过表达TLR4基因对胃癌细胞自噬的抑制作用及其机制

Inhibitory effect of TLR4 gene overexpression on autophagy of gastric cancer cells and its mechanism

目的:采用慢病毒过表达胃癌细胞中Toll样受体4 (TLR4)基因,探讨其对胃癌细胞自噬的作用,并阐明其作用机制。方法:处于对数生长期的胃癌BGC-823细胞和SGC-7901细胞分为未感染组、阴性对照病毒组和基因过表达组,分别给予无慢病毒感染、空载体慢病毒和过表达TLR4慢病毒稳定感染。采用Western blotting法检测正常胃黏膜上皮GES-1细胞及胃癌BGC-823细胞、SGC-7901细胞、HGC-27细胞和MGC-803细胞中TLR4蛋白表达水平。采用CCK-8法检测各组细胞增殖活性,实时荧光定量PCR (RT-qPCR)法分别检测各组细胞中TLR4、Beclin1和微管相关蛋白轻链3 (LC3) mRNA表达水平,Western blotting法检测各组细胞中磷脂酰肌醇-3激酶/蛋白激酶B(PI3K/Akt)、磷酸化PI3K/磷酸化Akt (p-PI3K/p-Akt)、哺乳动物雷帕霉素靶蛋白(mTOR)和磷酸化mTOR (p-mTOR)信号通路相关蛋白及细胞自噬相关蛋白(LC3、 Beclin1和p62)蛋白表达水平。结果:BGC-823细胞、HGC-27细胞和MGC-803细胞中TLR4蛋白表达水平高于胃黏膜上皮GES-1细胞。与未感染组和阴性对照病毒组比较,过表达TLR4组BGC-823细胞和SGC-7901细胞中TLR4蛋白表达水平明显升高(P<0.05)。与未感染组和阴性对照病毒组比较,过表达TLR4组BGC-823细胞和SGC-7901细胞增殖活性明显升高(P<0.05或P<0.01)。与阴性对照病毒组比较,过表达TLR4组BGC-823细胞和SGC-7901细胞中TLR4 mRNA表达水平明显升高(P<0.01),BGC-823细胞中Beclin1 mRNA表达水平降低(P<0.05),SGC-7901细胞中LC3和Beclin1 mRNA表达水平降低(P<0.05)。与未感染组和阴性对照病毒组比较,过表达TLR4组BGC-823细胞和SGC-7901细胞中LC3和Beclin1蛋白表达水平明显降低(P<0.05),p62、p-PI3K、p-Akt和p-mTOR蛋白表达水平明显升高(P<0.05)。结论:过表达TLR4基因可以通过调控PI3K/Akt/mTOR信号通路明显抑制胃癌细胞的自噬活性。

Objective:To investigate the effect of overexpression of Toll-like receptor 4(TLR4)gene by lentivirus on autophagy of the gastric cancer cells,and to clarify its mechanism. Methods:The gastric cancer cells BGC-823 and SGC-7901 at logarithmic growth phrase were divided into non-infection group,negative control virus group and over-expression TLR4 group;they were infected with lentivirus-free,empty vector and lentivirus that overexpress TLR4,separately. Western blotting method was used to detect the expression levels of TLR4 protein in gastric epithelial cells GES-1 and gastric cancer cells BGC-823,SGC-7901,HGC-27 and MGC-803. CCK-8 assay was used to detect the proliferation activities of cells in various groups,real-time fluorescence quantitative PCR(RT-qPCR)method was performed to determine the expression levels of TLR4,Beclin1 and microtubule-associated protein light chain 3(LC3)mRNA,the expression levels of phosphatidylinositol-3 kinase/protein kinase B(PI3K/Akt),mammalian target of rapamycin(mTOR), phosphorylated PI3K/phosphorylated Akt(p-PI3K/p-Akt) and phosphorylated mTOR(p-mTOR) signaling pathway-related proteins and autophagy-related proteins(LC3,Beclin1 and p62) were detected by Western blotting method. Results:The expression levels of TLR4 protein in BGC-823 cells,HGC-27 cells and MGC-803 cells were higher than that in gastric epithelial cells GES-1. Compared with non-infection and negative control virus groups,the TLR4 protein expression levels in BGC-823 and SGC-7901 cells in over-expression TLR4 group were significantly increased(P<0. 05). Compared with non-infection control and negative control virus groups,the proliferation activities of BGC-823 cells and SGC-7901 cells in over-expression TLR4 group were significantly increased(P<0. 05 or P<0. 01). Compared with negative control group,the expression levels of TLR4 mRNA in the BGC-823 cells and SGC-7901 cells in over-expression TLR4 group were significantly increased(P<0. 01),the expression level of Beclin1 mRNA in BGC-823 cells was decreased(P<0. 05),and the expression levels of LC3 and Beclin1 mRNA in SGC-7901 cells were decreased(P<0. 05). Compared with non-infection group and negative control group,the expression levels of LC3 and Beclin1 proteins in the BGC-823 cells and SGC-7901 cells in over-expression TLR4 group were significantly decreased(P<0. 05),while the expression levels of p62,p-PI3K、p-Akt and p-mTOR proteins were increaed(P<0. 05). Conclusion:The over-expression of TLR4 gene can significantly inhibit autophagy in gastric cancer cells via regulating the PI3K/Akt/mTOR signaling pathway.