人肝脏发育过程中CYP3A基因亚型选择性表达的表观遗传调控机制

Cytochrome P450 3A（CYP3A）metabolizes more than 60% of all clinically used drugs. Two enzymes, CYP3A4 and CYP3A7, are differentially expressed in liver at different developmental ages. The differences in catalytic activity and gene expression of CYP3A4 and CYP3A7 in liver lead to differences in drug disposition and action between pediatric and adult patients. Children are not "miniature adults". However, the mechanisms that control the developmental expression of the CYP3A genes in liver remain understood. The knowledge gap prevents us to develop strategies for age-dependent adjustments on drugs for pediatric patients. Our previous study showed that histone modifications such as dimethylation of histone H3 lysine 4 (H3K4me2) is associated with CYP3A4 and CYP3A7 mRNA expression during human liver development. Thus, we hypothesize that epigenetic mechanisms may be involved in transcriptional regulation of CYP3A4 and CYP3A7 ontogenic expression in liver. In this study, we plan to determine:1) the associations of histone modifications in the promoter region of CYP3A4 and CYP3A7 with nucler receptors (PXR, CAR, FXR, HNF4 and HNF1) or CYP3A4 and CYP3A7 expression in liver at different developmental ages; 2) the effects of siRNA-targeting nucler receptors (or histone-modifying enzymes) on CYP3A4 and CYP3A7 expression in liver at different developmental ages; 3) the interaction between histone-modifying enzymes and nucler receptors. This study will help us understand the molecular mechanisms underlying selective expression of CYP3A isoforms during human liver development in the field of epigenetics. Once the mechanisms are defined, the knowledge will help us to develop strategies for drug safe use in pediatric patients.

在个体发育不同阶段，药物代谢酶CYP3A4和CYP3A7在肝脏中基因表达和酶活性的不同，导致成人和儿童在药物代谢和效应上的差异，从而儿童用药不是成人"缩小版"。然而，调控CYP3A基因亚型选择性表达的分子机制仍不清楚。我们前期工作发现，组蛋白赖氨酸甲基化修饰如H3K4me2与人不同发育阶段肝组织中CYP3A4、CYP3A7基因表达相关。由此提出假说：表观遗传学机制参与CYP3A4、CYP3A7基因表达的转录调控。本课题拟研究：1)人肝脏发育不同阶段CYP3A4/3A7基因启动子区组蛋白修饰与核受体(PXR,CAR,FXR,HNF)、CYP3A4/3A7基因表达关系;2)siRNA干扰沉默核受体或组蛋白修饰酶基因对CYP3A4/3A7转录活性的影响;3)组蛋白修饰酶与核受体的相互作用。该课题从表观遗传学的角度阐明发育过程中CYP3A基因选择性表达的确切机制对儿童安全用药具有非常重要的意义。

在个体发育过程中，肝脏Ⅰ相药物代谢酶CYP3A4和CYP3A7的选择性表达以及Ⅱ相代谢酶UGT1A1的动态表达会导致成人与儿童在药物代谢和效应上的差异，从而影响儿童和成人的临床治疗。因此，阐明发育过程中药物代谢酶基因表达模式的确切机制对儿童患者安全用药有非常重要的意义。本研究旨在探讨人肝脏发育过程中CYP3A4/7在发育中表达转换以及UGT1A1发育表达的调控机制。结果表明：①人肝脏发育过程中存在CYP3A不同亚型的发育转换：出生前以CYP3A7表达为主，出生后以CYP3A4表达为主，UGT1A1在胎儿期低表达而出生后表达逐渐升高。②核受体PXR, CAR, RXR, CEBPB, PPARA, HNF1A及HNF4A表达在出生后显著增加，并且与 CYP3A4/3A7的选择性表达有关。成人肝脏中HNF4A对CYP3A4的基础表达发挥决定性作用，而胎儿肝脏中GR对CYP3A7的表达调控至关重要。③肝脏发育成熟过程中，组蛋白甲基化修饰H3K4me2、H3K27me3的动态转换参与CYP3A及UGT1A1的发育表达调控。HNF4A、GR介导的CYP3A4/3A7发育表达改变可能与靶基因启动子区及增强子区的H3K4me2高度富集有关。HNF1A可能通过募集组蛋白修饰酶MLL1激活UGT1A1的转录表达。④药物诱导的CYP3A4表达与组蛋白修饰机制有关：利福平通过激活PXR并招募组蛋白甲基转移酶相关因子NCOA6和组蛋白乙酰转移酶p300至CYP3A4基因启动子区，改变组蛋白甲基化和乙酰化修饰状态而调控CYP3A4的表达。. 本项目阐述了肝脏发育过程中CYP3A7、CYP3A4的选择性表达、UGT1A1的发育表达与核受体HNF4A 、GR和HNF1A的表达以及表观修饰H3K4me2、H3K27me3的密切关系；从表观遗传修饰角度入手，拓宽了CYP3A及UGT1A1发育表达调控机制的研究，筛选出调控CYP3A4/3A7和UGT1A1发育表达的重要组蛋白修饰位点和状态，为临床个体化用药的发展提供新的分子靶点，也为儿童患者发展个体化治疗策略提供新的理论依据。