微小RNAs差异表达对非酒精性脂肪性肝纤维化的影响及分子调控机制研究

The role and mechanism of miRNAs in non-alcoholic fibrosing steatohepatitis remain unclear. Previously, we have demonstrated that there were altered hepatic microRNAs expressions in non-alcoholic fibrosing steatohepatitis and up-regulation and/or activation of heme oxygeanse-1 (HO-1) protected against fibrogenesis in experimental non-alcoholic steatohepatitis. In this study, we aim to develop the experimental model of non-alcoholic fatty liver disease (NAFLD) with series of pathological change and explore the role and the molecular mechanism of miRNAs in non-alcoholic fibrotic steatohepatitis by the following investigations: ① hepatic abnormal expressed miRNAs in the mice with non-alcoholic fibrosing steatohepatitis were identified using the miRNAs arrays, and the target genes of the miRNAs were forecasted; ② the mice with non-alcoholic fibrosing steatohepatitis was administrated with HO-1 agonist and antagonist, and the changes of the identified abnormal miRNAs were analysed, respectively. This study is clarifying the significantly important effects of miRNAs in the evolution of non-alcoholic fibrosing steatohepatitis, and expound the mechanism of miRNAs in the protective role of HO-1, so as to provide new targets and scientific basis for gene diagnosis and gene therapy of non-alcoholic fibrosing steatohepatitis.

微小RNAs（miRNAs）在非酒精性脂肪性肝纤维化中的作用机制尚不清楚。我们前期研究证实，非酒精性脂肪性肝纤维化小鼠肝组织中存在差异表达的miRNAs，而血红素氧合酶-1（heme oxygeanse-1, HO-1）表达上调可阻止该病的发生与进展。本课题拟建立小鼠非酒精性脂肪性肝病（non-alcoholic fatty liver disease，NAFLD）系列病变模型进行下列研究：①筛选非酒精性脂肪性肝纤维化肝组织异常表达miRNAs，预测其关键靶基因并验证；②靶向调节HO-1，观察其对差异表达miRNAs及其关键靶基因表达的影响，探明miRNAs在非酒精性脂肪性肝纤维化中的作用及分子机制、HO-1对miRNAs调控在该病治疗中的重要作用，为该病的基因诊断及基因治疗提供科学依据。

微小RNA（microRNA，miRNAs）与非酒精性脂肪性肝纤维化发生发展密切相关，靶向调控关键miRNAs表达可能阻止或逆转肝纤维化。本研究结果包括以下三个方面：（1）应用miRNAs芯片技术筛选非酒精性脂肪性肝纤维化小鼠肝组织中差异表达miRNAs，发现37个miRNAs表达异常，其中19个（miR-15b-5p、miR-150-5p、miR-106a-5p等）表达上调， 18个（miR-146a-5p、miR-203-3p、miR-130a-3p等）表达下调，并应用实时定量PCR进行了验证；（2）经生物信息学方法分析及双荧光素酶报告基因检测证实，miR-146a-5p靶基因为Wnt1及Wnt5a；实时定量PCR结果显示小鼠原代肝星状细胞（hepatic stellate cell，HSC）活化过程中miR-146a-5p的表达逐渐降低；（3）在非酒精性脂肪性肝纤维化小鼠肝组织中，随着miR-146a-5p表达下调，其靶基因Wnt1、Wnt5a及其下游β-catenin、NFAT5及肝星状细胞活化相关因子α-SMA、Col-1表达显著增强，而GSK-3β表达显著下降；应用HO-1激动剂（血晶素）可上调miR-146a-5p表达，抑制Wnt1、Wnt5a及其下游信号分子、促肝纤维化因子的表达，肝组织学损伤随之改善。上述结果表明，在非酒精性脂肪性肝纤维化中，miR-146a-5p表达下调，HO-1激活可通过上调miR-146a-5p而抑制Wnt信号通路，进而有效阻止非酒精性脂肪性肝纤维化的进展。