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An investigation into anti-proliferative effects of microRNAs encoded by the miR-106a-363 cluster on human carcinoma cells and keratinocytes using microarray profiling of miRNA transcriptomes.

ABSTRACT: Transfection of human oral squamous carcinoma cells (clone E10) with mimics for unexpressed miR-20b or miR-363-5p, encoded by the miR-106a-363 cluster (miR-20b, miR-106a, miR-363-3p, or miR-363-5p), caused 40-50% decrease in proliferation. Transfection with mimics for miR-18a or miR-92a, encoded by the miR-17-92 cluster (all members being expressed in E10 cells), had no effect on proliferation. In contrast, mimic for the sibling miRNA-19a yielded about 20% inhibition of proliferation. To investigate miRNA involvement profiling of miRNA transcriptomes were carried out using deoxyoligonucleotide microarrays. In transfectants for miR-19a, or miR-20b or miR-363-5p most differentially expressed miRNAs exhibited decreased expression, including some miRNAs encoded in paralogous miR-17-92-or miR-106b-25 cluster. Only in cells transfected with miR-19a mimic significantly increased expression of miR-20b observed-about 50-fold as judged by qRT-PCR. Further studies using qRT-PCR showed that transfection of E10 cells with mimic for miRNAs encoded by miR-17-92 - or miR-106a-363 - or the miR-106b-25 cluster confirmed selective effect on expression on sibling miRNAs. We conclude that high levels of miRNAs encoded by the miR-106a-363 cluster may contribute to inhibition of proliferation by decreasing expression of several sibling miRNAs encoded by miR-17-92 or by the miR-106b-25 cluster. The inhibition of proliferation observed in miR-19a-mimic transfectants is likely caused by the miR-19a-dependent increase in the levels of miR-20b and miR-106a. Bioinformatic analysis of differentially expressed miRNAs from miR-106a, miR-20b and miR-363-5p transfectants, but not miR-92a transfectants, yielded significant associations to "Cellular Growth and Proliferation" and "Cell Cycle." Western blotting results showed that levels of affected proteins to differ between transfectants, suggesting that different anti-proliferative mechanisms may operate in these transfectants.

SUBMITTER: Khuu C

PROVIDER: S-EPMC4142865 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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An investigation into anti-proliferative effects of microRNAs encoded by the miR-106a-363 cluster on human carcinoma cells and keratinocytes using microarray profiling of miRNA transcriptomes.

Khuu Cuong C Jevnaker Anne-Marthe AM Bryne Magne M Osmundsen Harald H

Frontiers in genetics 20140825

Transfection of human oral squamous carcinoma cells (clone E10) with mimics for unexpressed miR-20b or miR-363-5p, encoded by the miR-106a-363 cluster (miR-20b, miR-106a, miR-363-3p, or miR-363-5p), caused 40-50% decrease in proliferation. Transfection with mimics for miR-18a or miR-92a, encoded by the miR-17-92 cluster (all members being expressed in E10 cells), had no effect on proliferation. In contrast, mimic for the sibling miRNA-19a yielded about 20% inhibition of proliferation. To investi ...[more]

PMID: 25202322

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Project description:MicroRNAs (miRs) have been identified as potent regulators of both normal development and the hallmarks of cancer. Targeting of microRNAs has been shown to have preclinical promise, and select miR-based therapies are now in clinical trials. Ewing Sarcoma is a biologically aggressive pediatric cancer with little change in clinical outcomes despite improved chemotherapeutic regimens. There is a substantial need for new therapies to improve Ewing Sarcoma outcomes and to prevent chemotherapy-related secondary sequelae. Most Ewing Sarcoma tumors are driven by the EWS/Fli-1 fusion oncoprotein, acting as a gain-of-function transcription factor causing dysregulation of a variety of targets, including microRNAs. Our previous studies, and those of others, have identified upregulation of miRs belonging to the related miR-17~92a, miR-106b~25, and miR-106a~363 clusters in Ewing Sarcoma. However, the functional consequences of this have not been characterized, nor has miR blockade been explored as an anti-cancer strategy in Ewing Sarcoma. To simulate a potential therapeutic approach, we examined the effects of blockade of these clusters, and their component miRs. Using colony formation as a read-out, we find that blockade of selected individual cluster component miRs, using specific inhibitors, has little or no effect. Combinatorial inhibition using miR "sponge" methodology, on the other hand, is inhibitory to colony formation, with blockade of whole clusters generally more effective than blockade of miR families. We show that a miR-blocking sponge directed against the poorly characterized miR-106a~363 cluster is a particularly potent inhibitor of clonogenic growth in a subset of Ewing Sarcoma cell lines. We further identify upregulation of miR-15a as a downstream mechanism contributing to the miR-106a~363 sponge growth-inhibitory effect. Taken together, our studies provide support for a pro-oncogenic role of the miR-106a~363 cluster in Ewing Sarcoma, and identify miR-106a~363 blockade, as well as miR-15a replacement, as possible strategies for inhibition of Ewing Sarcoma growth.

Dataset Information

An investigation into anti-proliferative effects of microRNAs encoded by the miR-106a-363 cluster on human carcinoma cells and keratinocytes using microarray profiling of miRNA transcriptomes.

Publications

An investigation into anti-proliferative effects of microRNAs encoded by the miR-106a-363 cluster on human carcinoma cells and keratinocytes using microarray profiling of miRNA transcriptomes.

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