MiR-214 Regulates The Growth And Invasion Of Human Cervical Cancer Hela Cells

[Objective]Cervical cancer is one of highly prevalent cancer that affects women across the world.It has been reported that the human papillomavirus(HPV)infection is a risk factor of cervical cancer,but there are some incidences without HPV infection remain to been elucidated.An accumulating body of evidence has implicated mutations,altered expression or activity of specific genes involving pathogenesis of cervical cancers,including microrRNA(miRNA).Therefore,identifying specific molecular markers that could predict the individual biological behavior of a tumor is important for understanding the molecular mechanisms of cervical carcinogenesis and developing effective diagnostic and therapeutic strategies.MiRNA is a novel class of small(-22 nt)noncoding RNAs that play important roles in the negative regulation of gene expression through base-pairing at complementary sites on the target mRNAs.Increasing evidence shows that miRNAs are aberrantly expressed or mutated in human cancers and involved in carcinogenesis,indicating their possible function as a novel type of oncogenes or tumor suppressors.Here,we aimed to investigate the functions of miR-214 in cervical cancers and their target genes.Through analyzing the effects of the target genes of miR-214 and the regulatory network of aberrant expression of miR-214,we try to elucidate the functions and molecular mechanisms of miR-214 on cervical carcinogenesis.[Methods]Real-time PCR was used to analyze the expression of miR-214 in 21 paired specimens of human cervical cancer and their adjacent normal parts.Through enforced expression of miR-214 in cervical cancer HeLa cells,we evaluated the effects on malignant phenotypes using a series of assays,including MTT,cell growth curve,colony formation assay,wound healing assay,transwell migration and invasion assays,cell survival assay,drug sensitivity test and TUNEL assays.Then the candidate target genes of miR-214 were screened by bioinformatics and miRNA profiling assays,and further verified by EGFP report assay,real-time PCR and Western Blot.Moreover,the functions of the target genes of miR-214 were investigated by RNA interference and cell phenotype assays.The Western Blot was further used to analyze some related molecules related with elevated miR-214 levels in HeLa cells.HeLa cells were treated with the demethylating agent and/or the histone deacetylase inhibitor,and we finally assessed the possible mechanisms regulating miR-214 expression.[Results]miR-214 was down-regulated in cercical cancer tissues and HeLa cells.Enforced expression of miR-214 in HeLa cells inhibited cell viability,cell growth and proliferation,cell migration and invasion capacity and cell survival ability.It can also render the sensitivity to chemotherapeutic drug cisplatin and promote cell apoptosis.Subsequently we screened and validate four genes targeted by miR-214,including Plexin-B1 and Bc1212.Knockdown of Plexin-B1 can inhibit cell growth and proliferation,migration and invasion.Down-regulation of Bc1212 can decrease cell survival and render the sensitivity to cisplatin.Western Blot also showed that the down-regulation of critical protein in apoptosis,including Bax、P53 and NF-κB1.Furthermore,increased expression of miR-214 and decreased expression of its target genes were detected when HeLa cells were treated by demethylating agent and/or the histone deacetylase inhibitor.[Conclusions]miR-214 is down-regulated in cervical cancers and functions as a tumor suppressor genes in HeLa cells through regulating their direct target gene——Plexin-B1 and Bc1212.Plexin-B1 is related with cell growth and proliferation,migration and invasion.And Bc1212 can affect cell survival and the sensitivity to cisplatin.We finally get a possible network including Bc1212,Bax,MEK3,JNK1,P53 and NF-κB1.Through these pathways,miR-214 can affect cell apoptosis.And we also found that miR-214 may be regulated by promoter methylation and histone deacetylation.All of these will provide new understanding into the function and mechanisms of miR-214 in cervical cancers and new theoretical clues for cervical carcinogenesis.