外泌体miR-106b靶向Vim在药物性肝损伤适应性反应中的调控机制研究

Adaptive response is a special phenomenon in drug-induced liver injury (DILI), which reflects the key role of restorative tissue repair in the outcome of liver injury. However, the underlying mechanisms of adaptive response in DILI have not been clarified. The results from our previous study suggested that the adaptive response was existed in the mouse model of toosendanin-induced liver injury (TILI) and exosomal miR-106b targeting Vim may play an important role in this response by promoting tissue repair. Therefore, this project will focus on investigating the regulatory mechanism of exosomal miR-106b targeting Vim in the adaptive response of DILI, using TILI as an example. First, the techniques, including luciferase reporter system and gene knockout, will be used to verify Vim is the key target gene for miR-106b to promote tissue repair. The effects of miR-106b on the Vim related pathways will be also determined. Second, the major source of exosomal miR-106b and exosome-mediated transmission of miR-106b between the liver cells will be elucidated. Third, by the in vitro, in vivo, and clinical studies, the relationship between miR-106b and the occurrence and prognosis of DILI will be clarified. This project will not only help to understand the mechanism of adaptive response in DILI, but also will promote the use of adaptive response to prevent DILI and other liver diseases.

适应性反应是药物性肝损伤中的特殊现象，它反映了恢复性组织修复在肝损伤转归中的关键作用，但其内在机制一直尚未明确。申请人前期研究发现川楝素引起的肝损伤具有适应性现象，且外泌体miR-106b可能通过靶向Vim在其中发挥促组织修复的关键作用。因此，本项目拟以川楝素肝损伤适应性反应为模式研究对象，采用荧光素酶报告系统、基因敲除等技术，首先确证Vim是miR-106b促组织修复的关键靶基因，明确miR-106b对Vim相关信号通路的调控作用，深入研究外泌体miR-106b的主要来源及其在细胞间的传递作用，阐明外泌体miR-106b介导促组织修复的确切分子事件及调控机制。并且，通过体内外及临床研究，辨析miR-106b与药物性肝损伤发生及预后的关系。这一创新研究将有助于深入认识药物性肝损伤适应性反应的内在机制，推动利用适应性反应治疗药物性肝损伤乃至其他肝脏疾病的研究进程。

目前利用适应性反应已成为防治疾病及靶器官损伤的新方向。药物性肝损伤中同样存在适应性反应，它反应了恢复性组织修复在肝损伤转归中的关键作用，但其内在机制仍未明确。申请人前期以小毒中药川楝子中的主要成分川楝素建立了与临床相符的药物性肝损伤适应性反应动物模型，并且发现血清外泌体miR-106b-5p可能是该反应中发挥促组织修复的关键作用分子。本研究在前期研究的基础上，深入探究了miR-106b-5p介导药物性肝损伤适应性反应的调控机制。研究揭示了miR-106b-5p通过抑制靶基因Vim的表达，从而促进肝细胞增殖与抑制肝细胞凋亡是川楝素药物性肝损伤适应性反应中介导恢复性组织修复的重要分子事件。并且，受损肝细胞可能是循环中高表达miR-106b-5p外泌体的主要来源，这些外泌体能被摄取进入靶肝细胞中，从而增加靶肝细胞内miR-106b-5p的水平。体内和体外研究发现敲除miR-106b-5p靶基因Vim也是对乙酰氨基酚等药物引起的肝损伤中促损伤修复的关键事件。此外，临床研究结果显示血清miR-106b-5p的表达水平与药物性肝损伤患者肝损伤的严重程度成负相关；Vim编码蛋白vimentin在健康小鼠肝细胞和健康人肝细胞中不表达，但在损伤的小鼠肝细胞和临床药物性肝损伤患者的肝细胞中表达，而miR-106b-5p能抑制肝细胞中vimentin的表达；单细胞RNA测序数据也显示，与川楝素组相比，miR-106b-5p能抑制Vim+特异性表达的肝细胞数量及其Vim的表达水平。本研究不仅有助于深入认识药物性肝损伤适应性反应发生发展的机制，为miR-106b-5p及其靶标vimentin作为药物性肝损伤预后生物标志物及潜在治疗靶点提供了支持，也有望进一步推动利用适应性反应治疗药物性肝损伤乃至其他肝脏疾病的研究进程。