MiR-630 Acts As A Tumor Suppressor In Cervical Cancer And Inhibits Epithelial-mesenchymal Transition In Cervical Cancer

ObjectiveCervical cancer is one of the most common malignant tumors in the world,the 5 year survival rate is still very low because of the invasion and metastasis of cancer cells.A large number of studies have shown that high-risk human papillomavirus(human papiuoma virus,HPV)infection(such as HPV16/18)is a necessary condition for the occurrence of cervical cancer.However,the pathogenesis of cervical cancer is a multi-stage process and the occurrence of cervical cancer is accomplished through the abnormal regulation of multiple genes which have synergistic or antagonistic effects on HPV E6/E7.In this study,microRNA gene chip by specific knockdown of HPV E6/E7 was constructed,related microRNA which were significantly regulated by E6/E7 in cervical cancer cells were chosen,among which microRNA-630(miR-630),draw our attention.The purpose of this study is to analyze the biological function of miR-630 in HPV16/18 positive cervical cancer during the occurrence and development of cervical cancer cells and the molecular mechanism of the expression of miR-630 in HPV16/18 positive cervical tissues,thus providing a new biomarker for diagnosis and prognosis marker of cervical cancer.MethodsSpecific knock down of HPV E6/E7 using RNA interference technology in cervical cancer cells,Caski and Ms751,sent to Shanghai Ou Yi biological Co.ltd for microarray construction.2.MiR-630,screened by MiRNA microarray and bioinformatics analysis technology,which is significantly regulated by the HPV16/18E6/E7 in cervical cancer cells,validated in cervical cancer cell lines using siRNA.3.RT-PCR was used to compare the expression of miR-630 in cervical normal tissues and cervical cancer tissues.4.The lentiviral vector was used to construct over expression and stable interference cell lines,and their effects on proliferation,migration and invasion of cervical cancer cell lines were detected.5.The altered expression of epithelial mesenchymal transition markers were detected by RT-PCR and Western blot.6.The use of bioinformatics analysis of the impact on the level of transcription.7.The results of bioinformatics analysis were verified by chromatin immunoprecipitation assay,and siRNA technology was used to further confirm in the cell lines.8.Luciferase gene reporter system was constructed to detect the target gene of miR-630 in cervical cancer.Result1.E6/E7 can regulate a wide rage of miRNA expression.A miRNA microarray analysis by knocking down E6/E7 in Caski and Ms751 cells was performed.The results showed that 8 miRNAs genes including miR-5194,miR-5088-5p,miR-192-5p,miR-331-5p,miR-183-3p were down-regulated in both cell lines and 23 miRNAs genes including miR-630,miR-29b-3p,miR-4758-3p,miR-6824-3p and miR-4436b-5p were up-regulated,among which miR-630 draw our attention.2.The expression of miR-630 was greatly elevated in Siha,Hela,Caski and Ms751 cells after silencing of E6/E7 by RT-PCR detection(P<0.05).3.Compared with the control group,the wound healing assay indicated that the relative migration distance of miR-630 silenced cells was significantly increased(P<0.01),while the relative migration distance of miR-630 overexpressed cells was obviously decreased(P<0.01).Furthermore,the transwell assays with and without matrigel showed that the migration and invasion rates increased when endogenous miR-630 was silenced by shmiR-630(P<0.01).In contrast,ectopic expression of miR-630 signifcantly inhibited the migration and invasion of Caski and Ms751cells(P<0.01).5.Cell Counting Kit-8(CCK8)assay showed that silencing of miR-630 had no effect on the proliferation of Siha and Hela cells in vitro(P>0.05).Consistent with this,overexpression of miR-630 had no influence on the proliferation of Caski and Ms751 cells in vitro.6.Histological examination of the pulmonary tissue showed that the number of pulmonary metastatic nodules was lower in the mice inoculated with the Ms751-miR630-mimics cells than in the mice inoculated with the Ms751-Vector cells(P<0.001).7.The silencing of miR-630 led to increased expression in Vimentin and N-cadherin,and decreased E-cadherin expression,whereas adverse conclusions was found in miR-630 overexpression group(P<0.05).The mRNA levels of Snail,Twist,Zebl and Zeb2,the key transcription factors that promote EMT,were determined by real-time PCR.Compared with the control group,the results showed that Snail,Zeb1 and Zeb2 were significantly increased in miR-630 silenced cells,while Twist remained unchanged,and Snail,Zeb1 and Zeb2 were significantly reduced in miR-630 overexpressed cells,8.Bioinformatics analysis,CHIP assay and cell experiments demonstrated p53 positively regulate miR-630 expression in cervical cancer cells.9.MiR-630 binds directly to the 3'-UTR of CTHRC1 using a dual-luciferase reporter system.Co-transfection with miR-630 expressing vector significantly inhibited luciferase activity driven by the reporter vector containing the wild-type CTHRC1 3'-UTR but not the mutant 3'-UTR.Overexpression of miR-630 decreased both mRNA and protein expression level of CTHRC in cervical cancer cell lines Siha and Hela(p<0.001),while downregulation of endogenous miR-630 increased CTHRC1 expression in cervical cancer cell lines Caski and Ms751(p<0.001).Conclusions:1.MiR-630 played a suppressive role on cervical cancer cells' invasion and migration both in vitro and vivo.2.MiR-630 could depress EMT in cervical cancer cells.3.MiR-630 could be regulated by P53.4.CTHRC1 was a potential target gene of miR-630 and miR-630 suppressed the expression of CTHRC1 in cervical cancer cells.