MicroRNA-145靶向SOX2调控胃粘膜细胞肠化生的机制研究

Gastric intestinal metaplasia (IM) is the most important precancerous lesion of gastric carcinoma.It is reported that loss of SOX2,which is specifically expressed in gastric epethelial cells, may be a key factor during the development of gastric intestinal metaplasia, but the mechanism remains to be fully elucidated. Our previous work confirmed that miR-145 inhibits SOX2 expression by directly binding to its 3'-UTR.We also found that expression of miR-145 and SOX2 in gastric IM are negtively correlated.Interestingly, our results showed that the upstream sequence of miR-145 promoter includes several potential binding sites of NF-kB, which was found to induce IM by directly activates CDX2, a intestinal specific transcription factor. These indicates that miR-145/SOX2 pathway may be regulated by NF-kB and plays vital roles in gastric IM. This project is going to explore the correlation of miR-145 and SOX2 expressions in clinical gastric IM tissues using in situ hybridization and immunohistochemisty;To investigate the functions of miR-145 and SOX2 in gastric IM in gain-of-function and loss-of-function model using lentiviral vector and siRNA;To explore the mechanism of NF-kB-induced miR-145 activation through Ch-IP,EMSA, and luciferase reporter gene assay. This project aims at elucidating the function and mechanism of miR-145/SOX2 pathway in gastric IM, further understanding intestinal metaplasia on microRNA and transcriptional factor level, and offering theoretical and experimental proof for the diagnosis and treatment of gastric precancerous lesion.

肠化生是胃癌最重要的癌前病变，胃转录因子SOX2的表达降低被认为是肠化生的关键因素, 但其机制尚不清楚。前期研究发现miR-145靶向负调控SOX2，其表达在肠化生组织中负相关；而miR-145上游存在多个NF-kB结合位点：提示miR-145/SOX2通路可能受NF-kB调控并对肠化生具有重要调控功能。本项目拟通过原位杂交、免疫组化等方法验证大规模样本肠化生组织miR-145同SOX2的相关关系；通过慢病毒载体、siRNA沉默等技术构建miR-145及SOX2的功能获得缺失模型；通过免疫印迹、PCR等技术检测肠化生相关分子表达研究miR-145的功能及SOX2对其功能的介导；最后采用染色质免疫共沉淀、DNA凝胶阻滞和报告基因等技术探索NF-kB激活miR-145的机制。本项目旨在阐释miR-145/SOX2通路在胃粘膜上皮肠化生中的调控作用及受调控机制，为胃癌癌前病变诊治提供新策略。

胃粘膜肠化生是胃癌重要的癌前病变，胃特异性转录因子SOX2表达下调和肠道特异性转录因子CDX2异常表达是胃粘膜肠化生发生发展的关键事件，但其上游机制未明。基于前期工作和文献回顾，我们推测，miRNA可通过多种途径调控SOX2/CDX2的表达，从而参与胆汁酸诱导的胃粘膜肠化生。.本项目主要研究结果：第一，明确了miR-21靶向SOX2促进肠化生的作用与机制。发现SOX2能够与CDX2形成复合物并抑制CDX2的转录活性，而miR-21能够靶向抑制SOX2的表达，胆汁酸同时诱导miR-21和CDX2的表达，miR-21通过靶向SOX2释放CDX2，最终促进胃粘膜肠化生。第二，发现miR-145/miR-143通过靶向MYO6促进胃癌细胞转移。通过报告基因试验发现miR-145可靶向SOX2的3’-UTR，但miR-145在胃上皮细胞内却不能调控SOX2的表达。进一步研究发现，miR-145/miR-143通过共同靶向MYO6增强胃癌细胞的迁移和侵袭能力。第三，明确了FXR/SHP/NF-kB和miR-92a/FOXD1/NF-kB介导胆汁酸对CDX2和肠化生的诱导作用。胆汁酸受体FXR作为转录因子直接启动SHP的表达，而SHP可以促进NF-kB的活性，后者转录激活CDX2的表达。胆汁酸可诱导miR-92a的表达，miR-92a靶向抑制FOXD1，从而激活NF-kB，促进CDX2和肠化生标志分子的表达。第四，发现了MicroRNA-7/NF-kB和cyclophilin B/STAT3/microRNA-520d-5p两个反馈环路参与幽门螺杆菌对胃粘膜的损伤与致癌作用，提示幽门螺杆菌和胆汁酸下游存在互作，共同促进胃粘膜病变进展。第五，明确了我国西北地区的胃粘膜肠化生危险因素。临床流行病学调查发现，年龄超过60岁、幽门螺杆菌感染、抽烟、胃癌家族史、高盐饮食和辛辣饮食是西北地区人群肠化生的相关危险因素。上述结果为阐明胃粘膜肠化生的分子机制和寻找治疗靶点提供了理论依据。.课题组共发表学术论文16篇，其中SCI论文7篇。培养博士研究生1名、硕士研究生5名。课题负责人2015年入选国家中青年科技创新领军人才，2016年入选国家万人计划，2017年当选中华医学会内科学分会委员，2018年当选中华医学会消化病学分会委员和消化肿瘤协作组组长，并荣获国之名医青年新锐。