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. 2015 Feb 5;16(1):40.
doi: 10.1186/s12864-015-1279-9.

miR-503 represses human cell proliferation and directly targets the oncogene DDHD2 by non-canonical target pairing

Damon Polioudakis  1 , Nathan S Abell  2 , Vishwanath R Iyer  3
Affiliations

miR-503 represses human cell proliferation and directly targets the oncogene DDHD2 by non-canonical target pairing

Damon Polioudakis et al. BMC Genomics. .

"V体育2025版" Abstract

Background: The pathways regulating the transition of mammalian cells from quiescence to proliferation are mediated by multiple miRNAs VSports手机版. Despite significant improvements in our understanding of miRNA targeting, the majority of miRNA regulatory networks are still largely unknown and require experimental validation. .

Results: Here we identified miR-503, miR-103, and miR-494 as negative regulators of proliferation in primary human cells. We experimentally determined their genome wide target profiles using RNA-induced silencing complex (RISC) immunoprecipitations and gene expression profiling V体育安卓版. Analysis of the genome wide target profiles revealed evidence of extensive regulation of gene expression through non-canonical target pairing by miR-503. We identified the proto-oncogene DDHD2 as a target of miR-503 that requires pairing outside of the canonical 5' seed region of miR-503, representing a novel mode of miRNA-target pairing. Further bioinformatics analysis implicated miR-503 and DDHD2 in breast cancer tumorigenesis. .

Conclusions: Our results provide an extensive genome wide set of targets for miR-503, miR-103, and miR-494, and suggest that miR-503 may act as a tumor suppressor in breast cancer by its direct non-canonical targeting of DDHD2. V体育ios版.

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Figures

Figure 1
Figure 1
miR-503, −103, and −494 inhibit proliferation. (A) miR-503, −103, and −494 transfection significantly reduces primary human fibroblast cell growth. Average cell number relative to 0 hr following miRNA or Control siRNA transfection is shown for each time point indicated. Error bars denote ± SD, n = 6. P-values were calculated by Student’s t-test comparing cell numbers following miRNA transfection to cell numbers following Control siRNA transfection at each time point. (B) miR-503 and −494 transfection significantly represses proliferation. The Y-axis indicates the relative percentage of primary human fibroblast cells expressing Ki67, measured by flow cytometry. Bars are the mean percentage of cells expressing Ki67 relative to Control RNA 2, and error bars denote ± SD, n = 3. (C) miR-503 and −103 transfection significantly inhibits progression through the cell cycle. The Y-axis indicates the percentage of primary human fibroblast cells found in each stage of the cell cycle measured using propidium iodide staining, and bars are the mean percentage the cell population found in each stage. Error bars denote ± SD, n = 3. (D) Inhibition of miR-503, −103, and −494 increases cell growth in quiescent fibroblasts. Average cell number relative to 0 hr following transfection of an LNA targeting miR-503, −103, or −494 or a LNA negative control is shown for each time point indicated. Error bars denote ± SD, n = 6. P-values were calculated by Student’s one tailed t-test comparing cell numbers following miRNA inhibitor transfection to cell numbers following Control inhibitor transfection at each time point. For B, P-values were estimated by Student’s t-test, and for C, P-values were estimated by Student’s paired t-test. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.
Figure 2
Figure 2
Strategy for genome-wide target identification of miRNAs using RIP and gene expression profiling. HeLa cells are transfected either with miRNAs or control duplexes. From each transfection, total RNA as well as RISC-associated RNAs are isolated by Ago2 immunoprecipitation. All populations of RNAs are profiled by deep sequencing or in come cases, by microarray hybridization (see text). RNAs showing RISC-association in a miRNA dependent manner are identified by comparing the immunoprecipitated RNAs between the miRNA and control RNA transfection. mRNAs showing repression by the miRNAs are identified by comparing the total RNAs between the miRNA and control RNA transfection.
Figure 3
Figure 3
Genome wide profiling of miR-503, −103, and −494 targets. (A) mRNAs enriched in the RIP-seq following miR-503, −103, or −494 transfection are also repressed. The Y-axis denotes the cumulative fraction of all mRNA transcripts profiled for each group of mRNAs denoted by line color, and the X-axis indicates the level of repression for each mRNA transcript profiled with positive values indicating increased repression. Orange: all mRNAs; Blue: mRNAs that contain the respective 7-mer miRNA seed match in their 3′UTR; Green: mRNAs that were 1.75 fold enriched in the RIP; Purple: mRNAs that were 1.75 fold enriched in the RIP and that contain the respective 7-mer miRNA seed match in their 3′UTR. Significance estimates were calculated with Student’s t-test. (B) mRNAs enriched in the RIPs had the highest frequency of the respective miRNA seed matches. The X-axis denotes consecutive groups of 250 genes, ranked from most enriched to least enriched in the RIP-seq. (C) For miR-103, and −494, mRNAs repressed in the gene expression experiments had the highest frequency of the respective miRNA seed matches. The X-axis indicates consecutive groups of 250 genes, ranked from most repressed to least repressed. (D) The highest ranked miRNA targets had the highest frequency of the respective miRNA seed matches. RIP enrichment and gene expression repression data were combined to rank miRNA targets. The X-axis denotes consecutive groups of 250 genes, from most highly ranked to least highly ranked. For B, C, and D, The line is the frequency indicated on the Y-axis of the respective 7-mer miRNA seed in the gene group compared to the frequency of the seed in all genes profiled. For B, C, and D, n = 3. E combines gene expression data, n = 3, with RIP-seq data, n = 3.
Figure 4
Figure 4
miRNA targeting utilizing pairing outside of the canonical miRNA 5′ seed region. (A) Enrichment of sequences pairing to the 3′ end of the miRNAs in the experimentally determined miRNA targetomes. The Y-axis indicates the frequency of a 6-mer in the experimentally determined miRNA target mRNAs relative to all mRNAs profiled. 6-mers are organized step-wise in 1 nucleotide increments along the X-axis from 5′ end to 3′ end of the mature miRNA. (B) Different types of miRNA-target pairing. (C) Frequency of different types of miRNA pairing in RIP-Seq enriched mRNAs. Bars are the frequency of mRNAs with the indicated type of miRNA-target pairing in the RIP enriched mRNAs, relative to the frequency of mRNAs with the indicated type of miRNA-target pairing in all mRNAs profiled. (D) RIP-seq enriched mRNAs containing a supplementary target site in their 3′UTR were significantly more repressed than all RIP-seq enriched mRNAs, but not more repressed than RIP-seq enriched mRNAs that contain a perfect 5′ seed in their 3′UTR (p = 0.04 and 0.06, respectively, Student’s t-test). Each box and whiskers plot indicates gene expression repression for genes that contain the type of miRNA-target pairing specified on the X-axis. Boxes extend from the 1st to 3rd quartile of gene expression repression, the band is the median, and whiskers denote the minimum and maximum excluding outliers. (E) There were no significant differences in miR-503 RIP enrichment in RIP enriched mRNAs with different types of miRNA-target pairing. Each box and whiskers plot indicates RIP enrichment for mRNAs that contain the type of miRNA-target pairing site specified on the X-axis. For C, D, and E, 5′ mismatch seed + 3′ mismatch (blue) corresponds to Compensatory in B; 5′ perfect seed match + 3′ mismatch (green) corresponds to Supplementary in B; and 5′ perfect seed match (purple and orange) correspond to Canonical seed in B. *P < 0.05.
Figure 5
Figure 5
Pairing of the 3′ and 5′ end of miR-503 is necessary for direct targeting by miR-503 of the proto-oncogene DDHD2. (A) Pairing by the 3′ and 5′ end of miR-503 to the 3′UTR of DDHD2 is necessary to repress expression of the luciferase reporter construct. The Y-axis denotes relative luciferase units from miR-503 transfected HEK293 cells normalized to control RNA transfected cells. The X-axis indicates the type of 3′UTR (either wild-type or deletion as indicated) Error bars denote ± SD, n = 3. In the diagram, vertical lines indicate base pairing, dashes show deleted base pairs, and numbers in parenthesis denote location in the 3′ UTR, not genomic coordinates. P-values were calculated by Student’s one tailed t-test comparing miRNA normalized to Control siRNA luciferase activity with intact 3′ UTRs, to normalized activity with the respective miRNA target site deleted 3′ UTRs. *P < 0.05. (B) miR-503 transfection decreased protein expression of DDHD2. DDHD2 protein levels following miR-503 transfection in fibroblasts and HeLa cells compared to Control siRNA transfection. Band intensities were quantified, normalized to GAPDH, and shown relative to the Control siRNA.
Figure 6
Figure 6
miR-503 involvement in breast cancer. (A) Low miR-503 expression correlates with a lower survival probability in ER+ breast cancer patients (p = 8.56e-06). Kaplan-Meier survival curves for patients with ER+ breast cancer expressing different levels of miR-503 were calculated using the MIRUMIR tool. Number of patients: n = 37. (B) DDHD2 is frequently amplified in breast cancer. The bars are the frequency of DDHD2 copy number alterations in the different types of cancer indicated on the X-axis. The frequency of patients with DDHD2 amplification was calculated using the cBio portal. Yellow: Deletion; Orange: Amplification. n = 1044. (C) Patients with copy number alterations in DDHD2 (purple line) have a lower survival probably than those without amplifications in DDHD2 (green line) (p = 0.027). Kaplan-Meier survival curves were was calculated using the cBio portal. n = 888.
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