3]
To test the efficiency of these different designs, the inhibitors were co -transfected with a miR-17 reporter that has the miR-17 binding site cloned after the luciferase gene into HEK 293FT cells (Figure 1d). [score:3]
The BMPR2 gene was identified as a target of miR-17 in PMIS-miR-17-18 bioinformatics analyses of transgenic mouse tissues. [score:3]
PMIS-miR-17 was specific for the inhibition of miR-17-5p and miR-106b-5p (Figure 8d). [score:3]
A similar IP used the Dicer antibody to pull down the inhibitor complex and Dicer was associated with PMIS-miR-17 (Figure 6b, lane 6). [score:3]
PMIS-miR-200c inhibits miR-200c to ~90% but does not affect miR-17 levels (Figure 2a). [score:3]
To determine if PMIS-miR-17 inhibited other miRs in cells, 293 cells were transduced with the lentiviral PMIS-miR-17 and randomly selected miRs were profiled by real-time PCR (q-PCR) (Taqman probes; Applied Biosystems, Waltham, MA, USA). [score:3]
Puromycin was added for selection and stable PMIS-miR-17 -expressing cells were used to analyze the stability of the PMIS. [score:3]
6 demonstrated 95% knockdown of miR-17 and 86% knockdown of miR-18a by RT-PCR (Figure 10a). [score:3]
A series of miR-17 antisense sequences (inhibitor) were constructed containing different flanking regions. [score:3]
Total RNA was extracted from 293FT cells stably expressing PMIS vector or PMIS-miR-17. [score:3]
Briefly, miR inhibitor of miR-17 with and without flanking double strand was produced by in vitro transcription. [score:3]
For AGO2 and DICER pull down, cell extract of 293FT cell stably expressing PMIS vector, PMIS-miR-200c or PMIS-miR-17 were incubated with AGO2 (Cell Signaling) and DICER (Abcam) antibodies. [score:3]
These results show that miR-17 and miR-18 levels were reduced by PMIS-miR-17-18 and this is consistent with other studies demonstrating miR degradation by inhibitors. [score:3]
To construct different designs of miR inhibitors for miR-17, we annealed and ligated the miR-17 binding site with a central bulge flanked by different sequences into pLL3.7 vector (Addgene, Cambridge, MA, USA) digested with HpaI and XhoI. [score:3]
Analyses of gene regulation by PMIS-miR-17. [score:2]
PMIS-miR-17-18 caused a twofold increase in luciferase activity, whereas miR-17 overexpression decreased luciferase activity 60% compared with controls (Figure 5c). [score:2]
Both miR-17 and miR-19 expression levels were decreased in the PMIS-miR-17-18-19-92 embryos compared with WT (Figure 11b). [score:2]
[37] Analyses of gene regulation by PMIS-miR-17To evaluate PMIS efficacy and specificity in a systematic analysis, we profiled whole-genome mRNA expression changes after transduction with PMIS-miR-17 in 293 cells. [score:2]
The miR-17 reporter was used as a control to show that PMIS-miR-200b-200a did not affect endogenous miR-17 activity. [score:1]
Sponge reporter was made by inserting six tandem binding sites for miR-17 into the psiCHECK2 vector. [score:1]
Furthermore, Bim transcripts levels were increased ~3-fold in PMIS-miR-17 cells (data not shown). [score:1]
These two PMIS mice show separation of the effects of the miR-17-92 cluster where miR-17 and -18 control skull width and miR-19-92 control width and anterior–posterior axis growth, but normal ossification. [score:1]
To evaluate PMIS efficacy and specificity in a systematic analysis, we profiled whole-genome mRNA expression changes after transduction with PMIS-miR-17 in 293 cells. [score:1]
The miR-17 reporter has a perfect miR-17 binding sequence used in previous reports. [score:1]
The complexes containing endogenous miR-17 and PMIS-miR-17 were pulled down by the AGO antibody (Figure 6a, lane 6). [score:1]
Cell miR Northern blot used 5′-end DIG (digoxigenin)-labeled mercury-locked nucleic acid miR detection probes for miR-17 and U6 from Exiqon (Woburn, MA, USA) according to the manufacturer's instructions. [score:1]
Distribution of mRNA fold change (PMIS-miR-17 vs PMIS vector) of these three groups was plotted with R using different colors. [score:1]
A miR sponge plasmid that has six tandem miR-17 binding sites was used as a control. [score:1]
For example, to construct the miR reporter for miR-17, two short oligos: miR-17 rf, 5′-TCGAATGACCCTACCTGCACTGTAAGCACTTTGCTCGAGCTGC-3′ and miR-17 rr, 5′-GGCCGCAGCTCGAGCAAAGTGCTTACAGTGCAGGTAGGGTCAT-3′ were annealed and cloned into psiCHECK2 vector digested by NotI and XhoI. [score:1]
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24
[+] score: 118
Mechanistically, we demonstrated that circ-ITCH up-regulates the expression of miR-17 and miR-224 target gene p21 and PTEN through ‘sponging’ miR-17 and miR-224, which suppressed the aggressive biological behaviors of BCa. [score:10]
We found that circ-ITCH partly rescued the inhibitory effect of miR-17 (or miR-224) on the expression of p21 and PTEN (Fig. 6k, l), which was agreed with the results of cell function. [score:5]
circ-ITCH acts as a tumor suppressor by a novel circ-ITCH/miR-17, miR-224/p21, PTEN axis, which may provide a potential biomarker and therapeutic target for the management of BCa. [score:5]
As shown in Fig. 5h, miR-17 or miR-224 mimics could partly attenuate the circ-ITCH overexpressing -mediated inhibition of proliferation in BCa cells (P < 0.05). [score:5]
Moreover, rescue experiments were conducted by co-transfecting circ-ITCH and miR-17 (or miR-224) mimics in BCa cells to assess whether the tumor-suppressing effect of circ-ITCH could be blocked by miR-17 (or miR-224) overexpression. [score:5]
Fig. 6Cir-ITCH modulated the expression of endogenous miR-17 and miR-224 targets p21 and PTEN. [score:5]
m Mode pattern of the cir-ITCH-miR-17/miR-224-p21/PTEN regulatory networkBesides, the combined transfection of circ-ITCH and miR-17 (or miR-224) was conducted to further assess p21 and PTEN expression. [score:4]
And we suggested a novel circ-ITCH/miR-17, miR-224/p21, PTEN signaling regulatory network in BCa, which may provide a potential biomarker and therapeutic target for the management of BCa. [score:4]
m Mode pattern of the cir-ITCH-miR-17/miR-224-p21/PTEN regulatory network Besides, the combined transfection of circ-ITCH and miR-17 (or miR-224) was conducted to further assess p21 and PTEN expression. [score:4]
Consequently, the findings proved that p21 and PTEN are direct targets of miR-17 (or miR-224). [score:4]
circ-ITCH modulated the miR-17 (or miR-224) targets p21 and PTEN. [score:3]
c and d A moderate negative correlation between the expression of cir-ITCH and miR-17/miR-224 was showed using Pearson correlation analysis. [score:3]
As shown in Fig. 5e, f, enforced expression of miR-17 or miR-224 significantly promoted the viability of cells (P < 0.05). [score:3]
k and l showed that mir-17 and miR-224 could partly decrease the protein expression level of p21 and PTEN which were promoted by cir-ITCH. [score:3]
MiR-17 and miR-224 are crucial molecules targeting anti-oncogenes (including p21 and PTEN) in multiple cancers [30– 33]. [score:3]
a and b miR-17 and miR-224 were up-regulated in BCa tissues as compared with adjacent normal tissues using qRT-PCR (n = 28, ** P < 0.01, Student’s t-test). [score:3]
Coincidently, by bioinformatic analysis (Starbase V2.0 [22], Circinteractome [23]), we found that both circ-ITCH and the 3′-untranslated region (UTR) of p21, PTEN share miRNA response elements (MREs) of miR-17 and mir-224, which suggested the association between circ-ITCH and p21/ PTEN in BCa. [score:3]
Above all, these data reflected that circ-ITCH suppressed BCa progression partly via abolishing the oncogenic effect of miR-17/miR-224. [score:3]
g and h Dual luciferase reporter assays demonstrated that p21 and PTEN are direct target of miR-17(* P < 0.05, ** P < 0.01, Student’s t-test). [score:3]
Further functional studies and luciferase reporter assay also verified that miR-17 and miR-224 promoted tumor progression by directly targeting p21 and PTEN in BCa. [score:3]
Correlation analysis revealed a moderate negative correlation between the expression of circ-ITCH and miR-17(r = − 0.50, P < 0.01) or miR-224 (r = − 0.55, P < 0.01) (Fig. 5c, d). [score:3]
On the contrary, transfection of miR-17 or miR-224 mimics significantly reduced the expression of p21 and PTEN (Fig. 6e, f). [score:3]
Given that p21 and PTEN share miRNA response elements with circ-ITCH, we next investigated whether miR-17 (or miR-224) targets p21 and PTEN and whether circ-ITCH exerts its anti-tumor effect by modulating the expression of p21 and PTEN (Fig.   6a). [score:3]
e and f miR-17 and miR-224 decreased the protein expression level of p21 and PTEN, individually. [score:3]
Taken together, these data demonstrated that circ-ITCH suppressed BCa progression by eliminating miR-17 (or miR-224) oncogenic effect and forming miR-17/miR-224-p21 axis (Fig. 6m). [score:3]
Therefore, we presented a novel regulatory axis formed by circ-ITCH-miR-17/miR-224-p21/PTEN in BCa. [score:2]
Circ-ITCH acts as a molecular sponge for miR-17 and miR-224. [score:1]
h miR-17 and miR-224 reversed the inhibitory effect of cir-ITCH on cell proliferation by CCK-8 assay (* P < 0.05, ** P < 0.01, compared to circ-ITCH, Student’s t-test. ) [score:1]
On the basis of the interaction of circ-ITCH and miR-17/miR-224, we next assessed the potential functional role of miR-17 and miR-224 in BCa by transfecting miRNA mimics. [score:1]
Meanwhile, FISH analysis in BCa cells showed that circ-ITCH was co-localized with miR-17 or miR-224 in the cytoplasm (Fig. 4h, i). [score:1]
Taken together, these results suggest that circ-ITCH acts as a sponge for miR-17 and miR-224 in BCa. [score:1]
b miR-17 was pulled down and enriched with cir-ITCH specific probe and then detected by qRT-PCR(** P < 0.01, Student’s t-test). [score:1]
Fig. 4Cir-ITCH acted as a sponge for miR-17 and miR-224. [score:1]
Nuclei was stained blue (DAPI), cir-ITCH was stained red, miR-17/miR-224 was stained green Additionally, the expression of miR-17 and miR-224 in 28 pairs of BCa tissues and adjacent normal tissues was measured and both miRNAs were increased in BCa tissues (P < 0.05, Fig.   5a, b). [score:1]
Nuclei was stained blue (DAPI), cir-ITCH was stained red, miR-17/miR-224 was stained greenAdditionally, the expression of miR-17 and miR-224 in 28 pairs of BCa tissues and adjacent normal tissues was measured and both miRNAs were increased in BCa tissues (P < 0.05, Fig.   5a, b). [score:1]
Analogously, biotin-coupled miR-17/miR-224 captured more circ-ITCH than biotin-coupled NC, indicating that miR-17/miR-224 could bind to circ-ITCH (Fig. 4d, f). [score:1]
a Putative miR-17/miR-224 binding sequence in the 3′-UTR of p21 and PTEN mRNA. [score:1]
We found that co-transfection of miR-17 (or miR-224) mimics and reporter plasmids strongly reduced the luciferase activity. [score:1]
Inversely, co-transfection of miR-17 (or miR-224) mimics and mutated vectors showed no obvious effect to the luciferase activity (Fig. 6g- j). [score:1]
Specific probes to circ-ITCH sequence and miR-17/miR-224 was used in situ hybridization. [score:1]
b Cir-ITCH arrested the EJ and T24 cell cycle at the G1/S phase, while miR-17 and miR-224 turned the G1/S transition. [score:1]
circ-ITCH Bladder cancer miR-17 miR-224 p21 PTEN Bladder cancer (BCa) has become one of the most prevalent malignancies worldwide, with an estimated 430,000 new cases diagnosed in 2012 [1]. [score:1]
In this study, by bioinformatic analysis (Starbase V2.0, Circinteractome), we found that circ-ITCH shares miRNA response elements (MREs) of miR-17 and mir-224, which might possibly bind with circ-ITCH in BCa. [score:1]
As shown in Fig.   4b, c, a specific enrichment of circ-ITCH, miR-17 and miR-224 was detected in the circ-ITCH pulled down pellet compared with control group, demonstrating that circ-ITCH could directly sponge miR-17 and miR-224. [score:1]
miR-17 and miR-224 promoted BCa cell proliferation. [score:1]
h and i RNA FISH for cir-ITCH and miR-17/miR-224 was detected in BCa cells, miR-17 and miR-224 were co-localized with cir-ITCH in cytoplasm (magnification, × 400). [score:1]
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[+] score: 114
As microRNA has to bind to its target gene for regulating cellular characteristics, we subsequently tested the expression of two direct target genes of miR-17, namely TCF3 and Smurf1. [score:7]
However, this effect disappeared after decrease of miR-17 expression by miR-17 inhibitor, which could be substantiated by gene expression analysis of osteoblast-related genes (Fig. 6E-F). [score:7]
Analyses on the molecular pathways involved in this repressive influence of p53 uncovered a reversion of the inhibitory effects of p53 on osteogenesis upon miR-17 overexpression with rescue of osteogenic differentiation of old BMMSCs and vice versa a restraint of miR-17 by p53 overexpression. [score:7]
Figure 5Up-regulation of miR-17 by miR-17 mimics reversed the effect of p53 on inhibiting osteogenic differentiation in old BMMSCs. [score:6]
We also elucidated the underlying mechanism that p53 restained the osteogenesis via modulating the trancription of pri-miR-17 and then affecting the expression of Smurf1, a direct target of miR-17. [score:6]
Since miR-17 were decreased obvious in old mice (bone, bone marrow and BMMSCs), we next used miR-17 mimics to up-regulated expression of miR-17 in old BMMSCs. [score:6]
After osteogenic induction of old BMMSCs for 14 d in vitro, alizarin red staining suggested that the osteogenic differentiation of BMMSCs was obviously enhanced after upregulating miR-17 expression (Fig. 5A). [score:6]
Rising of both p53 and miR-17 at the same time did not alter the level of Smurf1, demonstrate-ing that p53 regulates Smurf1 indirectly by inhibition of miR-17. [score:5]
MiR-17 is closely related to TCF3 and Smurf1, two direct target genes in the cells that miR-17 can bind and thus block these negative regulators of osteogenesis [25, 26]. [score:5]
On the basis of our previous results, miR-17 tends to target TCF3 in the normal microenvironment, while it aims to target Smurf1 under inflammatory conditions [25, 26]. [score:5]
Furthermore, the expression of pri-miR-17 and mature miR-17-92 family members significantly decreased upon overexpression of p53, suggesting that p53 can potently block the transcription of miR-17-92 (Fig. 4G, H). [score:5]
miR-17 was stable upregulated in BMMSCs by miR-17 mimics (= miR-17 mimics). [score:4]
Next, we transfected cells which were stably upregulated p53 with miR-17 mimics for following investigation of Smurf1 expression. [score:4]
Transfection of miR-17 mimics in stable upregulated p53 BMMSCs derived from young mice. [score:4]
These data of the staining results were supported by the transcriptional and protein analyses of the osteoblast-related genes Runx2, ALP and Osterix, which is illustrated in Fig. 5B-C. The in vitro results could be substantiated in vivoby the HA/TCP transplantation experiments earlier described, as after a transplantation perio d for 8 weeks, BMMSCs from old mice formed plenty of new bony structures around the HA/TCP granules when miR-17 was upregulated in the transplanted cells (Fig. 5D-E). [score:4]
Our previous research revealed that miR-17 acted as a negative regulator of osteogenesis in a physiological microenvironment, but contrarily as promoter for osteoblastic commitment of tissue-specific MSCs under inflammatory pathological conditions due to targeting different genes [25, 26]. [score:4]
Contrarily, TCF3 remained unaffected by any of the treatments, providing an indication that the target gene miR-17 binds to is dependent on the microenvironment and the cellular components involved. [score:3]
Importantly, the expression pattern of p53 and miR-17 were exactly opposite in BMMSCs from both 4 and 16-month-old mice during osteogenic differentiation (Fig. 4D-F). [score:3]
The western blot data showed that the Smurf1 level was almost unchanged (Fig. 6B), suggesting that p53 affects the expression level of Smurf1 mainly through miR-17, which is illustrated as an overview mo del in Fig. 7. Figure 6Smurf1 plays an important role in miR-17 -mediated osteogenic differentiation of BMMSCs. [score:3]
Our data showed that the transfection efficiencies of miR-17 mimics and inhibitor persisted at least for 14 d (Supplementary Fig. 3). [score:3]
As senescent cells have been shown to display elevated expression profiles of inflammatory molecules, the trend that miR-17 has an affinity for binding Smurf1 can be interpreted in this manner. [score:3]
The western blot data showed that the Smurf1 level was almost unchanged (Fig. 6B), suggesting that p53 affects the expression level of Smurf1 mainly through miR-17, which is illustrated as an overview mo del in Fig. 7. Figure 6Smurf1 plays an important role in miR-17 -mediated osteogenic differentiation of BMMSCs. [score:3]
Real-time PCR analyses showed a significant decrease of miR-17, miR-18a, miR-20a and miR-92a in bone tissues, reduction of all family members in bone marrow and reduced expression of miR-17, miR-18a, miR-19a, miR-20a and miR-92a could be observed in BMMSCs (Fig. 4A-C). [score:3]
Considering that senescence can be correlated to a chronic inflammatory microenvironment (Fig. 1E) and as our previous research revealed that miR-17 acts as positive regulator of osteogenesis in an inflammatory microenvironment [25], we investigated the expression pattern of miR-17-92 cluster in BMMSCs from young and old mice. [score:2]
MiR-17 mimics and inhibitor (Ribobio, Guangdong, China) were transfected into BMMSCs at a concentration of 50 nM with the siPORT NeoFX Transfection Agent (Ambion). [score:2]
Primary-miR-17 and GAPDH as endogenous control were commercially purchased (Invitrogen). [score:1]
Figure 7Schematic diagram of p53/miR-17/Smurf1 cascade. [score:1]
In addition, miR-17 mimics particular rescue the osteogenesis of old BMMSCs both in vitro and in vivo. [score:1]
Smurf1 plays an important role in the p53/miR-17 cascade acting on osteogenesis of BMMSCs. [score:1]
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[+] score: 105
Simulations of transcriptional networks were carried out using the Gillespie exact stochastic simulation algorithm, programmed and analysed using R based on a microRNA feed-forward mo del [8] to simulate CD69 protein expression in resting T cells (unfilled histogram), in a scenario where activation increases Cd69 mRNA but the expression of miR-17 and miR-20a remained the same as in resting T cells (activated without microRNA FFL, filled red histogram), and in a scenario where activation increases both Cd69 mRNA and miR-17 and miR-20a expression (activated with microRNA FFL, filled grey histogram). [score:7]
The coordinated regulation of miR-17, miR-20a and Cd69 in response to TCR signaling provides a potential mechanism for restricting cell-to-cell variability of microRNA target gene expression. [score:6]
Interestingly, this graded increase in Cd69 mRNA was accompanied by a proportional upregulation of miR-17 and miR-20a expression (Fig. 5A). [score:6]
Hence, activation signals of increasing strength induce a proportional upregulation of the microRNAs miR-17 and miR-20a and the target mRNA Cd69. [score:6]
This result is consistent with our experimental data where the mean and CV of activation -induced CD69 expression were significantly elevated in Dicer -deficient thymocytes: in the absence of a functional microRNA biogenesis pathway, the activation -driven increase in Cd69 mRNA was not balanced by increased miR-17 and miR-20a expression. [score:5]
B) Perceived signal strength varies among individual DP thymocytes and determines the expression of Cd69, miR-17 and miR-20a (microRNA expression is normalised to snoRNA-135 and snoRNA-202). [score:5]
Our data show that the expression of these microRNAs is induced together with Cd69 mRNA in response to TCR signals, and that the expression of CD69 protein, Cd69 mRNA and miR-17/miR20a is proportional in thymocytes. [score:5]
miR-17 and miR-20a form an incoherent positive feedforward loop with the target mRNA Cd69 miRNA expression responds to T-cell activation signals [34, 35, 39– 45]. [score:5]
Increasing expression of CD69 protein correlated with increasing Cd69 mRNA levels, and with incremental expression of miR-17 and miR-20a (Fig. 5B). [score:5]
The mo del predicts that thymocyte activation with co-regulation of Cd69 mRNA and miR-17/miR-20a reduces the mean (887 versus 1300) and the CV (10.2 versus 14.6) of CD69 expression compared to the regulation of Cd69 mRNA alone (P<10 [–4]). [score:4]
In contrast, induction of Cd69 mRNA without upregulation of miR-17/miR-20a results in a higher mean (1300) and increased CV (14.6%, activated without microRNA FFL, filled red histogram in Fig. 5D, P<10 [–4]). [score:4]
This mo del predicts that thymocyte activation results in mean CD69 expression of 887 with a CV of 10.2% when Cd69 mRNA and miR-17/miR-20a are induced together (activated with microRNA FFL, filled grey histogram in Fig. 5D). [score:3]
A dual fluorescence reporter system identifies endogenous microRNAs that target the Cd69 3'UTR in DP thymocytesThe Cd69 3'UTR contains predicted sites for miR-181, miR-130 and miR-17/20 (http://www. [score:3]
Mechanistically, T cell receptor signaling induces both Cd69 and miR-17 and miR-20a, two microRNAs that target Cd69. [score:3]
miR-17 and miR-20 form an incoherent positive feedback loop with the target mRNA Cd69. [score:3]
At a fixed extracellular signal of 1u/ml H57, the fold-change in miR-17 and miR-20 relative to CD69 negative DP and normalised to snoRNA-135 was proportional to the expression of CD69 (n = 3, mean ± SD). [score:3]
miR-17 and miR-20a form an incoherent positive feedforward loop with the target mRNA Cd69. [score:3]
A) The strength of activation signals (0.1, 1, 10 μg/ml H57) determines the expression of Cd69, miR-17 and miR-20a (normalised to snRNA-135 and -202, n = 2–3, mean ± SE). [score:3]
Next, we asked how miR-17 and miR-20a expression was related to the range of responses by individual cells to a uniform extracellular signal. [score:3]
However, deletion of the miR-130 and particularly the miR-17/20 site resulted in increased eGFP expression in wild type CD4+ T cells (Fig. 3C). [score:3]
Since the miR-17-92 cluster encodes microRNAs that target the Cd69 3'UTR, including miR-17 and miR-20a (Fig. 3), we investigated how the expression of miR-17 and miR-20a was affected by the activation of DP thymocytes. [score:3]
The predicted sites for miR-130 and miR-17/20 within the Cd69 3' UTR also affected eGFP reporter gene expression in DP thymocytes. [score:3]
org) and there is firm experimental evidence for Cd69 regulation by miR-181a, miR-130 and the miR-17-92 cluster (which encodes the microRNAs miR-17, -18, -19a, -19b, -20a, and -92 [34] in T lymphocytes [31– 33]. [score:2]
These findings suggest that miR-17, miR-20a and Cd69 are co-regulated. [score:2]
A different mechanism applies to the regulation of CD69 by miR-17 and miR-20a, two microRNAs of the miR-17-92 cluster. [score:2]
To implement a more specific mo del of CD69 regulation we estimated the mRNA copy numbers for Cd69 and the microRNA copy numbers for miR-17 and miR-20a in resting and activated T cells (see legend Fig. 5D). [score:2]
miReduce analysis [27] of 3'UTR motifs associated with post-transcriptional de-repression in Lck-Cre DP thymocytes (see GSE57511) showed enrichment for microRNAs miR-181, miR-17 and miR-142 (Fig. 1B). [score:1]
Based on reported cloning frequencies (89884 miR-181a-1/2 per 10 [6] microRNAs in DP, 1465 miR-17 per 10 [6] microRNAs in DP thymocytes, and 1050 miR-20a per 10 [6] microRNAs in DP [64]. [score:1]
The Cd69 3'UTR contains predicted sites for miR-181, miR-130 and miR-17/20 (http://www. [score:1]
As detailed in the legend to S4 Fig., the number of Cd69 mRNA copies was estimated as 0 in resting and 6 in activated cells, miR-17 and miR-20 were estimated as 6–12 copies per cell the resting state and 30–60 copies per cell after activation. [score:1]
This was confirmed by mo deling the experimentally estimated copy numbers of Cd69 mRNA and miR-17 and miR-20a in resting and activated T cells. [score:1]
DP thymocytes contain ∼6–12 copies of miR-17 and miR-20a per cell, and our quantitative PCR data show that this number increases by 5–10-fold in response to TCR signaling. [score:1]
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[+] score: 90
In parallel, p300 upregulation leads directly or indirectly to increased expression of miR-17∼92 cluster members that provides a brake on both processes and retards secondary maladaptive events. [score:8]
The 3′UTR regions of several p300 -upregulated angiogenic genes, including HIF1a [26], [27] as well as p300 itself, contain the shared target site for miR-17-5p and 20a. [score:6]
Interestingly, p300 overexpression in vivo was also associated with relative upregulation of several members of the anti-angiogenic miR-17∼92 cluster in vivo. [score:6]
Relative expression of most members of the 17∼92 cluster was similar in all 4 cardiac chambers and in other organs, however, significant downregulation of miR-17-3p and miR-20a occurred between 1 and 8 months of age in both wt and tg mice. [score:6]
This decline in miR-17∼92 cluster expression was accompanied by a steady rise in expression of VefgA at both mRNA (Figure 3B) and protein (Figure 3E) levels, suggesting that members of this cluster play a repressive role in myocardial vascular growth. [score:5]
Relative expression of miR-17∼92 cluster members and an unrelated microRNA, miR-199, in Adp300- vs AdGFP -expressing cardiac myocytes. [score:5]
Two members of the miR-17∼92 cluster, miR-17-3P and miR-20a, were upregulated in p300tg relative to WT myocardium in both young (1 month) and adult (9 month) mice (Figure 3A). [score:4]
Co-regulatory Expression of p300 and miR-17∼92. [score:4]
Confirming this finding, both miR-17-3p and miR-20a were upregulated in neonatal rat ventricular myocytes following adenoviral transduction of p300. [score:4]
Importantly, this positive effect is counterbalanced by upregulation of members of the anti-angiogenic miR17∼92 cluster [14], [15]. [score:4]
A. Relative upregulation of miR-17-3p and miR-20a in p300 transgenic mice. [score:4]
0079133.g004 Figure 4 A. Comparative expression of 7 members of the miR-17∼92 cluster in normal tissues. [score:3]
In addition, miR-20a has been recently reported to reduce apoptosis following hypoxia-reoxygenation of cardiomyocytes [36], and overexpression of the miR-17∼92 cluster modestly improved cardiac function in a mouse mo del of myocardial infarction [37], suggesting that members of this cluster may have more general protective effects during oxidative or biomechanical stress. [score:3]
Deletion of miR-17∼92 is embryonic lethal, due in part to defects in cardiac development [14], [44] which could arise from altered vasculogenic regulation. [score:3]
A. Comparative expression of 7 members of the miR-17∼92 cluster in normal tissues. [score:3]
Inverse Expression of miR-17∼92 and VegfA. [score:3]
B. Comparative miR-17∼92 cluster expression in normal murine heart. [score:3]
Impact of tissue type, p300 content and genomic context on miR-17∼92 cluster expression. [score:3]
All members of the miR-17∼92 cluster were expressed in all tissues examined (Figure 4A) and in all 4 chambers of the heart (Figure 4B) consistent with previous reports [25]. [score:3]
Transduction of miR-20a, but not a control miR, repressed expression of a luciferase construct containing the p300 3′UTR miR17-5p/miR-20a binding site (Figure 5G, left); mutagenesis of this site eliminated miR-20a repression (Figure 5G, right). [score:3]
Reciprocal regulation of myocardial VegfA and miR-17∼92 with age. [score:2]
Although we were unable to confirm direct regulation by p300, MiR-17∼92 harbors conserved MEF2 and GATA binding sites within 500 bp downstream of the transcriptional start site that may reflect the presence of a p300-responsive enhancer (Figure 4E). [score:2]
Members of the miR-17∼92 cluster can retard endothelial sprouting in Matrigel [41]; in addition, loss of miR-17∼92 is seen in several forms of cancer [15], [42], [43]. [score:1]
D. Gain of p300 induces multiple members of miR-17∼92 cluster. [score:1]
E. Genomic structure of miR17∼92 cluster. [score:1]
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[+] score: 76
Consistently with the up-regulation of the miR-17∼92 cluster in tumors, miR-92a is highly expressed in colon cancer tissues and targets the anti-apoptotic molecule BCL-2-interacting mediator of cell death (BIM) and the tumor suppressor PTEN [9], [10]. [score:10]
However, only miR-17 and miR-20a were shown to directly target TGF-β receptor II and miR-18a was reported to target the TGF-β down-stream signaling proteins Smad2 and Smad4 (for review see [4]). [score:6]
The polycistronic miR-17∼92 cluster, which comprises the mature miRNAs miR-17, -18a, -19a/b, -20a, and miR-92a, contributes to the pathogenesis of a variety of human diseases, including cancer, cardiovascular disease and congenital developmental defects [2], [4], [5]. [score:6]
The miR-92a [−/−] phenotype partially copies the previously reported skeletal development defects of miR-17∼92 cluster knock-out mice [6] and of humans with a reduced expression of the cluster [7]. [score:5]
Moreover, the expression of the closely related family members miR-17 (which only differs from miR-20a by 2 nucleotides) and miR-19a (which only differs from miR-19b by one nucleotide) was not significantly changed, and might compensate for the reduction in miR-20a and miR-19b expression, respectively. [score:5]
Whereas several reports showed that Shh stimulates the expression of the cluster [4], [19], [20], an interference of the miR-17∼92 cluster with the Shh pathway is only supported by indirect evidence showing that the tumorigenic effects of the miR-17∼92 cluster is dependent on the loss of the Shh receptor Patched [19]. [score:4]
These findings identify a regulatory function for miR-92a in growth and skeletal development, whereas miR-92a is not responsible for other defects in heart or B cell development that were observed in miR-17∼92 cluster mutants. [score:4]
Figure S1MiR-92a deficiency in mice moderately effects the expression of the other miR-17∼92 cluster members in muscle and skeletal tissue. [score:3]
Furthermore, mRNA expression of Runx2 and type I collagen was significantly lower in bones from miR-17–92 [+/Δ] mice [17]. [score:3]
Expression levels of the miR-17∼92 cluster members in lower leg muscles of the hind limbs (A) and femurs (B) of WT, miR-92a [+/−] and miR-92a [−/−] mice. [score:3]
Several key features of this phenotype were mimicked in mice harboring targeted deletion of the miR-17∼92 cluster [7]. [score:3]
The cluster is highly expressed in bone cells, and osteoblasts from miR-17–92 [+/Δ] mice showed a lower proliferation rate, alkaline phosphatase activity and less calcification in vitro [17]. [score:3]
The constitutive and conditional deletion of miR-92a-1, which is expressed by the miR-17∼92 cluster, was generated by homologous recombination in 129Sv/Pas embryonic stem (ES) cells by genOway (Lyon, France). [score:3]
Moreover, the miR-17∼92 cluster indirectly interacts with the Sonic Hedgehog (Shh) axes, a pathway that has been implicated in skeletal development [19], [20]. [score:3]
The mechanism by which the miR-17∼92 cluster affects skeletal development is not well explored. [score:2]
These data may suggest that the miR-17∼92 cluster regulates bone metabolism. [score:2]
Deletion of the miR-17∼92 cluster resulted in defects of heart and lung development, and homozygote mice postnatally died [6]. [score:2]
The miR-17∼92 cluster has been shown to modulate TGF-β signaling, one of the most important signaling pathways controlling skeletal development. [score:2]
Therefore, it is unclear whether there is a direct effect of miR-17∼92 loss of function on the activity of the Shh pathway. [score:2]
Thus, miR-92a [−/−] mice were smaller than their littermates, showed reduced skull size and tibia length and exhibit the typical shortening of the 5 [th] mesophalanx bone as it has been reported for miR-17∼92 [Δ/+] mice [7]. [score:1]
A germline hemizygous deletions of MIR17HG, encoding the miR-17∼92 polycistronic miRNA cluster, was observed in patients with Feingold syndrome [7], which is an autosomal dominant syndrome whose core features are microcephaly, relative short stature and digital anomalies, particularly brachymesophalangy of the second and fifth fingers and brachysyndactyly of the toes [8]. [score:1]
However, the contribution of miR-92a for the observed defects in miR-17∼92 cluster deficient mice has not been elucidated. [score:1]
MiR-92a [−/−] mice have no hematopoietic defectsSince miR-17∼92 cluster knock-out mice revealed defects in hematopoietic cell development, we characterized the hematopoietic phenotype of miR-92a [−/−] mice. [score:1]
Since miR-17∼92 cluster knock-out mice revealed defects in hematopoietic cell development, we characterized the hematopoietic phenotype of miR-92a [−/−] mice. [score:1]
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[+] score: 70
miR-20a and miR-17 directly bind to the 3′ untranslated region (UTR) of FBXO31 and inhibit FBXO31 mRNA and protein expression in human GC cells. [score:8]
Human miR-20a mimics, miR-17 mimics, control mimics, miR-20a inhibitors, miR-17 inhibitors and control inhibitors were synthesized from RiBoBio (Guangzhou, China). [score:7]
Our results indicated that miR-17 and miR-20a mimics inhibited, whereas miR-17 and miR-20a inhibitor increased, the expression of FBXO31. [score:7]
Therefore, the increased miR-17(20a) expression in GC tissue contributed to the down-regulation of FBXO31 partly. [score:6]
The overexpression of miR-17 and miR-20a contributed to the down-regulation of FBXO31 in GC tissues partly. [score:6]
Therefore,we transfected the mimics or inhibitor of miR-20a or miR-17 into GC cells BGC-823 and HGC-27 and used qRT-PCR and western blot to detect the expression of FBXO31. [score:5]
These results suggest miR-17(20a)-FBXO31-CyclinD1 pathway may be a potential therapeutic target of GC. [score:3]
We found that miR-20a or miR-17 mimics decreased, whereas miR-20a or miR-17 inhibitor increased, the mRNA and protein level of FBXO31, respectively (Fig. 3 A-F). [score:3]
Clinically,we found that the expression of miR-17 and miR-20a in tumor tissue was significantly higher than that in surrounding normal mucosa. [score:3]
FBXO31 expression is negatively associated with miR-20a and miR-17 in primary GC tissues. [score:3]
Therefore, we detected whether FBXO31 were regulated by miR-17 and miR-20a. [score:2]
Two miR-20a and miR-17 complementary sequences GCACTTT in the 3' UTR were mutated singly or together to remove complementarity by use of a QuikChange site-directed mutagenesis kit with pMIR-FBXO31/wt as the template. [score:2]
To further determine whether FBXO31 was a direct target of miR-20a and miR-17,we constructed a vector containing the 3'UTR of FBXO31 and luciferase reporter vector pMIR-REPORT (pMIR-FBX) and investigated the effect of miR-20a and miR-17 on the luciferase activity of pMIR-FBX. [score:2]
In all, 37/56 (66.1%) and 38/56 (67.9%)of the clinical GC specimens showed increased expression of miR-20a and miR-17,respectively, as compared with surrounding normal mucosa (Fig. 4A and 4B). [score:2]
Finally, we investigated whether miR-17 and 20a were up-regulated in primary GC tissues and associated with FBXO31. [score:2]
FBXO31 was negatively regulated by miR-17 and 20a. [score:2]
A highly significant negative correlation between miR-17 (20a) and FBXO31 was observed in these GC samples. [score:1]
Figure 4 (A and B) qRT-PCR analysis of miR-20a (A) and miR-17 (B) level in 56 paired human GC and adjacent normal gastric mucosa tissues. [score:1]
Therefore, the second region of the 3' UTR of FBXO31 is important in binding with the miR-20a and miR-17. [score:1]
The 217bp 3' -UTR sequence of human FBXO31 gene containing miR-20a and miR-17 binding sites was amplified and inserted into the SpeI/HindIII sites of the pMIR-REPORT luciferase vector (named as pMIR-FBXO31/wt). [score:1]
Statisticalanalysis showed that FBXO31 were highly correlated with miR-20a and miR-17 levels in GC samples (P<0.0001) (Fig. 4E and 4F). [score:1]
Figure 3 (A) miR-20a and miR-17 were analyzed with qRT-PCR in BGC-823 and HGC-27 cells transfected with miR-20a, miR-17 mimics or control mimics. [score:1]
Furthermore,the two members of miR-17-92 cluster, miR-17 and 20a, are important markers for GC [39, 40]. [score:1]
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[+] score: 69
Indeed, overexpression of miR-17 and miR-20a inhibited senescence in primary human fibroblasts by blunting the activation of p21 [WAF1], while inhibition of miR-17 caused senescence in anaplastic thyroid cancer cells (Takakura et al., 2008; Hong et al., 2010). [score:7]
Furthermore, miR-17–92 expression is consistently down-regulated in multiple mo dels of aging, i. e. after irradiation (Maes et al., 2008), p53 induction (Brosh et al., 2008), or stress -induced senescence (Li et al., 2009), and in old human skin, bone-marrow-derived mesenchymal stem cells, T cells (Hackl et al., 2010), and peripheral blood mononuclear cells (Noren Hooten et al., 2010). [score:6]
The pro-oncogenic activity of miR-17–92 partially involves the regulation of the ECM proteins CTGF and thrombospondin-1 (TSP-1) by the cluster members miR-18 and miR-19, through sequence-specific targeting within the 3′-untranslated region (3′-UTR) of these gene transcripts (Supporting information Fig. S1) (Dews et al., 2006). [score:6]
In conclusion, our study is the first to show that miRNA expression of the miR-17–92 cluster changes with cardiac aging and associates decreased miR-18a, miR-19a, and miR-19b expression with age-related remo deling in the heart. [score:5]
At 104 weeks of age, HF-prone mice had significantly reduced expression levels of miR-17, miR-18a, miR-19a, miR-19b, miR-20a, and miR-92a-1 as compared to 12-week littermates (Fig. 2C and Supporting information Table S1), coinciding with the observed increased presence of their targets TSP-1 and CTGF. [score:4]
Aging of HF-resistant mice, on the other hand, was accompanied by significantly enhanced expression of these miRNAs, except for miR-17 and miR-20a (Supporting information Table S1). [score:3]
Table S2 Fold change expression profiles of miR-17–92 cluster members in cardiomyocytes in vitro. [score:3]
In the present study, we showed extensive changes in the expression of the miR-17–92 cluster in a mo del of age-related heart failure in mice. [score:3]
CTGF and TSP-1 have been identified as target genes of the miR-17–92 cluster (Dews et al., 2006), more specifically of the cluster members miR-18a and miR-19a/b (Suarez et al., 2008; Ohgawara et al., 2009). [score:3]
RT-PCR analysis showed that the expression levels of all members of the miR-17–92 cluster were reduced in aged cardiomyocytes, except miR-92a-1 (Fig. 4D and Supporting information Table S2). [score:3]
Tumor suppressor mechanisms can induce cellular senescence and contribute to the aging process (Campisi, 2003), turning the miR-17–92 cluster into a potential mediator of aging. [score:3]
Table S1 Fold change expression profiles of miR-17–92 cluster members in HF resistant and HF prone mice at different ages. [score:3]
HF-prone hearts have more interstitial fibrosis and increased levels of CTGF and TSP-1. Opposite cardiac miR-17–92 cluster expression profiles in HF-resistant and HF-prone aging. [score:3]
Originally, the miR-17–92 cluster was linked to tumor genesis, and transcription of the cluster was found to be directly activated by the proto-oncogene c-Myc (He et al., 2005) [reviewed in(van Haaften & Agami, 2010)]. [score:2]
The miR-18/19 – CTGF/TSP-1 axis is regulated in aged cardiomyocytes in vitroTo gain further insight into the role of the miR-17–92 cluster in aging of cardiomyocytes, neonatal rat cardiomyocytes (NRCMs) were aged in vitro, and miRNA levels were determined. [score:2]
We found opposite expression profiles of the miR-17–92 cluster in HF-prone aging compared to aging with preserved cardiac function. [score:2]
The three miR-17–92 cluster members miR-18a, miR-19a, and miR-19b specifically target the ECM proteins CTGF and TSP-1. To investigate the role of these genes in human HF, we studied their expression profiles in cardiac biopsies of idiopathic cardiomyopathy (ICM) patients at old age with a moderately decreased or preserved systolic function (ejection fraction (EF) between 40 and 55%) (Paulus et al., 2007) and severely impaired cardiac function (EF < 30%) and compared them to young ICM subjects. [score:2]
Interestingly, cardiogenesis was severely hampered in mice deficient for miR-17–92, suggesting an important role for this cluster in cardiac development (Ventura et al., 2008). [score:2]
Initially, the miR-17–92 cluster was linked to cancer pathogenesis and was thought to be pro-tumorgenic because of its regulation by c-Myc (Dews et al., 2006). [score:2]
To gain further insight into the role of the miR-17–92 cluster in aging of cardiomyocytes, neonatal rat cardiomyocytes (NRCMs) were aged in vitro, and miRNA levels were determined. [score:1]
From the six members of the miR-17–92 cluster, miR-18a, miR-19a, and miR-19b were among the most strongly repressed miRNAs in aged cardiomyocytes and hearts of old failure-prone mice. [score:1]
This cluster encodes six miRNAs (miR-17, miR-18a, miR-19a, miR-19b, miR-20a, and miR-92a-1) that are located within an 800-base pair region of human chromosome 13. [score:1]
In addition, we demonstrate that the miR-17–92 cluster is part of the senescence signature of the aged cardiomyocyte. [score:1]
These reports are in line with our data showing repression of the miR17–92 cluster in old failing hearts. [score:1]
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[+] score: 66
The key reasons are as follows: Bim and Stat3 genes harbor miR-20a binding sites, and c-Kit and Socs3 genes harbor miR-19 binding sites, which are conserved across different phyla (ie, human, monkey, mouse, and rat) (Figures 8A, B and S7A, B); Bim is identified as direct targets of miR-17, 43, 44 miR-20a, [44] and miR-92a; [44] Stat3 is identified as direct targets of miR-17 45, 46 and miR-20a; 45, 46 Socs3 is identified as a direct target of miR-19a; [47] and Bim, Stat3, c-Kit, and Socs3 have been demonstrated to be implicated in the process of spermatogenesis. [score:10]
Yan HJ Liu WS Sun WH miR-17-5p inhibitor enhances chemosensitivity to gemcitabine via upregulating Bim expression in pancreatic cancer cells. [score:8]
Guo L Xu J Qi J MicroRNA-17-92a upregulation by estrogen leads to Bim targeting and inhibition of osteoblast apoptosis. [score:7]
Zhang M Liu Q Mi S Both miR-17-5p and miR-20a alleviate suppressive potential of myeloid-derived suppressor cells by modulating STAT3 expression. [score:7]
He M Wang QY Yin QQ HIF-1alpha downregulates miR-17/20a directly targeting p21 and STAT3: a role in myeloid leukemic cell differentiation. [score:7]
The miR-17-92 cluster and its 6 different mature microRNAs, including miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR-92a, play important roles in embryo development, immune system, kidney and heart development, adipose differentiation, aging, and tumorigenicity. [score:3]
Ventura A Young AG Winslow MM Targeted deletion reveals essential and overlapping functions of the miR-17 through 92 family of miRNA clusters. [score:3]
16– 19 The in situ hybridization analysis on adult testes revealed that the miR-17 is highly expressed in early stages of germ cells and greatly decreased as germ cells mature, and miR-20a is mainly detected in the spermatogonia and preleptotene spermatocytes. [score:3]
Patel V Williams D Hajarnis S miR-17∼92 miRNA cluster promotes kidney cyst growth in polycystic kidney disease. [score:3]
[18] Moreover, the in situ hybridization analysis shows that pri-miR-17-5p expression in human testes is lowest in a subset of spermatogonia and early spermatocytes close to the seminiferous tubule edge, and is increased with germ cell maturation proceeding toward lumen of the seminiferous tubule. [score:3]
Dakhlallah D Batte K Wang Y Epigenetic regulation of miR-17∼92 contributes to the pathogenesis of pulmonary fibrosis. [score:2]
The miR-17-92 gene cluster encodes 6 miRNAs of 4 miRNA families: the miR-17 family including miR-17-5p and miR-20a, the miR-18 family (miR-18a), the miR-19 family (miR-19a and miR-19b-1), and the miR-92 family. [score:1]
As shown in Figure S1A, to delete miR-17-92 in adult mice, the miR-17-92fl [/fl]Cre-ERT2 mice were obtained after 2 rounds of mating. [score:1]
To obtain miR-17-92fl [/fl]Cre-ERT2 mice, we identified the Cre gene after the first crossing, and then we identified homozygous miR-17-92fl [/fl] and Cre -positive mice after the second crossing. [score:1]
Currently, increasing evidence indicates that some members of miR-17-92 cluster may be critical players in spermatogenesis, including miR-17, miR-18a, and miR-20a. [score:1]
Deletion of miR-17-92 in AdultAs shown in Figure S1A, to delete miR-17-92 in adult mice, the miR-17-92fl [/fl]Cre-ERT2 mice were obtained after 2 rounds of mating. [score:1]
[21] The miR-17-92fl [/fl] mice (B6;129S4-Mirn17-92tm1.1Tyj/J: Stock Number: 008459) [23] were obtained from the Jackson Laboratory. [score:1]
[4] The miR-17-92 gene cluster encodes 6 miRNAs of 4 miRNA families: the miR-17 family including miR-17 and miR-20a, the miR-18 family (miR-18a), the miR-19 family (miR-19a and miR-19b-1), and the miR-92 family. [score:1]
UT: untreated with tamoxifen; D1, D7, D14, D21 and D28 represent the 1st, 7th, 14th, 21st, and 28th days after the last tamoxifen injection in miR-17-92fl [/fl]/hUb-Cre-ERT2 mice, respectively. [score:1]
Then, the 8-week-old miR-17-92fl [/fl] Cre-ERT2 mice were treated by intraperitoneal injection of 20 mg/mL tamoxifen in corn oil (0.2 mg/g body weight, once per day for 5 days) to conditionally delete miR-17-92 cluster in adult mice. [score:1]
Han YC Vidigal JA-O Mu P An allelic series of miR-17 approximately 92-mutant mice uncovers functional specialization and cooperation among members of a microRNA polycistron. [score:1]
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[+] score: 64
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]
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]
The circulating miRNA targets that were up-regulated following CLP belong not only to the miR-17∼92 cluster but also to its evolutionary paralogs, miR-106a∼363 (miR-106a, miR-18b, miR-20b, miR-19b-2, miR-92a-2, and miR-363) and miR-106b∼93 (miR-106b, miR-93, miR-25). [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]
miR-17, miR-20a, and miR-106a all specifically bind to the same seed sequence within the 3′-untranslated region (UTR) of signal-regulatory protein α (SIRPα), an essential signaling molecule that modulates leukocyte -mediated inflammatory responses and are inversely correlated with SIRPα expression in various cells [42]. [score:6]
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]
miR-17 and miR-20a belong to a group of commonly overexpressed miRNAs, the miR-17∼92 cluster, which is located on mouse chromosome 14 (13 in humans) and comprises 7 mature miRNAs (miR-17-5p and, miR-18a, miR-19a and b, miR-20a, and miR-92a). [score:3]
Both in vitro and in vivo assays demonstrate that miR-17, miR-20a, and miR-106a regulate macrophage infiltration, phagocytosis, and proinflammatory cytokine secretion by targeting SIRPα [42]. [score:3]
Numerous components of the “extended” ASK1 signalosome are potentially targeted by miRNAs encoded by the miR-17∼92 cluster [40]. [score:3]
This cluster is directly transactivated by c-Myc, a transcription factor initially linked to tumorigenesis [38], which has made the miR-17∼92 cluster the focus of attention in higher vertebrate research. [score:2]
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[+] score: 63
Both Th1 and Th2 cultured cells induced from STAT6 -deficient mice showed higher levels of miR-17-5p expression compared with corresponding WT Th cells, suggesting a novel critical role of IL-4R/STAT6-signaling in the down-regulation of miR-17 expression (Fig. 3C). [score:7]
Figure 4 Tumor bearing conditions down-regulate miR-17-5p expression in T cells. [score:6]
When healthy donor-derived CD4 [+ ]T cells were stimulated with rhIL-2, anti-CD3 and anti-CD28 mAbs, consistent with the mouse data, addition of rhIL-4 in the cultures suppressed expression of miR-17-5p (Fig. 4C). [score:5]
Interestingly, the tumor bearing condition did not suppress miR-17-5p expression by CD4 [+ ]T cells in STAT6 [-/- ]mice. [score:5]
Thus both IL-4 and GBM-bearing conditions suppress miR-17-5p expression in CD4 [+ ]T cells. [score:5]
Jurkat human T cell leukemia cells (American Type Culture Collection) were transduced by either one of the following pseudotype lentiviral vectors: 1) control vector encoding GFP; 2) the 17-92-1 expression vector encoding miR-17 18 and 19a, or 3) the 17-92-2 expression vector encoding miR 20, 19b-1, and 92a-1. All vectors were purchased from SBI. [score:5]
Jurkat cells were transduced by either one of the following pseudo typed lentivirus vectors: 1) control vector encoding GFP; 2) the 17-92-1 expression vector encoding miR-17 18 and 19a, or 3) the 17-92-2 expression vector encoding miR 20, 19b-1, and 92a-1. (A), Transduced Jurkat cells (5 × 10 [4]) in the triplicate wells were stimulated with PMA (10 ng/ml) and ionomycin (500 nM) for overnight and supernatant was harvested and tested for the presence of IL-2 by specific ELISA. [score:5]
The blockade of IL-4 up-regulated miR-17-5p and miR-92 significantly with p <. [score:4]
To determine whether there is an IL-4 dose -dependent suppression of miR-17-92 cluster, we next treated CD4 [+ ]T cells with increasing doses of IL-4 at 0, 10, 50 or 100 ng/ml and measured miR-17-5p expression by RT-PCR (Fig. 3B). [score:3]
Total RNA was extracted and analyzed by RT-PCR for miR-17-5p expression. [score:3]
To evaluate these aspects, we transduced Jurkat cells with lentiviral vectors encoding green fluorescence protein (GFP) and either the miR-17-92-1 expression vector encoding miR-17 18 and 19a, or the 17-92-2 expression vector encoding miR 20, 19b, and 92. [score:3]
CD4 [+ ]cells from TG/TG mice displayed a greater than 15 fold increase in miR-17-p5 expression as compared with controls. [score:2]
Indeed, CD4 [+ ]and CD8 [+ ]splenocytes (SPCs) derived from wild type C57BL/6 mice bearing B16 subcutaneous tumors expressed lower levels of miR-17-5p when compared with those derived from non-tumor bearing mice (Fig. 4A). [score:2]
Furthermore, CD8 [+ ]T cells in STAT6 [-/- ]mice demonstrated enhanced levels of miR-17-5p expression when these mice bore B16 tumors compared with non-tumor bearing mice. [score:2]
Although not statistically significant, CD8 [+ ]T cells demonstrated a trend towards decreased levels of miR-17-5p expression in GBM patients when compared with healthy donors (Fig. 4D). [score:2]
Interestingly, not only are STAT6 [-/- ]T cells resistant to tumor -induced inhibition of miR-17-5p, but CD8 [+ ]T cells in tumor bearing STAT6 [-/- ]mice exhibited higher levels of miR-17-5p when compared with CD8 [+ ]T cells obtained from non-tumor bearing STAT6 [-/- ]mice. [score:2]
The miR-17-92 transcript encoded by mouse chromosome14 (and human chromosome 13) is the precursor for 7 mature miRs (miR-17-5p, miR-17-3p, miR-18a, miR-19a, miR-20a, miR-19b and miR-92) [24, 25]. [score:1]
We isolated CD4 [+ ]splenocytes from these mice and evaluated the expression of miR-17-5p (Fig. 5A). [score:1]
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[+] score: 60
As reported in Figure 1b, MEF express the precursor of miR-20a, miR-17 and miR-106b, while they do not express the precursor of miR-106a. [score:5]
miRNA -expressing plasmids (p-miRs) were investigated for their ability to inhibit fluorescence and it was found that p-miR-20a (Figure 1d), p-miR-17 (Figure 1e) and p-miR-106b (Figure 1f) all inhibit in a dose dependent manner. [score:5]
J Biol Chem 24 Hossain A Kuo MT Saunders GF 2006 Mir-17-5p regulates breast cancer cell proliferation by inhibiting translation of AIB1 mRNA. [score:5]
The underlying mechanism of action appears to be the E2F1 down-regulation by two members of the cluster, miR-17 and miR-20a. [score:4]
0002542.g001 Figure 1a: Sequences of miRNAs belonging to miR-17 family; b: analysis of pri-miRNA expression in MEF. [score:3]
From an in silico analysis we found that zbtb7a 3′UTR is predicted to be the target of several miRNA families, among which is miR-17 family (Figure 1a). [score:3]
The PCR products of ∼500 bp length are pri-miRNAs of the miR-17 family; c: analysis of mature miR-20a expression in MEF. [score:3]
The relative expression of p- zbtb7a 3′UTR was obtained by the ratio of the mean fluorescence value of HEK293T cells transfected with p-miR-20a, p-miR-17 or p-miR-106b and the mean fluorescence value of HEK293T cells transfected with p-miR-26a control plasmid. [score:3]
Indeed, when miR-17 is over-expressed in breast cancer cells, a big decrease in ER -mediated signaling and in turn proliferation is observed and cells loose their ability to form colonies in soft agar [24]. [score:3]
Looking for other possible miR-20a targets, we focused our attention on E2F1, already known to be under miR-17 family control [20], [21], [23]. [score:3]
Before testing if mouse zbtb7a 3′UTR interacts with miR-17 family members, the expression of representative members of the family was ascertained by. [score:3]
a: Sequences of miRNAs belonging to miR-17 family; b: analysis of pri-miRNA expression in MEF. [score:3]
24 hours later, 140 ng of p- zbtb7a 3′UTR were cotransfected with 75–300 ng of p-miR-17, p-miR-20a, p-miR-106 (specific miRNAs) or p-miR-26a not predicted by any algorithm to target the 3′UTR of LRF. [score:3]
In this way, the expression plasmids nick-named p-miR-20a, p-miR-17, p-miR-106b and p-miR-26a were obtained. [score:3]
On the other hand, in breast cancer cells the most relevant target of miR-17 is not E2F1, but AIB1. [score:3]
Using a hybrid reporter assay, we were able to demonstrate that miR-17 family members, in particular miR-20a, interact directly with zbtb7a 3′UTR (Figure 1d,e,f,g). [score:1]
We then tested whether mouse zbtb7a 3′UTR interacts with miR-20a, miR-17 and miR-106b, using an. [score:1]
We focused our attention on the miR-17 family whose members are reported in Figure 1a [25]. [score:1]
p-miR-20a was tested against p- zbtb7a 3′UTR wild type or mutated at the two binding sites specific for miR-17 family. [score:1]
20 µg of total RNA was analyzed with miR-20a probe or valine tRNA control probe; d, e, f: Interaction between 3′UTR of mmu- zbtb7a mRNA and miR-17 family. [score:1]
Annealing temperatures were: 54.9°C for pri-miR-17, 54.2°C for pri-miR-20a, 57.6°C for pri-miR-106b, and 57°C for pri-miR-26a. [score:1]
Genomic fragments of about 500 bp which contained human pri-miR-17, pri-miR-20a, pri-miR-106a, pri-miR-106b or pri-miR-26a sequences were obtained by PCR. [score:1]
HEK293T cells were co -transfected with p- zbtb7a 3′UTR and increasing concentrations of p-miR-20a, p-miR-17 p-miR-106b or p-miR-26a control plasmid. [score:1]
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[+] score: 60
The well expressed miR-21, miR-155 and miR-146a clustered together as consistently upregulated, while the abundant microRNAs of the miR17~92 clusters (miR-19b, miR-20a and miR-92) showed a clear trend towards decreased expression in differentiated cells, as did miR-26a (Figure 2A). [score:8]
In contrast to the clear upregulation observed for miR-155, the expression of the miR-17~92 cluster (especially miR-17-3p and miR-20a) tended to decrease along differentiation. [score:6]
MiR-17-5p and miR-17-3p are expressed at lower levels (belong to group C), indicating intra-cluster differential expression. [score:5]
When compared to naive cells, miR-21 and miR-155 were indeed found upregulated upon differentiation to effector cells, while expression of the miR-17~92 cluster tended to concomitantly decrease. [score:5]
Expression levels of miR-155 (a), miR-142.3p and miR-142-5p (b) and of members of the miR-17~92 cluster (c) were determined by single specific qPCR in the indicated sorted human CD8 [+ ]T cell subsets (a), and are expressed as fold change relatively to levels in naive cells (n = 9; *: p < 0,05; **: p < 0,01). [score:5]
Transgenic overexpression of miR-17~92 cluster in mouse lymphocytes was shown to induce lymphoproliferative disease [16]. [score:5]
The miR-17~92 cluster tends to be downregulated during CD8 [+ ]T cell differentiationSince central memory (CM) CD8 [+ ]T cells are present at extremely low frequency in peripheral blood, a new sorting strategy was then designed to include this subset in our analysis. [score:4]
The miR-17~92 cluster tends to be downregulated during CD8 [+ ]T cell differentiation. [score:4]
Figure 3The miR-17~92 cluster is preferentially downregulated in differentiated CD8 [+ ]T cell subsets. [score:4]
On the whole, expression of several members of the miR-17~92 cluster appeared to be found preferentially during early memory differentiation. [score:3]
Along the same lines, miR-17-3p expression was significantly decreased in late effector memory cells. [score:3]
We found expression of a limited set of microRNAs, including the miR-17~92 cluster. [score:3]
Expression levels of miR-17-3p, miR-17-5p, miR-19b, miR-20a and miR-92 were therefore determined by single specific qPCR in differentiated CD8 [+ ]T cell subsets, and compared to the levels found in naïve cells. [score:2]
MiR-17-5p expression showed no association with CD8 [+ ]T cell differentiation. [score:2]
*: microRNA belonging to the miR-17~92 cluster. [score:1]
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[+] score: 59
For example, three of the Treg-enriched miRNAs, miR-17-3p, miR-19a-5p and miR-19b-5p are all predicted to target numerous signalling molecules, including multiple MAP kinases, serine/threonine kinases and phosphatases, as well as various transcription factors and cell cycle regulators. [score:4]
Clearly much work remains to understand the functions not only of the miR-17∼92a regulatory networks, but also of the numerous other miRNAs expressed in Tregs. [score:4]
However, in Mir17∼92a [fl/fl] animals, CreYFP expressing (miR-17∼92a deficient) cells only comprise <15% of the total Tregs. [score:3]
0088997.g001 Figure 1(A) Relative expression of the miR-17∼92a miRNAs species determined by Illumina high throughput sequencing. [score:3]
Indeed, we found that in older mice, the lack of miR-17∼92a expressing Tregs led to increases in CD4 [+] T cells with an effector memory CD44 [hi]CD62L [lo] phenotype (Figure 5A), and CD8 [+] T cells with an activated/central memory CD44 [hi]CD62L [hi] phenotype (Figure 5B). [score:3]
Thus, the expression of miR-17∼92a miRNAs in Tregs is necessary for their fitness and for general immunologic homeostasis. [score:3]
Although miR-17∼92a miRNAs are enriched in Tregs, they are still expressed at high levels in other T cell populations. [score:3]
0088997.g002 Figure 2(A) Foxp3 [+] (YFP [+]) cells were sorted from the spleens of Mir17∼92a [fl/fl] Foxp3 [CreYFP/CreYFP(Y)] (KO) and Mir17∼92a [fl/+] Foxp3 [CreYFP/CreYFP(Y)] control (WT) mice, then analyzed for miR-17 expression by Taqman -based RT-PCR. [score:3]
Although it is clear that the miR-17∼92a cluster is important, the precise molecular targets of these miRNAs in Tregs remain to be determine. [score:3]
The miR-17∼92a cluster is necessary for the homeostasis of regulatory T cells. [score:2]
The miR-17∼92a cluster of miRNAs are enriched in regulatory T cells. [score:2]
In control Mir17∼92a [fl/+] animals CreYFP expressing cells comprise ∼40% of the total Tregs. [score:2]
The regulation of Treg fitness by the miR-17∼92a cluster is but one important function of miRNAs in Tregs. [score:2]
Treg-specific miR-17∼92a deficient mice were generated by crossing the LoxP-flanked Mir17∼92a [fl] conditional allele [28] with an IRES-CreYFP allele knocked into the Foxp3 locus [29] to generate Mir17∼92a [fl/fl] Foxp3 [CreYFP/CreYFP(Y)] (KO) or Mir17∼92a [fl/+] Foxp3 [CreYFP/CreYFP(Y)] control mice. [score:2]
Increased apoptosis and decrease proliferation of miR-17∼92a deficient Tregs. [score:1]
Indeed, a previous study on the role of miR-17∼92a in IL-10-effector Tregs differentiation suggests this [34], but more work is still needed to understand the function of these miRNAs in Tregs under immunologic challenge. [score:1]
Older mice with Treg-specific miR-17∼92a deficiency display perturbations in Treg populations. [score:1]
Treg-specific miR-17∼92a deficiency results in immunologic abnormalities. [score:1]
Here, we report that miRNAs of the miR-17∼92a cluster are enriched in Tregs and that this cluster is important for controlling the fitness of these cells. [score:1]
miR-17∼92a deficient Tregs have a competitive disadvantage. [score:1]
The miR-17∼92 cluster of miRNAs are all derived from a single polycistronic transcript driven by a single promoter [32], [33]. [score:1]
0088997.g003 Figure 3(A) Female Foxp3 [CreYFP/+] heterozygous mice were analyzed on mir-17∼92 sufficient (fl/+) or conditional deficient (fl/fl) genetic background. [score:1]
To investigate the requirement of this miRNA cluster in Tregs, we generated Treg-specific miR-17∼92a deficient mice by intercrossing mice with a LoxP-flanked Mir17∼92a [fl] allele with a CreYFP allele expressed from the Foxp3 locus. [score:1]
A number of studies have now reported the impact of single miRNA gene deficiency in Tregs: miR-17∼92a (present study and [34]), miR-155 [24]– [26] and miR-146a [27]. [score:1]
In this study, we showed that the miR-17∼92a cluster of miRNAs is enriched in Foxp3 [+] Tregs. [score:1]
Thus, we conclude that the poor fitness of miR-17∼92a deficient Tregs is most likely due to a combination of increased apoptosis and decreased proliferation of Tregs. [score:1]
Consistent with the phenotype of Foxp3 [CreYFP/+] heterozygous females, miR-17∼92a deficiency reduced the competitive fitness of Tregs in mixed bone marrow chimeras. [score:1]
That is, the miR-17∼92a deficient Tregs were outcompeted by the miR-17∼92a sufficient Tregs. [score:1]
The miR-17∼92a cluster of miRNAs are enriched in Tregs. [score:1]
miR-17∼92a deficient Tregs display increased apoptosis and defective proliferation. [score:1]
This resulted in a loss of miR-17∼92a cluster miRNAs but not other miRNAs (Figure 2A). [score:1]
Immunologic abnormalities caused by Treg-specific miR-17∼92a deficiency. [score:1]
As mice aged, the reduced fitness of the miR-17∼92a deficient Tregs manifested as various immunologic abnormalities. [score:1]
We next wanted to determine if altered apoptosis or proliferation underlay the poor fitness of miR-17∼92a deficient Tregs. [score:1]
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[+] score: 59
In this study, when we compared expression level of miR-17∼92 in PMBL and DLBCL, we showed a down-regulation of every miRs in PMBL except for miR-92a, that was significantly overexpressed. [score:7]
In two genetically distinct B-cell lymphoma cell lines, miR-17∼92 transfection induced a down-regulation of different target genes: BIM in Raji cells, and p21 in SUDHL4 cells [27]. [score:6]
Here we compared the expression of each member of the miR-17∼92 oncogenic cluster in samples from 40 PMBL patients versus 20 DLBCL and 20 cHL patients, and studied the target genes linked to deregulated miRNA in PMBL. [score:5]
Here we compared the expression of each member of the miR-17∼92 cluster in PMBL human samples versus DLBCL and versus cHL, and we further studied the target genes linked to deregulated miRNA in PMBL. [score:5]
In AIDS-related Burkitt lymphoma and DLBCL human samples, the overexpression of miRNAs from the miR-17∼92 paralog clusters inhibited p21 [28]. [score:5]
In mantle cell lymphoma samples, the protein phosphatase PHLPP2, an important negative regulator of the PI3K/AKT pathway, was a direct target of miR-17∼92 miRNAs, in addition to PTEN and BIM [29]. [score:5]
We quantified the expression levels of each microRNA of miR-17∼92 cluster and its paralogs in 40 PMBL, 20 DLBCL, and 20 cHL human samples (Figure 1). [score:3]
Quantification of expression of the miR-17∼92 cluster and its paralogs. [score:3]
Increased miR-17∼92 expression is found in B-cell lymphomas[26] and solid tumors (breast [32], colon [33], lung [23], neuroblastoma [34]). [score:3]
In humans, an overexpression of the miR-17∼92 cluster and its paralogs has been associated with high proliferation in mantle cell lymphoma [19, 26]. [score:3]
Quantification of expression levels of each microRNA in the miR-17∼92 cluster and its paralogs in 40 PMBL, 20 DLBCL and 20 cHL patient samples. [score:3]
In PMBL and cHL, we found a similar expression profile for each microRNA of miR-17∼92 cluster and its paralogs. [score:3]
When DLBCL and cHL were compared, five miRNAs of the miR-17-92a cluster, but not miR-92a, and the miR-106a and miR-106b of the paralog clusters, were significantly overexpressed in DLBCL. [score:2]
No significant difference was found for miR-17. [score:1]
miR-17∼92 is a polycistronic miRNA cluster, with two paralogs, the miR-106a-363, and miR-106b-25 clusters [21], able to act as oncogenes [22]. [score:1]
The miR-17∼92 cluster has numerous biological roles [24]. [score:1]
The miR-17∼92 oncogenic cluster, located at chromosome 13q31, is a region that is amplified in DLBCL. [score:1]
The miR-17∼92 cluster is one of the most potent miRNA oncogenes, located at chromosome 13q31, a region amplified in Burkitt’s lymphoma, DLBCL, follicular and mantle cell lymphoma [31]. [score:1]
When we compared PMBL and DLBCL results for the miR-17∼92 cluster, we found that only miR-92a had a significantly higher level of expression in PMBL compared to DLBCL (PMBL median 4.64 (interquartile range (Q1-Q3), 2.47-10.75); DLBCL 1.92 (Q1-Q3, 1.08-2.87); P =< 0.001). [score:1]
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[+] score: 57
When the DLBCL cells were treated with OncomiR inhibitors, the expression levels of some target genes were changed, PTEN was upregulated by the inhibitors for miR-21, miR-125b and miR-155; p27kip1 was upregulated by the inhibitors for miR-21, miR-155 and miR-221; TIMP3 was upregulated by the inhibitors for miR-21, miR-155, miR-221 and miR-17; RECK was upregulated by all the tested inhibitors (Figure 4A). [score:27]
In addition, malignant B cell proliferation in miR-17~92 -overexpressing mice is associated with PTEN and BCL-2-related ovarian death gene (BIM) inhibition [30, 31], as well as PH domain and leucine rich repeat protein phosphatase 2 (PHLPP2) suppression and PI3K/AKT signaling pathway activation [32, 33]. [score:7]
Of these OncomiRs, the expression levels of miR-21, miR-155, miR-221/222, miR-17, miR-19a/19b, and miR-20a/20b were higher in OCI-Ly10 cells, whereas the expression levels of miR-21, miR-155, miR-125a-5p/125b, miR-146a/146b-5p, and miR-17 were higher in SUDHL-4 and DB cells (Figure 1C). [score:5]
For example, PTEN (phosphate and tensin homolog) is a tumor suppressor gene, which was regulated by several miRNAs, such as miR-21 [12], miR-155 [13], miR-221/222 [14], and miR-17~92 cluster [15]. [score:4]
The expression levels of miR-21, miR-155, miR-221/222, miR-125a-5p/125b, miR-146a/146b-5p, miR-17, miR-19a/19b, and miR-20a/20b were significantly higher in the OCI-Ly10, SUDHL-4, and DB cells than in the IM-9 cells. [score:3]
Overexpression of miR-17~92 cluster (miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR-92-1) induces lymphoma [29]. [score:3]
Based on the mechanisms of miRNA functions, we selected those that are highly expressed in DLBCL, including miR-21, miR-155, miR-221/222, miR-125a-5p/125b, and miR-146a/146b-5p, as well as the miR-17-92 family members miR-17, miR-19a/19b, and miR-20a/20b; subsequently, we generated tandem sequences of 10 copies of the antisense sequences to these miRNA seed sequences and synthesized an interfering long non-coding RNA (i-lncRNA). [score:3]
A tandem sequence containing 10 copies of complementary sequences to the seed sequences of highly expressed OncomiRs (miR-21, miR-155, miR-221/222, miR-125a-5p/125b, miR-146a/146b-5p, miR-17, miR-19a/19b, miR-20a/20b) in DLBCL (Table 1) was generated and used as the encoding sequence for i-lncRNA. [score:3]
The i-lncRNA-involved OncomiRs were not always decreased, miR-21 was decreased, miR-221 was increased, and the expression of miR-155, miR-17, miR-19a and miR-20a was not changed, compared with the control group. [score:2]
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[+] score: 55
To identify novel potential drivers in GBM subtypes, we performed in silico analysis of TCGA data and found that several in the miR-17˜92 cluster or in analogous clusters are highly upregulated in the PN subtype of GBMs, and these are known to be transcriptionally up-regulated by the E2F cell cycle and myc pathways [10– 12]. [score:7]
Figure 1 In silico analysis of the expression of selected miRNAs from the miR-17˜92 cluster and paralogs shows elevated expression in proneural GBM(A) Expression level of miR-17˜92 and paralog miRNAs in the four subtypes of GBM versus normal brain tissues. [score:7]
As described earlier, E2F1 drives expression of the miR-17˜92 family and its paralogs, which are highly expressed in PN GSCs. [score:5]
One report demonstrated that the miR-17˜92 cluster targets TGF-β signaling, a potential driver of mesenchymal GBM, and therefore over -expression of this miRNA cluster should push cancer cells toward a proneural or epithelial phenotype [38]. [score:5]
In silico analysis of the expression of selected miRNAs from the miR-17˜92 cluster and paralogs shows elevated expression in proneural GBM. [score:5]
We then analyzed the expression of the miR-17˜92 cluster and a paralog in the 13 GSCs using. [score:3]
Palbociclib decreases Rb1 phosphorylation and reduces miR-17˜92 family and paralog expression in the sensitive PN GSC lines. [score:3]
The E2F family of transcription factors and c-myc can bind directly to the promoters of the miR-17˜92 cluster and its paralogs to regulate their transcription [10, 11, 21]. [score:3]
Expression of PN markers and the miR-17˜92 family is elevated in a set of PN GSC lines. [score:3]
Palbociclib reduces Rb1 phosphorylation and miR17˜92 and paralog expression in the responder PN GSCs. [score:3]
Here, our in silico analysis showed the miR-17˜92 cluster and its paralogs to be elevated in PN GBM, potentially indicating an enhanced role for the E2F cell cycle pathway and potential sensitivity to a CDK4/6 inhibitor in PN GBM. [score:3]
The miR-17˜92 family and its paralogs are highly expressed in PN GSC lines. [score:3]
As predicted, the members of the miR-17˜92 cluster (miR-20a, -20b, -93, -106a, -130b, and -10b) and paralog clusters were over-expressed in seven of eight PN GSC lines (G44, 448, 464, 559, 578, 816 and 827; Figure 2C). [score:3]
The miR-17˜92 family and its paralogs are elevated in human PN GBM samples. [score:1]
Interestingly, miR-20a and -19a belong to the miR-17˜92 family, while miR-20b and -106a belong to the miR-106a˜363 family, a paralog of the miR-17˜92 cluster [20]. [score:1]
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[+] score: 48
[37] The upregulation of p38α (MAPK14) protein seen in the current human study may be owing to the decreased expression of miR-19a/b, members of the miR-17/92 cluster, or miR-185-5p, as they are predicted to target human MAPK14 (TargetScan release 7.0: http://www/targetscan. [score:12]
[37] Therefore, the protein expression levels of p38α in patient-derived neurospheres were predicted to be increased by the downregulation of miR-17/92 cluster members. [score:6]
[33] Therefore, the underexpression of miR-17/92 cluster members, miR-185-5p and miR-106a/b, and subsequent upregulation of p38α may underlie the observed size reduction of patient-derived neurospheres and the decreased neural differentiation efficiency in patient-derived neurospheres. [score:6]
34, 45 A recent study of the miR-17/92 cluster and miR-106a/b has shown that miR-19 and miR-92a repress PTEN and TBR2, and suppress the transition from radial glial cells to intermediate progenitors, [46] and that miR-17 and 106a/b repress p38α (MAPK14), leading to increased neurogenic and suppressed gliogenic competences in mice. [score:5]
It has been reported that (i) miR-17/106 targets the MAPK14 transcript (encoding the α-isoform of p38 protein kinase) in mice and (ii) the miR-17/106-p38 axis is a critical regulator of the neurogenic-to-gliogenic transition competence. [score:4]
[37] The patient-derived neurospheres showed reduced expression levels of miR-17/92 cluster members and miR-106a/b. [score:3]
[32] As the downstream targets of DGCR8, which may be causally linked to the observed phenotypes, we examined the miR-17/92 cluster and miR-106a/b. [score:3]
34, 35 The miR-17/92 cluster (Figure 3c) includes miR-17, miR-18a, miR-19a, miR-19b-1, miR-20a and miR-92a-1. Therefore, we set out to precisely quantify the expression levels of those eight miRNAs (miR-17, miR-18a, miR-19a, miR-19b-1, miR-20a, miR-92a-1 and miR-106a/b), all of which belong to the miR-17 family or the miR-17/92 cluster, using real-time quantitative RT-PCR with U6 snRNA as an internal control probe. [score:3]
In previous studies, the miRNAs of miR-17 family and miR-17/92 cluster have been reported to show abnormal expression levels in schizophrenic brains. [score:3]
The miR-17/92 have a general role in cell proliferation and survival during normal development and also during tumorigenesis. [score:2]
[33] The miR-17 family (Figure 3c) includes miR-17 (in the current study, hsa-miR-17-3p showed FC=0.7 and P=0.0449; Supplementary Figures 3a and 3b), miR-20a/b, miR-93 and miR-106a/b. [score:1]
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[+] score: 46
miR-17 targets Eos, a transcription factor that cooperates with Foxp3 to mediate suppressor gene expression (Figure 1D) (52). [score:7]
Thus, superior GVHD control may be acquired by increasing Treg fitness by upregulating miR-155 or reducing miR-17 or miR-146b expression. [score:6]
Strategically generating Tregs that have low miR-17, miR-16a/16, or miR-142-3p or high miR-146a or let-7d expression may represent an approach to increase Treg suppressor function. [score:5]
miR-17 directly targets TGF-β receptor II and the cAMP-responsive element binding protein 1, both implicated in bolstering Treg differentiation. [score:4]
miR-31 represses human Treg Foxp3 expression (36) while the miR-17–miR-92 cluster represses iTreg formation (37– 40). [score:3]
In line with this, transgenic miR-17 overexpression exacerbates pathology in a murine colitis mo del (52). [score:3]
It has been proposed that strong CD28 signals inhibit Foxp3 induction, which may be influenced by costimulatory signaling pathways that induce miR-17–miR-92 (41). [score:3]
By contrast, elevated miR-17–miR-92 in murine lymphocytes increased proliferation and reduced cell death (64), resulting in favored Treg accumulation in lymph nodes and non-lymphoid target tissues (63). [score:3]
For example, IL-6, which is an established mediator of acute GVHD in mice and patients (51) and required (with TGF-β) to induce Th17 cells, induces the expression of miR-17. [score:3]
Treg-specific miR-17–miR-92 deletion increased Treg apoptosis and reduced proliferation, causing loss of Foxp3 expressing Treg in aged mice (63). [score:3]
miR-31 and the miR-17–miR-92 cluster function as negative regulators of iTreg differentiation (29). [score:2]
The miR-17~92a cluster of microRNAs is required for the fitness of Foxp3+ regulatory T cells. [score:2]
Moreover, miRNA (miR-31, miR-17–miR-92, and miR-23–miR-27–miR-24) antagomir treatment of T cells in vitro may be exploited to support iTreg generation, while in vivo treatment may foster pTreg generation. [score:1]
miR-17–miR-92 also assists in maintaining Treg fitness (62). [score:1]
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[+] score: 40
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-16-1, 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-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
MiR-17-5p and miR-181d, which are downregulated in formaldehyde -treated human lung epithelial cells, were predicted to regulate the greatest number of genes in the brains of MAM -treated Mgmt [−/−] mice (Figure 3). [score:5]
Mir-17-5p regulates breast cancer cell proliferation by inhibiting translation of AIB1 mRNA. [score:5]
MiR-17-5p also has tumor-suppressing activity in hepatocellular, gastric, pancreatic, breast, and cervical cancer (Hossain et al., 2006; Yu et al., 2010; Chen et al., 2012; Wang et al., 2012) and is also thought to be involved in the regulation of APP expression (Hébert et al., 2009). [score:5]
Formaldehyde-responsive miRNAs predicted to modulate MAM -associated genes in mouse brains lacking MGMT include miR-17-5p and miR-18d, which regulate genes involved in tumor suppression, DNA repair, amyloid deposition, and glutamatergic and dopaminergic neurotransmission. [score:4]
These include miR-17-5p and miR-181d, which regulate genes involved in tumor suppression, DNA repair, amyloid deposition, and glutamatergic and dopaminergic neurotransmission. [score:4]
MiR-17-5p targets tumor protein P53 -induced nuclear protein 1 (TP53INP1), which suppresses cell growth and promotes apoptosis of cervical cancer cells (Wei et al., 2012). [score:4]
MicroRNA miR-17-5p is overexpressed in pancreatic cancer, associated with a poor prognosis, and involved in cancer cell proliferation and invasion. [score:3]
In particular, miR-17-5p (discussed later), mIR-20a, and miR-106b reduce endogenous APP expression in vitro (Hébert et al., 2009). [score:3]
MiR-17-5p regulates the expression of EPHA4, GNPDA2, and TXNIP. [score:3]
A comparison of all 89 formaldehyde-modulated miRNAs in human lung epithelial cells (Rager et al., 2011) with the miRNAs predicted here to regulate genes in the brains of MAM -treated Mgmt [−/−] mice show overlap of 6 miRNAs: miR-107, miR-152, miR-17-5p, miR-181d, and miR-454-3p. [score:2]
miR-17-5p as a novel prognostic marker for hepatocellular carcinoma. [score:1]
Circulating miR-17-5p and miR-20a: molecular markers for gastric cancer. [score:1]
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[+] score: 39
Furthermore, ALK may play a direct role in Xpcl1 paralog activation, as knockdown of ALK led to reduced expression of miRNA from both the miR-17~92 and miR-106a~363 clusters [53]. [score:5]
We were surprised to find that forced expression of miR-106a~363, alone, induced lymphomas, considering that overexpression of the paralogous cluster, miR-17~92, caused lymphoid hyperplasia, but not spontaneous lymphomas [14, 34]. [score:5]
On the other hand, miR-106b (which is also a miR-17 family member) targets the p21 Cdk inhibitor (Cdkn1a) and thereby enhances growth of mammary epithelial cells [17]. [score:5]
Forced expression of the miR-17~92 paralog, for example, required concurrent expression of Myc, to induce B cell lymphomas [14]. [score:5]
Constituents of the miR-106a~363 cluster, and its autosomal paralogs (miR-106b~25 and miR-17~92), include some of the most highly expressed miRNAs in blood cells [1]. [score:3]
Considering the compensatory activities of the paralogous miRNA clusters, it is possible that the mechanism of oncogenesis is not cluster-specific, but instead varies by cell type, and that forced expression of miR-17~92 would be equally oncogenic in T cells. [score:3]
These results are consistent with previously published miRNA sequence data, which indicated that expression of miR-17 family members, as a group, are high in DN thymocytes [2]. [score:3]
For example, miR-17~92, miR-106a~363, and miR-106b~25 exhibit sequential and overlapping waves of expression in developing B lymphocytes [15]. [score:3]
Xpcl1 paralog overexpression does not appear to be a general characteristic of T-cell lymphomas, to the contrary, PTCL/NOS showed significantly reduced expression of miR-17~92 and miR-106a~363 miRNAs compared to normal T-lymphocytes [54]. [score:2]
While genetic evidence of the oncogenicity of miR-106a~363 in human malignancies is lacking, amplifications of the miR-17~92 paralog has been documented in diffuse large B cell lymphomas [9]. [score:1]
Multiple miR-17 and miR-92 family members were amongst the miRNA that most effectively induced Th cell proliferation [18]. [score:1]
Interestingly, overexpression of miRNAs from miR-106a~363 and the paralogous cluster, miR-17~92, are both highly characteristic of ALK -positive ALCL [7]. [score:1]
Whereas the oncogenic potential of miR-17~92, was dependent on miR-19a and associated with impaired apoptosis, the Lx [+] transgene induced only minimal increases in miR-19b, with no appreciable reduction in apoptosis in Lx [+] thymocytes. [score:1]
Thus the oncogenic mechanism appears to be fundamentally different in Lx [+] mice than was previously reported for miR-17~92. [score:1]
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We selected, for experimental validation, targets to three of these miRNAs: hsa-miR-17-5p (one target: see below), hsa-miR-15a (two targets), and hsa-miR-324-3p (three targets). [score:9]
We found 777 genes (27.0%) with 2485 isoforms (31.8%) in which at least one predicted target site is reassigned to a different mRNA region in at least one isoform, with 6519 (9.8%) of predicted miRNA targets in this category; most of these cases involve a site found in the 5′UTR of one isoform but in the CDS of a second (e. g. the energetically most-favorable miR-17-5p target site on TNFSF12 mRNAs: Supplementary Figure S1), or in the CDS of one but in the 3′UTR of a second, although in a small number of isoform sets a binding site is reassigned among all three regions. [score:7]
For example, targets predicted for let-7a are shifted toward better free energies than are those predicted for both sets of controls (Figure 1B), whereas targets predicted for miR-17-5p and its controls show similar energy distributions (Figure 1C). [score:5]
For one of the experimentally validated miRNAs in this study, miR-17-5p, we predicted target sites in both known transcript isoforms of TNFSF12, but in the CDS of one and the 3′UTR of the other. [score:3]
Numbers of predicted target sites per miRNA and its control sequences for (A) miR-1 and its controls with WC nt 2–8; if miR-1 hybridized with perfect WC complementarity this would yield −30.8 kcal/mol (see Methods); (B) let-7a and imposing only the requirement of WC base pairs within nucleotide positions 2–8; let-7a perfect WC complementarity would yield −33.2 kcal/mol; (C) miR-17-5p and its controls with WC nt 2–8; perfect WC complementarity would yield −44.5 kcal/mol; (D) miR-324-3p and its controls with WC nt 2–8; perfect WC complementarity would yield −52.8 kcal/mol; and (E) miR-129 and its controls with WC nt 2–8; perfect WC complementarity would yield −41.4 kcal/mol. [score:3]
Panels B, C and D of Figure 1 show the increasingly better energy-score ranges for predicted targets of let-7a, miR-17-5p and miR-324-3p. [score:3]
Five of the target sites (hsa-miR-15a/TSPYL2, hsa-miR-15a/BCL2, hsa-miR-17-5p/TNFSF12, hsa-miR-324-3p/CREBBP and hsa-miR-324-3p/WNT9B) exhibit perfect WC complementarity in the seed regions, while has-miR-324-3p/DVL2 has one GU pair in the same region (Supplementary Figure S1). [score:3]
0005745.g001 Figure 1Numbers of predicted target sites per miRNA and its control sequences for (A) miR-1 and its controls with WC nt 2–8; if miR-1 hybridized with perfect WC complementarity this would yield −30.8 kcal/mol (see Methods); (B) let-7a and imposing only the requirement of WC base pairs within nucleotide positions 2–8; let-7a perfect WC complementarity would yield −33.2 kcal/mol; (C) miR-17-5p and its controls with WC nt 2–8; perfect WC complementarity would yield −44.5 kcal/mol; (D) miR-324-3p and its controls with WC nt 2–8; perfect WC complementarity would yield −52.8 kcal/mol; and (E) miR-129 and its controls with WC nt 2–8; perfect WC complementarity would yield −41.4 kcal/mol. [score:3]
This selection was made on the basis of functional association with cancer (hsa-miR-17-5p, hsa-miR-15a) or predicted targets in the Wnt signalling pathway (hsa-miR-324-3p) as described in Supplementary Table S7. [score:3]
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Both miR-17 and miR-20a also directly target TGFBRII [46], a gene well-known to be directly involved in the regulation of primordial follicle activation and ovarian aging [7]. [score:6]
The miR-17 family (mmu-miR-17, mmu-miR-20a and mmu-miR-93) was highly expressed within the ovaries and down-regulated with age exclusively in df/df mice. [score:6]
The miR-17 gene family is located at the same cluster (< 10 kb spaced on mouse chromosome 14) with mmu-miR-92a, the highest expressed ovarian miRNA in our samples, which was up-regulated in young df/df in comparison to young N mice. [score:6]
In fact, this same pattern of regulation was observed for other seven miRNAs (mmu-miR-29c, mmu-miR-296, mmu-miR-130b, mmu-miR-17, mmu-miR-434, mmu-miR-181c, mmu-miR-132), which further confirms the validity of our analysis and suggests miRNA regulation at the gene expression level [48]. [score:5]
miR-17 family miRNAs are expressed during early mammalian development and regulate stem cell differentiation. [score:5]
The miR-17 gene family is involved in the regulation of stem cell differentiation [45], and indeed signaling pathways regulating pluripotency in stem cells was one of the enriched pathways observed to be differentially regulated with aging in df/df mice. [score:4]
The miRNAs located in the miR-17/92 cluster are considered oncogenes and are frequently over-expressed in malignant cells, having the phosphatase and tensin homologue (PTEN) as one of their main targets [46]. [score:4]
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Supported by these considerations, we suggest that a functional loop between Shh deregulation and ionizing radiation exposure increases, via c/N-myc, the miR-17∼92 expression level immediately after irradiation, and this expression remains persistently upregulated in radio -induced MB compared with spontaneous tumors. [score:8]
It has been shown that the miR-17∼92 cluster is positively upregulated by c-Myc thereby favoring tumorigenesis by enhancing cell proliferation and inhibiting apoptosis [29]. [score:6]
The miR-17∼92 cluster is important in cell cycle, proliferation, apoptosis and other pivotal processes as well as in a wide array of diseases (i. e., in hematopoietic and solid cancers, and in immune, neurodegenerative and cardiovascular diseases) [25]. [score:5]
Importantly, it has been reported that Shh signaling pathway and miR-17∼92 collaborate during cerebellar development and in MB formation [26]; this functional interaction in GCPs is mediated in part by N-myc and C-Myc, both Shh effectors, which induce miR-17∼92 expression [27]. [score:4]
The Molecule Activity Predictor (MAP), based on significantly deregulated miRNAs, suggests the inhibition of senescence (blue) and a concurrent increase of cell survival and viability (light orange) and DNA damage (dark orange), mainly due to the miRNAs let-7a, mir-17, mir-21, mir-34a, mir-92, mir-133a, mir-181a and mir-486 (Figure 6). [score:4]
In absence of irradiation, here we show no difference in expression levels of three miRNAs belonging to miR-17∼92 cluster (miR-17, miR-19a, miR-20a; Figure 7), although at P2/3 GCPs are actively proliferating. [score:3]
Furthermore, results from Murphy and colleagues [28], showed the therapeutic potential of 8-mer LNA-anti-miRs in inhibiting miR-17, 20a, 106b, and 93 (anti-miR-17) and miR-19a and 19b-1 (anti-miR-19) in two murine SHH -driven MB mo dels. [score:3]
One of the miRNAs of interest is miR19a-5p, belonging to one of the best-known miRNA clusters, the miR-17∼92, which encodes six miRNAs (i. e., miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR-92-1) [24]. [score:1]
Second, we analyzed 3 members of the 17∼92 cluster, i. e., miR-17, miR-19a, miR-20a, involved in cell survival and viability. [score:1]
Bonferroni post-hoc tests, for unirradiated conditions, did not show any significant difference between WT and mutant cells in miR-17 and miR-19a, while a mild significance is observable in miR-20a (P < 0.01). [score:1]
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Other miRNAs from this paper: mmu-mir-106b, mmu-mir-19a
The miR-17~92 cluster family is expressed in proliferating GNPs [26] and up-regulated in SHH medulloblastoma [26– 28]. [score:6]
We previously showed that miR-17~92 expression is absolutely required for SHH medulloblastoma initiation [29] and that its silencing inhibits medulloblastoma progression [43]. [score:5]
Clearly other microRNAs besides those encoded by the miR-17~92 cluster family are expressed during cerebellar development. [score:4]
We here show that Dicer that processes pre-miRNAs into mature miRNAs, including those encoded by the miR-17~92 cluster, is required for both normal cerebellar development and medulloblastoma suppression. [score:4]
We previously reported that the miR-17~92 cluster family is expressed in the developing cerebellum [29]. [score:3]
To evaluate how the loss of Dicer affects the expression of mature microRNAs, we determined the expression of miR-19a (from the miR-17~92 cluster) and miR-106b (from the miR-106b~25 cluster) in the developing cerebella of control and Dicer c KO embryos at E14.5, E16.5 and E18.5 by Q-RT-PCR (Fig 5B and 5C, lanes 1–6). [score:3]
Therefore, the apoptosis observed at E15.5 Dicer c KO embryos is most likely due, only in part, to the loss of expression of microRNAs encoded by the miR-17~92 cluster family. [score:3]
We previously published that the miR-17~92 cluster family was required for proliferation of GNPs during postnatal cerebellar and medulloblastoma development [29, 43]. [score:2]
We will unable to correlate the phenotypes of Dicer c KO in embryos with the ones we observed with the mir17~92 cluster family because the effects of the loss of the mir17~92 cluster family are only revealed in the cerebellum after birth [29]. [score:1]
While loss of miR-17~92 leads to a relatively mild phenotype, it is absolutely required for SHH medulloblastoma formation [29]. [score:1]
In the present study, we used two of the microRNAs encoded by the miR-17~92 cluster family (miR-19a and miR106b) to determine the level of microRNA biogenesis. [score:1]
Co- deletion of miR-17~92 with its paralog miR-106b~25 in mice reduces proliferation of GNPs at postnatal age leading to a small cerebellum [29]. [score:1]
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[53] Some evidence suggests that this cluster regulates a broad spectrum of biological processes of T-cell immunity including a negative expression relationship between miR-17 and the synthesis of Sirp-α gene that effectively regulate macrophage inflammatory responses. [score:5]
Examples of this are Mtfp1 and Mllt11, both target genes for miR-17, and involved in positive regulation of apoptotic process. [score:4]
There are previous reports of the involvement of miRNAs in retinal development, function, and photoreceptors survival 1, 2 and also the involvement of different miRNAs in the pathogenesis of various diseases of the retina including miR-9, miR-34a, miR-125b, and miR-155 in macular degeneration, 17, 18 miR-146a and miR-195 in diabetic retinopathy, 19, 20 and miR-125a and miR-17 in retinoblastoma. [score:4]
[54] Among the retina-related miRNAs described above, we only found miR-17 and miR-29b to be inversely expressed with genes involved in pathways with a putative involvement in retinal degeneration. [score:3]
Zhu D, Pan C, Li L, MicroRNA-17/20a/106a modulate macrophage inflammatory responses through targeting signal-regulatory protein alpha. [score:3]
Lindberg RL, Hoffmann F, Mehling M, Altered expression of miR-17-5p in CD4+ lymphocytes of relapsing-remitting multiple sclerosis patients. [score:3]
MiR-17 is a member of the miR-17-92 cluster that has been implicated in inflammatory diseases, [51] Burkitt lymphoma, [52] and retinoblastoma. [score:3]
[57] Hence, increased expression of miR-17 and miR-29b could be part of survival mechanisms initiated to slow down the PR cell death process via the modulation of these genes. [score:3]
Robaina MC, Faccion RS, Mazzoccoli L, miR-17-92 cluster components analysis in Burkitt lymphoma: overexpression of miR-17 is associated with poor prognosis. [score:3]
Conkrite K, Sundby M, Mukai S, miR-17∼92 cooperates with RB pathway mutations to promote retinoblastoma. [score:2]
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The miRNA expression patterns in clinical specimens directly shows that most candidate Suppressive miRNAs, except for miR-31, are expressed at significantly lower levels than the candidate Oncogenic miR-17 (Figure 3). [score:8]
The expression of these miRNAs was validated in 15 low-passage primary cultures, with the mean level of miR-17 being higher than that of the Suppressive miRs, thus confirming the physiological relevance (Figure 3). [score:5]
In contrast, the mean expression of the candidate Oncogenic miR-17 among the clinical specimens was even higher than in HAG cells (Figure 3). [score:3]
miR-17 was cloned and stably over-expressed in the PAG cells. [score:3]
In agreement with these reports, ectopic expression of miR-17-5p in the PAG cells enhanced proliferation rate (Figure 6B). [score:3]
A 25-fold over -expression of miR-17 was verified by real time PCR (Figure 6A). [score:3]
0018936.g006 Figure 6(A) Verification of miR-17-5p over -expression in PAG transductants, as compared to mock-transduced cells. [score:2]
miR-17 was selected as an example for candidate Oncogenic miRNA (Figure 2C). [score:1]
Different roles of miR-17, miR-31 and miR-34a in cancer have been reported before, although never in cutaneous melanoma. [score:1]
Importantly, miR-17-transduced PAG cells displayed a significantly enhanced proliferative activity (Figure 6B) but not invasive ability (Figure 6C) or tube formation activity (data not shown). [score:1]
These results support the potentially oncogenic effects of miR-17 in melanoma. [score:1]
The oncogenic properties of miR-17-5p were discussed in earlier publications in other malignancies [13]. [score:1]
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[+] score: 32
In total, 114 miRNAs are significantly changed and can be classified into four groups (Figure 2A); 52 miRNAs, including the miR-30 family, are down-regulated during the first 8 days after infection (Figure 2B), 8 miRNAs are down-regulated before day 2 and up-regulated after day 2 after infection (Figure 2C), 2 miRNAs are up-regulated before day 2 and down-regulated after day 2 after infection (Figure 2D), and the remaining 52 miRNAs, including the miR-17 family, are up-regulated (Figure 2E). [score:19]
Based on the miRNA profile of early-infected MEF cells with OSKM, these up-regulated miRNAs such as the miR-17 family may be directly regulated by exogenous OSKM and play important roles in promoting iPS generation. [score:6]
All of these miRNAs are up-regulated in iPS cells, indicating the importance of miR-17 and miR-19 in the activation and maintenance of iPS pluripotency (Table 1). [score:4]
A strong Oct4 binding signal exists in the promoter region of the mir-17 cluster. [score:1]
Among 82 pre-miRNAs for these 73 mature miRNAs, which are commonly involved for activation and maintenance, we found all 7/8 members of the miR-17 family, all 3/3 members of the miR-19 family, 6/6 miRNAs in the cluster of miR-17-92 and 3/3 miRNAs in the cluster of miR-106-93. [score:1]
Two mean signal intensity plots are shown for this group and the miR-17 family. [score:1]
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miRNA Number of targets in miRNA-gene bigraph network P-value miR-20a 9 8.16E-09 miR-17 10 1.30E-07 miR-34a 9 2.78E-07 miR-155 14 2.16E-07 miR-18a 5 4.04E-06 miR-22 5 6.18E-06 miR-26a 6 9.29E-06 miR-101 5 3.30E-05 miR-106b 5 3.30E-05 miR-125b 8 8.37E-05 It is well known that AD is a complex disease and devastating neurodegenerative disorder without effective disease-modifying or preventive therapies. [score:7]
Moreover, the miR-17 and miR-20a bindings sites located in or near the APP 3'UTR variants T117C, A454G and A833C, and the A454G variant increased miR-20a binding (Delay et al., 2011), and miR-17 and miR-20a were down-regulated in age-related and senescence-related cellular processes (Hackl et al., 2010). [score:4]
APP expression was regulated by miR-20a (Hebert et al., 2009; Fan et al., 2010; Delay et al., 2011), miR-17 (miR-17-5p) (Delay et al., 2011), miR-106b (Hebert et al., 2009), and miR-101 (Vilardo et al., 2010; Long and Lahiri, 2011). [score:4]
In addition, microRNAs, including miR-20a, miR-17, miR-34a, miR-155, miR-18a, miR-22, miR-26a, miR-101, miR-106b, and miR-125b might regulate the expression of genes (nodes) in the sub-network, thereby disrupting the fine-tuning of genetic networks in SAMP8 mice. [score:4]
miR-17, miR-19b, miR-20a, and miR-106a are down-regulated in human aging. [score:4]
The top 10 miRNAs with P ≤ 8.37 e [5] were listed in Table 3. They are miR-20a, miR-17, miR-34a, miR-155, miR-18a, miR-22, miR-26a, miR-101, miR-106b, and miR-125b, indicating that these ten miRNAs could regulate the expression of nodes (genes) in the sub-network of SAMP8 mice and might be one cause inducing SAMP8 mice to exhibit significant nodes (or genes) and to display a distinct genetic sub-network in the brain. [score:4]
miRNA Number of targets in miRNA-gene bigraph network P-value miR-20a 9 8.16E-09 miR-17 10 1.30E-07 miR-34a 9 2.78E-07 miR-155 14 2.16E-07 miR-18a 5 4.04E-06 miR-22 5 6.18E-06 miR-26a 6 9.29E-06 miR-101 5 3.30E-05 miR-106b 5 3.30E-05 miR-125b 8 8.37E-05 Differentially expressed mRNA in the hippocampus and cerebral cortex of SAMP8 and SAMR1 mice at 2, 6, and 12 months were investigated using cDNA microarray (Cheng et al., 2007b). [score:3]
Based on the miRNA-gene bipartite graph network in the brain of SAMP8 mice, we identified the top 10 miRNAs with P ≥ 8.37E-05, including miR-20a, miR-17, miR-34a, miR-155, miR-18a, miR-22, miR-26a, miR-101, miR-106b, and miR-125b (Table 3). [score:1]
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In particular, the enforced expression of miR-17 reduced the expression of the tumor suppressor genes E2F5, TP53INP1, TRIM8 and ZBTB4, and protected CLL cells from apoptosis [1]. [score:7]
Here, we provide evidences that the abrogation of miR-17 expression by a specific antagomiR is sufficient to inhibit leukemic growth and progression both in-vitro and in-vivo. [score:5]
CLL MicroRNA miR-17 AntagomiR17 We have recently reported that microRNA from the miR-17 ~ 92 family may be responsible for the increased proliferation/survival in chronic lymphocytic leukemia (CLL) cells expressing unmutated (UM) IGHV genes and with high level of ZAP-70 [1]. [score:3]
This strategy could be extended to other lymphoproliferative disorders where miR-17 ~ 92 amplification and/or overexpression have a pathogenetic role [9, 10]. [score:3]
In MEC-1 cells, antagomiR17 transfection significantly reduced miR-17 expression respect to scrambled control, both at day 2 (mean fold change 0.84 ± 0.06; P = 0.049) and at day 4 (mean fold change 0.48 ± 0.14; P = 0.021; Figure  1b). [score:3]
Altogether, these data demonstrated that antagomiR17 administration effectively reduced the expression of miR-17 and cell proliferation. [score:3]
The MEC-1 cell line expressed miR-17 levels comparable to those of CLL samples in which proliferation is triggered by CpG-ODN (Figure  1a). [score:3]
Evidences reported here underline that miR-17 knockdown is sufficient to block CLL-like cells proliferation both in-vitro and in-vivo. [score:2]
MEC-1 CLL-like cell line was transfected with a molecule against miR-17 (hereafter antagomiR17), or scrambled control. [score:1]
miR-17 expression was evaluated by qRT-PCR at different time-points (2 and 4 days). [score:1]
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Using the software ‘TARGETSCAN’, “PICTAR” and ‘MIRANDA’, we found that miR-17 and miR-20a have the same seed sequences and two predicted target sites in the same region of the 3′UTR of mouse E2F1 (Fig.   4C). [score:5]
In addition, miR-17 and miR-20a could directly target the 3′UTR of E2F1 in PMCs and imped G1/S transition of PMCs. [score:4]
These results suggest that the effect of miR-17-92 on cell cycle control is dependent on miR-17 and miR-20a which directly target E2F1 in PMCs. [score:4]
This result indicates that E2F1 is a direct target of miR-17 and miR-20a in PMCs. [score:4]
After increase, miR-17 and miR-20a may negatively target E2F1, and thereby prevent the cells from excessive proliferation. [score:3]
The inhibition of miR-17 and miR-20a in cells transfected with miR-17-92 mimics decreased the proportion of cells in G0/G1 phase (Fig.   5C) while increasing the percent of cells in S phase (Fig.   5D). [score:3]
These results validate that E2F1 could direct bind to the promoter of miR-17-92 and promote the transcription of miR-17-92in PMCs. [score:2]
The luciferase activity in PMCs transfected with E2F1 WT constructs plus miR-17/20a mimics was significantly lower than that in PMCs transfected with either E2F1 WT or mutant constructs alone, and the scrambled miRNA did not affect the luciferase activity in either WT or mutant constructs transfected PMCs (Fig.   4D). [score:1]
Thus we generated pMIR-Report luciferase vectors of E2F1 for miR-17/20a. [score:1]
The 3′UTR recombinant construct of E2F1 was transfected into PMCs along with miR-17/20a mimics or scrambled miRNAs. [score:1]
The miR-17-92 cluster is conserved among vertebrates, comprising six miRNAs: miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR-92a-1 [8]. [score:1]
The seed sequences of miR-17/20a. [score:1]
To investigate whether this effect was dependent on miR-17 and miR-20a, the same cells were co -transfected with miRNA inhibitors against miR-17 and miR-20a respectively. [score:1]
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Thus, the miRNA -mediated down-regulation of Creb1 and Crtc3 in AAI, combined with the up-regulation of miR-17, could influence the secretion of the anti-inflammatory cytokine IL10 and could therefore have a beneficial impact on airway inflammation. [score:7]
Recently, miR-17/20 has also been shown to regulate pulmonary artery smooth muscle cell proliferation via targeting prolyl hydroxylase 2, leading to induction of hypoxia-inducible factor (HIF) 1α 42. [score:4]
As these results suggested that miRNA-17, -144, and -21 regulate Creb1 and its co-activators, we sought to verify this regulation during the development of OVA -induced AAI. [score:4]
A less stringent i n silico analysis with only two different target prediction tools identified miR-144 and miR-17 (homo sapiens only) binding sites in the 3′-UTRs of Crtc1 and Crtc3 (Table S1). [score:3]
Additionally, the levels of miR-17, miR-144 and miR-21 significantly correlated with total cell counts in bronchoalveolar lavage (BAL), indicating an increased expression of the miRNAs upon increasing inflammation (Fig. 3b). [score:3]
In vitro validation of predicted miRNA targetsThe functional interaction between Creb1 and miR-17, -144, and -22 was confirmed by luciferase reporter assays (Fig. 2a). [score:2]
So far, dysregulation of miR-17 has been mainly investigated in the context of cancer 38 39, but has also been proposed to regulate IL-10 in regulatory T cells 40. [score:2]
In total, the 3′UTR of Creb1 contains eight predicted binding sites for miR-17 (three sites), miR-144 (one site), miR-22 (two sites), and miR-181a (two sites) (Fig. 1b). [score:1]
Thus, it is intriguing to speculate that increased miR-17 levels in our mo del might induce HIF1α levels, contributing to allergic airway inflammation. [score:1]
miR-17 also decreased Crtc2 (Fig. S1a). [score:1]
Antagonism of miR-17 or -144 in vitro via transfection of anti-miRs slightly increased CREB1 mRNA levels (Fig. 2b). [score:1]
Ambion [®] Pre-miR Precursors (for miR-17 and miR-144), miRvana miRNA mimics (for miR-21) (Ambion, Austin, USA) or antimiRs (miR-17 and -144) (Ambion, Austin, USA) were transfected in duplicates to a final miRNA concentration of 20 nM per well in a murine lung epithelial cell line (MLE-12) or a human bronchial epithelial cell line (16-HBE14o [−]) 64. [score:1]
Antagonism of endogenous miR-17 and -144 (Fig. S1b) slightly increased CRTC1, -2, and -3 mRNA levels (Fig. S1b) levels. [score:1]
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Since loss of miR-17 group miRNAs (miR-17 and miR-20a) causes skeletal defects in mice [20], we tested whether miR-17 can suppress gene expression via the predicted target sequence of Tgfbr2 by luciferase reporter assay. [score:6]
The type II TGF-β receptor, Tgfbr2, a predicted target of miR-17 and miR-19 group miRNAs [19], was upregulated at both the protein and mRNA levels (Fig.   3a, d). [score:6]
miR-17 co-transfection significantly reduced the expression of a luciferase reporter construct with the wildtype, but not with a mutated, binding sequence (Fig.   3e). [score:3]
Mogilyansky E Rigoutsos I The miR-17/92 cluster: a comprehensive update on its genomics, genetics, functions and increasingly important and numerous roles in health and diseaseCell Death Differ. [score:3]
A renilla luciferase expression construct, a Tgfbr2-UTR reporter construct, and the miR-17 mimic or control mimic were co -transfected to primary limb bud cells isolated from E12.5 embryos using Attractene transfection reagent (Qiagen) according to manufacturer’s instruction. [score:3]
e for miR-17 regulation on Tgfbr2 binding site. [score:2]
A 350-bp-long DNA fragment containing the conserved miR-17 binding sequence (5′-GCACTTTA-3′) or a mutated sequence (5′-GC TCT ATA-3′) was PCR amplified using primers, Common R, 5′-GCAAGCTTCTCAGCTCCCTGGTCCATAA-3′ and Wt-F, 5′-GCACTAGTGATCAGCTAATTGACCAGATGCACTTTATTAATGCCTGTGTGTAAATACGAA-3′ or Mut-F, 5′-GCACTAGTGATCAGCTAATTGACCAGATGCTCTATATTAATGCCTGTGTGTAAATACGAA-3′ and subcloned into pMIR-REPORT miRNA Expression Reporter Vector System (AM5795, ThermoFisher) at the Hind III and Spe I sites. [score:2]
Primary limb bud cells were co -transfected with control miRNA mimic (Ctrl miR) or mmu-miR-17-5p (miR-17) and a luciferase reporter construct carrying a wildtype (Wt 3′UTR) or mutated 3′UTR (Mut 3′UTR) sequence of mouse Tgfbr2. [score:1]
Olive V miR-19 is a key oncogenic component of mir-17-92Genes Dev. [score:1]
miR-17 miRIDIAN microRNA mimic and a control mimic were purchased from Dharmacon. [score:1]
The DIG-labeled miR-17 oligo probe (mmu-miR-17-5p; YD00615470-BCG) was purchased from Qiagen. [score:1]
Section in situ hybridization for miR-17-5p was performed according to the standard protocol with minor modifications [38]. [score:1]
Han YC An allelic series of miR-17 approximately 92-mutant mice uncovers functional specialization and cooperation among members of a microRNA polycistronNat. [score:1]
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HEK 293T cells were seeded onto 24-well plates for 12 h. Afterwards, 0.2 μg of firefly luciferase reporter plasmid; 0.2 μg of β-galactosidase expression vector (Ambion); 0.2 μg of expression vector pcDNA3.1 -overexpressing miR-17-92 cluster; empty vector pcDNA3.1; or 10-, 20-, 50-nM miR-17, miR-20a, miR-19a, and miR-19b mimics were transfected into the cells. [score:7]
Our previous work demonstrated that miR-17 modulates neuron-astrocyte transition via inhibiting BMPR2 expression [19]. [score:5]
org) bioinformatics tools, we found that among those miRNAs in miR-17-92 cluster, miR-19a/b may target CNTFR and miR-17, miR-20a, and miR-19a/b may target glycoprotein 130 (GP130), respectively (Fig.   2a, b). [score:5]
Among these four identified targets, JAK2 and STAT3 have been proved to be regulated by miR-17 in several mo dels [26, 41, 42]. [score:4]
In addition, miR-17 and miR-20a are reported to target JAK2 and STAT3 [26, 27]. [score:3]
For quantitative analysis, the numbers of each type of cell scored in four random fields were averaged, utilizing the Image-Pro Plus image analysis software In our previous study, we demonstrated that miR-17 modulates the astrocytogenesis/neurogenesis transition during the mouse corticogenesis by targeting BMP signaling pathway [19]. [score:3]
b Sequence alignment of mature miR-19a, miR-19b, miR-17, and miR-20a revealed their seed sequences that were reverse complementary to the seed-matched sequence within the 3′ UTR of mouse CNTFR or GP130, respectively. [score:1]
Meanwhile, Naka-Kaneda et al. also reported the role of miR-17 in controlling neurogenic to gliogenic transition of NSCs [40]. [score:1]
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Interestingly, the inhibition of miR-17-5p and/or miR-106-5p leads to the recovery of TRIM8 -mediated p53 tumour suppressor activity, which in turn strongly inhibits MYCN -dependent cell proliferation. [score:7]
It has been demonstrated that miR-17-5p and miR-106b-5p directly target the 3′UTR of TRIM8 and both transcriptionally and post-transcriptionally repress the expression of TRIM8, indicating that TRIM8 and miR-17-5p/miR-106b-5p may be part of the same circuit involved in ccRCC and glioma pathogenesis [20, 24]. [score:6]
The TRIM8 deficit, observed in patients affected by ccRCC, was explained by the up-regulation of the miR-17-5p and miR-106b-5p members of the miR-17-92 family, whose overexpression has an oncogenic effect by promoting tumour cell proliferation [38]. [score:6]
An increasing number of papers report that miR-106b-5p and miR-17-5p, above all the microRNAs of the miR-17-92 family, are overexpressed in many different chemo/radio-resistant cancers, including ccRCC, glioma, CRC, and CLL cell lines [22, 38, 39, 40, 41]. [score:3]
Mogilyansky E. Rigoutsos I. The miR-17/92 cluster: A comprehensive update on its genomics, genetics, functions and increasingly important and numerous roles in health and diseaseCell Death Differ. [score:3]
Additionally, in human gliomas, a heterogeneous group of primary malignant brain tumours with strong resistance to chemotherapy and radiotherapy, the reduction of TRIM8 expression, correlates with high levels of miRNA-17-5p, leading to an unfavourable clinical outcome of the patients. [score:3]
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In Dicer c KO mutants, miR-18, -19, and- 92a were downregulated, whereas miR-17 and -20a were upregulated. [score:7]
The individual miRNAs of miR-17/92 cluster were all downregulated in DGCR8, whereas only miR-17, -18a, and -20b from this cluster were significantly downregulated in Drosha c KO mutants. [score:7]
In our previous study, we showed that disruption of DGCR8 in VSMCs leads to downregulation of the miR-17/92 cluster. [score:4]
In our previous work, we showed that loss of DGCR8 leads to significant downregulation of the miR-17/92 cluster in VSMCs of DGCR8 c KO mice compared to controls, which may partially contribute to reduced cell proliferation by attenuating ERK1/2 and AKT pathways. [score:3]
Although loss of DGCR8 in VSMCs leads to embryonic lethality, our recent work indicates that deletion of the miR-17/92 cluster in VSMCs does not lead to any developmental abnormality in mice(Figure S6). [score:2]
Figure S6 miR-17/92 VSMC-specific KO mice are developmentally normal. [score:2]
B. Three different genotypes of miR-17/92 c KO mice were detected by PCR. [score:1]
miR-17/92 c KO and control mice are maintained in B6/129S4 mixed genetic background. [score:1]
miR-17/92 VSMC specific c KO mice were generated by crossing miR-17/92 [loxp/loxp](Jackson Laboratory, Stock #008459) with SM22-Cre mice. [score:1]
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The mir-17 family is the one most enriched (p = 3.24 E-4; Table S6) and comprises mir-17, mir-18a, mir-19a, mir-20a, mir-19b-1 and mir-92-1. This family is expressed as polycistronic units, revealing a common regulatory mechanism [62], that is confirmed by the similarity of their expression profiles (Figure 4 D). [score:6]
The study of these miRNAs could be a good basis for the identification and analysis of potential immuno-modulatory effectors in immuno -mediated diseases like multiple sclerosis, where a down-regulation of miR-17 and miR-20a associated with T-cell activation was demonstrated [80], or like inflammatory myopathies. [score:6]
The over -expression of mir-17, 18, 19a, and 20 was demonstrated in tumors of the lung [79] and a second study reported the up-regulation of the miR-17-92 cluster in B-cell lymphomas [62]. [score:6]
We found that miRNAs with higher expression in WBCs includes different miRNA families: mir-15, mir-17, mir-181, mir-23, mir-27 and mir-29 families. [score:3]
The mir-17 family showed the most pronounced expression in WBC (Figure 4D and Table S6). [score:3]
We found, on the contrary, that mir-17, 18, and 20a are not expressed in normal lung (Figure 4 D). [score:3]
* indicates components of mir-17 family. [score:1]
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miR-17 and miR-20a inhibitors and mimics were used as positive controls, since these two miRNAs were previously shown to target HIF-1α. [score:5]
In silico prediction of miRNA: Hif-1α interaction miRNAs miRNAs conserved in rodent and human (predicted by at least 2 databases) miR-17 [[] [16] []], -18a [[] [16] []], -20a [[] [16] []], -20b-5p [[] [22] []], -135a, -135b, -138, -199a-5p, -203, -335, -338-3p, -93 MiRNA: Hif-1α target prediction was performed and 12 miRNAs, commonly predicted in at least 2 out of the 6 databases used, were selected. [score:3]
Ten of them, miR-17, -18a, -20a, -20b-5p, -135a/b, -203, -335, -338-3p and -93 were found to be differentially and significantly (p<0.05) expressed in our miRNA profiling analysis of eMCAo rat brain samples (Fig 1D). [score:3]
Among these miRNAs, miR-17 and miR-20a have been reported to directly regulate HIF-1α in lung cancer cells [16]. [score:3]
Among the 10 miRNAs, interaction of miR-17, -18a, -20a and -20b-5p with HIF-1a had been reported previously in cancer pathogenesis [16, 22] whereas the remaining six miRNAs, miR-135a/b, -203, -335, -338-3p and -93 had not been validated in any disease condition. [score:3]
Independent transfection of HeLa cells with anti miR-17/-20a/-335/-93 exhibited an increase in the relative luciferase activity, whereas introduction of miR-17/-20a/-335/-93 mimics resulted in a reduction in luciferase activity suggesting that, apart from miR-17 and miR-20a, miR-335 and miR-93 are also direct regulators of Hif-1α (Fig 2B). [score:3]
The search yielded 12 miRNAs (miR-17, -18a, -20a, 20b-5p, -93, -135a/b, -138, -199a-5p, -203, -335, -338-3p) as potential regulators (Table 1). [score:2]
miR-17 and -20a were also included in the validation study as positive controls for they were previously reported to be the strongest regulators of HIF-1α in cancer pathogenesis [16]. [score:2]
The predicted binding site(s) of selected miRNAs (miRNA-17, -20a, -135a, -203, -335, -338-3p and -93) to the 3'UTR of Hif-1α is mapped in this figure. [score:1]
Based on the in silico prediction, we identified miR-17, -20a, -135a, -203, -335, -338-3p and -93 to be predicted by at least two databases and to be conserved in both rodents and human. [score:1]
miR-93 was observed to share the same binding site as miR-17 and miR-20a while miR-335 had two binding sites. [score:1]
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Upregulation of miR-let-7c is expected to downregulate miR-17 via downregulation of c-myc [31]; miR-17 in turn is predicted to target Nr4a2 (Targetscan prediction; http://www. [score:14]
org/) so downregulation of miR-17 should upregulate Nr4a2; finally, upregulation of Nr4a2 should lead to upregulation of Kcnma1 [32] which could explain the larger K [+] current I [K,f] in Tc1 IHCs. [score:13]
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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-17, hsa-mir-24-1, hsa-mir-24-2, hsa-mir-25, mmu-let-7g, mmu-let-7i, mmu-mir-124-3, mmu-mir-9-2, mmu-mir-134, mmu-mir-137, mmu-mir-138-2, mmu-mir-145a, mmu-mir-24-1, hsa-mir-192, mmu-mir-194-1, mmu-mir-200b, hsa-mir-7-1, hsa-mir-7-2, hsa-mir-7-3, hsa-mir-215, hsa-mir-221, hsa-mir-200b, mmu-mir-296, mmu-let-7d, mmu-mir-106b, hsa-let-7g, hsa-let-7i, hsa-mir-124-1, hsa-mir-124-2, hsa-mir-124-3, hsa-mir-137, hsa-mir-138-2, hsa-mir-145, hsa-mir-9-1, hsa-mir-9-2, hsa-mir-9-3, hsa-mir-134, hsa-mir-138-1, hsa-mir-194-1, mmu-mir-192, mmu-mir-200a, 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-24-2, mmu-mir-346, hsa-mir-200c, mmu-mir-25, mmu-mir-200c, mmu-mir-221, mmu-mir-124-1, mmu-mir-124-2, mmu-mir-9-1, mmu-mir-9-3, mmu-mir-138-1, mmu-mir-7a-1, mmu-mir-7a-2, mmu-mir-7b, hsa-mir-194-2, mmu-mir-194-2, hsa-mir-106b, hsa-mir-200a, hsa-mir-296, hsa-mir-369, hsa-mir-346, mmu-mir-215, gga-let-7i, gga-let-7a-3, gga-let-7b, gga-let-7c, gga-mir-221, gga-mir-17, gga-mir-138-1, gga-mir-124a, gga-mir-194, gga-mir-215, gga-mir-137, gga-mir-7-2, gga-mir-138-2, gga-let-7g, gga-let-7d, gga-let-7f, gga-let-7a-1, gga-mir-200a, gga-mir-200b, gga-mir-124b, gga-let-7a-2, gga-let-7j, gga-let-7k, gga-mir-7-3, gga-mir-7-1, gga-mir-24, gga-mir-7b, gga-mir-9-2, dre-mir-7b, dre-mir-7a-1, dre-mir-7a-2, dre-mir-192, dre-mir-221, dre-mir-430a-1, dre-mir-430b-1, dre-mir-430c-1, dre-let-7a-1, dre-let-7a-2, dre-let-7a-3, dre-let-7a-4, dre-let-7a-5, dre-let-7a-6, dre-let-7b, dre-let-7c-1, dre-let-7c-2, dre-let-7d-1, dre-let-7d-2, dre-let-7e, dre-let-7f, dre-let-7g-1, dre-let-7g-2, dre-let-7h, dre-let-7i, dre-mir-7a-3, dre-mir-9-1, dre-mir-9-2, dre-mir-9-4, dre-mir-9-3, dre-mir-9-5, dre-mir-9-6, dre-mir-9-7, dre-mir-17a-1, dre-mir-17a-2, dre-mir-24-4, dre-mir-24-2, dre-mir-24-3, dre-mir-24-1, dre-mir-25, dre-mir-92b, dre-mir-124-1, dre-mir-124-2, dre-mir-124-3, dre-mir-124-4, dre-mir-124-5, dre-mir-124-6, dre-mir-137-1, dre-mir-137-2, dre-mir-138-1, dre-mir-145, dre-mir-194a, dre-mir-194b, dre-mir-200a, dre-mir-200b, dre-mir-200c, dre-mir-430c-2, dre-mir-430c-3, dre-mir-430c-4, dre-mir-430c-5, dre-mir-430c-6, dre-mir-430c-7, dre-mir-430c-8, dre-mir-430c-9, dre-mir-430c-10, dre-mir-430c-11, dre-mir-430c-12, dre-mir-430c-13, dre-mir-430c-14, dre-mir-430c-15, dre-mir-430c-16, dre-mir-430c-17, dre-mir-430c-18, dre-mir-430a-2, dre-mir-430a-3, dre-mir-430a-4, dre-mir-430a-5, dre-mir-430a-6, dre-mir-430a-7, dre-mir-430a-8, dre-mir-430a-9, dre-mir-430a-10, dre-mir-430a-11, dre-mir-430a-12, dre-mir-430a-13, dre-mir-430a-14, dre-mir-430a-15, dre-mir-430a-16, dre-mir-430a-17, dre-mir-430a-18, dre-mir-430i-1, dre-mir-430i-2, dre-mir-430i-3, dre-mir-430b-2, dre-mir-430b-3, dre-mir-430b-4, dre-mir-430b-6, dre-mir-430b-7, dre-mir-430b-8, dre-mir-430b-9, dre-mir-430b-10, dre-mir-430b-11, dre-mir-430b-12, dre-mir-430b-13, dre-mir-430b-14, dre-mir-430b-15, dre-mir-430b-16, dre-mir-430b-17, dre-mir-430b-18, dre-mir-430b-5, dre-mir-430b-19, dre-mir-430b-20, mmu-mir-470, hsa-mir-485, hsa-mir-496, dre-let-7j, mmu-mir-485, mmu-mir-543, mmu-mir-369, hsa-mir-92b, gga-mir-9-1, hsa-mir-671, mmu-mir-671, mmu-mir-496a, mmu-mir-92b, hsa-mir-543, gga-mir-124a-2, mmu-mir-145b, mmu-let-7j, mmu-mir-496b, mmu-let-7k, gga-mir-124c, gga-mir-9-3, gga-mir-145, dre-mir-138-2, dre-mir-24b, gga-mir-9-4, mmu-mir-9b-2, mmu-mir-124b, mmu-mir-9b-1, mmu-mir-9b-3, gga-mir-9b-1, gga-let-7l-1, gga-let-7l-2, gga-mir-9b-2
MicroRNA-17-92a upregulation by estrogen leads to Bim targeting and inhibition of osteoblast apoptosis. [score:7]
Mdm2 is negatively regulated by several miRNAs including miR-192 (Pichiorri et al., 2010), miR-194 (Pichiorri et al., 2010), miR-215 (Pichiorri et al., 2010), miR-221 (Kim et al., 2010), and miR-17 (Li and Yang, 2012) in different cellular contexts; however, whether these or other miRNAs regulate Mdm2 expression during the CNS development must be determined. [score:6]
Stress response of glioblastoma cells mediated by miR-17-5p targeting PTEN and the passenger strand miR-17-3p targeting. [score:5]
Although from the beginning of the spinal cord development the p2 progenitors express the pMN marker, Olig2, it is repressed by miR-17-3p through development progression, thus ensuring the proper specification of the pMN/p2 boundary and the production of V2 interneurons (Chen et al., 2011a). [score:5]
In this sense, mice lacking the miR-17/92 cluster present a dorsal shift in pMN/p2 boundary and incorrect production of V2 interneurons (Chen et al., 2011a). [score:1]
Mir-17-3p controls spinal neural progenitor patterning by regulating Olig2/Irx3 cross-repressive loop. [score:1]
Therefore, Olig2 repression mediated by miR-17-3p is crucial for the correct patterning of ventral spinal NPs domains and thus, it is possible that other miRNAs also participate in NPs specifications in different CNS regions. [score:1]
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[+] score: 25
Other miRNAs from this paper: hsa-mir-17, mmu-mir-142a, hsa-mir-142, mmu-mir-142b
The expression of as-miR-142-3p did not affect the expression of Fgf5 or Gata4, although as-miR-17 enhanced expression of Fgf5, as expected (Figures 1(g) and 1(h)). [score:7]
Data revealed that as-miR-142-3p, but not as-miR-17, suppressed the expression of T brachyury, which is expressed specifically in cells of the mesodermal lineage [22] (Figure 1(i)). [score:7]
An expression plasmid containing antisense sequence against miR-17, which is expressed at very high levels in undifferentiated iPS cells [3, 21], was used as a control. [score:5]
In contrast, the levels of miR-17 were rather reduced but not significantly by 5-aza-dC (Figure 2(c)), whereas the expression of neither miR-142-3p nor miR-17 was changed significantly by 5-aza-dC in undifferentiated iPS cells (Figures 2(d) and 2(e)). [score:3]
Plasmids containing antisense sequences of mature miR-142-3p or miR-17 expression plasmid were constructed as follows: double strand DNA, which encode antisense of mature miR-142-3p or miR-17, was inserted downstream of U6 promoter using BamHI and EcoRI sites of pMX retrovirus vector containing EGFP after 5′ LTR (Figure 1(b)). [score:3]
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Furthermore, pharmacological TNF inhibition of STAT3, using the STAT3 inhibitor Stattic, decreased miR-17~92 cluster expression in all of the ALK(+) ALCL cell lines tested (SUP-M2/TS, JB-6, L82, and KARPAS-299). [score:7]
It also induced the downregulation of the pro-apoptotic protein BIM, suggesting that the miR-17~92 cluster might mediate resistance to STAT3 knockdown by targeting BIM [34]. [score:7]
Several of the upregulated miRNA, such as the miR-17~92 cluster, are known to promote cell proliferation. [score:4]
The main findings of this study was that the forced expression of the miR-17~92 cluster could partially rescue STAT3 knockdown by sustaining the proliferation and survival of NPM/ALK(+) cells both in vitro and in a xenograft mouse mo del. [score:4]
The miR-17~92 Cluster. [score:1]
The miR-17~92 cluster fami