| ObjectivesHelicobacter pylori(H.pylori)is a Gram-negative bacterium that is mainly colonized on the surface of the gastric mucosa.It is mainly transmitted from person to person through the fecal-oral route.Long-term chronic infection of gastric epithelial cells by Helicobacter pylori is a major risk factor for the development of gastric cancer.DNA double-strand break damage and gene mutation in gastric epithelial cells caused by Helicobacter pylori infection are an important molecular mechanism in early gastric cancer.Previous studies have shown that various bacterial virulence factors secreted by H.pylori can cause DNA damage in gastric epithelial cells,including cytotoxin associated gene A(CagA)and vacuolating cytotoxin A(VacA).In mammals,there are mainly two ways to complete DNA damage repair after DNA double-strand breaks,accurate homologous recombination(HR)repair and inaccurate non-homologous end join(NHEJ)repair.Previous studies in gastric cancer cell lines showed that H.pylori infection reduces the expression of many DNA damage repair genes,allowing host cells to select more error-prone NHEJ repairs,resulting in increased genomic instability and cancer risks.There is no definitive study on the effect of Helicobacter pylori infection on HR repair at home and abroad,and the impact of its important virulence factor VacA on HR repair is still largely unknown.In the present study,we used Hp P12 and Hp P12 ?vacA strains to infect GES-1 cells and HEK-293(DR-GFP)cells to detect the effects of H.pylori infection on HR repair and the machanisms of VacA virulence factor on HR repair.Materials and Methods1.After infection of GES-1 cells with Hp P12 for 12 h at MOI 25,50,100,and 200,the expression of CagA in cells was detected by western blot to evaluate the effect of different MOI on Hp infection.2.Infected GES-1 cells with Hp P12 for 6h,12 h,and 24 h at MOI 100,then western blot was applied to detect the expression of HR repair key proteins(MRE11,CtIP)and CagA,and evaluate the effects of different infection time on HR repair.3.GES-1 cells were incubated with Hp P12 and its ?cagPAI,?cagA,?vacA,?cagL and ?cagE strains for 12 h at MOI=100.To analysis H.pylori virulence factors on DNA damage and HR repair,the expression of DNA damage marker protein ?H2AX,HR repair key proteins(CtIP,pCtIP,pMRE11,Rad51)and pChk2 were detected by western blot.4.GES-1 cells were infected with Hp P12 and Hp P12 ?vacA at MOI=100 for 12 h,then detect the DNA damage marker protein ?H2AX and HR repair key proteins CtIP and Rad51 by immunofluorescence.5.Infected HEK-293(DR-GFP)cells with Hp P12 or Hp P12 ?vacA at MOI 50 for 6h,then detect the proportion of GFP positive cells by flow cytometry to test the effect of HR repair.Results1.After Hp P12 infection of GES-1 cells,the expression of CagA was up-regulated compared with not-infected group(P<0.01),and the key proteins of HR repair(MRE11,CtIP)were up-regulated compared with not-infected group(P<0.01).2.After infected GES-1 cells with Hp P12 and its ?cagPAI,?cagA,?vacA,?cagL and ?cagE knockout strains,the expression of HR repair upstream regulatory proteins(pCtIP,pMRE11)and pChk2 were up-regulated(P<0.01),and the Hp P12 ?vacA-infected group was down-regulated compared with other groups(P<0.05).For the expression of HR repair key protein Rad51,Hp P12-infected group was up-regulated compared with not-infected group(P<0.01);?cagPAI,?cagA,?vacA,?cagE gene knockout strains infected groups were down-regulated compared with P12 standard strain infected group(P<0.01),the ?vacA gene knockout strain infected group was down-regulated compared with other strains(P<0.05);the ?cagL gene knockout strain infected group was up-regulated compared with the not-infected group(P<0.01),but there was no significant difference with the P12 standard strain infected group(P=0.6561).3.After infection of GES-1 cells with Hp P12 and Hp P12 ?vacA strains,the expression and recruitment of DNA damage marker protein ?H2AX was up-regulated in Hp P12-infected group compared with not-infected group(P<0.05),and the Hp P12 ?vacA-infected group was down-regulated compared with the Hp P12-infected group(P<0.05).Similarly,the expression and recruitment of HR repair key proteins(Rad51,pMRE11,CtIP,and pCtIP)were up-regulated in the Hp P12-infected group compared with the not-infected group(P<0.05),and down-regulated in the Hp P12 ?vacA-infected group compared to the Hp-P12-infected group(P<0.05).4.Furthermore,HEK-293(DR-GFP)cells transfected with I-SceI plasmid were infected by Hp P12 and Hp P12 ?vacA strains to verify the results above.The number of GFP-positive cells in Hp P12-infected group was down-regulated,and this tendency was more significantly in Hp P12 ?vacA-infected cells(P<0.05).Conclusions1.In GES-1 cells,Hp P12 infection cause DNA double-strand break damage and up-regulate the expression of HR repair key proteins as well as pChk2,and promote the recruitment of HR repair key proteins in DNA damage sites.It indicates that HR repair is promoted in the process of infection,and VacA virulence factor plays an important role.2.The mechanisms of Helicobacter pylori on HR repair are different in GES-1 and gastric cancer cell lines. |
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