三氯生间接靶向miR-125b异常表达的上游作用新通路及诱发斑马鱼NAFLD和肝脏线粒体凋亡的下游调控网络

Triclosan (TCS) is a kind of broad-spectrum bactericide, which is widely used in medical and chemical industries. Due to frequent use, it poses a great concern to aquatic organisms and human health. In the follow-up study of our previous NSFC (31270548), we found that when zebrafish (Danio rerio) were exposed to TCS, multi-fold abnormal expression occurred for miR-125b and its relative transcriptional factors, and an obvious fat accumulation as well as apoptosis appeared in liver. However, there were many different reports in the relationship between transcriptional factors and miR-125b, and the mechanism analysis on the regulation of fat metabolism and apoptosis. In this proposal, the molecular mechanisms due to TCS exposure to zebrafish will be disclosed by means of the following researches: (1) Using miR-125b as a breakthrough point, we will explore the new pathway due to abnormal expression of non-Era as the receptor, and confirm the origin and mechanism of miR-125b expression changes, i.e., the direct target molecules of TCS action; (2) Through the search and verification of target genes of downstream regulation, we will elucidate the relationship between abnormal changes of miR-125b and occurrence of non-alcoholic fatty liver diseases (NAFLD), hepatic mitochondrial apoptosis as well as liver cancer; (3) Further, we will infer the synergistic mechanisms on lipid metabolism disorder and apoptosis, and to define the downstream regulation network of miR-125b. This study can improve our understanding of the occurring mechanism on liver diseases due to environmental pollution, and also have an important practical significance in health prevention, early intervention, and gene therapy of drug-induced diseases.

三氯生被广泛应用于医药和化工等行业，其低剂量环境暴露所引发的慢性致毒效应对水生生物和人类健康构成了潜在风险。本项目在前基金的拓展研究中发现：三氯生慢性暴露斑马鱼引起miR-125b及相关转录因子均有多倍异常变化，肝脏有明显的脂肪堆积和凋亡发生。但以往研究miR-125b与有关转录因子的互作关系及其调控脂肪代谢和凋亡的机制分析存在诸多相悖之处。为此，本项目拟以miR-125b为切入点，探讨其异常表达非仅以ERα为受体的上游作用新通路，确证三氯生直接作用靶分子，率先在斑马鱼上阐明miR-125b变化的源头与机制；通过其下游调控靶基因的寻找与验证，阐明异常变化与诱发非酒精性脂肪肝、肝脏线粒体凋亡和肝癌的发生关系，揭示miR-125b作用的下游调控网络，进而推断脂肪代谢紊乱与凋亡的协同机制。本项目可揭示三氯生致毒的分子机理，增进对污染性肝脏疾病发生机制的认识，并对其预防、干预和治疗具有重要意义。

三氯生被广泛应用于医药和化工等行业，其低剂量环境暴露所引发的慢性致毒效应对水生生物和人类健康构成了潜在风险。本项目在前基金的拓展研究中发现：三氯生慢性暴露斑马鱼引起miR-125b及相关转录因子均有多倍异常变化，肝脏有明显的脂肪堆积和凋亡发生。但以往研究miR-125b与有关转录因子的互作关系及其调控脂肪代谢和凋亡的机制分析存在诸多相悖之处。为此，本基金项目(31770552)以miR-125b为切入点，在斑马鱼活体上阐明了TCS诱导脂质代谢紊乱的作用机制。通过TCS浓度梯度急、慢性暴露分别拟合出LC50，建立了亚致死剂量的染毒品系。研究亚致死剂量急、慢性暴露的典型表型畸形特征，幼鱼主要表现为卵黄、心包囊肿，在肝区、血管和脑部均有脂滴堆积；成鱼表现肥胖体征、肝脏指数升高，TG、TC显著增加，诱发肝炎和非酒精性脂肪肝发生。借助RNA-seq和miRNA-seq双重测序，挖掘出系列阳性差异miRNA和mRNA，通过生信分析与经典的分子生物学实验佐证，探讨了候选miRNAs包括miR-125、miR-30b、miR-27b与脂质合成、氧化、转运、代谢和摄取等脂肪代谢基因群，及脂肪细胞分化因子之间的靶向调控关系，在体呈现了TCS 间接靶向 miR-125b上调与线粒体凋亡通路激活、脂肪代谢紊乱、诱发NAFLD、肝肥大、肝炎、及肝细胞癌的关系，构建miR-125b调控下游脂质代谢功能靶基因的调控网络。上游溯源miR-125b上调源自pri-mir-125b1和b3的过表达，进一步追溯调控pri-mir-125b1和b3前体的转录因子Nrf2的磷酸化激活。逆向追踪与分子验证与表明：Nrf2的磷酸活化源于TCS靶向新型G蛋白偶联膜受体（GPER）而激活其下游信号通路MAPK/ERK 、PKC/Nrf2，进而导致转录因子Nrf2磷酸化后移入核内，影响miR-125b的转录，确证TCS诱导脂代谢紊乱的直接作用靶是激活了GPER。从miRNA前体转录调控的角度确证了miR-125b 变化的源头与机制。研究成果揭示了TCS致代谢毒性的分子机理，增进对污染性肝脏疾病发生机制的认识、预防、干预和诊疗具有一定的现实意义。