Trop2过表达对人胃黏膜上皮细胞GES-1迁移能力的影响及机制

Effects of over-expression of Trop2 on migration of human gastric mucosal epithelial cells and its mechanism analyses

目的 :观察Trop2过表达对人胃黏膜上皮细胞GES-1迁移能力的影响,并对其作用机制进行初步探讨。方法 :realtime PCR和Western blot检测GES-1细胞中Trop2 m RNA和蛋白的表达水平;构建过表达Trop2质粒及空载对照质粒并转染GES-1细胞,检测转染效率;细胞划痕实验和Transwell迁移实验检测过表达Trop2后GES-1细胞迁移能力的变化情况;Western blot检测过表达Trop2后GES-1细胞上皮-间质转化(epithelial-mesenchymal transition,EMT)相关分子[上皮细胞标志E-钙黏附蛋白(E-cadherin),间皮细胞标志纤维连接蛋白(fibronectin)、波形蛋白(vimentin)]的变化情况。结果:相比胃癌细胞株BGC823和MGC803,Trop2在GES-1细胞中的m RNA和蛋白表达水平均较低;过表达Trop2的质粒转染GES-1后,Trop2的m RNA表达水平与未转染组相比提高(6.18±0.52)倍,而空载对照组和未转染组相比无明显变化,蛋白水平的变化情况和m RNA相似;细胞划痕实验表明,转染72 h后,过表达组细胞迁移率达到95%,空载对照组细胞迁移率为50%,两组相比差异有统计学意义(P﹤0.05);Transwell迁移实验发现,转染24 h后,过表达组穿膜细胞数为(87±6)个,而空载对照组为(41±6)个,两组相比差异有统计学意义(P﹤0.05);Western blot检测过表达Trop2后,随着Trop2蛋白表达量增高,GES-1细胞E-cadherin表达下调,fibronectin和vimentin表达上调。结论 :Trop2过表达可以提高胃黏膜上皮细胞的迁移能力,其机制与Trop2促进胃黏膜上皮细胞EMT有关。

Objective：To study the effecs of human trophoblast cell surface glycoprotein（Trop2） overexpression on migration of the human gastric mucosal epithelial cells and preliminary mechanism analyses. Methods：We explored m RNA and protein expressionlevels of Trop2 in GES-1 cell line by real-time RT-PCR and Western blot. OE-Trop2 and VE-Trop2 plasmids were constructed and then transfected into GES-1 cell line,and the efficiency of transfection was detected. Cell wound healing assay and Transwell migration assay were performed to detect the change of the migration in GES-1 after transfected OE-Trop2 plasmids. The change of surface markers（E-cadherin,fibronectin,and vimentin）of epithelial-mesenchymal transition（EMT）in GES-1 cell line were detected by Western blot after Trop2 overexpression. Results：Compared with gastric cancer cell lines BGC823 and MGC803,the m RNA and protein expression levels of Trop2 in GES-1 were lower. After OE/VE-Trop2 plasmid transfected in GES-1,the m RNA expression level of Trop2 in the OE-Trop2 group was 6.18 ± 0.52 times higher than that of the control group,whereas no differences were detected between the control group and the VE-Trop2 group. Meanwhile,the protein expression model of Trop2 was similar to that of the m RNA expression. After transfected plasmids 72 h,cell wound healing assay showed that the migration rate of GES-1 was 50% in the VE-Trop2 group and 95% in the OE-Trop2 group（P〈0.05）. After transfected OE-Trop2 plasmids 24 h,transwell migration assay showed that the number of migration cell was 41 ± 6 in the VE-Trop2 group,whereas 87 ± 6 in the OE-Trop2 group（P〈0.05）.Western-blot assay showed that the expression level of E-cadherin in GES-1 was down-regulated;meanwhile,fibronectin and vimentin were up-regulated after transfected OE-Trop2. Conclusion：Trop2 overexpression could promote migration of GES-1 cell line through inducing EMT phenomenon.