The Role Of Transcriptional Coactivator MED1 In CC1₄-induced Liver Fibrosis

OBJECTIVE:Liver fibrosis is the result of dynamic and highly integrated cells to chronic liver injury.It is the result of excessive accumulation of scar tissue caused by liver cell inflammation,it will eventually lead to liver cirrhosis and even liver cancer.As a key member of the Mediator complex,MED1 interacts with transcription factors to promote the formation of transcription initiation complex,control the activity and extension of Pol II,and involvement the regulation of histone code,high-level structure of chromatin and phase separation.It has been reported that MED1 plays an important role in liver regeneration and lipid metabolism but its role and mechanism in liver fibrosis is not yet clear.In order to study the role and molecular mechanism of MED1 in liver fibrosis,we used the GEO database to screen liver fibrosis samples in mice,and GEO2R to perform quality control,filtration,and differential expression analysis in 10 samples of health and disease.After determining the average fluorescence value of MED1expressed in the control group and the experimental group,we used the liver MED1specific knockout mouse as a model and injected CCl₄ to produce fibrosis in mouse liver.By using H&E staining,immunohistochemistry,RT-PCR and Western Blot we explored the role and molecular pathway of MED1 in CCl₄-induced liver fibrosis.METHODS:1.Using the GEO database to analyze the expression of MED1 in liver fibrosisUsing NCBI?https://www.ncbi.nlm.nih.gov/?GEO database and taking liver fibrosis as a keyword to screen gene expression data?Mus musculus?,obtained?GSE55747?10 samples of health and disease status.Use GEO2R to perform quality control,filtering and differential expression analysis on these 10 samples.2.Generation of MED1 specific knockout mouse liver fibrosis model MED1^fl/fl wild type mice were hybridized with MED1^?liv knockout mice to obtain the offspring.The tail at 3 weeks mice were cut to extract genomic DNA.PCR was used to obtain 401 bp of cre+in MED1^?liv mice,others were MED1^fl/fl mice.At the 8th week of age,CCl₄ was injected intraperitonealy continuously for 6 weeks,3 times a week,to generate liver fibrosis model.3.Biochemical and pathological testingAST and ALT were used to detect the level of transaminase in MED1^?liv and MED1^fl/fll/fl mice.Liver injury and inflammation were observed by H&E staining.Sirius red and Masson were used to observed collagen deposition.By using Immunohistochemistry we detect the molecular marker of hepatic stellate cell activation??-SMA?.4.Molecular testingRT-PCR and Western Blot were used to detect liver fibrosis molecular markers?IL-1?,TGF-?,MMPs,Colla1,TIMPs,etc.?mRNA and FN-1,Egr1,?-SMA and other protein expression levels.RESULTS:1.GEO data shows that compared with healthy samples,MED1 and its family members in liver fibrosis samples.The fluorescence value increased significantly?P<0.01?,and GO analysis showed that the MED family is involved in the regulation of cell proliferation in liver fibrosis.2.PCR identification showed that the 401bp bands are MED1^?liv mice and the others are MED1^fl/fll/fl mice,Mice were injected with CCl₄ intraperitoneally for the last time,weighed after 48 hours,and then killed.Compared with MED1^fl/fl mice,MED1^?liv had bright liver,soft texture,without solid nodules on the surface,and significant differences in body weight?P<0.01?,indicating liver fibrosis.So there was successful generation of chemically damage mouse model.3.The ALT and AST levels of MED1^?liv were significantly lower than those of MED1^fl/fll/fl mice?P<0.01?,indicating that the liver damage of MED1^?liv mice was reduced.H&E staining of MED1^?liv mice did not form bridge lesions around the central vein,hepatic lobule structure was intact,and hepatocyte inflammation and infiltration increased;Sirius Red and Masson results showed that MED1^?livliv mice collagen deposition was significantly reduced?P<0.01?,indicating that accumulation of ECM in liver of MED1^?livliv mice was reduced and immunohistochemistry further showed that the expression of?-SMA in MED1^?livliv mice was reduced indicating that the activity of hepatic stellate cells was suppressed and liver fibrosis was reduced.4.RT-PCR results showed that the expression levels of MMP2 and MMP13 mRNA and TIMP1 in MED1^?liv mice were significantly reduced indicating that the loss of MED1 affected the expression of matrix metalloproteinases.The expression of TNF?,MIP2,and Colla1 in MED1^?liv mice was decreased,indicating that liver fibrosis in MED^?livmouse is reduced.Western Blot results showed that the expression of TGF?1,?-SMA and FN1 decreased,which further proved that the degree of liver fibrosis in MED1^?livliv mice was lesser.CONCLUSION:In the CCl₄-induced liver fibrosis model,specific loss of MED1 in the liver can inhibit the activation of HSCs and reduce the generation of fibers,which can reduce the degree of liver fibrosis.