幽门螺杆菌CagA与细胞eEF1A1相互作用对细胞增殖、凋亡及细胞周期的影响及其机制

CagA is the important virulent protein of helicobacter pylori(Hp), and is closely related to the carcinogenesis of gastric cancer. Our preliminary study found that CagA can interact with the eEF1A1 protein in gastric epithelial cells. Based on the findings that eEF1A1 protein participates in translation, and functions in regulating the cell cycle, proliferation and apoptosis, we can therefore speculate that CagA, by interacting with eEF1A1, could interfere with the biological functions of eEF1A1 including protein translation, cell proliferation, apoptosis and cell cycle. In the present study, we aimed to 1) determine the binding domains between CagA and eEF1A1; 2) detect the changes in cell cycle, proliferation and apoptosis in the gastric epithelial cells infected by CagA recombinant adenoviruses together with the overexpression or knocking down of eEF1A1; 3) examine the influence of CagA on eEF1A1 mediated translation and the related changes of the protein expression profiles; 4) detect the influence of CagA on the eEF1A1-mediated signaling pathway of pAKT- HDM2- P53, which lead to the changes of the cell proliferation, apoptosis and cell cycle. The results will be helpful to reveal carcinogenic mechanism of CagA, and provide new insight in prevention and treatment of the helicobacter pylori associated gastric cancer.

CagA是幽门螺杆菌重要的毒力蛋白，与胃癌的发生密切相关。本课题组前期研究发现CagA能与胃黏膜上皮细胞eEF1A1蛋白相互作用。鉴于eEF1A1参与蛋白的翻译，并具有调节细胞周期、增殖及凋亡功能，推测CagA可通过与eEF1A1相互作用，影响eEF1A1介导的蛋白翻译、细胞增殖、凋亡及细胞周期改变。因此，本研究拟进行：1）确定CagA与eEF1A1相互作用各自的作用位点 ；2）将CagA 作用于eEF1A1高表达或沉默的胃黏膜上皮细胞，检测细胞周期、增殖及凋亡的改变；3）检测CagA与eEF1A1结合对eEF1A1介导的翻译功能影响以及由此导致的蛋白表达谱改变；4）检测CagA与eEF1A1结合对eEF1A1介导pAKT- HDM2- P53信号通路的影响，揭示二者结合引起细胞增殖、凋亡和细胞周期改变的信号通路。本研究将进一步揭示CagA的致癌机制，为幽门螺杆菌相关胃癌的防治提供新思路。

CagA是幽门螺杆菌重要的毒力蛋白，与慢性消化性疾病及胃癌的发生密切相关。本课题组前期研究发现CagA能与胃黏膜上皮细胞eEF1A1蛋白相互作用。鉴于eEF1A1参与蛋白的翻译，并具有调节细胞周期、增殖及凋亡功能，推测CagA可通过与eEF1A1相互作用，影响细胞的功能。因此，本研究拟在确定CagA与eEF1A1相互作用及其作用位点的基础上探究 CagA与eEF1A1互作对细胞功能的影响及其机制，研究内容包：1）确定幽门螺杆菌CagA与eEF1A1相互作用及各自的作用位点；2）CagA与eEF1A1蛋白相互作用对细胞增殖、凋亡及细胞周期的影响；3）CagA与eEF1A1蛋白相互作用对胃癌上皮细胞IL-6表达的影响；4）信号通路CagA-eEF1A1-PKCδ-p-STAT3S727-IL-6在IL-6表达中的作用；5）CagA 与 eEF1A1 蛋白相互作用对 eEF1A1 介导的蛋白表达的影响。研究结果：确定了幽门螺杆菌CagA与eEF1A1在胃上皮细胞内可相互作用并确定了其结合区域是CagA的M段（769-2616）与 eEF1A1蛋白的（1-240）区段。证明CagA对细胞增殖无影响；可抑制细胞凋亡；对细胞G0/G1期和S期无影响、但缩短细胞G2期；可抑制细胞蛋白的翻译，但其对凋亡、细胞周期及蛋白翻译的影响与CagA和eEF1A1相互作用无关。CagA-eEF1A1互作诱导胃癌上皮细胞IL-6表达，其机制是CagA通过eEF1A1招募PKCδ，进而调控p-STAT3S727磷酸化影响胃癌上皮细胞IL-6表达。提示CagA-eEF1A1-PKCδ-p-STAT3S727-IL-6是H. pylori相关胃癌细胞中炎症通路，丰富了CagA这一重要毒力蛋白的致病机制，具有重要的理论价值。