DNA结合抑制因子ID4在H.pylori相关胃癌发展中的关键调控机制研究

启动子区甲基化导致的抑癌基因失活,是调控肿瘤发生发展的关键因素。幽门螺杆菌(H.pylori)在胃癌恶性进展中行使重要功能,寻求H.pylori相关胃癌中甲基化的关键抑癌基因成为亟待解决的重要问题。我们前期在胃癌临床标本、小鼠胃炎模型及胃癌细胞株中发现H.pylori通过甲基化抑制了DNA结合抑制因子ID4的表达；过表达ID4抑制了胃癌细胞增殖、迁移及肿瘤干细胞的形成；DNA甲基转移酶DNMT3B可能是H.pylori诱导的ID4甲基化的关键分子。本课题拟通过体内外功能实验及临床标本,深入探讨H.pylori通过DNMT3B诱导ID4的甲基化的机制,结合ChIP-seq和细胞分子生物学技术，明确ID4通过何种关键靶分子和效应通路调控了H.pylori相关胃癌的恶性进展。本研究证明的这种H.pylori通过DNA甲基化沉默ID4表达促进胃癌恶性进展的机制，可为胃癌诊断和治疗提供新思路。

Infection with Helicobacter pylori (H. pylori) has been considered a major risk factor for the development of gastric cancer. Several recent studies have demonstrated that H pylori infection reduces tumor suppressor gene expression by increased promoter methylation. However, the functional significance of H.pylori epigenetically deregulated genes in gastric carcinogenesis remains unclear. The aberrant expression of transcription factors governed selective temporal and spatial activities of their target genes, which contributed to tumorigenesis. Consequently, we focused our attention on H. pylori dependent alterations in the transcriptional factor promoter methylome. Our research has been performed to jointly analyze information of both gene methylation profiling microarray (GSE99553) and gene expression profiling microarray (TCGA) in the H. pylori positive gastric cancer. The core tumor suppressor genes were restricted to transcriptional factors. We have shown that promoter hypermethylation of transcriptional factors ID4(Inhibitor of DNA binding，ID4) is an epigenetic event that correlates with progressive loss of expression in the H.pylori positive gastric cancer. Sequenom MassArray EpiTYPER platform results confirmed that DNA methylation of the promoter region of ID4 was increased in H. pylori positive gastric cancer. Methylation of ID4 is involved in H.pylori-induced gastritis in mice and accompanied by decreased ID4 expression. We further determined the tumor-suppressive functions of ID4 in gastric cancer cells. Aberrant DNA methylation mediated by deregulation of DNA methyltransferases (DNMTs) is a key hallmark of tumor. We used RNA-seq to analyze key DNMTs involved in H. pylori -positive gastric cancer. We observed a significant higher expression of DNMT3B in H. pylori infected gastric cancer cells, which were further confirmed by TCGA. Furthermore, ID4 expression was increased by DNMT3B depletion. The demethylation effect of DNMT3B inhibitor on the CpG island of ID4 was confirmed by MSP. Gli1, the key regulatory factor of Hedgehog signaling pathway, was also found highly expressed in H. pylori infected cells. Overexpression Gli1 increased expression of DNMT3B. Therefore, we propose ID4 is silenced by promoter methylation in gastric cancer by Gli1/DNMT3B pathway. Transcriptional activity of ID4 is based on dominant negative inhibition of basic helix-loop-helix (bHLH) transcription factors. We found DEC1 (Bhlhe40) should be an ID4 potential target gene in H. pylori -positive gastric cancer cells. In this research, RNAi, Gene over expression, inhibitor will be implemented for molecular mechanism investigation. Target genes were identified by ChIP-seq and luciferase reporter analyses. We use gastritis model induced by H.pylori in mice to detect promoter methylation of ID4. We will over expression ID4 and DNMT3B expression and determine the tumorigenic capacity in nude mice. The treatment effects of DNMT3B inhibitor will be examined to detect whether it can restore ID4 levels to suppress the growth of gastric cancer. We will also analyze epigenetic ID4 silencing in gastric tissues from patients with H pylori infected gastric tumors, or without disease. In the clinical cohort level, we will analyze the correlation between them, revealing the clinical significance and how ID4 acts in the development and prognosis of H. pylori related gastric cancer. Our project intends to address the key mechanism of ID 4 in the malignant progression of H. pylori related gastric cancer. We hope that some of our studies may help us understand the new epigenetic regulation mechanism of ID4, and have significant clinical implications in gastric cancer management.