MicroRNA 31在决定药物性肝损发生的关键炎症信号通路中调控机理研究

Drug-induced liver injury (DILI) is a common liver disease and an important adverse effect of drug administration, which may result in acute hepatic failure or patient death in its severe cases. However, the mechanisms of DILI are complicated, recent study has confirmed that IL-17-mediated inflammatory damage being one of the final common events involved in the occurrence and development of DILI. Our previous study showed that miR-31could be regulated by IL-17 in a DILI model; meanwhile, miR-31 could also upgrade IL-17-dependent NF-κB signaling pathway. Based on these findings, we hypothesize that miR-31 acts as a key positive feedback molecule in DILI-associated inflammation, and may contribute to over-activation of inflammatory signaling pathway in hepatic tissue, which finally leads to inflammation injury in liver. In this study, we will investigate the roles of miR-31 in DILI process by a miR-31 conditional knockout mice model, and illustrate the network molecules and regulatory mechanisms of miR-31 in the pathogenesis of DILI by systemic biological tools, as well as its potential translational significance in clinical DILI cases. Our project may provide a better understanding of DILI-associated inflammation and its regulatory mechanisms, as well as new approaches to prevention, early diagnosis and treatment of DILI.

药物性肝损伤(DILI) 是临床常见的肝脏疾病及重要的药物不良反应，重症者危及患者生命。DILI的发生机制复杂，最新研究证实IL-17介导的免疫炎症损伤是DILI发生的最后共同事件之一。我们前期研究发现DILI发生相关的miR-31能被IL-17调节，同时也能上调IL-17介导的NF-κB通路。基于此我们提出以下假说： miR-31作为炎症通路的一个关键的正反馈调节分子，其表达上调可导致肝组织炎症通路过度活化，造成肝组织的炎症损伤，从而参与DILI的发生发展。本项目将利用miR-31的条件敲除小鼠来研究miR-31在DILI中的作用，并结合系统生物学手段，阐明miR-31调节的分子调控网络，同时探索miR-31表达调控的分子机制，以及miR-31在临床转化研究中的潜在意义。本项目的预期结果将有助于更好地理解DILI病理的炎症发生和调控机制，为发展新的DILI诊断治疗手段打下基础。

项目的背景：对乙酰氨基酚是最常用的解热镇痛非处方药，也是引起药物性肝损最常见的原因，严重者甚至引起不可逆转的急性肝衰竭。微小RNA是长度为19-22nt的非编码RNA，在代谢、肿瘤、细胞增殖、炎症浸润调控等方面发挥重要作用。目前一些研究分析了DILI相关的miRNA表达谱，但miRNA具体调控机制目前依然缺乏。.主要研究内容：使用APAP 300mg/kg构建稳定的肝损模型；肝组织行miR-31原位杂交观察肝脏miR-31的表达分布； qPCR测量肝组织，肝实质细胞，肝非实质细胞miR-31的表达量；使用miR-31基因敲除小鼠构建肝损模型，肝功病理检测，流式检测炎症细胞浸润探索miR-31在肝损中所起作用；构建骨髓移植嵌合小鼠，探索miR-31在肝实质细胞和免疫系统中所起的作用；对肝组织行RNA-seq，同时对肝实质和肝非实质细胞的Ago2蛋白行iRIP-seq测序；对收集的人药物性肝损样本进行miR-31表达检测。.重要结果：APAP 300mg/kg可构建出稳定的肝损模型，肝脏损伤坏死面积和肝功变化趋势相近；肝损过程，miR-31在肝实质细胞和肝非实质细胞中均有表达变化，原位杂交结果显示miR-31在肝损时主要在中央静脉周围表达增高；miR-31基因敲除小鼠转氨酶高于野生型小鼠，HE病理染色肝损面积大于野生型小鼠，表明31-KO小鼠肝损重于WT小鼠；骨髓移植嵌合小鼠结果提示肝损时，miR-31在肝实质细胞和免疫系统中均起到调控作用；对31-KO和WT肝损组织RNA表达谱测序结果进行KEGG富集分析，提示MAPK信号通路等方面有显著差异，进一步的western-blot也验证了JNK蛋白磷酸化增强，提示miR-31可通过调控JNK/MAPK信号通路影响肝损。iRIP-seq是寻找miRNA靶点的有效方法，测序出的数据里存在JNK/MAPK信号通路上游调控基因。人药物性肝损肝组织相比与正常人肝组织，miR-31表达显著升高。.关键数据：相比于正常肝组织，在药物性肝损引起的人体肝衰竭标本中miR-31显著增高；AILI小鼠肝组织miR-31表达呈动态变化； 31-KO小鼠肝损显著重于WT小鼠，31-KO p-JNK蛋白表达显著高于WT小鼠。.科学意义：本项目的结果将有助于更好地理解miRNA参与DILI的机理，为发展特异性药物干预治疗手段和新的生物标志物打下基础。