Zhou Jianjiang, Wang Wenling, Xie Yuan, Zhao Yan, Chen Xian, Xu Wenjie, Wang Yan, Guan Zhizhong
Molecular Biology Key Laboratory, Guizhou Medical University, Guiyang, Guizhou, China.
Department of Clinical Laboratory, Affiliated Hospital of Guizhou Medical University, Guiyang, Guizhou, China.
PLoS One. 2016 Jan 8;11(1):e0146521. doi: 10.1371/journal.pone.0146521. eCollection 2016.
Helicobacter pylori (H. pylori) is a spiral-shaped Gram-negative bacterium that causes the most common chronic infection in the human stomach. Approximately 1%-3% of infected individuals develop gastric cancer. However, the mechanisms by which H. pylori induces gastric cancer are not completely understood. The available evidence indicates a strong link between the virulence factor of H. pylori, cytotoxin-associated gene A (CagA), and gastric cancer. To further characterize H. pylori virulence, we established three cell lines by infecting the gastric cancer cell lines SGC-7901 and AGS with cagA+ H. pylori and transfecting SGC-7901 with a vector carrying the full-length cagA gene. We detected 135 differently expressed proteins from the three cell lines using proteome technology, and 10 differential proteins common to the three cell lines were selected and identified by LC-MS/MS as well as verified by western blot: β-actin, L-lactate dehydrogenase (LDH), dihydrolipoamide dehydrogenase (DLD), pre-mRNA-processing factor 19 homolog (PRPF19), ATP synthase, calmodulin (CaM), p64 CLCP, Ran-specific GTPase-activating protein (RanGAP), P43 and calreticulin. Detection of the expression of these proteins and genes encoding these proteins in human gastric cancer tissues by real-time PCR (RT-qPCR) and western blot revealed that the expression of β-ACTIN, LDH, DLD, PRPF19 and CaM genes were up-regulated and RanGAP was down-regulated in gastric cancer tissues and/or metastatic lymph nodes compared to peri-cancerous tissues. High gene expression was observed for H. pylori infection in gastric cancer tissues. Furthermore, the LDH, DLD and CaM genes were demethylated at the promoter -2325, -1885 and -276 sites, respectively, and the RanGAP gene was highly methylated at the promoter -570 and -170 sites in H. pylori-infected and cagA-overexpressing cells. These results provide new insights into the molecular pathogenesis and treatment targets for gastric cancer with H. pylori infection.
幽门螺杆菌（H. pylori）是一种螺旋形革兰氏阴性菌，可引发人类胃部最常见的慢性感染。约1%-3%的感染者会罹患胃癌。然而，幽门螺杆菌诱发胃癌的机制尚未完全明晰。现有证据表明，幽门螺杆菌的毒力因子细胞毒素相关基因A（CagA）与胃癌之间存在紧密联系。为进一步表征幽门螺杆菌的毒力，我们通过用携带全长cagA基因的载体转染胃癌细胞系SGC-7901，并将cagA+幽门螺杆菌感染胃癌细胞系SGC-7901和AGS，建立了三个细胞系。我们运用蛋白质组技术从这三个细胞系中检测到135种差异表达蛋白，选取了三个细胞系共有的10种差异蛋白，通过液相色谱-串联质谱法（LC-MS/MS）进行鉴定，并通过蛋白质免疫印迹法进行验证：β-肌动蛋白、L-乳酸脱氢酶（LDH）、二氢硫辛酰胺脱氢酶（DLD）、前体mRNA加工因子19同源物（PRPF19）、ATP合酶、钙调蛋白（CaM）、p64 CLCP、Ran特异性GTP酶激活蛋白（RanGAP）、P43和钙网蛋白。通过实时定量聚合酶链反应（RT-qPCR）和蛋白质免疫印迹法检测这些蛋白及其编码基因在人胃癌组织中的表达，结果显示，与癌旁组织相比，β-肌动蛋白、LDH、DLD、PRPF19和CaM基因在胃癌组织和/或转移淋巴结中的表达上调，而RanGAP基因的表达下调。在胃癌组织中观察到幽门螺杆菌感染的高基因表达。此外，在幽门螺杆菌感染和cagA过表达的细胞中，LDH、DLD和CaM基因在启动子-2325、-1885和-276位点分别发生去甲基化，而RanGAP基因在启动子-570和-170位点发生高度甲基化。这些结果为幽门螺杆菌感染所致胃癌的分子发病机制及治疗靶点提供了新见解。
