MOLECULAR MECHANISMS OF DIET-INDUCED CARCINOGENESIS

流行病学和临床研究表明，人体缺乏叶酸可能导致对某些类型癌症的易感性。 补充叶酸可逆转癌前发育不良。 因此，阐明叶酸缺乏和癌症易感性的分子机制对于确定叶酸和其他膳食元素在癌症预防中的作用至关重要。一个很好的大鼠模型系统可用于研究低蛋氨酸，胆碱和叶酸的饮食（脂肪营养素缺乏或LD饮食）在没有任何外源性物质的情况下诱导肝癌发生的作用。 在LD诱导的肝癌中，一个关键的抑癌基因（p53）发生甲基化。 利用这个模型系统，我们将（a）通过亚硫酸氢盐基因组测序和Ms-SNuPe探索（甲基化敏感的单核苷酸引物延伸）在肝癌发生的不同阶段p53启动子上的CpG二核苷酸的甲基化状态（B）通过瞬时转染试验研究特定CpG二核苷酸的甲基化在p53启动子失活中的作用（c）研究甲基化的机制（d）通过瞬时转染试验研究特定CpG二核苷酸的甲基化在p53启动子失活中的作用。介导的染色质结构的改变，并确定参与p53启动子随肿瘤发生进展而随之沉默的关键因素，通过限制性内切酶可及性分析和染色质免疫沉淀（ChIP）与甲基CpG结合蛋白（MeCP）特异性抗体（d）研究不同DNA甲基转移酶同工酶的表达和活性的调节（e）鉴定与从头甲基化酶相互作用的蛋白质（f）克隆基因，特别是通过RLGS技术检测的在癌前肝中甲基化的三个基因，鉴定它们，研究这些基因在肿瘤发生不同阶段的表达水平，并探讨这些基因沉默在肿瘤发生中的功能意义。 希望这项研究将产生重要的信息，叶酸/甲基缺乏症的关系，区域超甲基化的生长/肿瘤抑制基因或基因编码的蛋白质，抑制生长调节基因在肿瘤发生的不同阶段，和他们的沉默，导致肿瘤的形成。

Epidemiological and clinical studies have demonstrated that folate deficiency in humans could lead to susceptibility to certain types of cancers. Premalignant dysplasia could be reversed by folate supplementation. Elucidation of the molecular mechanisms underlying folate deficiency and predisposition to cancer is, therefore, of critical importance in determining the role of folate and other dietary elements in cancer prevention. An excellent rat model system is available to study the role of diet low in methionine, choline and folate (lipotrope-deficient or LD diet) on the induction of hepatocarcinogenesis in the absence of any exogenous xenobiotic agents. It is known that a key tumor suppressor (p53) gene is methylated in the hepatoma induced by LD diet. Using this model system, we will (a) explore by Bisulfite genomic sequencing and Ms-SNuPe (Methylation- sensitive single nucleotide primer extension) the methylation status of CpG dinucleotides on p53 promoter at different stages of hepatocarcinogenesis (b) investigate the role of methylation of specific CpG dinucleotides in p53 promoter inactivation by transient transfection assay (c) investigate the mechanism of methylation-mediated alteration in chromatin structure and identify the key factors involved in consequent silencing of p53 promoter with progression of tumorigenesis, by restriction endonuclease accessibility assay and chromatin immunoprecipitation (ChIP) with antibodies specific to methyl CpG binding proteins (MeCPs) (d) study the regulation of expression and activity of different DNA methyltransferase isozymes (involved in maintenance and de novo methylation) during hepatocarcinogenesis induced by LD diet (e) identify the proteins that interact with de novo methylases (f) clone the genes, specifically the three genes that are methylated in preneoplastic liver as detected by RLGS technique, identify them, investigate their expression levels at different stages of tumorigenesis and study functional significance of silencing of these genes in tumorigenesis. It is hoped that this study will yield important information concerning the relationship of folate/methyl deficiency to regional hypermethylation of growth/tumor suppressor genes or genes encoding proteins that suppress the growth regulatory genes at different stages of tumorigenesis, and their silencing that lead to tumor formation.