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Expression of microRNA-380-5p in cervical cancer tissues and cell lines and its inhibition on proliferation and migration of C33A cells by down-regulating RHOA
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Abstract:
[Abstract] Objective: To investigate the expression of microRNA-380-5p (miR-380-5p) in cervical cancer tissues and cell lines, and to explore the mechanism of miR-380-5p inhibiting the proliferation and migration of cervical cancer cells. Methods: 16 pairs of cervical cancerous tissues and corresponding para-cancerous tissues were collected from the Department of Obstetrics and Gynecology, the Affiliated Wuhan Central Hospital of Tongji Medical College from December 2016 to July 2017; in addition, cervical cancer cell lines (HCC94, C33A, Hela, SiHa) and human cervical epithelial immortalized H8 cells were also collected for this study. The expression of miR-380-5p in above mentioned tissues and cell lines was detected by Real-time quantitative polymerase chain reaction (qPCR). miR-380-5p mimic (experimental group) and miR-NC (negative control group) were transiently transfected into C33A cells by lipofection,and qPCR was used to detect the expression of miR-380-5p in the transfected cells. Cell proliferation and migration were evaluated by cell counting kit (CCK-8) and Transwell assay. Bioinformatics software TargetScan predicted the downstream genes of miR-380-5p,and dual luciferase reporter assay was used to verify the binding of miR-380-5p to the downstream gene RHOA (Ras homolog gene family member A). qPCR and Western blotting were used to detect the expression of miR-380-5p downstream gene-RHOA. Results:The expression level of miR-380-5p in cervical cancer tissues and cell lines was significantly lower than that in para-cancerous tissues and normal cervical epithelial H8 cells (P<0.01); and the expression in C33A cells was the lowest (P<0.01). Compared with the negative control group, the miR-380-5p mimic transfection singnificantly inhibited the proliferation (P<0.05) and migration ability of C33A cells (P<0.01), and down-regulated protein expressions of RHOA, ROCK1, ROCK2, CDK2 and N-cadherin (all P<0.01). Bioinformatics software predicted that RHOA may be a downstream gene of miR-380-5p, and dual luciferase reporter assay proved the specific binding of miR-380-5p to the 3'UTR of RHOA (P<0.01). miR-380-5p could significantly down-regulate RHOA gene expression (P<0.01). Conclusion: miR-380-5p is low-expressed in cervical cancer cell lines. Over-expression of miR-380-5p may inhibit the proliferation and migration of cervical cancer C33A cells by down-regulating the expression of RHOA gene and its downstream proteins.
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