过/降表达KIF20A的宫颈癌细胞迁移、侵袭、上皮—间质转化能力变化及其分子机制

Effects of KIF20A overexpression or knockdown on migration,invasion and epithelial-mesenchymal transformation of cervical cancer cells and their molecular mechanisms

目的观察过/降表达KIF20A的宫颈癌细胞迁移、侵袭、上皮—间质转化(EMT)能力的变化,并探讨其可能的分子机制。方法培养人宫颈永生化鳞状细胞系ECT及人宫颈癌细胞系C33A、MS751、Hela、SiHa、CasKi,以RT-qPCR法检测细胞中KIF20A mRNA表达水平。选取KIF20A表达量最高的CasKi细胞并分为对照组和敲降组,分别转染sh-NC、sh-KIF20A;选取KIF20A表达量最低的Hela细胞分为对照组和过表达组,分别转染Vector、KIF20AOE。应用划痕实验和Transwell小室实验分别测定四组细胞迁移和侵袭能力,Western blotting法检测四组细胞中EMT相关蛋白Vimentin、E-cadherin;利用细胞组分分离实验结合Western blotting法检测两组Hela细胞细胞质膜及细胞核中的β-catenin,免疫共沉淀实验结合Western blotting法检测两组Hela细胞中β-catenin免疫共沉淀的Ecadherin蛋白水平。结果划痕12、24、48 h时,CasKi细胞中敲降组划痕愈合率均低于对照组,Hela细胞中过表达组划痕愈合率均高于对照组(P均<0.05)。CasKi细胞中敲降组细胞迁移数少于对照组,Hela细胞中过表达组细胞迁移数多于对照组(P均<0.05)。CasKi细胞中,敲降组细胞中Vimentin蛋白表达量低于对照组,E-cadherin蛋白表达量高于对照组(P均<0.05);Hela细胞中,过表达组Vimentin蛋白表达量高于对照组,E-cadherin蛋白表达量低于(P均<0.05)。过表达组细胞膜中β-catenin蛋白表达量高于对照组,细胞核中β-catenin蛋白表达量低于对照组(P均<0.05)。与对照组比较,过表达组β-catenin免疫共沉淀的E-cadherin蛋白表达量减少(P<0.05)。结论过表达KIF20A能促进宫颈癌细胞迁移、侵袭,诱导EMT相关蛋白Vimentin表达并抑制E-cadherin表达;降表达KIF20A后,作用与之相反。该调控作用可能与KIF20A抑制E-cadherin/β-catenin复合物形成来诱导β-catenin激活有关。

Objective To observe the changes in migration,invasion and epithelial-mesenchymal transition(EMT)abilities of cervical cancer cells overexpressing/downgrading KIF20A,and to explore the possible molecular mechanisms.Methods The human cervical immortalized squamous cell line ECT and human cervical cancer cell lines C33A,MS751,Hela,SiHa,CasKi were cultured,and the KIF20A mRNA expression levels in the cells were detected by RT-qPCR.CasKi cells with the highest KIF20A expression were selected and divided into the control group and knockdown group,which were transfected with sh-NC and sh-KIF20A,respectively.Hela cells with the lowest KIF20A expression were divided into the control group and overexpression group,which were transfected with Vector and KIF20A-OE,respectively.The migration and invasion abilities of cells in the four group were determined by Scratch experiment and Transwell assay,respectively.Western blotting was used to detect the EMT-related proteins Vimentin and E-cadherin.The cell fraction separation assay combined with Western blotting were used to detect the β-catenin in the cytoplasmic membrane and nucleus of Hela cells in the two groups.Immunoprecipitation assay combined with Western blotting were used to detect Ecadherin protein immunoprecipitated byβ-catenin in Hela cells of the two groups.Results At 12,24 and 48 h of scratching,the scratch healing rate of CasKi cells was lower in the knockdown group than in the control group,and the scratch healing rate of Hela cells was higher in the overexpression group than in the control group(both P<0.05).The number of migration cells in the knockdown group was smaller than that in the control group,and the number of migration cells was larger in the overexpression group than in the control group(all P<0.05).In CasKi cells,the expression of Vimentin protein in the knockdown group was lower than that in the control group,and the expression of E-cadherin protein was higher than that in the control group(both P<0.05).In Hela cells,the expression of Vimentin protein in the overexpression group was higher than that in the control group,and the expression of E-cadherin protein was lower than that in the overexpression group(both P<0.05).The expression of β-catenin protein in the cell membrane of the overexpression group was higher than that of the control group,and the expression of β-catenin protein in the nucleus of the cells was lower than that of the control group(both P<0.05).The E-cadherin protein immunoprecipitated by β-catenin was reduced in the overexpression group as compared with that of the control group(P<0.05).Conclusions Overexpression of KIF20A promotes the migration and invasion of cervical cancer cells,induces the expression of EMT-related protein Vimentin and inhibits E-cadherin expression.The opposite effect is observed after knockdown of KIF20A.This regulatory effect may be related to the inhibition of E-cadherin/β-catenin complex formation by KIF20A to induce β-catenin activation.