RNA干扰沉默claudin-4基因表达对子宫内膜癌细胞生物学行为的影响

摘要

目的:采用RNA干扰技术沉默claudin-4基因在子宫内膜癌细胞Ishikawa中的表达,探讨claudin-4表达下调对Ishikawa细胞增殖力和侵袭力的影响;比较RNA干扰前后差异表达的蛋白质,探讨claudin-4基因在子宫内膜癌发病机制中的作用.方法:设计靶向claudin-4的小干扰RNA (siRNA),转染人子宫内膜癌细胞Ishikawa.采用CCK分析法检测干扰前后细胞增殖能力的变化,Transwell小室实验检测干扰前后细胞侵袭能力的变化.应用双向凝胶电泳和质谱分析检测RNA干扰前后蛋白质表达的差异.结果:claudin-4基因沉默可以显著降低子宫内膜癌细胞的增殖能力和侵袭能力(P＜0.05).应用蛋白质组学技术比较claudin-4 siRNA干扰前后蛋白质表达的差异,共鉴定出 5种差异蛋白质.结论:claudin-4基因具有促进子宫内膜癌细胞增殖和细胞侵袭的作用,这种作用可能通过改变差异蛋白的表达得以实现.%Objective:To detect the efficacy of downgrading claudin-4 expression on cell proliferation and invasion of Ishikawa endometrial adenocarcinoma cell line in response to silence claudin-4 gene expression by RNA interference technique.To compare the difference of protein expression and to discuss the effect of claudin-4 gene on the pathogenesis of endometrial cancer.Methods:Small interfering RNA (siRNA) was designed to target claudin-4 and transfected into human endometrial cancer cells-Ishikawa cells.The prolifera tion and invasion capability of Ishikawa cells before and after the interference were evaluated by CCK and transwell analysis.The difference of protein expression was also detected before and after the interference by 2D gel and mass spectrometry.Results:Effective knockdown of claudin-4 suppressed the proliferation and reduced the invasiou capability of Ishikawa cells.Further proteomics analysis revealed that USP7 and APT1 were up-regulated while LASP1,ERP29 and PRDX5 were down-regulated by silence of claudin-4.Conclusion:Knockdown of claudin-4 suppressed cell proliferation and reduced the invasion capability of Ishikawa cells by down-regulation of key proteins involved in cell proliferation and invasion.