幽门螺杆菌介导孤儿核受体NURR1过表达促进恶性转化的机制研究

Helicobacter pylori (H. pylori) mediated chronic inflammation malignant transformation is a key scientific problem. Applicants first discovered that nuclear receptor Nurr1, a member of nerve growth factor-induced clone B (NGFI-B) family, is elevated and promotes cell proliferation during the malignant transformation associated with H. pylori infection. Nurr1 over-expression is associated with the prognosis of gastric cancer. Based on this, the applicant put forward the following scientific questions: How does H. pylori infection mediate elevated Nurr1 expression? How does Nurr1 mediate the spread of inflammatory cancer and determine the prognosis of the disease? Based on previous studies, applicants proposed scientific hypotheses for this project: 1. Helicobacter pylori up-regulates Nurr1 expression via NF-κB signaling pathway 2. Nurr1 promotes the malignant proliferation of gastric mucosal cells through transcriptional activation of RASD1 3. Nurr1 can promote gastric mucosal cells to secrete nerve growth factor NGF, and then stimulate the release of acetylcholine in local microenvironment. 4.acetylcholine can bind with its receptor, activate Wnt signaling pathway, and generate feedback effect on Nurr1 expression in gastric cells. Combined with the previous work and scientific hypothesis, The applicants intend to analyze the specific role of Nurr1 in the malignant transformation mediated by H. pylori infection from the molecular mechanism and the overall organizational level in order to provide a new prevention and treatment target for the malignant transformation associated with H. pylori infection.

幽门螺杆菌感染介导的胃组织炎癌转化是关键的科学问题。申请者首次发现幽门螺杆菌感染相关的癌化过程中，核受体神经生长因子诱导克隆B（NGFI-B）亚家族成员Nurr1表达升高且与疾病预后相关。据此，申请者提出如下科学问题：幽门螺杆菌如何介导Nurr1表达升高？ Nurr1如何介导炎癌跨越并决定疾病预后？基于前期研究，申请者提出本项目科学假设：1.幽门螺杆菌通过NF-κB信号通路上调Nurr1表达；2.Nurr1可转录激活RASD1促进胃粘膜细胞的恶性增殖；3.Nurr1可促进胃黏膜细胞分泌神经生长因子NGF，继而上调局部乙酰胆碱含量；4.乙酰胆碱可作用于胃上皮细胞表面受体，激活Wnt信号通路，对细胞内Nurr1表达产生反馈性效应。结合前期工作及科学假设，申请者拟从分子机制及整体组织水平解析Nurr1在幽门螺杆菌感染介导恶性转化中的具体作用，旨在丰富幽门螺杆菌感染相关恶性转化理论以及防控新靶点。

幽门螺杆菌感染是胃组织炎癌转化的重要外界环境因素，其感染相关的具体恶性转化机制是实现早期胃癌发生干预的科学基础。本课题针对孤儿核受体Nurr1介导幽门螺杆菌相关恶性转化的调控机制进行了相关探讨及研究。本研究主要完成工作如下：1. 临床组织层面：（1）通过在临床标本中进行基因芯片分析发现，Nurr1在胃癌组织中表达显著高于在萎缩性胃炎组织中的表达，免疫组化、实时定量PCR分析亦验证了上述结果；（2）通过对三个GEO数据库（GSE51105, GSE62254, GSE14210）分析发现，Nurr1高表达组患者的整体生存期显著低于Nurr1低表达组患者。2.细胞水平层面：（1）克隆形成、CCK8分析显示，Nurr1干扰组胃癌细胞的增殖能力和成瘤能力明显减弱；（2）实时定量PCR检测、Western blot、双荧光素酶和染色质免疫共沉淀实验发现Nurr1可直接结合到细胞周期调控分子CDK4的启动子区域，通过促进CDK4的转录达到促进胃癌细胞增殖的效用；（3）通过幽门螺杆菌感染胃癌细胞实验证实幽门螺杆菌可通过PI3K/AKT/-SP1信号通路调控Nurr1及下游基因的表达。3.动物水平层面：（1）构建Nurr1稳定敲除的胃癌细胞进行裸鼠皮下成瘤，Nurr1敲除组肿瘤成瘤能力减弱；（2）幽门螺杆菌感染的小鼠模型中，Nurr1、SP1、CDK4、Ki67的表达均显著升高。通过上述工作的完成，以期为早期胃癌提供新的筛查标志物及胃癌靶向性治疗提供有力靶点。