The Role Of TFF1/GKN2 In DNA Damage Of Gastric Mucosal Epithelial Cells By Hp Infection

Objective: Gastric cancer is a common malignancy.H.pylori(Hp)infection is an important risk factor in the occurrence of gastric cancer.Our previous study showed that the incidence of gastric cancer was significantly higher in patients with Hp infection and serum pepsinogen abnormality.CagA,the most pathogenic protein in Hp infecton,could induce cytokines production,apoptosis and proliferation,and then play a crucial role in the development of gastric cancer.Previous studies have found that Hp infection can cause cell double stranded DNA damage(DSBs),resulted in cell death or tumor occurrence.The Gastrokines(GKNs)family,a group of gastric-specific proteins with auto-stabilization and tumor suppressor genes,could inhibit the growth of gastric cancer.The trefoil factor family(TFF)is mainly secreted in the human gastrointestinal tract cells.Some data showed that TFF could promote the movement of gastric mucosal epithelial cells,prevent the occurrence of inflammation,and regulate the differentiation of gastric epithelial cells.It is a protective factor of gastric mucosa.Our previous experimental results showthat the expressions of TFF1 and GKN2 were decreased in gastric mucosal with Hp infection,indicating that TFF1 and GKN2 are closely related to the divelopment of gastric cancer.However,the interaction between Hp infection and gastric mucosal protective factors,and the role of TFF1/GKN2 in DNA damage repair by Hp infection is still unclear.Methods: 1.The expression of the DNA damage marker protein γH2AX was detected in gastric mucosal immortalized cell(GES-1)with Cag A treatmentat different concentrations and different times using cell culture method.2.The expressions of γH2AX and DNA damage repair factor(Ku70 and Ku80)were investigated using transfection,Western Blot and Real-time PCR to analyze the effect of TFF1/GKN2 overexpression on DNA damage repair induced by Hp infection.Results: 1.Effect of CagA on Double-stranded DNA damage in GES-1 CellsGES-1 cells were stimulated with 2.5 μg/ml,5 μg/ml,and 10 μg/ml CagA.Western blot results showed that the expression of γH2AX in GES-1 cells was significantly higher at 24 h,48 h,72 h and 96 h in each concentration group than in the control group.It is suggested that Hp infection can cause cell double stranded DNA damage.2.Effect of overexpression of TFF1 and GKN2 on DNA damage repair induced by HpOn the basis of our previous studies that Cag A could significantly reduce the expression of GKN2 and TFF1,the effect of TFF1/GKN2 on DNA damage repair induced by Hp was further analyzed using transfection of GKN2 and TFF1 overexpression.2.1 Effect of GKN2 overexpression in CagA-induced DNA damage repairAfter transfected with the overexpression of GKN2 plasmid for 72 h,GKN2+CagA treatment could decrease γH2AX expressioncompared with CagAtreatmentgroup(0.550±0.083vs0.722±0.094,P<0.05).However,the expressions of DNA damage repair protein,Ku70 and Ku80,were significantly higher in GKN2+CagA group than in CagA group(0.749±0.105vs0.558±0.088,0.343±0.057vs0.208±0.047,P<0.05).2.2Effect of TFF1 lentivirus transfection in CagA-induced DNA damage repairWestern blot results showed that the expression of γH2AX in the TFF1+CagA group was significantly lower than that of the CagA alone group(0.752±0.064vs1.124±0.172,P<0.05).Moreover,the expression of Ku70 protein was significantly increased in TFF1+CagA group compared with CagAtreatment group(0.536±0.047vs0.288±0.033,P<0.05).However,the expression of Ku80 protein was not significantly changed.Conclusion:1.Hp CagA can cause double-stranded DNA damage in GES-1 cells.2.GKN2 and TFF1 can reduce DNA damage of Hp CagA,and improve its repair ability,and then protect the gastric mucosal cells.