The Effect Of MiR200c On The Invasion, Migration And Proliferation In The Epithelial Ovarian Cancer Cells

Epithelial ovarian cancer（EOC）is one of the most common femalegynecological malignancy. Due to the lack of sensitive and effective tumormarkers, early diagnosis rate of ovarian cancer is very low. Despite theapplication of anticancer drugs and tumor cytoreductive surgery，the five-yearsurvival rate of the patients with advanced stage is still low at about30%.Recurrence and resistance become the biggest problems in the treatment ofovarian cancer.Therefore，the development of new treatment of EOC is urgentproblem.The late research has found that the expression of miR200family alteredin the human tumors，including EOC. Many studies about canine kidney cells,human pancreatic cancer, colorectal cancer, prostate cancer and breast tumortissue show that miR200can inhibit the expression of ZEB1.Epithelial-mesenchymal transition (EMT) is closely related to the migration,invasion and resistance. E-cadherin is one of calcium-dependenttransmembrane adhesion protein, which is mainly distributed on the surface ofepithelial cells to mediate intercellular adhesion effects. Vimentin is the mostimportant intermediate filament of cell cytoskeleton，which is considered asthe key protein of mesangial cells. Zinc finger E-box binding homeobox (ZEB)can combine with Zinc finger structure of E-cadherin gene promoter to inhibitthe expression of E-cadherin. Mesothelin is a tumor surface antigen, whichexpresses higher in EOC tissue than normal ovarian tissue. CA125is the onlyreceptor of mesothelin. Studies have shown that mesothelin enhances theproliferation, adhesion and invasion ability of ovarion cancer cells. Domesticreports about the impact of miR200c on the proliferation, adhesion andinvasion ability of ovarion cancer cell are rare.Objective: By transient transfection miR200c mimic, we raised the miR200c expression of EOC cell lines SKOV3and HO-8910to observe thechange of cell proliferation, migration and invasion, and to study theregulation mechanisms of cell proliferation, migration and invasion.Methods:1The SKOV3and HO-8910cells were divided into blank control group,miR200c mimic group and negative control group. RT-PCR was carried out totest the relative expression of miR200c in each group.2MTT assay tested the effect on proliferation of SKOV3and HO-8910after transfecting miR200c mimic with different concentrations (0nmol/L、25nmol/L,50nmol/L,100nmol/L and200nmol/L) at different times (24h,48h,72h).3Transwell chamber was used to measure the ability of invasion andmigration.4ZEB1, E-cadherin and Vimentin expression were analysied by Westernblotting.5CA125and mesothelin expression were detected by enzyme-linkedimmunosorbent assay.6Statistical Methods: SPSS13.0statistical software was used to evaluatthe date. Each assay was performed at least three times.The measurement datawas expressed as mean±standard deviation. Single factor analysis of variance(One-Way ANOVA) was used for data analysis among groups.α=0.05is theinspection level.When P＜0.05, the difference was statistical significant.Results:1RT-PCR analysis the relative expression of miR200c in each group.ThemiR200c expression of SKOV3-mimic and HO-8910-mimic weresignificantly higher than that in SKOV3and HO-8910(P<0.05). The miR200cexpression of SKOV3-neg and HO-8910-neg was not increased significantlycompared with that in SKOV3and HO-8910cells (P>0.05).2The proliferation rate of SKOV3and HO-8910cells which were treatedwith miR200c was determined by MTT assay.Transfected with miR200cmimic at different concentrations, the proliferation rate of SKOV3and HO-8910was not changed significantly (P>0.05). Transfected with the sameconcentration of miR200c mimic to cultivate different time, the proliferationrate of SKOV3-mimic and HO-8910-mimic had no obvious change (P>0.05).3The result of Transwell for invitation showed that the number ofSKOV3-mimic and HO-8910-mimic got through the maxtrix film wassignificantly reduced (P<0.05). The number of SKOV3-neg and HO-8910-negcompared with blank control group was not statistically significant (P>0.05).This indicates that miR200c significantly inhibites the invasion ability ofSKOV3and HO-8910cells.4The result of Transwell for migration showed that the number ofSKOV3-mimic and HO-8910-mimic cells got through the filter membranewas significantly higher than blank control groups (P<0.05). It indicates thatthe migration ability of SKOV3-mimic and HO-8910-mimic was significantlyenhanced.The number of SKOV3-neg and HO-8910-neg cells had notsignificantly changed (P>0.05).5Western blotting was used to detect the relative expression of ZEB1,Vimentin and E-cadherin.The expression of ZEB1and Vimentine inSKOV3-mimic and HO-8910-mimic were significantly higher than that inSKOV3and HO-8910cells and the expression of E-cadherin was significantlylower(P<0.05). The expression of ZEB1， Vimentin and E-cadherin inSKOV3-neg and HO-8910-neg was not significantly changed (P>0.05).6The result of Enzyme-linked immunosorbent assay showed that theexpression of CA125and mesothelin in SKOV3-mimic and HO-8910-mimicwere significantly decreased than that in blank control group (P<0.05).Theexpression of CA125and mesothel in SKOV3-neg and HO-8910-neg cellswas not significantly decreased (P>0.05).Conclusion:1Raising miR200c can significantly inhibit the migration and invasion ofthe SKOV3and HO-8910cells，but there is no significant effects on theproliferation. 2Transfected with50nm/L miR200c mimic could significantlyup-regulats the miR200c expression of ovarian cancer line SKOV3andHO-8910.We found that the expression of ZEB1and Vimentin could besignificantly decreased and E-cadherin expression significantly raised. Itsuggests that miR200c may regulate migration and invasion of ovarian cancervia miR200c/ZEB1/E-cadherin pathway.3The expression of CA125and mesothelin was reduced significantly. Itindicates that miR200c may affect the cells migration and invasion viamesothelin/CA125pathway，but specific regulatory mechanism needs furtherstudy.