MiR-23b/27b/24-1 Cluster Directly Downregulates The Expression Of Hepatic Fibrosis Related Genes:Smad3,TGFβ2 And ITGα2/5

Objective Hepatic fibrosis is a key pathological process in chronic liver disease.When liver is damaged under the stimulation of inflammation and cytokines,hepatic stellate cells（HSCs）activate and secrete large numbers of extracelluar matix（ECM）mainly collagen,the imbalance between deposition and degradation of ECM result in hepatic fibrosis.Our previous research showed that miR-23b/27/24-1 cluster targets Gremlin1 and LOX directly,it also downregulates the expression of COLIα1,COLIα2 andα-SMA significantly,but the molecular mechanism remains to be explored.This study aims at verifying that hepatic fibrosis related genes could be directly targeted by the miR-23b/27b/24-1 cluster:the targeting effects of miR-23b on TGFβ2,TGFβr2,Smad3and COL4α1;the targeting effects of miR-27b on TGFβr1,Smad2,MMP13,ITGα2 and ITGα5;the targeting effect of miR-24-1 on MMP14.Methods（1）Screening several genes which could be targeted by miR-23b/27b/24-1cluster with the use of bioinformatics.（2）Luciferase reporter plasmids were constructed by inserting parts of 3′UTR which contain the binding sites of miR-23b/27b/24-1 cluster or its mutants into PMIR-REPORT^TMLuciferase plasmid.（3）Luciferase reporter assay was used to screen hepatic fibrosis related genes which could be directly targeted by miR-23b/27b/24-1 cluster.（4）The effects of miR-23b/27b/24-1 cluster on specific target genes need further verification by Western Blot.Results（1）miR-23b downregulates the expression of TGFβ2 and Smad3 directly,miR-23b can’t target TGFβr2 and COL4α1 directly.（2）miR-27b downregulates the expression of ITGα2 and ITGα5 directly,miR-27b can’t target Smad2,TGFβr1 and MMP13 directly.（3）miR-24-1 can’t target MMP14 directly.（4）miR-23b/27b/24-1 cluster downregulates the protein levels of Smad3 and ITGα2 as a whole.Conclusion In HSC-T6,TGFβ2 and Smad3,as the direct targets of miR-23b,can be downregulated by miR-23b;ITGα2 and ITGα5,as the direct targets of miR-27b,can be downregulated by miR-27b;the protein levels of Smad3 and ITGα2 could be downregulated by miR-23b/27b/24-1 cluster;miR-23b/27b/24-1 cluster inhibits the activation of HSC-T6 and its molecular mechanism is related to silence the expression of Smad3,TGFβ2,ITGα2 and ITGα5.