细胞色素P4502C19特异性表达的表观遗传学调控机理

Epigenetics is the study of heritable changes in gene expression or cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence. DNA methylation, which is the most common epigenetic phenomenon, alters gene expression by way of causing changes in chromatin structure, DNA conformation and DNA-protein interactions. Studies have shown that multiple genes achieve tissue-specific expression or participate in tumorigenesis by DNA methylation. CYP2C19 is an important human drug-metabolizing enzyme with its expression under the regulation of a number of transcriptional factors. It has been confirmed that CYP2C19 is highest expressed in hepatoma tissue and followed by normal liver tissue, which indicates a certain relationship between the increasd CYP2C19 level and carcinogenesis. Tissue-specific expression is one of its traits since CYP2C19 is not expressed in any other tumor or normal tissue. However, current knowledge of CYP2C19 is not sufficient to give explaination to such phenomenon. Cytochrome P 450 superfamily is a large group of enzymes which is under control of epigenetic mechanisms. Combining with the rencent epigenic studies on CYPs, we speculate that DNA methylation also plays an important role in the regulation of CYP2C19. In this study, hepatoma and other cancer cells cultured in vitro will be treated with 5-aza-dC while a variety of newly collected tumor tissue and paired adjacent normal tissue samples are analyzed. We will take advantage of qRT-PCR, bisulfite sequencing PCR（BSP）and other experimental methods to reveal the relationship between CYP2C19 level and DNA methylation.In addition, we will also endeavour to identify the key sites of DNA methylation so that the regulatory factors of CYP2C19 could be further clarified.

表观遗传调控是一种不涉及DNA序列变化的可遗传的基因表达方式的改变，DNA甲基化修饰是最常见的表观遗传现象之一，已证实多种基因通过DNA甲基化实现组织特异性表达或参与肿瘤发生。CYP2C19是人体内重要的药物代谢酶之一，研究证实CYP2C19只在肝脏与肝癌组织中表达，并且其表达量的提高与肝癌的发生有一定的关系，但目前对CYP2C19的表达调控研究尚不能对上述现象做出解释。CYP450家族是受表观遗传机制调控的一大类基因，结合近年来CYP450家族的表观研究现状，我们推测DNA甲基化在CYP2C19的表达调控中发挥着重要的作用。本研究将进行体外细胞培养，并采集新鲜的癌组织和癌旁组织标本进行分析，通过5-aza-dC处理细胞、qRT-PCR、BSP等实验手段比较CYP2C19的mRNA与DNA甲基化水平之间的关系，并确定关键的甲基化位点或区域，以阐明DNA甲基化对CYP2C19的表达调控作用。

CYP2C19是CYP450超家族的重要成员，其组织特异性表达模式明显，主要在肝脏表达，在十二指肠也有少量表达。此外，肝细胞性肝癌（HCC）肿瘤组织中CYP2C19基因的表达水平相对于癌旁组织显著降低，说明CYP2C19表达异常与HCC发生也有一定关联。表观遗传学包括DNA甲基化和组蛋白修饰等机制，广泛参与基因的表达调控，参与细胞分化、肿瘤形成等生命进程。.在本研究中，我们首先分析不同组织的WGBS和ChIP-seq数据，发现CYP2C19转录起始位点附近CpG位点的甲基化水平在肝脏组织中要明显低于其他组织；、此外，肝脏CYP2C19启动子的H3K9ac和H3K27ac水平显著高于其他组织。这些差异修饰位点可能与CYP2C19组织特异性表达有关。.为了考察DNA甲基化和组蛋白乙酰化对CYP2C19的调控作用，我们分别用组蛋白去乙酰化酶抑制剂TSA和DNA甲基化酶抑制剂5-aza-dC处理原代肝脏细胞，CYP2C19都能被诱导上调。此外，5-aza-dC作用下转录因子CAR也被上调。考察给药组和对照组细胞的DNA甲基化修饰发现，给药组细胞基因组中CYP2C19启动子和CAR启动子区域DNA甲基化水平降低，证实在正常肝脏中，组蛋白乙酰化和DNA甲基化是CYP2C19转录的可能调控机制，CAR与DNA甲基化调控CYP2C19表达过程可能有关。.在此基础上，我们接着研究HCC中CYP2C19表达抑制机制。qRT-PCR检测发现，肿瘤组织中CYP2C19和CAR的表达水平相对于癌旁组织显著降低。BSP发现，CYP2C19启动子甲基化水平在肿瘤和癌旁组织中不存在差异，但CAR转录起始位点（TSS）附近e-box的 CpG位点甲基化水平存在显著差异，这一位点的甲基化水平与CYP2C19和CAR的表达水平呈反相关。报告基因实验证实该位点甲基化水平升高可抑制CAR的表达，从而间接抑制下游CYP2C19的表达。接着，我们又考察了组蛋白乙酰化是否也是HCC中CYP2C19表达水平下降的因素。ChIP结果显示，在肿瘤组织中，CYP2C19的H3K9ac有所下调，但H3K27ac水平无明显差异。.综上，本文初步揭示了DNA甲基化和组蛋白乙酰化修饰是CYP2C19组织特异性表达的可能机制。CYP2C19在HCC中的异常表达也受到这些表观遗传机制的调控，然而具体通路可能有所区别，有待进一步研究。