Role And Mechanism Of LincRNA-ROR In Regulating HERC5-mediated ISG Modification Of P53 Protein Through MiR-145/ZEB2 In Liver Fibrosis

Objective:Liver fibrosis is a pathological process,which is the abnormal proliferation of connective tissue in the liver caused by various etiologies.The process of liver fibrosis exists in the process of repair and healing after all liver injuries,and the continuous process of fibrosis will develop into liver cirrhosis.Hepatic stellate cells are the main effector target cells for hepatic fibrosis therapy,and their activation and abnormal proliferation lead to massive extracellular matrix deposition.p53 has a wide range of effects on the biological functions of HSCs,the key cells in the formation of liver fibrosis.Regulation of p53 gene function is an effective way to treat liver fibrosis.The stability of p53 protein is a prerequisite for its function,and the ISG modification mediated by HREC5 can regulate the level of p53 protein in the tumor state;but in the pathogenesis of liver fibrosis and the process of HSC from quiescence to activation,whether the existence of ISGylation and the degradation pathway of p53 protein dependent on ISGylation are still unclear.In this paper,we study the changes of ISG15 and its modification system in the formation of liver fibrosis from the animal and cell levels,reveal the degradation pathway of p53 protein dependent on ISG in the course of liver fibrosis,and explore the regulation of p53 by LINC-ROR through miR-145/ZEB2 the mechanism of protein ISG modification involved in the course of hepatic fibrosis opens up a new way for the prevention and treatment of hepatic fibrosis.Methods:In vitro and in vivo models of liver fibrosis were established by CCl4-induced mice and TGF-β1 stimulated LX2 cells,and mice with liver-specific expression of miR-145 were established by AAV9-miR-145;pathology was observed by HE staining,Sirius red staining,Masson staining and immunohistochemistry;serum alanine aminotransferase（ALT）and aspartate aminotransferase（AST）activities were measured to evaluate liver injury;the content of hyaluronic acid（HA）,one of the evaluation indicators of serum liver fibrosis,was measured by ELISA;the protein expression of ZEB2,ISG15,HERC5,P53,α-SAM and Collal was detected by WB;the expression of p53,LINC-ROR and miR-145 was detected by RT-qPCR;the co-localization of α-SMA with ZEB2 and HERC5 was detected by immunofluorescence double staining;cell proliferation was detected by CCK-8 and EDU,and the relationship between LINC-ROR and miR-145 was explored by dual-luciferase gene report;the interaction of ZEB2 and HERC5 was detected by Co-IP;Results:1.In CC14-induced mouse liver fibrosis and TGF-β1-stimulated LX-2 cells,the protein expressions of ISG15,HERC5,α-SAM and Collal were significantly increased,and the protein expression of P53 was significantly decreased.HERC5 siRNA significantly increased the expression of p53 protein and decreased the protein expression of α-SAM and Collal.2.Co-immunoprecipitation and protein analysis showed the interaction between HERC5 and ZEB2.ZEB2 siRNA significantly reduced the protein expression of HERC5,α-SAM and Collal and increased the expression of p53 protein.In LX-2 cells,miR-145 mimics significantly reduced the protein expression of ZEB2,HERC5,α-SAM and Collal,increased the expression of p53 protein,and inhibited the proliferation of HSC;the effect of miR-145 inhibitor was similar to that of miR145 mimics are the opposite.In mice with liver-specific overexpression of miR-145,the protein expressions of ZEB2 and HERC5 were significantly inhibited,the expression of p53 protein was significantly increased,and liver fibrosis markers and liver injury were significantly inhibited.3.In LX-2 cells,after silencing LINC-ROR,the mRNA level of miR-145 was significantly increased,the protein expressions of ZEB2,HERC5,α-SAM and Collal were significantly inhibited,the expression of p53 protein was significantly increased,and the proliferation of HSCs capacity is significantly reduced.Overexpression of LINC-ROR results in the opposite of silencing LINC-ROR.Conclusion:The role and mechanism of lincRNA-ROR regulating p53 protein ISG modification through miR-145/ZEB2 in liver fibrosis.