人类DNA甲基化酶DNMT2在胃癌发病中的功能机制

DNMT2，a member of DNA methyltransferase family, while containing DNA methyltransferase domain, mainly methylates eukaryotic tRNA. One of DNMT2 polymorphisms, which led to H101T mutation, was found to be significantly associated with susceptibility of gastric cancer. So far, the detailed mechanisms of DNMT2 association with gastric cancer has never been studied. It is unclear how DNMT2 recognizes tRNA substrates and whether DNMT2 methylation mediates the onset and progression of gastric cancer. In this study, on one hand,we aim to address the methylation mechanisms of wild type (WT) DNMT2 and its H101T mutant using kinetic approach，and to examine the effect on cell carcinogenesis by overexpressing the WT DNMT2 and H101T mutant in cell culture. On the other hand, we propose to search DNMT2 binding partners and to measure the level of DNMT2 expression and tRNA methlylaion under normal and cancerous conditions, using cell lines, mouse tissue and cancer tissue from gastric cancer patients. Additionally, we intend to monitor the changes of DNMT2 binding partner and its expression, as well as tRNA methylation in response to stress stimulus.The proposed work will uncover the molecular details relating DNMT2 to gastric cancer, and will provide a new potential target, strategy and theoretical basis for early cancer detection and prevention as well as anticancer drug development.

DNA甲基化转移酶 DNMT2含有DNA甲基化活性区域，但主要甲基化tRNA。文献报道其分子多态性导致H101T突变并与胃癌发病率显著相关，但是二者关联的分子机制国际国内尚无报道；此外DNMT2甲基化tRNA机制及其是否介导胃癌的发生发展也不清楚。我们将通过生物化学与分子生物学等技术，研究野生型和H101T突变型DNMT2对tRNA底物甲基化作用的差别及其对细胞癌化的影响，并通过分析在正常与癌变细胞、正常与癌变小鼠及临床胃癌病人癌变组织中DNMT2表达水平、其甲基化tRNA产物水平及与其结合的蛋白和/或核酸，并观测这些指标对应激状态的变化，来探讨DNMT2在胃癌发病中的分子机制。研究结果将为胃癌的早期诊断和干预手段提供新的潜在的靶点和思路，并为抗胃癌药物的研制提供新的线索和理论依据。

DNMT2是DNA甲基转移酶家族的一员。虽然含有DNA甲基转移酶结构域，但DNMT2主要甲基化tRNAAsp、tRNAVal、tRNAGly等的38位胞嘧啶，防止tRNA在应激状态被剪切。同时，DNMT2的单核苷酸多态性引起的H101Y有义突变报道与汉族人胃癌发病显著相关。但是，tRNA结构中对DNMT2 甲基化的重要决定因素及H101Y突变与胃癌关联的分子机制国际国内尚未见报道。利用生化与生物物理手段，我们发现tRNA的D-与T-环茎结构都对DNMT2甲基化具有重要作用，但反密码子环中C36，A37U，C32，C38对DNMT2甲基化影响极其显著，C38与C32是tRNA结构中影响DNMT2活性的最重要决定因素。利用生化、细胞与分子生物学手段，我们发现癌组织中DNMT2比癌旁组织表达低，而H101Y突变体表达降低能促进ITG3、MMP9与MMP12表达，从而促进癌细胞的迁移。我们的研究结果阐明了DNMT2甲基化tRNA的生化机制，揭示了H101Y的DNMT2突变体与胃癌相关的分子机制。. 其次，调整与拓展研究结果揭示高表达DNMT2可以对抗仔猪断奶应激与动物热应激，为农业生产实践提供了解决农业动物应激的新方法和新思路；此外还发现我国兽医临床分离得到的解没食子酸链球菌巴氏亚种多重耐药包括大环内酯高水平耐药，其耐药性由甲基转移酶ermB/T的共同诱导耐药导致，为大环内酯类抗菌增效剂的研发奠定了理论基础；同时，我们还发现小鼠成熟精细胞中tRNA裂解与DNMT2的缺失有关，为精细胞成熟提供了更多的分子细节。