TGF-β1/smad Signaling Pathway Is Involved In Epithelial-mesenchymal Transition(EMT)in Gastric Cancer Cells Induced By TNF-α-inducing Protein Of Helicobacter Pylori

Background:Gastric cancer is a common malignant tumor,which is the fifth most common cancer in the world with mortality rate as high as 75%.Therefore,it is of great significance to investigate the mechanism and development of gastric cancer.Development of gastric cancer is closely associated with Helicobacter pylori(H.pylori)infection,and which has become the focus of attention.Tipα,a specific protein secreted by H.pylori,is a newly identified carcinogenic factor associated with the development of gastric cancer.Recent researches have demonstrated that Tipα is involved in the pathogenic process of H.pylori by the induction of proinflammatory reaction and epithelial-to-mesenchymal transition(EMT)in gastric epithelial cells.However,its promotion on inflammation and carcinogenesis and the detailed mechanism remain to be elucidated.So,investigating the effect and mechanism of Tipα on EMT in gastric cancer cells will provide new targets for the clinical diagnosis and targeted therapy of gastric cancer.Objective: In this study,we will use the model of recombinant Tipα protein-treated gastric cancer cell lines,to explore the effects of Tipα on proliferation,apoptosis,migration and invasion of gastric cancer cells,and to clarify the signaling pathways involved in the Tipα-induced EMT.Methods:(1)The prokaryotic expression vector containing Tipα gene was highly expressed with the induction of IPTG in Escherichia coli,then the recombinant Tipα protein was purified by affinity chromatograp Hy and subjected to an endotoxin removal test.The Tipα protein was thereafter identified by Western-blot and the concentration was determined.(2)SGC7901 cells were stimulated by Tipα and the morp Hological changes were observed under a microscope.The effect of Tipα on cell proliferation and apoptosis was detected by CCK8 and flow cytometry.(3)ELISA method was used to detect the effect of different concentration of Tipα treatment on the secretion of TGF-β1 in SGC7901 at different time points.TGF-β1/Smad signaling pathway was blocked by TGF-β receptor inhibitor LY2109761,and then q RT-PCR and Western blot were employed to detect the m RNA and protein expression of the EMT-related markers E-cadherin,α-SMA and pathway related TGF-β1,Smad3 and p Smad3 respectively.(4)After the blocking of TGF-β1/Smad signaling pathway,the effect of Tipα on the cell migration and MMP9 expression were also analyzed by wound-healing,q RT-PCR and Western blot respectively.Results:(1)The results showed that Tipα protein could induced the elongation of SGC7901 cells after treatment for 12 h.The cells become relatively loose and had pseudopodia at the same time.(2)Tipα could enhance the proliferation and inhibit the apoptosis of SGC7901 cells.(3)Tipα(100μg/m L protein was the most significant)could induced the secretion of TGF-β1 in SGC7901 cells,with decreased E-cadherin expression and increased α-SMA expression.On the contrary,the expression of E-cadherin was increased and the expression of α-SMA was reduced when TGF-β1/Smad signaling pathway was blocked.The expression of TGF-β1 and Smad3 there was no significant difference between the control and blocking gruop,but the expression of p Smad3 was dcreased.(4)The Tipα stimulation could enhance the migration of SGC7901 cells and the expression of MMP9.The abilities of Tipα promote gastric cancer cells migration and invasion were significantly inhibited by the TGF-β1/Smad signaling pathway blocking.Conclusions:(1)Tipα could enhance the proliferation,migration,invasion and inhibit the apoptosis of SGC7901 cells.(2)TGF-β1/Smad signaling pathway was involved in the H.pylori Tipα-induced EMT in gastric cancer cells.