Study On The Mechanism Of SOX9 Regulating Hepatocyte LncRNA-H19 In Liver Fibrosis

Liver fibrosis is a common pathological process of liver cirrhosis and liver cancer.Therefore,it is of clinical significance.SOX9,as a highly conserved HMG box transcriptional regulator associated with SRY(sex determination region of Y chromosome),not only plays a key role in development,but also activates silencing hepatic stellate cells to become myofibroblast during liver fibrosis.Myofibroblast s secrete extracellular matrix,thereby breaking the normal dynamic balance of collagen synthesis and degradation,allowing collagen deposition and promoting the development of liver fibrosis.The function of lncRNA(long non-coding RNA)involves various aspects of cell biology.It is known that the expression change of lncRNA is closely related to the occurrence of various diseases,and has significant tissue and cell specificity.The implication of lncRNA in the course of cirrhosis is still not fully understood.It has been reported that in the BDL fibrosis model,the expression of lncRNA-H 19 in liver tissue is increased.As one of the earliest discovered lncRNAs,lncRNA-H19 has hitherto been reported in many diseases,but its role in liver fibrosis is not entirely clear.We isolated liver and hepatocytes from CC14-and BDL-induced mice liver fibrosis respectively.The expressions of SOX9 in fibrotic liver and hepatocytes were significantly up-regulated as measured by qRT-PCR.To investigate whether downregulation of hepatic SOX9 might inhibit hepatic fibrosis,we injected SOX9 antisense oligonucleotide(ASO)into the tail vein to reduce SOX9 expression in the liver of mice and found that it significantly inhibited CC14-induced liver fibrosis and liver inflammation.Bioinformatics software analysis showed that SOX9 can bind to the lncRNA-H19 promoter region and promote its expression in hepatocytes.To verify the results,we transfected hepatocytes with the plasmid PCI-SOX9 to overexpress SOX9.The results showed that transient overexpression of SOX9 promoted the upregulation of lncRNA-H19 expression.In addition,we also constructed stable transgenic cells overexpressing SOX9 to detect the expression of lncRNA-H19 and found similar results.The promoter region of H19 was further cloned and a luciferase reporter vector was constructed.Luciferase assay results showed that SOX9 can indeed bind to the lncRNA-H19 promoter region and promote its expression.Subsequently,we examined the lncRNA-H19 content in liver and hepatocytes in CCl4-induced liver fibrosis and found that the expression of lncRNA-H19 in fibrotic liver and hepatocytes was significantly up-regulated,indicating that the expression of H19 may be related to the fibrosis process.To investigate whether the down regulation of liver lncRNA-H19 expression may affect liver fibrosis,we injected lncRNA-H 19 antisense oligonucleotide(ASO)into the tail vein to reduce the expression of liver lncRNA-H19 and found that CCl4-induced liver fibrosis and liver inflammation were significantly inhibited in mice.Hepatocyte injury is the underlying cause of hepatic fibrogenesis.We found that the expression of SOX9 and lncRNA-H 19 was significantly increased in hepatocyte treated by adriamycin or H2O2.Moreover,knockdown of lncR-NA-H19 could significantly reduce hepatocyte cell necrosis rate.We also found that lncRNA-H 19 is related to the proliferation rate of hepatocytes.Overexpression of lncRNA-H 19 can effectively inhibit cell proliferation.Finally,to verify whether the effect of SOX9-mediated hepatic fibrosis depends on the expression of lncRNA-H 19 at the animal level,we overexpressed lncRNA-H 19 in mouse liver,and then analyzed the effect of SOX9ASO on CCl4-induced liver fibrosis in mice.The experimental results showed that knockdown of SOX9 did not inhibit the occurrence of hepatic fibrosis after the overexpression of lncRNA-H 19 in hepatocyte,indicating that the role of SOX9 in regulating liver fibrosis depends on the expression of lncRNA-H19.Finally,we performed RNA-sequence analysis of AML 12 cells that stably overexpressed lncRNA-H19.The results showed that there are indeed many deregulated genes involved in biological processes,cell components,and molecular functions after stable expression of lncRNA-H19 in hepatocytess.Taken together,the results of this study illustrate that during hepatic fibrosis,SOX9 increase in hepatocytes promotes the expression lncRNA-H 19 that inhibits hepatocytes proliferation and promotes hepatocytes apoptosis and necrosis during liver fibrosis.