Dataset Information

The identification of novel targets of miR-16 and characterization of their biological functions in cancer cells.

ABSTRACT:

Background

In eukaryotes, miR-16 is an important microRNA (miRNA) that is involved in numerous biological processes. However, it is not fully understood how miR-16 executes its physiological functions. In the present study, we aimed to identify novel miR-16 targets and study their biological functions.

Methods

Candidate target genes of miR-16 were screened by microarray analysis of mRNA levels in several cancer cell lines with enhanced miR-16. Three bioinformatics algorithms, including TargetScan, PicTar, and miRanda, were used in combination to calculate the miR-16 targets. The expression levels of miR-16 and target mRNA were examined by relative quantification RT-PCR, and the expression levels of target protein were detected by Western blot. Luciferase reporter plasmids were constructed to confirm direct targeting. The effect of miR-16 and target gene on cell viability was evaluated using MTT assays. The effects of miR-16 and target gene on apoptosis and cell cycle distribution were evaluated by flow cytometry analysis.

Results

By overexpressing miR-16 in several cancer cell lines and measuring global mRNA levels using microarray analysis, we identified 27 genes that may be regulated by miR-16. After the bioinformatics filtering process, 18 genes were selected as candidate miR-16 targets. Furthermore, we experimentally validated three of these candidates, MAP7 (microtubule-associated protein 7), PRDM4 (PR domain containing 4) and CDS2 (CDP-diacylglycerol synthase 2), as direct targets of miR-16. Finally, we demonstrated that miR-16 targeting MAP7 played a critical role in regulating proliferation but not apoptosis and cell cycle progression in cancer cells.

Conclusion

In summary, the present study identifies several novel miR-16 targets and illustrates a novel function of miR-16 targeting MAP7 in modulating proliferation in cancer cells.

SUBMITTER: Yan X

PROVIDER: S-EPMC3751425 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Publications

The identification of novel targets of miR-16 and characterization of their biological functions in cancer cells.

Yan Xin X Liang Hongwei H Deng Ting T Zhu Kegan K Zhang Suyang S Wang Nan N Jiang Xueyuan X Wang Xueliang X Liu Rui R Zen Ke K Zhang Chen-Yu CY Ba Yi Y Chen Xi X

Molecular cancer 20130814

<h4>Background</h4>In eukaryotes, miR-16 is an important microRNA (miRNA) that is involved in numerous biological processes. However, it is not fully understood how miR-16 executes its physiological functions. In the present study, we aimed to identify novel miR-16 targets and study their biological functions.<h4>Methods</h4>Candidate target genes of miR-16 were screened by microarray analysis of mRNA levels in several cancer cell lines with enhanced miR-16. Three bioinformatics algorithms, incl ...[more]

PMID: 23941513

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Project description:Identification of protein targets for microRNAs (miRNAs) is a significant challenge due to the complexity of miRNA-mediated regulation. We have previously demonstrated that miR-193b targets estrogen receptor-? (ER?) and inhibits estrogen-induced growth of breast cancer cells. Here, we applied a high-throughput strategy using quantitative iTRAQ (isobaric tag for relative and absolute quantitation) reagents to identify other target proteins regulated by miR-193b in breast cancer cells. iTRAQ analysis of pre-miR-193b transfected MCF-7 cells resulted in identification of 743 unique proteins, of which 39 were down-regulated and 44 up-regulated as compared with negative control transfected cells. Computationally predicted targets of miR-193b were highly enriched (sevenfold) among the proteins whose level of expression decreased after miR-193b transfection. Only a minority of these (13%) showed similar effect at the mRNA level illustrating the importance of post-transcriptional regulation. The most significantly repressed proteins were selected for validation experiments. These data confirmed 14-3-3? (YWHAZ), serine hydroxyl transferase (SHMT2), and aldo-keto reductase family 1, member C2 (AKR1C2) as direct, previously uncharacterized, targets of miR-193b. Functional RNAi assays demonstrated that specific combinations of knockdowns of these target genes by siRNAs inhibited growth of MCF-7 cells, mimicking the effects of the miR-193b overexpression. Interestingly, the data imply that besides targeting ER?, the miR-193b effects include suppression of the local production of estrogens and other steroid hormones mediated by the AKR1C2 gene, thus provoking two separate molecular mechanisms inhibiting steroid-dependent growth of breast cancer cells. In conclusion, we present here a proteomic screen to identify targets of miR-193b, and a systems biological approach to mimic its effects at the level of cellular phenotypes. This led to the identification of multiple genes whose combinatorial knock-down likely mediates the strong anti-cancer effects observed for miR-193b in breast cancer cells.

Dataset Information

The identification of novel targets of miR-16 and characterization of their biological functions in cancer cells.

Background

Methods

Results

Conclusion

Publications

The identification of novel targets of miR-16 and characterization of their biological functions in cancer cells.

Similar Datasets

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