LncRNA-Tug1 Promotes Hepatic Fibrosis By HSCs Activation Via Down-regulating MiR-29b

Liver fibrosis refers to the abnormal proliferation of intrahepatic fibrous connective tissue caused by a variety of liver injury factors,which leads to the imbalance between the generation and degradation of extracellular matrix in the liver,and then leads to the death of patients with liver cirrhosis and liver failure.Early liver fibrosis can be reversed by treatment,so finding the key factors involved in the occurrence of liver fibrosis and its mechanism of action is of great significance for the treatment of liver fibrosis.A growing number of studies have shown that long noncoding RNAs(lnc RNAs)are involved in a wide range of biological processes and can regulate gene expression at multiple levels.In this study,we performed gene microarrays analysis in normal and fibrotic mouse livers and selected one up-regulated lnc RNAs :lnc RNA-Tug1 and then investigate its function and mechanism in hepatic fibrosis.Thus providing new ideas for clinical prevention and treatment of liver fibrosis.Methods:1.Construction of mice model of liver fibrosis: Mouse liver fibrosis model was established by intraperitoneal injection of olive oil and carbon tetrachloride(CCl4).Two groups of mouse liver tissues were treated with HE staining,Sirius red staining and immunohistochemical staining,q RT-PCR and Western Blot was used to detect liver fibrosis-related genes to ensure successful model establishment.2.Screening of lncRNA: we use microarray analysis to select the lncRNA-Tug1 which is up-regulated in liver fibrosis and has homology in mice and human.3.The liver tissue was extracted from normal and cirrhotic patients,and the expression of lnc RNA-Tug1 was detected by q RT-PCR.The total RNA of liver tissue of mice with liver fibrosis induced by CCl4 and BDL was extracted,lnc RNA-Tug1 expression was detected by q RT-PCR.4.Primary hepatic stellate cells and primary hepatocytes were extracted from normal and liver fibrosis model mice.q RT-PCR was used to detect the expression of lnc RNA-Tug1.At the same time,LX-2 cells and AML-12 cells were treated with TGF-?.The expression of lnc RNA-Tug1 is detected by q RT-PCR.The role of lnc RNA-Tug1 in activated hepatic stellate cells as well as hepatocytes was revealed and verified.5.Primary hepatic stellate cells of normal mice were extracted,treated with lentiviral vector knocking down or overexpressing lnc RNA-Tug1,q RT-PCR was used to detect the expression of lnc RNA-Tug1 and fibrosis-related genes,and Western Blot was used to detect the expression of profibrotic genes.LX-2 cells were treated with a lentiviral vector knockdown of lnc RNA-Tug1,then treated with TGF-? and detect the expression of lnc RNA-Tug1 and profibrotic genes.Explore the role of lnc RNA-Tug1 in liver fibrosis.6.To verify whether lnc RNA-Tug1 can affect the expression of mi R-29 b,the primary hepatic stellate of normal mice is extracted.The cells were treated with a lentivirus knocking down and overexpressing lnc RNA-Tug1,and the RNA was extracted to detect the expression level of mi R-29 b.7.To determine whether mi R-29 b is involved in the up-regulation of lnc RNA-Tug1-mediated profibrotic genes,the extracted mouse primary hepatic stellate cells were treated with lentiviral vector overexpressing lnc RNA-Tug1,and then treated with mi R-29 b mimic and negative control,q RT-PCR and Western Blot were used to detect the expression level of fibrosis-related genes.It demonstrated that lnc RNA-Tug1 can promote the development of liver fibrosis by down-regulating mi R-29 b.Results:1.In this study,we used microarray analysis to screen out a lncRNA with increased expression and homology in liver fibrosis model mice,which is lnc RNA-Tug1.2.The expression level of lnc RNA-Tug1 was significantly higher in liver tissue samples from patients with cirrhosis than in normal subjects.In the liver tissues of mice with liver fibrosis induced by CCl4 and BDL,lnc RNA-Tug1 was also detected.3.The primary hepatic stellate cells of normal and CCl4-induced liver fibrosis model mice were extracted,and the expression of lnc RNA-Tug1 and a-SMA was detected.The results showed that the expression of lnc RNA-Tug1 was increased.After TGF-? treatment of LX-2 cells,the expression level of lnc RNA-Tug1 was also increased.It revealed that lnc RNA-Tug1 is expressed in activated hepatic stellate cells.However,there was no significant change in the expression of lnc RNA-Tug1 in primary hepatocytes and mouse hepatocyte cell line AML-12,suggesting that lnc RNA-Tug1 does not affect hepatocyte function.4.Normal mouse hepatic stellate cells were treated with knockdown or overexpression of lnc RNA-Tug1 lentiviral vector.q RT-PCR and Western Blot showed that knockdown of lnc RNA-Tug1 reduced fibrosis gene expression and overexpression of lnc RNA-Tug1 increased expression of fibrosis gene;LX-2 cells was transfected with lnc RNA-Tug1 knockdown lentivirus vector,and then treated with TGF-?,q RT-PCR and Western Blot showed that knockdown of lnc RNA-Tug1 reduced profibrotic genes expression.It demonstrated that lnc RNA-Tug1 can promote the expression of profibrotic genes in hepatic stellate cells.5.Primary hepatic stellate cells from normal mice were extracted and treated with lentiviral vector knocking down and overexpressing lnc RNA-Tug1.q RT-PCR showed that mi R-29 b expression was negatively correlated with lnc RNA-Tug1.After overexpression of lnc RNA-Tug1 and then treated with mi R-29 b mimic,q RT-PCR and Western Blot results showed that mi R-29 b abolished the effect of lnc RNA-Tug1 on profibrotic genes in hepatic stellate cells.Conclusion:In this study,we screened a lncRNA-Tug1 that is elevated in liver fibrosis and has human and mouse homology by microarray analysis.It is up-regulated in CCl4-induced and BDL-induced liver fibrosis in mice.The expression in liver tissue of patients with cirrhosis is also up-regulated.The dates showed that lnc RNA-Tug1 was expressed in activated hepatic stellate cells and promoted the expression of profibrotic genes in hepatic stellate cells,but had no effect on the function of hepatocytes.On the mechanism,it was found that lnc RNA-Tug1 negatively regulates the expression of mi R-29 b,and then promote the expression of hepatic fibrosis gene by down-regulating mi R-29 b during liver fibrosis.