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