MiR-200c-3p Inhibits Epithelial-mesenchymal Transition By Targeting ZEB2 In Prostate Cancer Cell Lines

Background:MicroRNAs(miRNAs)are a broad class of single non-coding RNAs with highly conserved sequences which exist in eukaryotes.In common,miRNAs regulate the expression of its target genes at the post transcriptional level by binding to the 3' untranslated region(3'-UTR)of these targets.An enormous amount of research has indicated that miRNA plays a crucial role in various biological processes,especially in tumorigenesis,proliferation,apoptosis,tumor invasion and metastasis.Previous studies have demonstrated that miR-200c-3p,as an important member of miR-200 family,exhibits ectopic expression in multiple human cancers.However,the underlying mechanism of miR-200c-3p regulation in prostate cancer(PCa)still remains unclear.The present study is carried out with an aim to further investigate the function of miR-200c-3p in castration resistant PCa cells,PC3 and DU 145,and identify the regulating mechanism of miR-200c-3p and Zinc Finger E-Box Binding Homeobox 2(ZEB2)in epithelial-mesenchymal transition(EMT)pathway.Martials and methods:Quantitative real time PCR method(qRT-PCR)was applied to detect the expression level of miR-200c-3p in 16 paired samples of primary PCa and adjacent normal tissues.The expression level of ZEB2 protein in 11 paired primary PCa and adjacent normal tissue samples was measured by Western blot analysis.PC3 and DU145 cell lines were transfected by endogenous miR-200c-3p mimics respectively,and qRT-PCR method was used to test the miR-200c-3p expression level in the transfected and non-transfected PCa cell lines,PC3 and DU 145.Western blot analysis was utilized to assay the expression levels of ZEB2 and EMT related proteins,E-cadherin and Vimentin,in transfected and control groups,and the effect of elevated miR-200c-3p expression on PCa cells was evaluated by cell invasion and wound scratch assay.Besides,dual luciferase reporter assay was applied to identify the specific binding site of miR-200c-3p and ZEB2 3'-UTR.Results:Compared to 16 paired adjacent normal tissue samples,miR-200c-3p expression was significantly down regulated in 16 PCa tissues.ZEB2 expression level was increased in primary PCa tissues compared with that in adjacent normal tissues.The inverse correlation between the expression of miR-200c-3p and ZEB2 suggested the interaction between the two factors which might play an important role in tumorigenesis and progression of PCa.In vitro,compared to negative control(NC)group,the expression level of ZEB2 protein in PC3 and DU 145 cell lines has significantly decreased after a 72-hour transfection of miR-200c-3p mimics(P<0.05).We also observed an up-regulation of E-cadherin expression level,which was statistically significant(P<0.05),and the expression level of Vimentin significantly decreased(P<0.05).In addition,elevated miR-200c-3p expression significantly reduced the invasive and migratory abilities of PCa cells in vitro(P<0.05).Dual luciferase reporter assay suggested that ZEB2 was a direct target of miR-200c-3p.Conclusions:Our findings demonstrate that miR-200c-3p function as a potential tumor suppressor through targeting ZEB2 in PCa.Overexpression of miR-200c-3p in PCa cell lines,PC3 and DU145,results in significant down regulation of the ZEB2 expression level and further leads to the inhibition of EMT process.Therefore,miR-200c-3p plays a vital factor in PCa and has important research significance as a molecular marker for the diagnosis of PCa.