The Mechanism Of MiR-23b/27b/24-1 Gene Cluster Regulating HSC-T6 Activation By Genes Related To Hepatic Stellate Cell Activation

Background Liver fibrosis is a chronic reversible wound healing reaction caused by various injury factors,which eventually leads to the pathological process of cirrhosis and even liver cancer,and is mainly characterized by massive deposition of extracellular matrix.The proliferation and activation of hepatic stellate cells play a central role in the occurrence and development of hepatic fibrosis,and anti-fibrosis therapy targeting activated hepatic stellate cells has become a hot spot in this field.Objective Our research group previously demonstrated that the degree of liver fibrosis was significantly reduced after the overexpression of miR-23b/27b/24-1 gene cluster through in vivo experiments in liver fibrosis modeling mice,and it could exert its anti-fibrosis effect in vivo and inhibit the liver fibrosis induced by CCl₄in mice.In this study,molecular biological techniques were used to confirm that miR-23b/27b/24-1 gene cluster could effectively down-regulate the expression of TGFβ2,Gremlin1,ITGα2,ITGα5 and other target genes of liver fibrosis by the high expression of miR-23b/27b/24-1 gene cluster in the protein level of rat hepatic stellate cell line（HSC-T6）.The specific mechanism of miR-23b/27b/24-1 gene cluster inhibiting HSC-T6 activation was studied by analyzing RNA-seq data of rat primary hepatic stellate cells（HSCs）.Methods（1）By analyzing the data of six types of differentially expressed genes related to liver fibrosis in the gene expression of 18000 m RNAs of the activated primary HSCs infected by p CDH-miR-23b/27b/24-1 lentivirus particles activated by TGF-β1 stimulation using RNA-seq technique,through the online enrichment analysis results for the enrichment of David Gene Ontology analysis of differentially expressed genes function of enrichment of clustering,KEGG looking for signaling pathway enrichment analysis;The different gene sets were obtained through the web tool String,and the functional modules were constructed by clustering with the MCODE plug-in.The Cyto Hubba plug-in was used to calculate the degree of interaction between nodes.Genemania protein interaction software was used to explore the correlation between different proteins to study the specific mechanism of action.（2）The plasmid p CDNA3.1-ALK5（a continuously activated receptor expressing TGF-β1）was transfected into HSC-T6 cells instantly with Turbofect transfection reagent.After 24 hours,36hours and 48 hours of culture,the time required to stimulate HSC-T6 tocomplete activation was explored.（3）Instantly transfected p CDH-miR-23b/27b/24-1 plasmid into fully activated HSC-T6.At the same time,transfected p CDH-PURO was set as no-load control group.After overexpression,the expression of target gene protein and the expression of hepatic fibrosis related genes were detected by Western Blot.Results（1）The time required for ALK5 to stimulate HSC-T6 cells to complete activation was about 36 hours.（2）At the level of HSC-T6 protein,miR-23b/27b/24-1 gene cluster stimulated by ALK5 could down-regulate the expression of ITGα2,ITGα5,Grem Lin1and TGFβ2.（3）Compared with the no-load group,the expression of COL1A1,COL3A1,COL1A2,COL4A5,COL4A4,COL4A2,COL5A3,COL5A2,COL5A1,COL6A1,COL6A3,COL6A2,COL17A1,COL7A1,COL18A1,FBLN5,MFAP4,LUM,FN1,LAMB1 in the collagenous group infected with miR-23b/27b/24-1 was down-regulated,and the expression of VTN was up-regulated.ACVR2A,BMP6,CTGF,DPP7,FZD4,GDF1,GDF6,GDF11,GREM1,GREM2/PRDC,INHBB,NBL1,NEO1,NOG,RGMA,TGFB2,TGFB3,TGFBR1were all down-regulated in TGF-βs.In Intergrin,the expression of ITGA1,ITGA2,ITGA3,ITGA5,ITGA6,ITGA7,ITGA8,ITGA9,ITGA11,ITGAL,ITGAV,ITGAX,ITGB1,ITGB3 and ITGB5 was down-regulated,and the expression of ITGB7 was up-regulated.The expressions of LOX,LOXL1,LOXL2 and LOXL3 were all down-regulated in LOX.MMPs:the expression of MMP was down-regulated in MMP2/8/16/17/19/23 and up-regulated in MMP3/13.TIMPS:TIMP1/2/3 was down-regulated;（4）Through GO enrichment analysis,it was found that miR-23b/27b/24-1 played its role by influencing the binding of integrin and collagen through the integrin-mediated signaling pathway and the adhesion of cell matrix,and by changing the composition of extracellular stroma,extracellular proteins and basement membrane.Through KEGG enrichment analysis,we speculated that miR-23b/27b/24-1 mainly affected the extracellular matter-receptor interaction,local adhesion,PI3K-Akt and other signaling pathways.Through protein interaction analysis,it was found that miR-23b/27b/24-1 gene cluster targeted to down-regulate the expression of its target genes mainly affected the expression of other liver fibrosis related proteins through shared protein domain,co-expression and co-localization.Conclusion miR-23b/27b/24-1 gene cluster can target to down-regulate the expression of ITGα2/5,Grem Lin1 and TGFβ2,and inhibit the activation of HSC-T6 through the extracellular matrix receptor interaction and other signaling pathways.