幽门螺杆菌感染向恶性转化演进中网络调控节点分子HNF4a的致病机制研究

Helicobacter pylori (H. Pylori) infection leads to abnormal gene regulation of the host cells, which is one of the most important internal driving forces that promote the malignant transformation of gastric epithelial cells. For the first time, we have found that HNF4α, one of the nuclear receptor super-family molecules, situated on the node of the regulatory network and play some biological effect on the inflammation to malignant transformation started with H. Pylori infection in gastric mucosal by using systems biology techniques and preliminary functional studies. Since the heterogeneity of tissue function and the differences of pathogenic mechanisms have been identified for HNF4α, our project will focus on the role of HNF4α in the process of malignant transformation induced by H. Pylori infection with the theory of cellular microbiology. The following scientific questions will be explored specifically: (1) to determine the relevance between HNF4α and malignant transformation associated with H. Pylori infection in the sentinel mutated mice (HNF4α mutation in gastric epithelial cells) and Chinese gastric tissue specimens; (2) to examine the mechanism through which H. Pylori infection activates the abnormally high expression of HNF4α, and thus check what kinds of target genes are involved in HNF4α-mediated inflammation to malignant transformation in gastric mucosal tissue in molecular and cellular levels. This work will provide a theoretical basis for the early intervention strategies based on HNF4α as the regulatory node in gastric cancer.

幽门螺杆菌感染导致宿主细胞基因调控异常是促进胃粘膜上皮细胞恶性转化的内在动力。我们运用系统生物学技术和初步功能学研究首次发现核受体超家族分子HNF4α处于幽门螺杆菌启动胃粘膜感染炎症向恶性转化调控网络的重要节点位置并发挥生物学效应。由于HNF4α存在组织功能的异质性和致病机制的差异性，本项目将以细胞微生物学理论为指导,研究HNF4α在幽门螺杆菌感染向恶性转化过程中的作用机制，具体探索以下研究内容：(1) 应用胃粘膜组织HNF4α基因定点突变小鼠和中国人胃组织病理标本，在整体和组织层面确定HNF4α与幽门螺杆菌感染促进恶性转化的关联性；(2)在分子和细胞水平层面解析幽门螺杆菌感染通过何种机制激活HNF4α异常高表达，由此调控何种靶基因介导胃粘膜组织细胞由感染炎症向恶性转化。通过上述工作，为基于HNF4α为调控节点的胃癌早期干预策略提供理论依据。

幽门螺杆菌感染导致宿主细胞基因调控异常是促进胃粘膜上皮细胞恶性转化的内在动力。本研究发现核受体超家族分子HNF4α处于幽门螺杆菌启动胃粘膜感染炎症向恶性转化调控网络的重要节点位置并发挥生物学效应。我们首先通过qRT-PCR和免疫组化实验发现萎缩性胃炎标本中HNF4α表达水平显著低于胃癌标本。胃癌细胞系中HNF4α表达明显高于永生化胃黏膜上皮细胞。体外克隆形成实验和体内裸鼠成瘤实验发现胃癌细胞系中HNF4α被敲除后增殖能力减低。然后我们用qRT-PCR和western blot实验证实不同菌株来源的幽门螺杆菌感染均可诱导胃癌细胞HNF4α表达。这种调控作用依赖于其毒力因子cagA。幽门螺杆菌在小鼠体内亦可使HNF4α表达上调。Ensemble数据库提示HNF4αP1及P2启动子区存在两个NF-κB信号通路关键转录因子p65的结合元件。使用NF-κB信号通路抑制剂或p65 siRNA均可逆转幽门螺杆菌感染导致的HNF4α表达上调。双荧光素酶及CHIP实验证实，p65可结合于HNF4α两个启动子区。因此，幽门螺杆菌可通过NF-κB信号通路激活HNF4α表达。此外我们通过全基因组表达芯片对胃组织中的细胞表面受体分析发现，IL-1R1在胃炎向胃癌的转化过程中表达上调最为显著。收集临床标本进行qRT-PCR检测发现IL-1R1与HNF4α表达存在正相关性。Western blot及细胞免疫荧光证实干扰HNF4α表达IL-1R1表达随之下调，反之亦然。双荧光素酶实验证实HNF4α可结合于IL-1R1上游启动子区。HNF4α对IL-1R1的靶向性调控，可增敏胃部细胞对外源性细胞炎性因子IL-1β的反应。IL-1β与IL-1R1结合可进一步激活NF-κB信号通路，进而诱导HNF4α表达，形成正反馈环路促进胃部细胞恶性转化。通过上述工作，为基于HNF4α为调控节点的胃癌早期干预策略提供了理论依据。