吉林大学学报(医学版) ›› 2022, Vol. 48 ›› Issue (5): 1238-1246.doi: 10.13481/j.1671-587X.20220518

• 基础研究 • 上一篇    

过表达TLR4基因对胃癌细胞自噬的抑制作用及其机制

张蒨,李静()   

  1. 锦州医科大学附属第一医院消化内科,辽宁 锦州 121001
  • 收稿日期:2021-12-04 出版日期:2022-09-28 发布日期:2022-11-15
  • 通讯作者: 李静 E-mail:jz7203@139. com
  • 作者简介:张 蒨(1994-),女,四川省宜宾市人,在读硕士研究生,主要从事消化肿瘤基础和临床方面的研究。
  • 基金资助:
    辽宁省科技厅民生科技计划项目(2021JH2/10300042);辽宁省科技厅自然科学基金项目(20170540391)

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

Qian ZHANG,Jing LI()   

  1. Department of Gastroenterology,First Affiliated Hospital,Jinzhou Medical University,Jinzhou 121001,China
  • Received:2021-12-04 Online:2022-09-28 Published:2022-11-15
  • Contact: Jing LI E-mail:jz7203@139. com

摘要:

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。

关键词: 胃肿瘤, Toll样受体4, 细胞自噬, 细胞增殖

Abstract:

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.

Key words: Stomach neoplasms, Toll-like receptor 4, Autophagy, Cell proliferation

中图分类号: 

  • R735.2