叶酸与哈萨克族食管癌的关系及机制研究

Many environmental risk factors such as dietary deficiency could increase the individual susceptibility of cancer through changing the individual epigenetic characteristic property especially DNA methylation pattern. The Kazakh residents of Xinjiang have a higher morbidity of esophagus cancer in China. Our research proved that esophageal cancer of Kazakh was associated with DNA methylation related to folic acid metabolism. But comprehensive and systematic direct evidences of relationship between folic acid and esophageal cancer of Kazak nationality based on case-control study and laboratory studies still lack.In our study, we will discuss diet intervetion for esophagus cancer of kazakh from the entry point of epigenetics and with the methyl group donor nutrients-folic acid as starting point.We will select patients with esophageal cancer of Kazakh and matched controls for each 200 cases by hospital-based case-conrol study , and analyze the relationship between folic acid level and realted DNA methylation status.Meanwhile, we will establish the DNMT1 overexpression Kazak esophageal epithelial cells on basis of Kazakh normal esophageal epithelial cells, and adopt many molecular biologic methods such as methylation immunofluorescence,MSP and etc to analyze DNA methylation of genes related metabolsim of folic acid and cancer such as MTHFR、CBS、ALDH1L1、GNMT、 p16、FHIT、RASSFlA、MGMT，and DNA damage of cells, and related singnaling pathway under Nitrosamine compounds(MNNG) in culture medium with adequate and lack of folic acid. The project will expolore deeply the role of folic aced in esophageal cancer of Kazakh and its epigenetcs mechanism by population-based study and vitro experiment, and it is important significance for prevention and mechanism of suscepbility for esophagus cancer of Kazak nationality.

多种环境因素如膳食营养素缺乏可通过表观遗传特性特别是DNA甲基化改变机体对肿瘤的易感性。新疆哈萨克族居民是我国食管癌高发民族。前期研究表明，哈萨克族食管癌与叶酸代谢相关基因异常甲基化相关，但仍缺乏叶酸与哈萨克族食管癌关系的全面系统的人群病例对照研究和实验室直接证据。本项目以饮食参与干预食管癌发生的表遗传特征改变为切入点，以重要甲基基团供体营养素-叶酸为着眼点，选择哈萨克族食管癌患者及匹配对照各200例，分析体内叶酸水平与相关基因DNA甲基化状况关系；同时，在前期建立的哈萨克族食管正常上皮细胞系的基础上拟构建DNMT1高表达细胞，应用甲基化免疫荧光等技术检测细胞在叶酸充足和缺乏情况下，亚硝胺类化合物染毒后对相关DNA甲基化、DNA损伤的影响及涉及的信号途径。本研究通过人群和细胞实验两个方面深入探讨叶酸在哈萨克族食管癌发生中的作用及其表观遗传调控机制，对食管癌预防及易感性机制研究具有重要意义。

多种环境因素如膳食营养素缺乏可通过表观遗传特性特别是DNA甲基化改变机体对肿瘤的易感性。新疆哈萨克族居民是我国食管癌高发民族。前期研究表明，哈萨克族食管癌与叶酸代谢相关基因异常甲基化相关，但仍缺乏叶酸与哈萨克族食管癌关系的全面系统的人群病例对照研究和实验室直接证据。本项目采用TALE技术成功构建了哈萨克族食管上皮DNA甲基化转移酶1（DNMT1）高表达细胞株模型，以及哈萨克族食管上皮DNMT1高表达细胞的恶性转化细胞模型，为今后致癌机制研究提供了重要的研究工具。同时，采用营养流行病学研究方法，以病例-对照研究为基础探讨哈萨克族食管癌患者与正常对照人群体内叶酸及相关基因甲基化水平等，发现哈萨克族食管癌患者叶酸摄入严重不足，随血清叶酸水平升高，食管癌发病风险降低；随血清DNMT1水平升高，食管癌发病风险增加。MTHFR、CBS、MGMT、P16、FHIT及RASSF1A基因甲基化均与哈萨克族食管癌发生密切相关，肿瘤浸润程度、区域淋巴结转移、TNM分期、CBS和RASSF1A基因甲基化是影响食管癌预后的独立危险因素，而血清叶酸是保护因素。另外，通过体外细胞实验表明，哈萨克族食管上皮DNMT1高表达细胞较食管上皮细胞更易受到MNNG影响，且随染毒时间延长细胞损伤程度加重，表现为细胞形态学改变、DNA损伤和表观遗传学指标改变，而高浓度叶酸可降低MNNG对细胞的损伤作用，可逆转表观遗传学指标改变，对细胞的不良反应起到一定的拮抗作用；同时，充足的叶酸可抑制MNNG对PI3K-AKT信号通路和Wnt/β-catenin信号转导通路的活化，从而起到抑制细胞不良反应的作用。本项目通过体外细胞实验及人群研究，全面系统地提供了叶酸与哈萨克族食管癌发生的直接证据，同时探讨了叶酸通过表观遗传修饰作用逆转不利基因表达模式和表型的作用及相关调控机制，对食管癌预防及易感性机制研究具有重要价值。