叶酸对肝癌发生的影响及其表观遗传学调控机制

The incidence of primary liver cancer is very high in our country, and more than half of new liver cancer cases worldwide were estimated to occur in China. Among primary liver cancers, hepatocellular carcinoma (HCC) represents the major histological subtype. Although some advances have been achieved in the diagnosis and treatment of HCC, the long-term outcome for patients with HCC is still very poor. Therefore，the prevention of HCC becomes an important clinical problem to be solved. Folate (or Folic acid) plays a central role in the one-carbon metabolism, and insufficient intake of folate or impairments in folate-mediated metabolism are associated with several common diseases including cancers. Previous epidemiologic studies indicated that dietary folate intake and blood folate levels are inversely associated with HCC risk, but the exact mechanism is not known and no experimental data support this conclusion. In this project, the role of folic acid in hepatocarcinogenesis will be investigated by in vivo experiments using chemical-induced HCC and HBx transgenic mouse models, and the key factors affecting hepatocarcinogenesis will be screened by microarray and chromatin immunoprecipitation (ChIP) assays. On the basis of these findings, the role of folic acid in HCC prevention will be demonstrated and its epigenetic mechanism that modified the DNA and (or) histone methylation status of the key factor promoter will be clarified. Our study will broaden knowledge on the efficacy of folic acid as a potential HCC chemopreventive agent, and it will also provide valuable references for cancer prevention in our country.

原发性肝癌是我国高发肿瘤，全球每年新发肝癌病例中有一半以上集中在中国，因此如何预防肝癌的发生是当前亟待解决的重大临床问题。已有充分证据表明，叶酸摄入不足与代谢异常会对人体基因组和表观基因组产生各种损伤，导致包括肿瘤在内的多种疾病的发生。近年来的流行病学资料显示体内叶酸缺乏是肝癌发病风险增加的高危因素，但是缺少后续实验支持。本项目组前期研究结果提示叶酸能够干预实验动物肝脏病变的进程，表观遗传修饰在其中起了重要作用。据此项目组拟通过化学诱癌、HBx转基因小鼠动物模型进一步实时观察叶酸干预对肝脏病变及肝癌发生的影响；利用生物学芯片、染色质免疫共沉淀等手段筛选叶酸影响肝癌发生的关键因子，在此基础上阐明叶酸通过调控关键因子启动子区DNA甲基化和(或)组蛋白甲基化进而干预肝癌发生的表观遗传学作用机制，为叶酸是肝癌化学预防的候选分子提供扎实的理论和实验基础，也为我国制定相应的肝癌预防策略与措施提供依据。

原发性肝癌在我国发病率很高，肝细胞癌（HCC）是其主要类型。尽管近年来肝癌的诊断与治疗都取得了长足进步，但术后生存率并没有明显改善。因此，从疾病预防角度考虑降低肝癌发生率就显得很有意义。叶酸摄入不足与代谢异常会对人体基因组和表观基因组产生损伤，导致包括肿瘤在内的多种疾病的发生，流行病学资料显示体内叶酸缺乏是肝癌发病风险增加的高危因素，但缺少后续实验支持。. 本项目首先通过模型动物体内实验观察叶酸干预对肝癌发生的影响，构建二乙基亚硝胺（DEN）诱导的小鼠肝癌模型，实验数据表明叶酸干预可降低DEN诱发的小鼠肝癌发生。这一过程与上皮－间质转化（EMT）相关，补充叶酸阻止了肝癌发生中肝细胞的EMT进程，叶酸通过上调上皮标志分子E-cadherin启动子区组蛋白H3K4Me2水平进而抑制EMT，且这种作用依赖于去甲基化酶LSD1。. 体外实验研究叶酸对正常肝细胞功能的影响，结果提示叶酸缺乏使正常肝细胞获得了恶性转化的能力。在此基础上进行叶酸处理细胞的转录组测序，选取差异表达的癌基因LCN2进行研究，叶酸通过提高LCN2基因启动子区组蛋白H3K9Me2甲基化水平进而抑制了LCN2的表达。临床组织样本中，癌组织叶酸水平普遍低于癌旁组织，LCN2基因表达水平显著高于癌旁组织，二者呈负相关。综上实验结果表明叶酸缺乏是肝癌发生的潜在危险因子，叶酸通过调控关键基因启动子区组蛋白甲基化进而影响肿瘤发生，叶酸可作为肝癌化学预防的候选分子。同时上述研究丰富了叶酸的表观遗传学调控机制。. 在以上基础上，我们进一步拓展了项目研究内容。我室前期构建的结直肠癌标本库在项目研究后期已成熟，而结直肠癌是最易受环境因素（如饮食结构改变）影响的肿瘤。甲基测序的基础上我们选取叶酸代谢通路关键酶基因CBS作为研究对象。结果显示外周血中CBS启动子游离DNA(cfDNA)甲基化水平能够独立指示结直肠癌患者的复发率和总体生存率，患者血浆中CBS启动子的cfDNA低甲基化与不良临床结果相关。以上研究表明CBS启动子区DNA低甲基化可作为一潜在的循环分子标志物用于肿瘤的预后判断。