microRNA在微囊藻毒素致白鲢肝毒性及其解毒中的调控作用研究

Microcystins (MCs) are a group of monocyclic heptapeptide hepatotoxins produced mainly by the cyanobacterium Microcystis. They usually have potent hepatotoxicity to most freshwater fish and even induce hepatitis and promote liver cancer in human being exposed at low dose for long term through cyanobacteria-contaminated drinking water. MicroRNAs (miRNAs) are small non-coding RNAs (21-23 nucleotides) that can negatively regulate the target gene by binding to recognition sequences on 3'-untranslated regions of their mRNAs, resulting in miRNA-mediated mRNA decay or translational repression. They not only control a wide range of biological processes but also regulate gene expression induced by environmental chemicals. Currently, increasing experimental evidences indicate that miRNA may be involved in controlling the hepatotoxicity and detoxification of microcystins. In the present project, the regulatory functions of microRNA in the hepatotoxicity and detoxification of microcystin in silver carp were studied by using in vitro over expression and expression inhibition to predict the target genes of microRNA in MAPK pathway (MAPK, c-fos, c-jun, and c-myc) and the MC detoxification enzymes (CYP3A and GST) and their receptor PXR. Then the predicted genes were identified by using dual luciferase reporter gene assay in vitro. Furthermore, in vivo antisense RNA was conducted to verify the results in vitro and to identify the drug target of liver disease. This project aims to determine the regulatory function of microRNA in the hepatotoxicity and detoxification of microcystin in silver carp and to find the drug target of molecular interference in the control and treatment of liver disease of freshwater fish. The results of this project will be valuable for the discovery of drug target in the liver disease preventing and treatment of freshwater fish. In addition, this project will also provide key fundament for liver disease preventing and treatment of freshwater fish and the development of new medicine.

微囊藻毒素是一类由蓝藻产生的肝毒素，对大部分淡水养殖鱼具有肝毒性，而且长期慢性低剂量暴露可引起易感人群患肝炎和肝癌。目前微囊藻毒素的肝毒性及其解毒机理国内外已有较多报道，但miRNA对其调控作用则报道甚少。申请人前期研究发现，miRNA可能与微囊藻毒素肝毒性及其解毒相关。本项目拟选择在自然水体中摄食蓝藻的白鲢为实验对象，采用毒理学和分子生物学方法，通过体外肝细胞系miRNA表达抑制和过表达实验，预测miRNA对微囊藻毒素肝毒性的信号通路（MAPK）和肝细胞解毒酶 (CYP3A、GST) 及其受体PXR调控的靶基因并用报告基因验证；再通过白鲢体内反义RNA实验验证miRNA调控的靶基因；以期最终揭示miRNA在微囊藻毒素肝毒性及其解毒中的调控作用，寻找鱼肝病预防和治疗的药物靶点。该项目研究结果不仅有助于发现肝炎预防和治疗药物靶点，同时也为鱼肝病预防控制和新型鱼药研发提供重要的理论依据。

微囊藻毒素（MC）是一类由蓝藻产生的肝毒素，对大部分淡水鱼具有肝毒性，而且低剂量长期慢性暴露可引起易感人群患肝炎和肝癌。目前微囊藻毒素的肝毒性及其解毒机理国内外已有较多报道，但microRNAs对其调控作用则报道甚少。本项目首先通过全转录组测序，发现和解析了MC致白鲢肝毒性的信号通路和靶基因，并进行了体内和体外实验验证，初步揭示了MC致白鲢肝毒性的分子机制。同时，转录组分析发现，microRNAs在MC致白鲢肝毒性中可能有重要调控作用。发现miR-16在MC致白鲢肝毒性中高表达，有望成为肝毒性新的分子标志物。研究还发现，低剂量MC慢性暴露可引起肝细胞炎性反应，而且ROS、促炎性因子和NFкB信号通路与该毒素导致的炎性反应相关，为低剂量MC暴露导致肝炎、肝癌的机理研究提供了理论支持。研究还发现，MC致肝细胞凋亡主要是通过产生过量的ROS、激活线粒体信号途径而介导细胞凋亡，并首次发现PUMA 和survivin 在该过程中发挥作用。另外，本研究还发现，MC暴露引起肝细胞I相和II相部分代谢酶及外排泵基因表达或酶活性的改变，说明MC染毒可能扰乱这些代谢酶的正常功能。MC染毒明显抑制小鼠肝脏CYP1A1和CYP3A11酶活性，但显著提升了CYP2E1酶活性。该结果说明，MC的肝毒性及其解毒可能与P450家族有关。项目组按照本项目申请书和计划书展开工作，完成了该项目的主要研究内容和目标任务。项目执行情况良好，获得了一批具有一定学术价值的科研成果。本项目执行期间共发表13篇SCI论文，其中SCI二区共4篇，对促进本领域研究工作具有一定的推动作用。该项目的研究结果为淡水养殖鱼肝病预防控制和新型鱼药研发提供重要的理论支撑，对于保护MC易感人群健康也具有一定的科学价值。