Hu Jiangfeng, Chen Chao, Liu Qidong, Liu Baohai, Song Chenlin, Zhu Songchen, Wu Chaoqun, Liu Su, Yu Hongyu, Yao Dingkang, Kang Jiuhong, Zhu Liang
*Department of Gastroenterology, Changzheng Hospital, Second Military Medical University, Shanghai, China.
‡‡Department of Pathology, Changzheng Hospital, Second Military Medical University, Shanghai, China.
Clin Sci (Lond). 2015 Aug;129(4):305-17. doi: 10.1042/CS20140012.
The miRNAs are small, non-coding RNAs that regulate various biological processes, including liver fibrosis. Hepatic stellate cells (HSCs) play a central role in the pathogenesis of liver fibrosis. By microarray profiling and real-time PCR, we noted that miR-31 expression in HSCs from rats, mice and humans was significantly increased during HSC activation in culture. Overall, miR-31 expression levels were unchanged in the whole-liver RNA extracts from fibrotic rat and human samples. Nevertheless, we found that miR-31 was particularly up-regulated in HSCs but not in hepatocytes during fibrogenesis. Thus, we hypothesized that miR-31 may mediate liver fibrosis. In the present study, we found that inhibition of miR-31 expression significantly inhibited HSC activation, whereas its over-expression obviously promoted HSC activation. Moreover, over-expression of miR-31 promoted HSC migration by enhancing matrix metalloproteinase (MMP)-2 expression whereas inhibition of miR-31 has an opposite effect. The biological function of miR-31 during HSC activation might be through targeting FIH1, a suppressor of hypoxia-inducible factor (HIF-1), because a knockdown of FIH1 by shRNA could mimic the effects of miR-31. In addition, primary rat HSCs were isolated and treated with different cytokines, such as transforming growth factor β (TGF-β), vascular endothelial growth factor and platelet-derived growth factor-BB, to evaluate upstream regulators of miR-31. We found that only TGF-β, a pivotal regulator in liver fibrosis, remarkably increased miR-31 expression in HSCs. And the effects of TGF-β on HSCs can be partially counteracted by inhibition of miR-31. In addition, chromatin immunoprecipitation experiments and the luciferase reporter assay demonstrated that Smad3, a major TGF-β-downstream transcription factor, stimulated the transcription activity of miR-31 by binding directly to miR-31's promoter. In conclusion, the miR-31/FIH1 pathway associates with liver fibrosis, perhaps by participation in the TGF-β/Smad3 signalling of HSCs.
微小RNA（miRNA）是一类小的非编码RNA，可调节包括肝纤维化在内的各种生物学过程。肝星状细胞（HSC）在肝纤维化的发病机制中起核心作用。通过微阵列分析和实时定量PCR，我们注意到在培养过程中，大鼠、小鼠和人类的肝星状细胞中miR-31的表达在肝星状细胞激活期间显著增加。总体而言，纤维化大鼠和人类样本的全肝RNA提取物中miR-31的表达水平没有变化。然而，我们发现miR-31在肝纤维化形成过程中在肝星状细胞中特别上调，而在肝细胞中则不然。因此，我们推测miR-31可能介导肝纤维化。在本研究中，我们发现抑制miR-31的表达可显著抑制肝星状细胞的激活，而其过表达则明显促进肝星状细胞的激活。此外，miR-31的过表达通过增强基质金属蛋白酶（MMP）-2的表达促进肝星状细胞迁移，而抑制miR-31则具有相反的作用。miR-31在肝星状细胞激活过程中的生物学功能可能是通过靶向FIH1实现的，FIH1是缺氧诱导因子（HIF-1）的一种抑制剂，因为用短发夹RNA（shRNA）敲低FIH1可以模拟miR-31的作用。此外，分离原代大鼠肝星状细胞并用不同的细胞因子处理，如转化生长因子β（TGF-β）、血管内皮生长因子和血小板衍生生长因子-BB，以评估miR-31的上游调节因子。我们发现，只有TGF-β（肝纤维化中的关键调节因子）能显著增加肝星状细胞中miR-31的表达。抑制miR-31可部分抵消TGF-β对肝星状细胞的影响。此外，染色质免疫沉淀实验和荧光素酶报告基因检测表明，Smad3（一种主要的TGF-β下游转录因子）通过直接结合miR-31的启动子刺激miR-31的转录活性。总之，miR-31/FIH1通路可能通过参与肝星状细胞的TGF-β/Smad3信号传导与肝纤维化相关。
