Twist基因对结肠癌细胞系侵袭转移能力影响的体外研究

摘要

目的:探讨Twist基因在SW480、HCT116和HT29结肠癌细胞系上皮-间质转化(EMT)中的作用,明确其对恶性肿瘤侵袭转移能力的影响.方法:利用重组质粒pTracer-CMV/BSD-Twist和pGenesil1.2-Twist-shRNA转染SW480、HT29和HCTI 16;流式细胞术检测转染率;Real time-PCR和Western blot检测Twist、E-cadherin和Vimentin转录及蛋白表达水平;Transwell实验检测细胞的迁移和侵袭能力.结果:转染高表达Twist质粒后,各结肠癌细胞系中Twist和Vimentin表达水平显著升高(P＜0.05),E-cadherin显著降低(P＜0.05);转染低表达Twist质粒后,SW480和HT29中各指标均无显著变化(P＞0.05),HCT116中Twist和Vimentin表达水平显著降低(P＜0.05),E-cadherin无显著变化(P＞0.05);Transwell实验表明抑制HCT116细胞系Twist表达后,其侵袭和迁移能力明显减弱(P＜0.01).结论:上调Twist表达可以促进EMT;抑制HCT116中Twist表达能减弱结肠癌细胞的侵袭和转移能力.%Objective:To explore the role of Twist gene in epithelial-mesenchymal transition (EMT) in SW480,HCT116 and HT29,and then study the effect of Twist on invasion and metastasis of malignant tumors.Methods:Recombinant plasmids pTracer-CMV/BSD-Twist and pGenesill.2-Twist-shRNA were used to transfect SW480,HCT116 and HT29.Transfection efficacy of the plasmid in each cell line was confirmed by flowcytometry.The mRNA transcription level and protein expression level of Twist,E-cadherin and Vimentin were detected by RT-PCR and western blot,respectively.The migration and invasive analysis was done by transwell assay.Results:After transfected by recombinant high-expressed twist plasmid,the mRNA and protein expression levels of Twist and Vimentin increased significantly (P＜0.05),whereas E-cadherin was inhibited (P＜0.05).After transfected by recombinant low-expressed twist plasmid,all the parameters in SW480 and HT29 cell lines were statistically insignificant (P＞0.05),the mRNA and protein levels of Twist and Vimentin were prominently inhibited in HCT116 cell line (P＜0.05) and the level of E-cadherin was not statistically significant (P＞0.05).Significant reduction of invasion and migration was observed in transwell assay after inhibiting the Twist expression in HCT116 cell line (P＜0.01).Conclusion:Up-regulation of Twist gene expression can promote the EMT and inhibiting the Twist gene expression can lessen the migration and invasion of HCT116.