摘要
目的 探讨微小RNA-7(miR-7)对幽门螺杆菌(Hp)诱导的胃上皮细胞(GES-1)自噬及上皮细胞间质转化(EMT)的影响.方法 体外将Hp悉尼株(SS1)与GES-1 细胞共培养建立GES-1 细胞转化模型;实验分为空白对照组(NC组)、Hp组、miR-7模拟物(mimics)组、Hp+miR-7 mimics组.qRT-PCR验证转染效果;MTT法、划痕实验、Transwell实验检测各组GES-1细胞增殖能力、迁移能力和侵袭能力;透射电镜下观察细胞自噬体;免疫印迹法检测各组GES-1 细胞自噬相关蛋白[RM-9106 微管相关蛋白1 轻链3-Ⅰ(LC3-Ⅰ)、LC3-Ⅱ]及EMT相关蛋白[E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)]表达情况.结果 与NC组比较,Hp组GES-1转化细胞增殖抑制率、p62 蛋白、E-Cadherin蛋白水平降低(P<0.05),迁移率、侵袭数、自噬体数量及自噬泡/胞质总面积、LC3-Ⅱ/LC3-Ⅰ比值、N-Cadherin和Vimentin蛋白水平均升高(P<0.05),miR-7 mimics组GES-1细胞增殖抑制率、p62 蛋白、E-Cadherin蛋白水平升高(P<0.05),迁移率、侵袭数、自噬体数量及自噬泡/胞质总面积、LC3-Ⅱ/LC3-Ⅰ比值、N-Cadherin和Vimentin蛋白水平均降低(P<0.05);与miR-7 mimics组比较,Hp+ miR-7 mimics组转化细胞增殖抑制率、p62 蛋白、E-Cadherin蛋白水平降低(P<0.05),迁移率、侵袭数、自噬体数量及自噬泡/胞质总面积、自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ比值、N-Cadherin和Vimentin蛋白水平均升高(P<0.05).结论 miR-7 下调Hp诱导的GES-1细胞自噬,降低细胞侵袭及迁移能力,抑制EMT.
Abstract
Aim To investigate the effect of microRNA-7(miR-7)on Helicobacter pylori(Hp)-induced autophagy and epi-thelial mesenchymal transition(EMT)in gastric epithelial cells(GES-1).Methods The Hp Sydney strain(SS1)was co cul-tured with GES-1 cells in vitro to establish a GES-1 cell transformation model.The experiment was divided into blank control group(NC group),Hp group,miR-7 mimics group,and Hp+miR-7 mimics group.The transfection effect was verified by qRT-PCR.MTT assay,scratch assay and Transwell assay were used to detect the proliferation,migration and invasion of GES-1 cells in each group.Western blotting was used to detect the expression of autophagy related proteins(RM-9106 microtubule related protein 1 light chain 3-Ⅰ(LC3-Ⅰ),LC3-Ⅱ)and epithelial mesenchymal transformation(EMT)related proteins(E-cadherin,N-cadherin,Vim-entin)in GES-1 cells in each group.Results Compared with the NC group,the Hp group showed a decrease in the proliferation inhibition rate,p62 protein,and E-Cadherin protein levels of GES-1 transformed cells(P<0.05),while the migration rate,invasion number,number of autophagosomes,total area of autophagic vesicles/cytoplasm,ratio of LC3-Ⅱ/LC3-Ⅰ,N-Cadherin and Vimentin protein levels increased(P<0.05).The miR-7 mimics group also showed an increase in the proliferation inhibition rate,p62 pro-tein,and E-Cadherin protein levels of GES-1 cells(P<0.05),and the migration rate The number of invasions,the number of autoph-agosomes,the total area of autophagic vesicles/cytoplasm,the ratio of LC3-Ⅱ/LC3-Ⅰ,and the levels of N-Cadherin and Vimentin proteins all decreased(P<0.05);Compared with the miR-7 mimics group,the Hp+miR-7 mimics group showed a decrease in the in-hibition rate of transformed cell proliferation,p62 protein levels,and E-Cadherin protein levels(P<0.05),while the migration rate,invasion number,number of autophagosomes,total area of autophagic vesicles/cytoplasm,ratio of autophagy related protein LC3-Ⅱ/LC3-Ⅰ,and levels of N-Cadherin and Vimentin proteins all increased(P<0.05).Conclusion miR-7 down-regulates Hp-in-duced GES-1 autophagy,reduces cell invasion and migration,and inhibits EMT.
基金项目
海南省自然科学基金高层次人才项目(822RC862)
维普
万方数据