肝细胞外泌体lncRNA调控HSC自噬及EMT促进肝纤维化的机制研究

Increased expression of exosomal lncRNA-lncRNA MALAT1 and HOTAIR from hepatocyte treated by TGFβ1 has been found in our previous work, and both of the lncRNA could contribute to the enhanced level of autophagy and EMT of HSC. In view of the important role of exosome in intercellular signal transduction, we speculate that hepatocyte exosomal lncRNA MALAT1 and HOTAIR might act as the important mediators to promote HSC activation and hepatic fibrosis by regulating autophagy and EMT of HSC. In the current study, we will further focus on the association of differential expression of MALAT1 and HOTAIR with the process and prognosis of hepatic fibrosis. Then we will change the level of MALAT1 and HOTAIR in HSC using purified hepatocyte exosomal lncRNAs and siRNA to investigate the effect of MALAT1 and HOTAIR on activation and biological characteristics of HSC. At the same time, we will further explore the regulatory mechanism of MALAT1 and HOTAIR on the down-stream miRNA, related signaling pathways of autophagy and EMT in HSC activation and hepatic fibrosis. Finally, the regulatory effect and mechanism of MALAT1 and HOTAIR on hepatic fibrogenesis would be explored in fibrotic models, which would demonstrate the important role of the crosstalk between hepatocyte and HSC in hepatic fibrosis and provide potential target for anti-fibrotic therapy.

前期研究发现，TGFβ1刺激的原代肝细胞外泌体lncRNA MALAT1、HOTAIR表达增加，与HSC共孵育后，HSC自噬及EMT相关指标上调。鉴于外泌体在细胞间信号转导中的重要地位，我们推测肝细胞外泌体MALAT1及HOTAIR可通过调控HSC自噬及EMT促进HSC活化及肝纤维化进展。本课题拟进一步研究上述外泌体表达水平与肝纤维化程度及预后的相关性；利用提取纯化的TGFβ1刺激的原代肝细胞外泌体lncRNA、siRNA等调控HSC内上述lncRNA表达，研究其对HSC活化和生物学活性的影响，明确外泌体lncRNA对下游miRNA、EMT及自噬相关通路的调控作用，探讨相应机制；构建不同肝纤维化模型，体内研究调控肝细胞外泌体lncRNA对肝纤维化的影响，进一步深入探讨肝细胞-HSC的交互作用，为肝纤维化诊治提供新策略。

前期研究发现，TGFβ1刺激的原代肝细胞外泌体lncRNA MALAT1、HOTAIR表达增加，与HSC共孵育后，HSC自噬及EMT相关指标上调。鉴于外泌体在细胞间信号转导中的重要地位，我们推测肝细胞外泌体MALAT1及HOTAIR可通过调控HSC自噬及EMT促进HSC活化及肝纤维化进展。本课题首先证实肝纤维化动物模型及肝硬化患者血清中外泌体lncRNA MALAT1、HOTAIR表达上调，且与肝纤维化程度相关。利用提取纯化的TGFβ1刺激的原代肝细胞外泌体lncRNA孵育原代HSC，可刺激HSC增殖活化、促进HSC自噬及EMT。合成抑制MALAT1、HOTAIR的siRNA，转染HSC，发现抑制lncRNA MALAT1、HOTAIR表达后，HSC增殖活化减慢，自噬减弱，EMT部分逆转。进一步机制研究发现lncRNA MALAT1调控肝纤维化的机制为通过lncRNA MALAT1/miR-194-5p/ATG5轴促进HSC自噬和EMT。构建含有抑制MALAT1表达的siRNA序列shMALAT1的肝细胞外泌体，可在体内外抑制HSC活化、自噬及EMT，并在CCl4诱导的肝纤维化模型中发挥良好的抗肝纤维化作用。上述工作不仅进一步证实了肝细胞-HSC交互作用对肝纤维化的调控作用，丰富了肝纤维化的机制研究；也有可能为肝纤维化的无创诊断和治疗提供新的方法和策略。通过本课题研究，培养硕士研究生1名，博士研究生1名；发表SCI论文3篇。