腺病毒载体介导CagA基因表达对原代胃癌细胞糖酵解代谢酶表达的影响  

Effects of CagA gene expression on expression of glycolytic enzymes in primary gastric cancer cells by adenovirus

作　　者：杨丽萍 贾岑岑 龙妮娅 骆梅 杨丹 王珺 谢渊[1,2] 赵艳 王琴容[1,2] 周建奖 YANG Li-ping;JIA Cen-cen;LONG Ni-ya;LUO Mei;YANG Dan;WANG Jun;XIE Yuan;ZHAO Yan;WANG Qin-rong;ZHOU Jian-jiang(Key Laboratory of Endemic and Ethnic Diseases,Ministry of Education,Guizhou Medical University,Guiyang,Guizhou,China 550004;Key Laboratory of Molecular Biology,Guizhou Medical University;Clinical Research Center,Guizhou Medical University Hospital,Guiyang;Clinical Laboratory,Guizhou Medical University Hospital)

机构地区：[1]贵州医科大学地方病与少数民族疾病教育部重点实验室,贵州贵阳550004 [2]贵州医科大学分子生物学重点实验室 [3]贵州医科大学附属医院临床医学研究中心 [4]贵州医科大学附属医院临检科

出　　处：《中国病原生物学杂志》2019年第11期1297-1302,1311,共7页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.31560326,31660031);贵州省科技厅黔科合平台人才项目[2017]5652;贵州市科技局筑科合同项目[2017]5-16;贵州省卫计委科技基金项目(No.gzwjkj2015-1-010)

摘　　要：目的研究过表达CagA对原代胃癌细胞糖酵解代谢酶表达的影响。方法构建CagA重组腺病毒载体pAdeno-MCMV-CagA-3Flag-IRES2-EGFP(pAdeno-CagA),转染HEK293细胞,包装腺病毒,以感染复数(Multiplicity of infection, MOI) 50感染原代胃癌细胞72 h,并用幽门螺杆菌(Helicobacter pylori,Hp)东亚株GZ7(CagA^+)以MOI 50感染原代胃癌细胞24 h,通过Western blot检测CagA和已糖激酶2(HK2)、葡萄糖转运蛋白1(GLUT1)、烯醇化酶1(ENO1)、丙酮酸激酶1/2(PKM1/2)、乳酸脱氢酶A(LDHA)糖酵解代谢酶的蛋白表达,采用流式细胞术检测胃癌细胞周期分布。结果成功构建了CagA重组腺病毒载体pAdeno-CagA,用pAdeno-CagA腺病毒和Hp GZ7感染原代胃癌细胞后糖酵解代谢酶HK2、ENO1、GLUT1的表达与空载组或Hp非感染组比较均显著下调(P<0.05)。与空载组比较,pAdeno-CagA感染胃癌SGC-7901细胞24、48、72 h,其G2/M期细胞百分比显著增加(P<0.05),感染72 h的空载组和CagA腺病毒组G2/M期的比值分别为16.90%和65.93%。结论 Hp通过CagA使原代胃癌细胞糖酵解代谢酶表达下调,可能与DNA损伤使细胞阻滞在G2/M期有关。Objectives To investigate the effect of CagA over-expression on the expression of glycolytic enzymes in primary gastric cancer cells.Methods The recombinant adenovirus vector pAdeno-MCMV-CagA-3 Flag-IRES2-EGFP(pAdeno-CagA)was constructed and transfected into HEK293 cells to package adenovirus.This adenovirus and Helicobacter pylori GZ7(H.pylori GZ7,CagA^+),an East Asian strain of H.pylori,were respectively used to infect primary gastric cancer cells for 72 and 24 hours at a multiplicity of infection(MOI)of 50.The expression of CagA,hexokinase 2(HK2),glucose transporter 1(GLUT1),enolase-1(ENO1),pyruvate kinase1/2(PKM1/2),and lactate dehydrogenase A(LDHA)protein were evaluated using Western blot analysis,and the cell cycle of infected cells was detected using flow cytometry.Results The recombinant vector pAdeno-CagA was successfully constructed.Compared to the cells infected with the empty vector or cells not infected with H.pylori,expression of HK2,GLUT1,and ENO1 protein was significantly down-regulated(P<0.05)in primary gastric cancer cells infected with pAdeno-CagA or H.pylori GZ7.The percentage of cells in the G2/M phase increased significantly in gastric cancer SGC-7901 cells infected with pAdeno-CagA for 24,48,or 72 h(P<0.05).The ratio of cells in the G2/M phase was 16.90%in the empty vector group and 65.93%in the pAdeno-CagA group after 72 h.Conclusion H.pylori induced the down-regulated expression of glycolytic enzymes by CagA in primary gastric cancer cells.This action may be related to DNA damage that arrests cells in the G2/M phase.

关 键 词：幽门螺杆菌 腺病毒 CAGA 胃癌 糖酵解 细胞周期 

分 类 号：R378.911[医药卫生—病原生物学]