CTEN促进非小细胞肺癌上皮间质转化及迁移侵袭的作用及机制研究

摘要

目的:探讨CTEN对非小细胞肺癌(NSCLC)细胞上皮间质转化(epithelial-to-mesenchymal transition,EMT)以及迁移侵袭的影响及其机制.方法:应用Lipofectamine 2000将CTEN高表达质粒pcmv-CTEN和对照质粒pcmv转染至A549细胞,分别为高表达组和对照组,分别用定量PCR和Western blot检测CTEN、TGF-β1和EMT标记分子mRNA及蛋白水平的变化,应用细胞划痕实验和Transwell实验检测细胞迁移和侵袭能力;应用Lipofectamine 2000将TGF-β1高表达质粒pcmv-TGF-βl和对照质粒pcmv转染至A549细胞,分别为高表达组和对照组,分别用定量PCR和Western blot检测TGF-β1和EMT标记分子mRNA及蛋白水平的变化,应用细胞划痕实验和Transwell实验检测细胞迁移和侵袭能力;应用Lipofectarnine 2000将TGF-β1干扰质粒siTGF-β1和对照质粒siNC转染至A549细胞,分别为干扰组和对照组,分别用定量PCR和Western blot检测两组细胞中TGF-β1 mRNA及蛋白水平的表达情况,然后再分别应用Lipofectamine 2000将CTEN高表达质粒pcmv-CTEN转染人两组细胞,分别用定量PCR和Western blot检测CTEN和EMT标记分子mRNA及蛋白水平的变化,应用细胞划痕实验和Transwell实验检测细胞迁移和侵袭能力.结果:与对照组相比,高表达CTEN组的TGF-β1表达升高,上皮标记分子表达下降,间质标记分子表达升高,促进EMT的发生,细胞迁移和侵袭至下室的细胞数明显增多;与对照组相比,高表达TGF-β1组的上皮标记分子表达下降,间质标记分子表达升高,细胞迁移和侵袭至下室的细胞数明显增多;在A549细胞中干扰TGF-β1后再过表达CTEN,其促进EMT的作用明显减弱,细胞迁移和侵袭能力的增加也明显减弱.结论:CTEN具有促进NSCLC细胞A549的EMT和迁移侵袭的作用,其发生机制可能与TGF-β1有关.%Objective:To explore the effects and mechanism of CTEN on epithelial-to-mesenchymal transition,migration and invasion.Methods:The A549 cells were divided into high expression group and control group,then transfected with pcmv-CTEN and pcmv with lipofectamine 2000,CTEN,TGF-β1 and EMT biomarkers were detected by qRT-PCR and Western blot.The migration and invasion ability of cells were detected through would scratch assay and Transwell assay.The A549 cells were transfected with pcmv-TGF-β1 and pcmv with lipofectamine 2000,respectively,TGF-β1 and EMT biomarkers were detected by qRT-PCR and Western blot.The migration and invasion ability of cells were detected through would scratch assay and Transwell assay.TGF-β1 silenced and control cell lines were constructed in A549 cells by the transfection of siTGF-β1 and siNC with lipofectamine 2000.TGF-β1 expression was detected by qRT-PCR and Western blot,then pcmv-CTEN and pcmv were transfected with lipofectamine 2000 in A549 cells in which TGF-β1 was silenced,then CTEN and EMT biomarkers were detected by qRT-PCR and Western blot.The migration and invasion ability of cells were detected through would scratch assay and Transwell assay.Results:Compared with control group,the expression of TGF-β1 and mesenchymal markers in CTEN high expression group were enhanced,and the epithelial marker was descended,which induced the EMT process.Meanwhile,would scratch assay showed that the migrated distance of high CTEN expression group were more than the control group.Transwell assay showed that the number of cells that migrated and invaded through the membrane were obviously more than those in control group.The expression of mesenchymal markers in TGF-β1 high expression group were enhanced,and the epithelial marker was descended,which induced the EMT process.Meanwhile,would scratch assay showed that the migrated distance of high TGF-β1 expression group were more than the control group.Transwell assay showed that the number of cells that migrated and invaded through the membrane were obviously more than those in control group.The enhanced EMT and metastasis induced by CTEN could be reversed by interfering TGF-β1.Conclusion:CTEN induce epithelial-to-mesenchymal transition,migration and invasion ability of human lung cancer A549 cells,which may act through activating TGF-β1.