The Role And Mechanism Of Long Non-coding RNA MIAT In Liver Fibrosis

Research objects:Liver fibrosis is a pathological change of liver tissue caused by chronic liver injury,and continuous liver injury will eventually lead to cirrhosis.At present,there are few effective targets and drugs for the diagnosis and treatment of liver fibrosis.Myocardial Infarction-Associated Transcript(MIAT)is a long noncoding RNA that is abnormally expressed in a variety of diseases and plays a role in promoting fibrosis-related diseases such as myocardial fibrosis and renal fibrosis.Whether MIAT plays a role in liver fibrosis has not been reported.Therefore,this study conducted a preliminary study on the role of MIAT in liver fibrosis and related molecular mechanisms,which is of great significance for the diagnosis and treatment of liver fibrosis-related diseases.Methods:At the cellular level,the activation of hepatic stellate cells-LX2 was induced by TGF-β1,and the expression of MIAT was detected by RT-qPCR.In LX2 cells,CRISPR-dCas9 was used to activate the expression of MIAT,and then,the expression of fibrosis-related genes is detected by RT-qPCR and Western blot.siRNA and shRNA were transfected into LX2 cells to knock down the expression of MIAT,and the expression of fibrosis-related marker genes is then tested.In LX2 cells with stable MIAT knockdown,the effect of MIAT knockdown on cell proliferation was detected by CCK8.In terms of mechanism research,through whole transcriptome sequencing of knockdown and overexpression cells,the differentially expressed gene TGFB2 was identified.Next,we use DIANA tools,miRCODE,Starbase,Targetscan,GEO and other bioinformatics platforms to screen out miRNAs related to MIAT and TGFB2,miR-16-5p,and verify the relation among these three genes through the dual luciferase reporter gene experiment.The expression of miR-16-5p in LX2 stimulated by TGF-β1 and TGF-β2 and in LX2 cells overexpressing MIAT were verified by RTqPCR.Subsequently,miR-16-5p was overexpressed in LX2 cells,the expression of MIAT,TGFB2 and liver fibrosis related genes was verified by RT-qPCR,and the effect of miR-16-5p on cell proliferation was detected by CCK8 and SRB.Finally,we use RTqPCR and Western blot to detect the expression of TGFB2 in overexpression and knockdown MIAT LX2 cells,and use RT-qPCR to detect the expression of MIAT in LX2 cells stimulated by TGF-β2,then the regulatory mechanism between MIAT and TGFB2 was verified.Conclusion:The results of cell experiments suggest that MIAT is highly expressed in LX2 stimulated by TGF-β1;overexpression of MIAT will up-regulate the expression of fibrosis-related genes,while knocking down MIAT leads to the opposite result.In addition,knock-down of MIAT inhibits cell proliferation;Reporter gene experiment results show that miR-16-5p has binding sites on MIAT and TGFB2;overexpression of miR-16-5p will down-regulate the expression of MIAT,TGFB2 and fibrosis-related genes,while inhibiting cell proliferation;the expression of miR-16-5p and MIAT are decreased and increased in LX2 cells stimulated by TGF-β2,respectively.Based on the above research results,it is preliminarily elucidated that MIAT can regulate liver fibrosis by endogenously competing with TGFB2 with miR-16-5p,which provides new targets and ideas for the treatment of liver fibrosis.