MiR-20b-5p负向调控JAK2/STAT3/HIF-1α在肝内型门脉高压症中的作用和机制研究

Hepatic stellate cells (HSC) and the multicellular factors involved in the process of liver fibrosis and inter- and intra-hepatic abnormal angiogenesis are an important pathological feature and intervention targets of portal hypertension due to liver cirrhosis. Our previous studies have shown that inhibition of JAK2/STAT3 pathway can significantly inhibit the pathological process and improve portal hypertension. On this basis, we found that miR-20b-5p can effectively inhibit the activation of STAT3 protein in HSC. Studies showed miR-20b-5p also has a negative regulatory role in hypoxia inducible factor-1α (HIF-1 alpha), another important promotor of the activation of HSC and angiogenesis. So we conclude that miR-20b-5p may participate in portal hypertension by negatively regulating of STAT3 and HIF-1α. The molecular biology and cell biology method will be used in the study to simulately upregulate the expression of miR-20b-5p in HSC cells and animal model of portal hypertension, to observe the effect on the related cytokines, the function of HSC cells and the animal model of portal hypertension respectively from the level of cell and animal. Clinical significance will be determined with the detection the expression of miR-20b-5p in clinical human liver samples from patients of portal hypertension with cirrhosis. This study will explore the mechanism of miR-20b-5p in the regulation of JAK2/STAT3/HIF-1α in portal hypertension suffered from cirrhosis. It will further clarify the intrahepatic portal hypertension pathogenesis, providing effective target for diagnosis and treatment of portal hypertension.

以肝星状细胞（HSC）为中心的、多细胞因子参与的肝纤维化和肝内外血管异常增殖是肝内型门脉高压症（PHT）的重要病理特征和干预靶点。我们前期研究证实抑制JAK2/STAT3通路可明显抑制这一病理过程从而改善PHT。在此又发现miR-20b-5p可有效抑制HSC中STAT3活化，同时对HSC另一重要调控因子低氧诱导因子-1α（HIF-1α）具有负向调控作用，因此我们推断miR-20b-5p可能通过负向调控STAT3和HIF-1α在PHT中发挥重要作用。本题拟模拟上调HSC及PHT动物体内miR-20b-5p表达，分别从细胞和动物水平观察对相关细胞因子、HSC细胞功能及对模型动物的影响，最后检测其临床大样本中表达以明确可能的临床意义。本题将以miR-20b-5p为切入点，明确其在PHT中调控JAK2/STAT3/HIF-1α具体机制，以为PHT诊断治疗提供可能有效靶点。

以肝星状细胞（HSC）为中心的、多细胞因子参与的肝纤维化和肝内外血管异常增殖是肝内型门脉高压症（PHT）的重要病理特征和干预靶点。我们前期研究证实抑制JAK2/STAT3通路可明显抑制这一病理过程从而改善PHT。在此基础上又发现miR-20b-5p可有效抑制HSC中STAT3活化，对HSC中STAT3具有负向调控作用，因此我们推断miR-20b-5p可能通过负向调控STAT3在肝硬化PHT中发挥重要作用。本课题明确了：①在细胞水平上miR-20b-5p可通过靶向抑制HSC中STAT3及其下游相关分子抑制HSC活化促进HSC凋亡。PCR检测发现miR-20b-5p 在IL-6活化的HSC中表达显著降低，且随着作用时间延长表达进一步下降；通过miR-20b-5p mimics模拟上调HSC内表达量后可显著降低活化HSC数量、降低HSC增殖能力，促进HSC凋亡。基于targetscan生物信息分析及双荧光素酶报告基因检测，明确了STAT3是miR-20b-5p潜在作用靶点。miR-20b-5p通过降低HSC中STAT3蛋白及其磷酸化蛋白水平抑制其下游MMP-2、α-SMA、cyclin D1、VEGF蛋白表达水平，因此从细胞水平上证实miR-20b-5p 可通过靶向抑制STAT3抑制HSC活化及降低HSC促纤维化、促血管化相关分子表达，为改善肝纤维化提供了细胞学基础。②动物水平上明确miR-20b-5p可改善纤维化水平，抑制肝内外血管异常增殖进而有效降低门静脉压力。通过建立小鼠肝硬化门静脉高压模型，明确了在纤维化肝脏中miR-20b-5p表达显著降低，miR-20b-5p agomir 模拟上调小鼠体内miR-20b-5p 含量后可显著抑制肝内STAT3通路及下游相关分子蛋白表达，进而抑制小鼠肝内HSC活化水平及降低肝纤维化程度。同时miR-20b-5p 可通过抑制STAT3显著降低肠系膜内VEGF及CD31蛋白水平降低肠系膜内异常血管增殖。从动物学水平证实miR-20b-5p通过靶向抑制STAT3改善肝内纤维化水平、降低肝内外异常血管增殖从而有效改善门静脉压力。因此本课题明确了MiR-20b-5p负向调控JAK2/STAT3在肝内型门脉高压症中的作用和机制，丰富了肝硬化门静脉高压的病理基础，最终可能为临床诊断和治疗提供有效靶点。