The Mechanism Of MiR-30 And LincRNA-p21 Regulating Liver Fibrosis

The direct expression of hepatic fibrosis at the tissue level is the excessive deposition of extracellular matrix (matrix extracellular, ECM) and the destruction of normal liver structure. And at the cellular level, the direct cause of liver fibrosis is generally considered to be a large number of ECM secreted by excessive activation of hepatic stellate cells. However, the sustained necrosis and apoptosis of hepatocytes can cause early inflammation of liver tissue and activate hepatic stellate cells directly. Therefore, hepatocytes apoptosis plays a key role in the process of hepatic fibrosis. Despite a number of studies have indicated that hepatic stellate cell activation and hepatocyte apoptosis are closely related to TGF-β signaling pathway, the current understanding of the specific molecular mechanism that TGF-β signaling regulates the activity of these cells is still very limited. Therefore, more comprehensive and in-depth to clarify the relationship between hepatic stellate cell activation and hepatocyte apoptosis and the regulation of TGF-β signaling pathway is still the key scientific problems to be solved. Sufficient experimental evidences have demonstrated that microRNA (miRNA) and long non-coding RNA (lncRNA) as the most important two types of non-coding RNA are involved in the occurrence and development of many diseases. They participate in the regulation of various signaling pathways as well as a series of cellular biological processes as very important functional molecules. However, miRNA and lncRNA take part in the regulation of TGF-β signaling pathway and the relationship between them in the process of liver fibrosis remain to be elucidated. Therefore, based on the above scientific problems, we carried out in-depth analysis and discussion on the relationship between microRNA and lncRNA and TGF-(3 signaling pathway and the process of they involving in regulating hepatic stellate cell activation and hepatocyte apoptosis.First, we explored the regulatory mechanism of microRNA in TGF-β signaling pathway in activated hepatic stellate cells in liver fibrosis. Using a mouse model of carbon tetrachloride (CCl4)-induced liver fibrosis, we observed significant downregulation of miR-30 in the activated HSCs during liver fibrogenesis. Meanwhile, the upregulation of KLF11, which is the early transcription factor in TGF-β signaling pathway, was observed in the HSCs isolated from fibrotic liver. Next, we investigated the interaction between miR-30 and its potential target KLF11 and their effects on hepatic stellate cell activation in liver fibrosis animal models and hepatic stellate cells respectively. Adenovirus-mediated hepatic stellate cell specific expression of miR-30 was under the control of a-SMA promoter, showing that the increasing of miR-30 in HSCs greatly reduced CCl4-induced liver fibrosis, and effectively restrained KLF11 expression and increased the level of Smad7 in HSCs in mice. Further exploration of the mechanism confirmed that KLF11 was the direct target of miR-30, and miR-30 blunted the profibrogenic TGF-β signaling in HSC by suppressing KLF11 expression and thus enhanced the negative feedback loop of TGF-β signaling imposed by Smad7. Subsequent investigation found that using KLF11 overexpression vector or siRNA to raise or lower the level of KLF11 in HSCs can promote or inhibit the activation of hepatic stellate cells. At the same time, it is proved that miR-30 can inhibit HSCs proliferation and migration to achieve the purpose of facilitating the reversal of activated HSCs to a quiescent state by using miR-30 precursors or inhibitors. In conclusion, our results uncover that as an inhibitor of liver fibrosis, miR-30 could supress the TGF-β signaling in hepatic stellate cells by inhibiting the expression of KLF11. And the research also provides useful insights into the mechanisms underlying liver fibrosis.Although lncRNAs have important biological functions, it is still unknown whether and how it is involved in the regulation of hepatic fibrosis. Here, we first detected the expression status of lncRNAs in CCl4-induced liver fibrosis mouse hepatocytes by NGS(Next Generation Sequencing). The result showed that hepatocytes from fibrotic liver compared with normal liver,63 lncRNAs raised significantly and 48 lncRNAs were significantly downregulated at the same time. Further screening found that the expression of one upregulated lncRNA called lincRNA-p21 was increased about 4 times and its specific mechanism of action in liver fibrosis is still blank. Therefore, we investigated the effect of lincRNA-p21 on hepatocyte apoptosis and hepatic fibrosis in liver fibrosis mouse models respectively. First of all, we monitored the expression pattern of lincRNA-p21 in liver tissue and different cell types in the process of liver fibrosis by using Northern blot, qRT-PCR, RT-PCR and other methods. And we found in the process of CCl4-induced liver fibrosis in mice, the expression of lincRNA-p21 in hepatocytes is specifically increased. Next, we used the adenovirus-mediated hepatocyte-specific interference of lincRNA-p21 expression to study the biological function of lincRNA-p21. The in vivo experiment revealed that reducing lincRNA-p21 levels in mouse hepatocytes has a significant therapeutic effect on liver fibrosis, and downregulation of lincRNA-p21 can effectively inhibit apoptosis of hepatocytes and promote proliferation of them. Then we explored the molecular mechanism of lincRNA-p21 regulating hepatocyte apoptosis, and we found lincRNA-p21 sequence exists miR-30 family conserved binding site by using bioinformatics analysis combined with luciferase reporter assay. At the same time, we labeled miR-30 and lincRNA-p21 with biotin in vitro respectively. The RNA pulldown and RNA precipitation experiments were carried out and we found lincRNA-p21 combines miR-30 specifically. Finally, we also discovered the curative effect of lincRNA-p21-knockdown adenovirus in liver fibrosis mouse model was offset by interference of miR-30 expression in hepatocytes. Consequently, our study starts from the perspective of hepatocytes, revealed that lincRNA-p21 can be induced and participate in the process of liver fibrosis by competitively binding with miR-30. Moreover, it identified lincRNA-p21 can be used as a novel competitively endogenous non-coding RNA involved in the regulation of hepatocytes apoptosis and proliferation in the process of hepatic fibrosis, and also provided a new target and research approach for the treatment of liver fibrosis.