MiR-20A Promotes Invasion And Migration By Regulating TNKS2in Human Cervical Cancer Cells

[objective] MicroRNAs (miRNAs) are a class of small non-coding RNAs that include18-24nucleotides, which post-transcriptionally regulate gene expression. miRNAs are involved in many physiological and pathological progress. Recent evidence indicates that many miRNAs function as oncogenes or tumor suppressors and play an important role in cancer initiation and progression by regulating their target genes. Using real-time PCR assay, we found that miR-20a were significantly up-regulated in human cervical cancer tissue samples. In this study, we focused on the effects of miR-20a on the phenotypes of cervical cells as well as the identification of their direct target genes, in order to illuminate the molecular mechanisms of miR-20a in the initiation and progression of human cervical cancer.[Methods] We detected the differencial expression of miR-20a in human cervical cancer and adjacent normal tissues by real-time PCR assays. miR-20a was overexpressed in18pairs of tissues, and the changes of cell phenotypes were detected both with MTT assay,colony formation assay, migration assay and invasion assay. Subsequently, we combined bioinformatics and identified the candidate target genes for miR-20a. Then fluorescent reporter assay was performed to confirm the reliability of the direct regulaiton genes. Furthermore, the mRNA levels and protein levels of TNKS2genes in miR-20a overexpressed human cervical cancer cells or tissues were detected with real-time PCR and western blot, in order to confirm the regulative role of miR-20a on TNKS2expression. Finally, the target gene was knocked down using RNA interference and changes of cell phenotypes were detected both with MTT assay, colony formation assay, migration assay and invasion assay.[Results] Real-time PCR results showed that the expression of miR-20a in human cervical cancer tissues was higher than those in adjacent normal tissues. We report that after overexpression of miR-20a, the short-term proliferation was not affected and the long-term proliferation, migration and invasion of human cervical cancer cell were enhanced. And downexpression of miR-20a got the opposite results. After that, we identified oncogene tankyrase2(TNKS2) as a candidate target genes for miR-20a. The TNKS2mRNA (3’UTR) contains the potential binding site of miR-20a. The fluorescent reporter assay also confirmed that miR-20a can directly bind to the specific site of TNKS2mRNA3’UTR and regulate the gene expression. When miR-20a function was overexpressed in cervical cancer cells, mRNA level and protein level of TNKS2were both upregulation and the mRNA level of TNKS2in human cervical cancer tissues was higher than those in adjacent normal tissues. We also discovered that knockdown of TNKS2has the same result as downexpression of miR-20a.[Conclusions] Our study demonstrated that miR-20a was upregulated in cervical cancer tissues.Overexpression of miR-20a in cervical cancer-derived cell lines, HeLa and C-33A, enhanced long-term cellular proliferation, migration and invasion, whereas inhibition of miR-20a suppressed those functions. We also confirmed that oncogenic TNKS2is directly upregulated by miR-20a. Furthermore, suppression of TNKS2expression could inhibit colony formation, migration and invasion of cervical cancer cells. Therefore, we concluded that miR-20a can promote migration and invasion of cervical cancer cells through the upregulation of TNKS2.