G蛋白偶联胆汁酸受体1促进胃癌细胞的增殖、迁移和侵袭的实验研究

目的:探讨G蛋白偶联胆汁酸受体1(G-protein coupled bile acid receptor 1,GPBAR1/TGR5)对胃癌细胞增殖、迁移和侵袭的影响。方法:免疫组织化学染色方法(Immunohistochemistry,IHC)检测胃癌及癌旁组织芯片中TGR5表达情况;qRT-PCR及Western blot检测胃癌细胞系中TGR5表达水平;小干扰RNA处理AGS、MKN-45胃癌细胞后构建TGR5敲减细胞系,慢病毒载体转染胃癌SGC-7901细胞构建TGR5过表达细胞系;CCK-8实验、平板克隆形成实验、裸鼠皮下移植瘤实验检测TGR5对细胞增殖的影响;流式细胞仪检测TGR5对细胞周期及凋亡的影响;Tanswell实验检测TGR5对胃癌细胞迁移及侵袭的影响;Western blot检测上皮间充质转化(Epithelial-mesenchymal transition,EMT)相关分子β-连环蛋白(β-catenin)、锌脂蛋白转录因子(Snail)、E盒结合锌指蛋白(Zinc finger E-box binding homeobox 1,ZEB)1在AGS、MKN-45及SGC-7901胃癌细胞中的表达。结果:TGR5在胃癌及癌旁组织中均有表达,胃癌组织TGR5高表达率(41.0%)显著高于癌旁组织(9.5%),伴肠化生癌旁组织TGR5高表达率(50%)显著高于不伴肠化生的癌旁组织(0%),胃癌组织TGR5表达与肿瘤大小相关。TGR5在正常人胃上皮永生化细胞株GES-1及各胃癌细胞系中均有表达。TGR5表达敲低的AGS和MKN-45细胞增殖能力减弱、凋亡率显著升高、侵袭和迁移能力显著降低。过表达TGR5的SGC-7901细胞增殖能力增强、克隆形成能力提高、凋亡率明显减低、侵袭和迁移能力显著升高。此外,TGR5过表达显著上调了间质细胞标志物β-catenin、Snail、ZEB1的表达水平。结论:TGR5能够增强胃癌细胞增殖及迁移能力,并抑制细胞凋亡。TGR5可能通过EMT途径介导胃癌细胞转移。

The Study of G Protein-Coupled Bile Acid Receptor 1 in Promoting the Proliferation, Migration and Invasion of Gastric Cancer Cells

Objective: To explore the influence of G-protein coupled bile acid receptor 1(GPBAR1/TGR5)on malignant phenotypes of gastric cancer cells. Methods: The expression of TGR5 in gastric tissue microarray was analyzed with immunohistochemistry. qRT-PCR and Western blot was used to detect TGR5 expression in gastric cancer cell lines. MKN-45 and AGS gastric cancer cells were transfected with TGR5 si RNA to knockdown TGR5 expression. Ectopic expression of TGR5 in SGC-7901 cells was achieved by infection with recombinant lentivirus. CCK-8 assay, colony-forming assay and subcutaneous tumor model were used to evaluate cell proliferation. Flow cytometry was exploited to observe cell cycle and apoptosis. Tanswell assay was used to test cell migration and invasion. The protein levels of β-catenin, Snail and ZEB1 were examined by Western blot. Results: TGR5 expression was presented in both gastric cancer and adjacent tissues, and strong expression of TGR5 was displayed in 41.0 % of gastric cancer tissues as well as 9.5 % of adjacent tissues. In addition, strong expression of TGR5 was found in 50 % of adjacent tissues with intestinal metaplasia as well as 0 % of adjacent tissues without intestinal metaplasia. The expression level of TGR5 in gastric cancer tissues was correlated to tumor size. Western blot and qRT-PCR revealed that TGR5 was expressed in both GES-1 and gastric cancer cell lines. Knock-down of TGR5 in AGS and MKN-45 cells resulted in decreased proliferation, increased apoptosis and suppressed migration and invasion. In the contrary, over-expression of TGR5 in SGC-7901 cells promoted proliferation, colony-forming ability, migration and invasion but inhibited apoptosis. Furthermore, increased expression of β-catenin, Snail and ZEB1 was exhibited in SGC-7901 cells with ectopic TGR5 expression. Conclusions: TGR5 could enhance the proliferation, migration and invasion of gastric cancer cells as well as inhibit cell apoptosis. TGR5 may take a part in metastasis of gastric cancer cells through regulation of EMT pathway.