Table_1_MicroRNA-98 Inhibits Hepatic Stellate Cell Activation and Attenuates Liver Fibrosis by Regulating HLF Expression.docx

Liver fibrosis is a major endpoint of patients with chronic liver diseases. The molecular mechanisms behind liver fibrosis remain largely unknown. Many studies have indicated the role of microRNA (miRNA) in hepatic tumorigenesis. But the role of miRNA in liver fibrosis is little known. Activated hepatic stellate cells (HSCs) can secret extracellular matrix proteins (ECM) and are the major contributors to liver fibrosis/cirrhosis. Here, a microarray assay of quiescent and transforming growth factor β1 (TGF-β1) activated HSCs indicated that miR-98 might play a crucial role in liver fibrosis. We found that miR-98 was significantly downregulated in activated HSCs. miR-98 overexpression inhibited HSCs activation. Furthermore, we hypothesized that miR-98 regulated hepatic leukemia factor (HLF) expression by binding to the 3′ UTR of its mRNA directly, as evidenced by luciferase reporter assay. HLF overexpression increased HSCs activation by inducing hypoxia inducible factor-1 alpha (HIF-1α) expression, resulting in the activation of TGF-β/Smad2/3 signaling pathway. Besides, low expression of miR-98 was also found in liver tissues from various fibrotic murine models, including carbon tetrachloride (CCl4), bile duct ligation (BDL), and high-fat diet (HFD)-induced liver fibrosis. miR-98 overexpression in vivo by ago-miR-98 injection could attenuate CCl4-, BDL-, and HFD-induced murine hepatic fibrosis. Meanwhile, miR-98 overexpression suppressed HLF expression and reduced fibrosis marker expression. Collectively, our study demonstrates that miR-98 suppress HSCs activation by targeting HLF directly and interacting with HIF-1α/TGF-β/Smad2/3 signaling pathway, which may be an effective therapeutic target for liver fibrosis.

肝纤维化（Liver fibrosis）是慢性肝病患者的主要临床终点。目前肝纤维化的分子机制仍未完全阐明。多项研究已证实微小RNA（microRNA, miRNA）在肝脏肿瘤发生过程中发挥调控作用，但miRNA在肝纤维化中的功能仍鲜为人知。活化的肝星状细胞（hepatic stellate cells, HSCs）可分泌细胞外基质（extracellular matrix, ECM）蛋白，是肝纤维化/肝硬化的主要致病细胞。本研究通过对静息状态及转化生长因子β1（transforming growth factor β1, TGF-β1）活化的HSCs进行微阵列分析，发现miR-98可能在肝纤维化进程中发挥关键调控作用。本研究发现，活化HSCs中miR-98的表达水平显著下调；上调miR-98的表达可抑制HSCs的活化。进一步通过荧光素酶报告基因实验（luciferase reporter assay）证实，miR-98可通过直接结合肝白血病因子（hepatic leukemia factor, HLF）mRNA的3'非翻译区（3′ UTR）调控其表达。上调HLF的表达可通过诱导缺氧诱导因子-1α（hypoxia inducible factor-1 alpha, HIF-1α）的表达增强HSCs的活化，进而激活TGF-β/Smad2/3信号通路。此外，在多种纤维化小鼠模型的肝组织中同样检测到miR-98的低表达，这些模型包括四氯化碳（carbon tetrachloride, CCl4）诱导、胆管结扎（bile duct ligation, BDL）以及高脂饮食（high-fat diet, HFD）诱导的肝纤维化模型。通过注射ago-miR-98在体内上调miR-98的表达，可减轻CCl4、BDL及HFD诱导的小鼠肝纤维化。与此同时，上调miR-98的表达可抑制HLF的表达，并降低纤维化标志物的表达水平。综上，本研究证实miR-98可通过直接靶向HLF，并调控HIF-1α/TGF-β/Smad2/3信号通路以抑制HSCs的活化，该机制或可成为肝纤维化治疗的有效靶点。