肝癌发生中芳香烃/雄甾烷核受体AhR/CAR/CYP450酶代谢通路表观遗传调控机制的研究

The clinical profiles of gender differences have been observed and confirmed in different sexual hepatocellular carcinoma patients. However, whether the nuclear transcription factors including aromatic hydrocarbon receptor (AhR), constitutive androstane receptor (CAR) and theirs downstream target gene cytochrome P450 (CYP450) metabolic enzymes influence the level of sex steroid hormones, and how affect the clinical therapeutic outcomes, moreover, the exact epigenetic modulating mechanism on the above metabolic pathway in hepatocarcinogenesis is still unknown. The objective of the present study is to investigate and compare the distribution frequencies of DNA methylation and/or hydroxymethylation in the AhR/CAR/CYP450s genomic and promoter CpG sites, and evaluate the effects of these DNA methylation and variant genotypes on the pharmacokinetic phenotypes of sex hormones including androgen (testosterone), estrogen (estradiol and estrone) and progestogen (progesterone), in the peripheral blood samples between the different sexual Chinese healthy volunteers and liver cancer patients, the tissue samples of hepatocellular carcinoma and paracancerous, the embryonic samples of hepatic and extrahepatic tissues in human, the hepatocarcinogenesis stimulated by diethylnitrosamine (DEN) plus phenobarbital (PB) in rats, and transgenetic AhR and/or CAR hepatoma HepG2 cells in vivo and in vitro. The present research will be performanced by the MALDI-TOF-MS, RFLP-PCR, LC-MS/MS and pyrosequencing methods, respectively. The present results will be not only able to provide the new explain for etiology of hepatocarcinogenesis, but also might be expected to elucidate the epigenetic modulating mechanisms of AhR/CAR/CYP450s/sex steride hormones metabolic pathyway, and then might be discovered more specific and selective drug target for inhibiting hepatocarcinogenesis and tumor metastasis, provided novel therapeutic strategy and experimental evidences.

肝癌的发生发展呈现显著的男女性别差异性特点，但核转录因子芳香烃/雄甾烷受体AhR/CAR/CYP450酶代谢通路如何影响性甾体激素水平变化与肝癌临床转归、及其确切的表观遗传调控机制尚知之甚少。本研究拟在前期工作的基础上，以不同性别啮齿类动物肝癌整体模型、人肝癌AhR/CAR转基因细胞模型、临床肝癌患者癌组织与外周血、人胎肝及肝外胚胎组织为对象，采用MALDI-TOF-MS、RFLP-PCR、LC-MS、焦磷酸测序等方法，考察肝癌发生进程中，AhR/CAR/CYP450基因组中DNA甲基化分布频率的异同点；性甾体激素应激/氧化/硝化应激对基因启动子区CpG岛甲基化或羟甲基化的影响，及对雄(睾酮)/雌(雌二醇与雌酮)/孕(孕酮)激素代谢表型的影响。研究旨在阐明AhR/CAR/CYP450代谢通路的表观遗传调控分子机制，以期为肝癌发生的病因学提供新的解释，为相关新药研发提供新靶点和实验依据。

中国人群是肝癌高发的易感人群，且肝癌发生的病理生理进程呈现显著的男女性别差异性特点。但核转录因子芳香烃/雄甾烷受体AhR/CAR/CYP450酶代谢通路如何影响性甾体激素水平变化与肝癌临床转归、及其确切的表观遗传调控机制尚知之甚少。本研究在国家自然科学基金面上项目（81773809）的资助和支持下，以中国汉族及多民族人群（健康者与肝癌患者）临床样本、肝癌动物整体及体外细胞模型为对象，采用MALDI-TOF-MS、RFLP-PCR、LC-MS、Sequenom技术、焦磷酸测序等方法，考察了肝癌进程中，AhR/CAR/CYP450基因组中DNA甲基化分布频率的异同点；探讨了性甾体激素应激/氧化/硝化应激对基因启动子区或全基因组CpG岛甲基化水平的影响，及对雄(睾酮)/雌(雌二醇与雌酮)/孕(孕酮)激素代谢表型的影响。探讨了性激素水平与DNA甲基化定性定量分析的方法，及不同性别肝癌的表观遗传调控机制。研究结果为阐明AhR/CAR/CYP450代谢通路的表观遗传调控分子机制提供了新的视角，为肝癌发生的病因学提供了新的解释，为新药研发提供了新靶点和实验依据，故具有潜在的临床应用前景。