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/cortactin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-shRNA);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 MHCC-97H by downregulating EMSl/cortactin, and to explore the possible function of EMSl/xontactin within cell. Methods ShRNA eukaryotic expression plasmid targeting EMSl( EMS1-PSilencer4. 1-GFP-shrank, EMSl-shRNA ) was constructed and transfected into MHCC-97H after treatment with Lipofectamine 2000 . Transfection 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 EMSl-shRNA was constructed successfully. GFP within the transfected cell was obviously observed under immunofluorescence microscopy and the fluorescence intensity peak at 48 h with transfection efficiency of 84. 76%. As compared with untreated group, the levels of EMS1 gene and cortactin protein in transfected cells decreased 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 EMSl/cortactin inhibited migration and endocytosis of MHCC-97H, suggested it may contribute to the invasive potential of HCC.
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