lnc-HNF1A/HNF1A通路调控CYP3A4表达的分子机制

As part of the epigenetics, long non-coding RNAs (lncRNA) have been showed valuable roles in the regulation of gene expression. However, no study has revealed the effect of lncRNA on the regulation of drug metabolism enzymes. In preliminary studies, we have found that the expression of HNF1A related lncRNAs (lnc-HNF1A) were positively related with the expression of both HNF1A and CYP3A4. Besides, ChIP-seq data showed that the H3K4me2 level in CYP3A4 promoter was associated with the increase expression of CYP3A4. Based on these studies, we hypothesize that lnc-HNF1A may be a coactivator and interact with HNF1A to change the histone modifications and chromatin conformation in the promoter of CYP3A4 through recruiting histone methyltransferases and binding to specific sites in CYP3A4 promoter to activate the expression of CYP3A4. The aim of this study is to research the epigenetic mechanism in the regulation of CYP3A4 expression by identifying the role of lnc-HNF1A. Firstly, the effect of lnc-HNF1A on CYP3A4 expression in liver cells will be determined with gain-of-function and loss-of-function assays. Then, how lnc-HNF1A interacts with HNF1A and regulates the histone modifications will be studied. Finally, the changes of histone modifications and chromatin condensation of CYP3A4 promoter will be revealed. This study on lncRNA-controlled gene regulation shall not only improve mechanistic understanding of CYP3A4 individual variability but also provide novel insights into developing more effective therapeutic strategies.

长链非编码RNA(lncRNA)作为重要的表观调控因子在基因表达调控中发挥重要作用，但目前尚未见关于其调控药物代谢酶的研究。我们前期研究发现肝脏转录因子HNF1A相关lncRNA（lnc-HNF1A）的表达与HNF1A及肝脏代谢酶CYP3A4的表达均呈正相关，且CYP3A4的高表达与其基因启动子区组蛋白甲基化（H3K4me2）相关。由此假设：lnc-HNF1A作为HNF1A的辅激活因子参与CYP3A4表达调控。因此，本课题拟研究：1)采用过表达、RNA干扰明确lnc-HNF1A/HNF1A对CYP3A4表达的影响；2)研究lnc-HNF1A与HNF1A蛋白以及其他表观修饰酶的相互作用；3)探讨lnc-HNF1A/HNF1A通路对CYP3A4基因启动子区组蛋白修饰状态及染色质浓缩状态的影响。该课题从lncRNA调控的角度解释CYP3A4表达个体差异的分子机制，为临床个体化治疗提供新思路。

细胞色素P450酶（CYPs）的表达和活性存在明显的个体差异，影响临床药物效应及不良反应的发生。CYP3A4是肝脏中最重要的药物代谢酶，50%以上的临床药物经其代谢。目前影响CYP3A4表达调控的机制仍未完全阐明。本研究从lncRNAs调控基因表达的角度研究了以下内容：① lncRNA HNF1A-AS1参与人肝组织和肝细胞系中CYP3A4等8种CYPs表达的调控作用；② HNF1A-AS1调控肝细胞中CYP3A4表达的双重功能的研究；③ lncRNA HNF4A-AS1对CYPs的表达调控作用及机制；④ HNF1A-AS1和HNF4A-AS1在药物诱导肝损伤中的作用。研究结果表明：①HNF1A-AS1参与HNF1A介导的人肝组织和肝细胞（Huh7和HepaRG细胞）中CYPs(如CYP1A2、3A4、2C8、2C9、2C19、2D6、2E1)和核受体（如PXR、CAR、AhR）表达的调控。② HNF1A-AS1作为RNA支架与精氨酸甲基转移酶（PRMT1）和PXR相互作用，HNFA-AS1促进二者的相互作用并通过组蛋白修饰实现PXR的反式激活和CYP3A4转录改变；此外，HNF1A-AS1与HNF1A结合，阻断后者与TRIM25的相互作用，从而阻止HNF1A泛素化和蛋白质降解，并进一步调控CYP3A4的表达。③ HNF4A-AS1负调控HNF4A介导的CYPs（包括1A2、2E1、2B6、2D6、2C8、2C9、2C19和3A4）和转录因子（PXR）的基础及利福平诱导表达；HNF4A-AS1反向调控CYP3A4的表达与其基因启动子区组蛋白修饰H3K4me3和HNF4A富集的改变有关。④ HNF4A-AS1和HNF1A-AS1通过影响CYP3A4启动子区PXR的结合及组蛋白修饰状态而调控CYP3A4表达，在利托那韦诱导的肝毒性中发挥重要作用。本研究首次证实了lncRNA HNF1A-AS1和HNF4A-AS1参与CYP3A4等CYPs的表达调控，深入探讨了HNF1A-AS1对CYP3A4表达调控的双重功能，进一步考察了这两个lncRNAs在PXR激活CYP3A4介导的利托那韦肝毒性中的作用。该研究为建立能够预测和评价CYP3A4个体差异及药物效应的分子标志物提供了新思路，也为指导临床用药提供方向。