NF-κB诱导YAP磷酸化调控mTOR通路参与胃黏膜炎症恶性转化分子机制研究

Intestinal metaplasia and intraepithelial neoplasia are the precancerous lesions of gastric cancer. They are the key nodes of chronic gastritis transforming to gastric cancer or be cured, but the specific mechanism is unknown. Previous studies of our research group revealed that inflammatory factor TNF-α can directly or through NF-κB activate YAP to promote the inflammatory malignant transformation of gastric mucosa. Interfering YAP can significantly inhibit this event, suggesting that YAP plays an important role in gastric carcinogenesis. IRF3 is the positive regulator of YAP, and the target inhibition of IRF3/YAP can apparently inhibit the growth of gastric transplant tumors. Therefore, it is speculated that inflammatory factors can promote the inflammatory malignant transformation of gastric mucosa by phosphorylating YAP and regulating IRF3/YAP by mTOR through autophagy. This study is to construct the gastric mucosa inflammatory malignant transformation cell lines and PDX mice models, using lentiviral interference and chromatin immunoprecipitation method, to clarify the complex network regulation mechanism among Hippo-YAP, mTOR-YAP and NF-κB/IRF3-YAP, at mRNA level, protein level and post-translational modification levels. Our research will focus on the activation of Hippo and mTOR by compounds and inhibitors to interfere this regulating network, or IRF3/YAP, to determine the effect on intestinal metaplasia / intraepithelial neoplasia, and provide new ideas and theoretical basis for the treatment of gastric precancerous lesions.

胃黏膜肠上皮化生及上皮内瘤变为胃癌前病变，是慢性胃炎转化成胃癌或被治愈的关键节点，但具体作用机制不明。项目组前期研究发现：炎症因子TNF-α可直接或通过NF-κB激活YAP促进胃黏膜炎症恶性转化，干扰YAP可显著抑制此事件，提示YAP在致癌变中具重要地位。IRF3是YAP正调控因子，靶向抑制IRF3/YAP能显著抑制胃癌移植瘤生长。故推测炎症因子通过磷酸化YAP以及mTOR通过自噬调控IRF3/YAP促进胃黏膜炎症恶性转化。本研究拟构建胃黏膜炎症恶性转化细胞系与PDX小鼠模型，用慢病毒干扰、染色质免疫沉淀等方法，在转录水平、蛋白水平及翻译后修饰水平研究Hippo-YAP、mTOR-YAP及NF-κB/IRF3-YAP复杂网络调控机制，重点探索化合物或抑制剂激活Hippo和mTOR干扰该调控网络，或干扰IRF3/YAP，对肠上皮化生/上皮内瘤变的作用，为治疗胃癌前病变提供新的思路和理论依据。

本项目重点研究Hippo-YAP在胃黏膜癌前病变到癌变过程中的作用机制。通过测定GC和萎缩性胃炎组织和细胞中miR-365、TLR4、CDX2和IRF3表达。转染shRNA和过表达质粒，检测细胞活力、凋亡和炎症因子的改变。采用生物信息学分析、Co-IP、双荧光素酶报告基因分析以及动物实验等方法，分析miR-365与TLR4以及IRF3和YAP在胃黏膜癌前病变到癌变过程中的作用机制。研究发现miR-365的过表达能抑制胃癌前病变，microRNA-365通过抑制TLR4降低IRF3磷酸化，抑制YAP介导的CDX2转录，从而抑制胃癌前病变进展。通过RNA高通量测序筛选慢性非萎缩性胃炎、胃黏膜低级别/高级别上皮内瘤变以及胃癌组织之间共同表达的差异lncRNA，分别是lncRNA H19、LINC00895、lnc-SRGAP2C-16、lnc-HLA-C-2、lnc-APOC1-1、lnc-B3GALT2-1，这6种lncRNA有望成为胃癌早期诊断的分子标志物。探讨胃癌发生中LINC00857、TFAP2C、非典型钙粘蛋白1（FAT1）和AP-1信号之间相互作用。分析FAT1表达与GC患者总生存率之间的相关性，体外和体内测定以评估FAT1在GC细胞中的生物学作用。蛋白质印迹分析、萤光素酶报告基因、ChIP、RIP和RNA分析鉴定FAT1的上游和下游分子。研究发现LINC00857招募TFAP2C来提高FAT1的表达，从而增强GC细胞的恶性生物学特性。体内试验表明，敲除LINC00857可减缓肿瘤生长并逆转上皮-间质转化过程。提示LINC00857可能通过招募TFAP2C上调FAT1表达，然后升高AP-1表达，从而促进GC的进展。发现LncRNA PVT1能通过YAP促进胃癌细胞增殖、迁移和侵袭。研究发现胃癌细胞系与胃癌组织基因表达有差异。在体外和计算机中比较了原发性胃癌、健康组织和细胞系中的差异表达基因（DEG）。在队列研究和实验验证中分析了七个报告的基因，这些基因通过DNA甲基化在GC中表达降低。但发现报告的癌症相关下调基因仅在癌细胞系中发现，而在活检组织中未发现，提示胃癌细胞系与胃癌组织基因不同。YWHAQ和THBS1的增加与胃癌患者的低生存率相关，在GC活检中观察到YWHAQ和THBS1的表达升高，这可能是GC诊断的潜在生物标志物。