自发性胃癌高发小鼠模型中ISG15调控炎症微环境和肿瘤形成的机制研究

Inflammation mediates cancer initiation and progression, but the underlying molecular mechanisms are largely unclear. Our preliminary studies first identified a mice model of spontaneous gastric cancer with high-incidence, namely the Collaborative Cross (CC) 036 mice, in which, the expression of many inflammation-related genes as well as the level of T- and B-lymphocytes in peripheral blood were remarkably increased. Bioinformatics analysis revealed that the expression level of human homologous genes to such mouse inflammation-related genes were inversely correlated with the overall survival of patients with gastric cancer. ISG15, an inflammation-related gene that was found to be highly expressed in CC036 mice, may regulate inflammation pathway via ubiquitin-like modification of STAT1, and its protein expression was increased in human gastric adenocarcinoma in our studies. Taken together, these results indicated that the development of spontaneous gastric adenocarcinoma in CC036 mice might be closely associated with inflammation. This project aims to understand the possible correlation of high incidence of gastric cancer with inflammation; to elucidate the role of ISG15 as a driver gene in gastric cancer development by generating an ISG15 knockout in CC036 mice using CRISPR; to comprehensively explore the mechanism(s) by which ISG15 promotes tumorigenesis through regulating inflammation pathway at molecule, cell, animals and clinical levels, and to further explicit the application value of the CC036 model. All of these will finally provide scientific basis for fighting against gastric cancer via inhibition of inflammatory responses.

炎症介导肿瘤的发生发展, 但其分子机制尚不清楚。我们的前期工作在国际上首次发现了自发性胃癌高发小鼠模型，即复杂性状遗传CC036小鼠，其胃组织中诸多炎症相关基因的表达以及外周血T和B淋巴细胞水平均显著增高。生物信息分析揭示,对应于这些小鼠炎症基因的人同源基因的表达水平与胃癌病患的总生存率呈逆相关。CC036中高表达的炎症基因ISG15通过类泛素化STAT1调控炎症通路并在人胃腺癌中其蛋白表达升高。这些结果提示CC036自发性胃腺癌的发生发展可能与炎症密切相关。本课题拟以此小鼠模型为平台，通过胃癌诱发和炎症抑制正反两方面阐明其胃癌高发和相伴炎症之间的关系；应用CRISPR技术敲除CC036中ISG15，明确其在胃癌发生的驱动作用，并从分子、细胞、动物、临床样本多角度综合探讨ISG15通过调控炎症通路促肿瘤发生的机制，进一步明确CC036模型的应用价值，并为抑制炎症反应抗胃癌发生提供科学依据。

干扰素刺激基因15(ISG15)及类泛素化在炎-癌转化中起重要作用但具体机制未明。我们在国际上首次发现了自发性胃癌高发小鼠模型，其胃组织中ISG15表达及外周血淋巴细胞水平均显著升高，遗传分析发现此基因为NFκB炎症活化通路的下游靶标。前期结果显示ISG15过表达促进胃癌细胞增殖和迁移;生物信息学及免疫共沉淀发现NFκB通路活化分子PCSK9为ISG15靶蛋白，应用免疫共沉淀结合质谱筛选到PCSK9和ISG15之间可能存在相互作用。免疫共沉淀检测证实ISG15能够与PCSK9相互结合。进一步的细胞实验发现过表达ISG15后，细胞内PCSK9的类泛素化水平升高、泛素化水平降低。另外，Western结果显示过表达ISG15可以显著促进NFκB经典信号通路的活化。我们猜想ISG15可通过类泛素化提高PCSK9的稳定性，进而调控NFκB信号通路，同时后者可正反馈强化ISG15表达，促进胃癌的发生发展。本项目从细胞、基因改造动物和临床病人的不同水平，明确ISG15及类泛素化在胃癌发生发展中的作用;阐明ISG15-PCSK9-NFκB相互作用的分子机制;分析人胃癌组织中ISG15与PCSK9、NFκB活化的相关性及临床意义。从新的视角探索了胃炎-胃癌序贯发生的分子机制，为胃癌防治提供新的有效靶点。