microRNA-34a/SIRT1信号调控在原发性硬化性胆管炎中的作用及其机制研究

Primary sclerosing cholangitis is one kind of the most common cholestatic diseases, which is characterized by chronic inflammation, progressive fibrosis and biliary stricture, eventually developing into cirrhosis and hepatic failure. The patients with PSC are prone to cholangiocarcinoma. The mechanisms of PSC are not fully elucidated and there is still lack of effective therapy for PSC. Our preliminary data demonstrated that microRNA-34a (miR-34a) which is associated with cellular senescence was expressed much higher in the liver of multiple drug resistance gene 2 knockout (Mdr2-/-) mice which is a typical model of PSC than that in control mice, but the expression of Sirtuin 1(SIRT1) targeted by miR-34a was decreased compared to control mice, suggesting that miR-34a may be involved in the development of PSC. Therefore, this project will first observe the changes of liver function and fibrosis due to inhibition or overexpression of miR-34a in Mdr2-/- mice. Then, the mechanism related to the promotion effect of miR-34a on liver injury in PSC will be further explored, especially for the effect of down-regaulation of SIRT1 caused by increased expression of miR-34a on cellular senescence in the liver and inflammation mediated by NF-κB signaling pathway. Finally, the regulation effect of miR-34a/SIRT1 axis in the development of PSC will be elucidated, which can provide the novel theoretic basis and a promising target for the therapy of PSC.

原发性硬化性胆管炎（PSC）是最常见的胆汁淤积性疾病，以慢性炎症、进行性纤维化和胆管狭窄为特征，最终导致肝硬化和肝功能衰竭，且极易诱发胆管癌的发生。PSC的发病机制尚未阐明而临床治疗措施非常有限。我们的前期实验发现，与细胞衰老密切相关的microRNA-34a（miR-34a）在PSC的动物模型—多药耐药基因2敲除（Mdr2-/-）小鼠肝脏的表达较对照组显著升高而其调控的去乙酰化酶1（SIRT1）显著降低，提示miR-34a可能参与了PSC的发生。因此，本项目首先观察抑制或促进miR-34a的表达后对Mdr2-/-小鼠肝损伤和纤维化的影响，然后探讨miR-34a促进PSC肝损伤的机制是否与miR-34a下调SIRT1引起肝内细胞衰老的改变和NF-κB信号通路介导的炎症反应改变有关，最终阐明miR-34a/SIRT1信号轴在PSC发生发展中的调控作用，为PSC的治疗提供新的靶点和理论基础。

项目的背景：原发性硬化性胆管炎（PSC）是最常见的胆汁淤积性疾病，microRNA参与了PSC的发生发展。本研究旨在探讨microRNA-34a（miR-34a）/SIRT1信号轴在PSC发生发展中的作用及其机制。.研究内容：本研究以PSC的动物模型—多药耐药基因2敲除（Mdr2-/-）小鼠、肝星状细胞、胆管细胞为研究对象，探讨抑制或促进miR-34a的表达后，是否通过miR-34a/SIRT1信号轴影响胆汁淤积性肝损伤的发生及其机制。.重要结果：本研究通过GEO数据库分析，发现PSC病人肝脏miR-34a的表达显著高于健康人。在PSC病人的肝组织样本，miR-34a的表达，衰老和纤维化相关基因的表达均较健康人明显增加。在Mdr2-/-小鼠腹腔给予miR-34a抑制剂后，肝脏SIRT1的表达较Mdr2-/-小鼠升高，肝脏损伤和纤维化有所减轻；而且肝脏炎症、纤维化和衰老相关因子的表达也显著降低。在肝细胞特异性敲除miR-34a的胆总管结扎（BDL）小鼠中发现，特异性敲除miR-34a增加了BDL小鼠肝细胞SIRT1的表达，减少了BDL小鼠肝脏的胆管增生，纤维化和衰老相关因子的表达。体外培养的肝星状细胞（LX-2）给予甘氨胆酸（GCA）刺激后，miR-34a的表达减低，SIRT1的表达显著升高，抑制miR-34a的表达降低了脂多糖诱导的肝细胞纤维化和衰老基因的表达。.关键数据：获得了抑制miR-34a的表达对Mdr2-/-小鼠肝功能、肝组织形态、炎症、纤维化和衰老相关基因和蛋白表达影响的数据；获得了肝细胞特异性敲除miR-34a后对BDL小鼠肝功能和肝纤维化影响的数据；也获得了胆酸刺激肝星状细胞后miR-34a和SIRT1的表达的数据，以及抑制miR-34a的表达对脂多糖刺激的肝细胞纤维化和衰老相关因子表达影响的数据。.研究结论：miR-34a/SIRT1信号轴参与了胆汁淤积性肝损伤的发生，抑制或沉默miR-34a的功能可通过减轻炎症因子，纤维化相关因子的表达减轻胆汁淤积小鼠的肝损伤和肝纤维化；胆酸刺激可引起肝星状细胞miR-34a的下调和SIRT1的上调，抑制miR-34a的表达可减少肝细胞纤维化和衰老基因的表达。.科学意义：本研究证实了miR-34a/SIRT1可能促进胆汁淤积性肝损伤的发生，对PSC的发生机制增添了新的理解，也为PSC的诊断和治疗提供了潜在的靶点。