MiR-191 Regulates Primary Human Fibroblast Proliferation and Directly Targets Multiple Oncogenes

<div><p>miRNAs play a central role in numerous pathologies including multiple cancer types. miR-191 has predominantly been studied as an oncogene, but the role of miR-191 in the proliferation of primary cells is not well characterized, and the miR-191 targetome has not been experimentally profiled. Here we utilized RNA induced silencing complex immunoprecipitations as well as gene expression profiling to construct a genome wide miR-191 target profile. We show that miR-191 represses proliferation in primary human fibroblasts, identify multiple proto-oncogenes as novel miR-191 targets, including CDK9, NOTCH2, and RPS6KA3, and present evidence that miR-191 extensively mediates target expression through coding sequence (CDS) pairing. Our results provide a comprehensive genome wide miR-191 target profile, and demonstrate miR-191’s regulation of primary human fibroblast proliferation.</p></div

miR-191 represses proliferation.

<p>(A) miR-191 transfection reduces the rate of cell growth. Average cell number relative to 0 hr following miR-191 or control siRNA transfection is shown for each time point indicated. Error bars denote ± SD, n = 4 (independent biological replicates). P-values were calculated by Student’s t-test comparing cell numbers following miR-191 transfection to cell numbers following Control siRNA transfection at each time point. (B) miR-191 transfection represses proliferation. The Y-axis indicates the relative percentage of cells expressing Ki67. Bars are the mean percentage of cells expressing Ki67 relative to Control RNA 2, and error bars denote ± SD, n = 3. (C) miR-191 transfection slows progression through the cell cycle. Cell-cycle profiles of transiently transfected fibroblasts following treatment with nocodazole. Representative histograms shown are the median for each treatment of 3 biological replicates. The Y-axis denotes cell number and the X-axis DNA content. Numbers in each histogram indicate percentage of cells in G1 or G2. For the bar graph, the Y-axis denotes the percentage of cells found in each stage of the cell cycle. Error bars indicate ± SD, n = 3. (D) Inhibition of miR-191 increases cell growth in fibroblasts serum starved into quiescence. Average cell number relative to 0 hours post transfection of an LNA targeting miR-191 or a LNA negative control is shown for each time point indicated. Error bars denote ± SD, n = 6. P-values were estimated by Student’s one tailed t-test comparing cell numbers following miR-191 inhibitor transfection to cell numbers following the Control inhibitor transfection at each time point. For B, and C, P-values were calculated by Student’s one tailed t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.</p

Genome wide profiling of miR-191 targets.

<p>(A) Transcript levels of mRNAs enriched in the RIP-seq following miR-191 transfection were decreased in the RNA-seq expression profiles. The Y-axis indicates the cumulative fraction of mRNAs profiled for each group of mRNAs denoted by line color, and the X-axis shows the level of repression for each mRNA profiled with positive values indicating increased repression. mRNAs that contain a miR-191 miRNA 7-mer seed match were significantly more repressed than all mRNAs profiled (p = 4.65e-24). mRNAs enriched in the RIP were significantly more repressed than mRNAs with a miR-191 7-mer seed match (p = 2.54e-12). mRNAs enriched in the RIP that contain a 7-mer miR-191 seed match were significantly more repressed than mRNAs enriched in the RIP (p = 3.97e-17) Significance estimates were calculated with Student’s t-test. (B) mRNAs with a miR-191 seed match were significantly enriched in the RIP-seq compared to all mRNAs profiled (p = 4.65e-24, Student’s t-test). The Y-axis shows the cumulative fraction of mRNAs profiled for each group of mRNAs denoted by line color, and the X-axis indicates the amount of enrichment in the RIP with positive values indicating increased enrichment. (C) mRNAs most enriched in the RIP-seq had the highest frequency of miR-191 seed matches. The X-axis denotes consecutive groups of 250 genes, ranked from most enriched to least enriched in the RIP-seq. The line is the frequency indicated on the Y-axis of the miR-191 7-mer seed match in the 3’ UTRs of the group relative to the frequency of the seed match in all mRNAs profiled. (D) mRNAs repressed in the gene expression experiments had the highest frequency of miR-191 7-mer seed matches. The X-axis indicates consecutive groups of 250 genes, ranked from most repressed to least repressed. The line is the frequency indicated on the Y-axis of miR-191 7-mer seed matches in the 3’ UTRs of the group relative to the frequency of miR-191 seed matches in all mRNAs profiled. (E) The highest ranked miR-191 targets had the highest frequency of miR-191 seed matches. RIP-seq enrichment and gene expression repression data were combined to rank miR-191 targets. The X-axis denotes consecutive groups of 250 genes, from most highly ranked to least highly ranked. The line is the frequency indicated on the Y-axis of miR-191 7-mer seed matches in the 3’ UTRs of the group relative to the frequency of seed matches in all mRNAs profiled. For A-D, n = 3. E combines gene expression RNA-seq data, n = 3, with RIP-seq data, n = 3.</p

miR-191 directly targets multiple proto-oncogenes.

<p>(A) Enriched Gene Ontology terms for the experimentally identified set of miR-191 targets. BP: Biological process. Numbers indicate gene counts. (B) Luciferase reporter assays showed miR-191 directly targets the 3’ UTRs of the genes indicated on the X-axis. The Y-axis denotes relative luciferase units from miR-191 transfected HEK293 cells normalized to Control siRNA transfected cells. Purple bars: 3’ UTRs with the putative miR-191 target site entirely deleted. Green bars: Intact 3’ UTRs. P-values were estimated by Student’s one tailed t-test comparing miR-191 normalized to Control siRNA luciferase activity with intact 3’ UTRs to normalized luciferase activity with miR-191 target site deleted 3’ UTRs. (C) miR-191 transfection significantly decreased <i>AGO2</i>, <i>BCL2</i>, <i>CDK6</i>, <i>CDK9</i>, <i>NOTCH2</i>, and <i>RPS6KA3</i> transcript levels in fibroblasts. Fold changes are indicated on the Y-axis relative to the Control siRNA transfection. GAPDH was used to normalize input RNA levels. P-values were estimated by Student’s one tailed t-test comparing normalized transcript levels following miR-191 transfection to normalized transcript levels following Control siRNA transfection. (D) miR-191 transfection in fibroblasts decreased protein expression of CDK9, NOTCH2, and RPS6KA3 compared to Control siRNA transfection. Band intensities were quantified, normalized to GAPDH, and shown relative to the Control siRNA. For B and C, bars indicate the mean, and error bars denote ± SD, n = 6 and 3 respectively. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.</p

Experimental setup used to profile RISC association and repression of gene expression.

<p>Experimental setup used to profile RISC association and repression of gene expression.</p