表观遗传调控的微小RNA-9-5p通过转化生长因子β受体1和转化生长因子β受体2抑制肝星状细胞的激活。

Epigenetically-Regulated MicroRNA-9-5p Suppresses the Activation of Hepatic Stellate Cells via TGFBR1 and TGFBR2.

作者信息

Yu Fujun, Chen BiCheng, Fan XuFei, Li Guojun, Dong Peihong, Zheng Jianjian

机构信息

Department of Gastroenterology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.

Key Laboratory of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.

出版信息

Cell Physiol Biochem. 2017;43(6):2242-2252. doi: 10.1159/000484303. Epub 2017 Oct 27.

DOI:10.1159/000484303

PMID:29073595

Abstract

BACKGROUND/AIMS: Recently, microRNAs (miRNAs) have been demonstrated to act as regulators of activation of hepatic stellate cells (HSCs). It is well known that the main profibrogenic inducer transforming growth factor-β1 (TGF-β1) contributes to HSC activation, which is a key event in liver fibrosis. Increasing studies show that miR-9-5p is down-regulated in liver fibrosis and restoration of miR-9-5p limits HSC activation. However, the role of miR-9-5p in TGF-β1-induced HSC activation is still not clear.

METHODS

miR-9-5p expression was quantified using real-time PCR in chronic hepatitis B (CHB) patients and TGF-β1-treated LX-2 cells. In CHB patients, histological activity index (HAI) and fibrosis stages were assessed using the Ishak scoring system. Effects of miR-9-5p on liver fibrosis in vivo and in vitro were analyzed. Luciferase activity assays were performed to examine the binding of miR-9-5p to the 3'-untranslated region of type I TGF-β receptor (TGFBR1) as well as TGFBR2.

RESULTS

Compared with healthy controls, miR-9-5p was reduced in CHB patients. There was a lower miR-9-5p expression in CHB patients with higher fibrosis scores or HAI scores. miR-9-5p was down-regulated by TGF-β1 in a dose-dependent manner. TGF-β1-induced HSC activation including cell proliferation, α-SMA and collagen expression was blocked down by miR-9-5p. Notably, miR-9-5p ameliorates carbon tetrachloride-induced liver fibrosis. As determined by luciferase activity assays, TGFBR1 and TGFBR2 were targets of miR-9-5p. Further studies demonstrated that miR-9-5p inhibited TGF-β1/Smads pathway via TGFBR1 and TGFBR2. Interestingly, promoter methylation was responsible for miR-9-5p down-regulation in liver fibrosis. The relationship between miR-9-5p expression and methylation was confirmed in CHB patients and TGF-β1-treated cells.

CONCLUSION

Our results demonstrate that miR-9-5p could inhibit TGF-β1-induced HSC activation through TGFBR1 and TGFBR2. Loss of miR-9-5p is associated with its methylation status in liver fibrosis.

摘要

背景/目的：最近，微小RNA（miRNA）已被证明可作为肝星状细胞（HSC）激活的调节因子。众所周知，主要的促纤维化诱导因子转化生长因子-β1（TGF-β1）有助于HSC激活，这是肝纤维化中的关键事件。越来越多的研究表明，miR-9-5p在肝纤维化中表达下调，miR-9-5p的恢复可限制HSC激活。然而，miR-9-5p在TGF-β1诱导的HSC激活中的作用仍不清楚。

方法

使用实时PCR对慢性乙型肝炎（CHB）患者和TGF-β1处理的LX-2细胞中的miR-9-5p表达进行定量。在CHB患者中，使用Ishak评分系统评估组织学活动指数（HAI）和纤维化分期。分析了miR-9-5p在体内和体外对肝纤维化的影响。进行荧光素酶活性测定以检查miR-9-5p与I型TGF-β受体（TGFBR1）以及TGFBR2的3'-非翻译区的结合。

结果

与健康对照相比，CHB患者中miR-9-5p降低。纤维化评分或HAI评分较高的CHB患者中miR-9-5p表达较低。TGF-β1以剂量依赖性方式下调miR-9-5p。miR-9-5p可阻断TGF-β1诱导的HSC激活，包括细胞增殖、α-SMA和胶原蛋白表达。值得注意的是，miR-9-5p可改善四氯化碳诱导的肝纤维化。通过荧光素酶活性测定确定，TGFBR1和TGFBR2是miR-9-5p的靶标。进一步研究表明，miR-9-5p通过TGFBR1和TGFBR2抑制TGF-β1/Smads信号通路。有趣的是，启动子甲基化是肝纤维化中miR-9-5p下调的原因。在CHB患者和TGF-β1处理的细胞中证实了miR-9-5p表达与甲基化之间的关系。