HINT1调控DSB修复参与胃癌发生发展分子机制研究

Hint1 was discovered to be one of candidate tumor suppressor genes recently while the functions and mechanisms of Hint1 are still unclear in gastric carcinogenesis. Our previous study revealed that low expression of Hint1 was closely associated with human gastric carcinogenesis. We also found the positive correlation between the expression of HINT1 and acetyl-ATM, the key enzyme of DNA double strand break ( DSB ) repair. On the basis, we hypothesize that HINT1 may involve in gastric carcinogenesis by regulating DSB repair from the prospect of DNA damage repair. RNA interference,over expression vector and related molecular biological techniques would be applied in the study to explore the molecular mechanisms of HINT1 in gastric carcinogenesis by detecting the changes of DNA damage repair, the specific protein binding, the signaling protein's expression and activity in gastric cancer cell lines after inducing DSB. Furthermore, through in vitro malignant transformation of gastric mucosal cell, tumor-bearing nude mice and clinical specimens we would verify its functions and mechanisms in gastric carcinogenesis. The ultimate goal of this study is to reveal the potential anticancerous effect and mechanisms of Hint1 in gastric cancer,and provide laboratory evidence for the early-stage screening, individualized diagnosis and treatment of gastric cancer.

Hint1是近年新发现的候选抑癌基因，但在胃癌发生发展中的功能和机制尚不明确。本课题组前期工作发现Hint1低表达与人胃癌发生发展密切相关，且HINT1与DNA双链断裂（DSB）修复关键酶ATM乙酰化表达呈正相关。在此基础上，本课题组从DNA损伤修复角度入手，提出HINT1调控DSB修复参与胃癌发生发展的分子机制假说。本课题组拟以胃癌细胞株为模型，使用RNA干扰、过表达载体及相关分子生物学技术，检测DSB诱导后DNA损伤修复、特定蛋白结合、信号蛋白表达及活性的变化，探讨HINT1调控DSB参与胃癌发生发展的分子机制，并通过胃粘膜细胞体外恶性转化实验、裸鼠移植瘤模型以及临床标本进行相关功能和机制验证。通过研究以期揭示Hint1在胃癌中潜在的抑癌作用及机制，为今后胃癌早期筛查、个体化诊断及治疗等临床工作的开展提供实验依据。

Hint1是近年新发现的候选抑癌基因，但在胃癌发生发展中的功能和机制尚不明确。本课题组前期工作发现Hint1低表达与人胃癌发生发展密切相关，且HINT1与DNA双链断裂（DSB）修复关键酶ATM乙酰化水平呈正相关。在此基础上，本课题组拟从DNA损伤修复角度入手，探索HINT1参与胃癌发生发展的分子机制。本研究发现沉默Hint1后胃癌细胞株AGS和SGC7901细胞增殖能力明显提高，初步验证了Hint1在胃癌中潜在的抑癌作用。分子机制研究显示Hint1蛋白可通过与乙酰化酶Tip60结合促进ATM乙酰化活化，激活ATM磷酸化，并进一步激活下游效应蛋白如Chk2参与DSB修复。在此基础上，我们进一步研究Hint1对胃癌细胞放疗敏感性的影响，然而体内体外研究显示Hint1沉默并未增强胃癌细胞对放疗的敏感性。相比于对照组，沉默Hint1反而有助于SGC7901细胞放疗相关DNA损伤的总体修复，这一结果提示Hint1参与DSB修复的机制并不在肿瘤放疗应答中起决定性作用。进一步研究显示Hint1为ERK信号通路的负调控因子，ERK信号通路在肿瘤发生发展中占有十分重要的作用，Hint1表达下降可导致ERK信号过度激活，激活NF-kb信号通路，并可促进细胞周期蛋白Cyclin D1过表达，进一步诱导癌基因Rb磷酸化活化，抑制细胞凋亡，增强胃癌细胞放疗耐受性，本研究首次发现Hint1调控ERK信号通路的作用，进一步完善了Hint1的分子机制，本研究工作为今后胃癌早期筛查、个体化诊断及治疗等临床工作的开展提供实验依据。