Research On The Regulatory Mechanism Of MicroRNAs Down-regulate The Expression Of Gremlin1in Activated HSC

Objective: miRNAs play a critical role in regulation the progression of development,cell differentiation, apoptosis and organ developing. Previous studies of our group hadvalidated that down-regulating the expression of Gremlin1could suppress the activation ofHSC or reverse hepatic fibrosis induced by CCL4in SD rat. Our objective is to screenmiRNAs which may suppress the expression of Gremlin1in HSC-T6and decide theirability to inhibit the activation of HSC.Methods:(1) Applying bioinformatic software to screen miRNAs that could suppressthe expression of Gremlin1;(2) Overall and section length of3′UTR of Gremlin1mRNAare transfected into HSC-T6by luciferase reporter gene analysis, respectively. Luciferaseactivity values is used to screen which segment of Gremlin1mRNA is the binding site withmiRNAs that can reduce the expression of Gremlin1.(3) Double screening decidemiRNAs may reduce the expression of Gremlin1by bioinformatic software and luciferasereporter gene analysis.(4) After synthesizing screened miRNA Mimics, we evaluated theirtransfection efficiency when these miRNA Mimics were transfected into HSC-T6withfluorescent tag. Western blotting is used to certificate whether the miRNA have the effectthat can down-regulate the expression of Gremlin1and fibrosis related gene in activatedHSC, such as α-SMA, TGF-β, col1agen after miRNA Mimics are transfects intoHSC-T6.Results:(1) There are6types of conserved miRNAs that may suppresse theexpression of Gremlin1by applying bioinformatic software.（2）Luciferase reporter geneanalysis have screened the binding site with miRNA that can be down-regulated by somemiRNAs.(3) Four conservative miRNAs that can reduce the expression of Gremlin1havebeen filtrated by bioinformatic software and luciferase reporter gene analysis, includingmiR-23a/b, miR-27a/b, miR-181a/b/c/d, miR-182.(4) Successful screened miRNA mimichad been synthesized by Guangzhou ribobio company and then they are transfected intoHSC-T6showed that miR-23b-3p, miR-27b and miR-182can suppress the expression ofGremlin1and miR-181b-1-3p can reduce the expression of col1a1and colla2（.5）miR-27bcan reduce the protein expression of Gremlin1, α-SMA, TGF-β, Col1α1and Col1α2in HSC-T6.Conclusion: Gremlin1promote the activation of HSC-T6by regulated theBMP-7/TGF-β-Smad signaling pathways, thus, suppressing the expression of Gremlin1 can inhibit the activation of HSC-T6. MiR-27b, miR-23b-3p and miR-182can reduce theexpression of Gremlin1and miR-27b can inhibit the activation of HSC-T6.