下调EMS1/cortactin对人高转移潜能肝癌细胞株迁移和内吞的影响  

Downregulating EMS1/cortactin inhibits migration and endocytosis of MHCC-97H

作　　者：周家名[1,2] 陈莉[1] 叶蕴瑶[1] 孙树刚 王桂兰[1] 吴圆圆[1] 

机构地区：[1]南通大学医学院病理系,南通226001 [2]南通大学杏林学院,南通226001 [3]南通市瑞慈医院,南通226001

出　　处：《临床与实验病理学杂志》2012年第11期1235-1238,1242,共5页Chinese Journal of Clinical and Experimental Pathology

基　　金：江苏高校优势学科建设工程资助项目;江苏省研究生科技创新立项课题;南通大学研究生科技创新课题(201229);江苏省卫生厅课题(H201052);南通市课题(K2009060)

摘　　要：目的观察下调人高转移潜能肝癌细胞株(MHCC-97H)中EMS1/cortactin对细胞迁移和内吞的影响,探讨EMS1/cortac-tin在细胞内可能存在的功能。方法构建靶向EMS1的shRNA真核表达质粒(EMS1-PSilencer4.1-GFP-shRNA,EMS1-shR-NA);通过Lipofectamine 2000处理后将EMS1-shRNA转染MHCC-97H细胞,免疫荧光显微镜下观察转染效率;采用RT-qPCR和Westen blot分别检测EMS1 mRNA和cortactin蛋白表达水平;利用划痕、Transwell小室实验、转铁蛋白(transferrin,Tfn)内吞实验分别研究细胞迁移和内吞作用。结果成功构建EMS1-shRNA。免疫荧光显微镜下见转染细胞中明显的绿色荧光蛋白(green fluorescence protein,GFP),荧光强度在48 h达到峰值,其转染效率达84.76%。与未处理组相比,转染EMS1-shRNA细胞中EMS1 mRNA水平下降至19.00%,cortactin蛋白水平下降至36.74%、细胞相对迁移距离下降至55.52%、相对穿膜细胞数下降至27.79%、细胞内吞荧光标记Tfn水平显著减弱(P均<0.05)。结论下调EMS1/cortactin后,MHCC-97H细胞迁移、内吞能力明显下降,提示EMS1/cortactin与原发性肝癌(primary human heptatocellular,HCC)转移的生物学行为有关。Purpose To observe its effect on migration and endocytosis of MI-ICC-97H by downregulating EMS1/cortactin, and to ex- plore the possible function of EMS1/xontaetin within cell. Methods ShRNA eukaryotie expression plasmid targeting EMS1 （ EMS1- PSileneer4. 1-GFP-shrank, EMSI-shRNA） was constructed and transfected into MHCC-97H after treatment with Lipofectamine 2000 . Transfeetion efficiency was evaluated under immunofluorescence microscopy. The expression EMS1 mRNA and protein were detected u- sing real-time quantitative RT-qPCR and western blotting. The migration and endocytosis were tested through wound-healing assay, tr- answell invasion and transferrin endocytosis assay, respectively. Results EMSI-shRNA was constructed successfully. GFP within the transfected cell was obviously observed under immunofluorescenee microscopy and the fluorescence intensity peak at 48 h with transfec- tion efficiency of 84. 76%. As compared with untreated group, the levels of EMS1 gene and eortaetin protein in transfeeted cells de- creased to 19. 00% , 36. 74% , respectively. The relative migration distance were reduced to 55. 52% and the relative tranverse cell numbers were reduced to 27.79% （P 〈 0. 05, respectively）. Conclusion Downregulating EMS1/eortaetin inhibited migration and endocytosis of MHCC-97H, suggested it may contribute to the invasive potential of HCC.

关 键 词：MHCC-97H细胞 EMS1 CORTACTIN 核酸干扰 迁移 内吞 

分 类 号：R735.7[医药卫生—肿瘤]