幽门螺杆菌细胞毒素相关基因A蛋白不同片段对胃癌细胞γH2AX表达的影响

Effects of different fragments of Helicobacter pylori cytotoxin-related gene A protein onγH2AX expression in gastric cancer cells

目的探讨幽门螺杆菌(H.pylori)细胞毒素相关基因A(CagA)蛋白不同片段对胃癌细胞磷酸化组蛋白H2AX(γH2AX)表达的影响。方法取前期分离的H.pylori东亚株GZ7及其CagA敲除株H.pylori GZ7ΔCagA感染的癌细胞株AGS,采用免疫荧光方法检测DNA双链断裂标志物γH2AX的表达;构建不同序列的CagA表达载体(载体a、b、c、d、e),转化大肠杆菌,提取质粒后测序鉴定,将鉴定后的载体转染AGS,采用免疫荧光方法检测γH2AX的表达,并通过流式细胞术检测细胞周期。结果成功鉴定了含不同片段的CagA载体;H.pylori GZ7感染的胃癌细胞AGS核内γH2AX的表达高于H.pylori GZ7ΔCagA感染的细胞(P<0.05);与NC组比较,不同序列CagA载体转染后胃癌细胞AGS中γH2AX表达均有增加(P<0.05或P<0.01),且载体b、c及d表达增加较明显(P<0.01);载体a和c转染后的胃癌细胞AGS的G0/G1期细胞比例升高(P<0.05),载体b、c、d及e转染后胃癌细胞AGS的G2/M期比例明显升高(P<0.01)。结论CagA是H.pylori感染诱导胃癌细胞DNA双链断裂的重要因素,CagA中的EPYIA基序是这种DNA损伤的重要结构。

Objective To investigate the effect of different fragments of Helicobacter pylori(H.pylori)cytotoxin-related gene A(CagA)protein on the expression of histone protein H2AX(γH2AX)in gastric cancer cells.Methods Gastric cancer cell line AGS was transfected with H.pylori GZ7(East Asian strain)and its CagA-knockout strain H.pylori GZ7(CagA that was previously isolated by our research group.The expression ofγH2AX,a marker of DNA double-strand breaks,was measured by immunofluorescence.Meanwhile,the plasmids containing different regions(vector a,b,c,d,e)of the CagA gene were constructed and transfected into competent E.coli cells.Subsequently,these plasmids were extracted and identified by sequencing.The identified vectors were used to transfect AGS,and thenγH2AX expression in AGS cells and the cell cycle were measured by immunofluorescence and flow cytometry,respectively.Results CagA vectors containing different fragments were successfully identified;the expression ofγH2AX in the nucleus of gastric cancer AGS cells infected with H.pylori GZ7 was higher than that of cells infected with H.pylori GZ7(CagA(P<0.05);compared with the NC group,the expression ofγH2AX in gastric cancer AGS cells increased after transfection(P<0.05 or P<0.01),and the expression of vectors b,c,and d increased significantly(P<0.01).The proportion of gastric cancer AGS cells transfected with vectors a and c in the G0/G1 phase increased(P<0.05),and the proportion of gastric cancer AGS cells transfected with vectors b,c,d,and e in the G2/M phase increased(P<0.01).Conclusion CagA is a crucial factor for H.pylori-induced DNA double-strand breaks,and the EPYIA motif of CagA is an important structure for this DNA damage.