hsa-miR-766通过抑制NQO1表达调控马兜铃酸I致肝毒性的机制研究

Aristolochic acid (AA) and AA-containing plants are type I carcinogens, and aristolochic acid I (AAI) is the most potent and abundant component. Recent studies showed that AA is significantly linked to liver damages, but the underlying mechanism remains unclear. miRNA can modulate the metabolism and toxicity of toxicants by downregulating the expression of metabolizing enzymes through targeting the 3’UTR of the genes which leads to translation termination or mRNA degradation. In order to investigate how miRNA would affect AAI-induced hepatotoxicity, in this study, we first selected NQO1, the enzyme most relevant to AAI hepatotoxicity. Next, we bioinformatically identified that hsa-miR-766 can regulate NQO1 expression. We will demonstrate the validity using multiple techniques such as FR-EMSA and luciferase reporter assay. We also identified the transcription factors which target the promoter region of miR-766, which we will validate using similar methods. We plan to investigate the relationship between transcription factors and miRNA using AAI-exposed HepaRG cells and C57BL/6 mice, and to study the effect of miRNA in liver cell damage and liver injuries using miR-766 transfected cells and miR-766 injected mice. This study proposes to unravel the mechanism of miRNA mediating AAI-induced hepatotoxicity through suppressing the expression of NQO1, and to identify potential biomarkers and therapeutic targets for the prevention and clinical therapeutic decision of liver damage at early stage.

马兜铃酸是明确的1类致癌物，其中毒性最强的成分是马兜铃酸I（AAI）。近期研究发现马兜铃酸与肝损伤的发生显著相关，但目前对其致肝毒性的调控机制还不清楚。miRNA通过与代谢酶基因的3’UTR结合，引起翻译终止或mRNA降解，下调代谢酶的表达，从而调节毒物代谢和毒性。为了研究miRNA通过调控代谢酶表达影响AAI致肝毒性中的机制，本项目首先筛选出与AAI肝损伤最具相关性的代谢酶NQO1，利用生物信息学手段预测出可靶向调控其表达的hsa-miR-766，并验证其靶向调控关系；同时预测并验证可与miR-766启动子区结合的转录因子。继而我们利用经AAI染毒的细胞和小鼠，分析转录因子与miRNA的表达相关性；并通过细胞转染miRNA和尾静脉注射miRNA的方式，观察miRNA对细胞和肝脏损伤的保护作用。研究结果将阐明miRNA调控AAI致肝毒性的作用机制；为肝损伤早诊早治提供潜在靶点。

马兜铃酸(AA)作为一种新兴的中草药或农作物污染物，自20世纪90年代以来被认为是引起肾病的主要原因。在过去十年中，越来越多的证据表明AA与肝损伤相关;然而，其机制尚不清楚。microrna响应环境应激并介导多种生物学过程，因此具有预测或诊断的生物标志物潜能。在本研究中，我们研究了miRNAs在AA诱导的肝毒性中的作用，特别是调节NQO1，负责AA生物活化的关键酶。计算机模拟分析显示，hsa-miR-766-3p和hsa-miR-671-5p与AAl暴露和NQO1诱导显著相关。在20mg/kg AA暴露的28 d大鼠实验中，NQO1增加了3倍，同源miR-671下降了近50%，并伴有肝损伤，这与计算机模拟预测一致。在Huh7细胞中进一步的机制研究表明，hsa-miR-766-3p和hsa-miR-671-5p均能直接结合并下调NQO1的基础表达。此外，在70 μM的细胞毒性浓度下，这两个mirna均可抑制马兜铃酸诱导的Huh7细胞中NQO1的上调，从而减轻马兜铃酸诱导的细胞效应，包括细胞毒性和氧化应激。综上所述，这些数据表明miR-7663p和miR-671-5p可减轻马兜铃酸诱导的肝毒性，因此具有监测和诊断的潜力。