近日，卫生毒理学研究团队在酒精性肝病中药物代谢酶CYP2B6的非编码RNA调控机制领域取得重要进展。相关研究成果以“A novel epigenetic mechanism unravels hsa-miR-148a-3p-mediated CYP2B6 downregulation in alcoholic hepatitis disease”为题，在线发表在期刊Biochemical Pharmacology（IF=4.96，药学1区，DOI: 10.1016/j.bcp.2021.114582）上。
细胞色素P450 （CYP）在药物转化过程中起关键作用，包括CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, and CYP3A5在内的12种CYP是目前大多数临床药物的主要代谢酶。饮酒会影响药物代谢和药物活性，在酒精性肝炎（alcoholic hepatitis, AH）患者肝脏中总CYPs的活性显著降低，但是药物代谢CYP的异常表达的原因还不清楚。miRNAs是一种内源性非编码小RNA，调节许多重要的生物学过程，其发挥作用的经典模式是通过靶向mRNA的3’UTR区导致靶基因转录本的降解或翻译水平的抑制。大量研究表明miRNA在调节药物代谢CYPs表达方面起重要作用，有潜力成为药物疗效和安全性的指标。但是在酒精性肝炎中，miRNA依赖的CYPs表达调控机制尚不清楚。
在本研究中，我们从GEO数据库提取了AH患者肝样本中关键药物代谢CYP的表达变化，对靶向表达失调的CYP转录本的miRNA进行了计算机预测，并阐明了一种肝细胞中由miRNA介导的CYP正向调控的非经典机制。经预测，有9个显著失调的miRNA靶向CYP1A2，CYP2A6，CYP2B6，CYP2C8，CYP2C19，CYP2J2和CYP3A4的转录本，其中hsa-miR-148a-3p被选为案例研究。随后的生化和分子实验结果表明，miR-148a通过直接与CYP2B6 3'UTR序列结合而增加mRNA的稳定性，从而促进CYP2B6的表达，并且这种转录后正向调控是AGO1/2依赖性的。此外，萤光素酶报告基因实验和RNA二级结构分析表明， miR-148a介导的CYP2B6上调取决于非种子区序列，而不是种子区序列。此外，我们通过EMSA和染色质免疫沉淀实验鉴定出HNF4A是MIR-148A的肝脏特异性转录因子。我们的结果表明，乙醇通过下调HNF4A从而降低了肝细胞中的miR-148a的表达，最终导致CYP2B6的表达降低。我们的发现将有助于了解AH患者的药物代谢失调，并阐明了一种CYP的表观遗传调控的非经典机制。
于典科教授为本文的通讯作者，博士后罗娇和硕士研究生谢梦玥为本文的共同第一作者。上述研究得到国家自然科学基金(81903354,91943301,91743113)、中国博士后科学基金(2020M672008)、国家重点研发计划(2017YFC1600201)、山东省博士后创新项目和青岛市博士后应用研究项目资助。
Cytochrome P450 (CYP) enzymes play critical roles in drug transformation, including CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, and CYP3A5. CYPs are also the major enzymes responsible for metabolism of most clinical drugs. Alcohol drinking affects the drug metabolism and drug activities. The amounts and activities of total CYPs are markedly decreased in alcoholic hepatitis (AH), but the mechnism for the abnormal expression of drug metabolizing CYP is still unclear. miRNAs are endogenous small noncoding RNAs that regulate many essential biological processes. Generally, miRNAs regulate gene expression by targeting the 3 'UTR region of mRNA to facilitate mRNA degradation or inhibit translation. Many researches have shown that miRNAs play crucial roles in regulating drug-metabolizing CYPs and have the potential to be an indicator of drug efficacy and safety. However, knowledge concerning miRNA regulation of CYPs in AH disease is limited.
Here we presented the changes of key CYPs in liver samples of AH patients retrieved from GEO database, performed in silico prediction of miRNAs potentially targeting the dysregulated CYP transcripts, and deciphered a novel mechanism underlying miRNA mediated CYPs expression in liver cells. Nine miRNAs were predicted to regulate CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2J2, and CYP3A4, among which hsa-miR-148a-3p was selected as a case study. Biochemical and molecular evidences demonstrated that miR-148a promoted CYP2B6 expression by increasing mRNA stability via directly binding to the 3’UTR sequence, and that this positive posttranscriptional regulation was AGO1/2-dependent. Further, luciferase reporter gene assay and RNA secondary structure analysis illustrated that the seedless target site, not the seed target site, controlled miR-148a-mediated CYP2B6 upregulation. Moreover, we identified HNF4A as a liver-specific transcription factor of MIR-148A through EMSA and chromatin immunoprecipitation experiments. In conclusion, ethanol downregulated miR-148a in hepatocytes through HNF4A regulation, which eventually decreased CYP2B6 expression. Our finding will benefit the understanding of dysregulated drug metabolism in AH patients and highlight an unconventional mechanism for epigenetic regulation of CYP gene expression.
Dr. Dianke Yu is the corresponding author of the paper. Dr. Jiao Luo and postgraduate students Mengyue Xie are the co-first authors. This work was supported by grants from the National Natural Science Foundation of China (81903354, 91943301, and 91743113),China Postdoctoral Science Foundation (2020M672008), National Key R&D Program of China (2017YFC1600201), Postdoctoral Innovation Project of Shandong Province, and the Qingdao Postdoctoral Application Research Project.
