Hes1 Decoy ODN Inhibits The Activation Of Hepatic Stellate Cells By Interfering With The Regulatory Loop Between Hes1 And MiR-23b/27b/24-1

Objective The study aims at verifying the existence of regulatory ring between Hes1 and miR-23b/27b/24-1 cluster.To explore whether the aptamer AC4 WJF delivers Hes1 decoy ODN into rat hepatic stellate cell line(HSC-T6)and primary rat hepatic stellate cells(HSCs).To investigate the effects of Hes1 decoy ODN on hepatic fibrosis-related genes in HSCs and the inhibition of Hes1 decoy ODN on HSC activation.Methods(1)Targetscan was used to predict the target binding sites of miR-23b/27b/24-1 in Hes1-3′UTR and JARSPAR was used to predict the target binding sites of Hes1 on the promoter of the miR-23b/27b/24-1.(2)The promoter of miR-23b/27b/24-1 was cloned and the luciferase reporter plasmid of p GLuc-miR-23 b cluster promoter was constructed.Luciferase activity was analyzed to explore the target effects of Hes1 on p GLuc-miR-23 b cluster promoter.(3)Luciferase reporter plasmids of p MIR-Luc-Hes1,p MIR-Luc-Hes1-mutant23 b and p MIR-Luc-G3-mutant27 b were constructed by inserting full length rat Hes1-3′UTR or its mutants into p MIR-REPORTTM Luciferase(p MIR-Luc)plasmid respectively.Luciferase activity was analyzed to explore the direct target effects of miR-23 b and miR-27 b on Hes1-3′UTR;Plasmids p CDH-miR-23b/27b/24-1 was constructed,western blot was used to explore the effects of miR-23b/27b/24-1 on Hes1.(4)Confocal Microscope was used to explore whether Aptamer AC4 WJF deliver Decoy ODN into HSC-T6 and primary HSCs which were separated and extracted from elder SD rats,TGF-β1was used to stimulate their activation.(5)Western blot was used to investigate the effects of Hes1 decoy ODN on hepatic fibrosis-related genes in HSC-T6 and primary HSCs.(6)RT-PCR was used to explore the effects of Hes1 decoy ODN on miR-27 b in HSC-T6.Results(1)It was confirmed that Hes1 downregulated the expression of miR-23b/27b/24-1 cluster.(2)It was demonstrated that miR-23b/27b/24-1 downregulated the expression of Hes1 directly.(3)It was confirmed that aptamer AC4 WJF delivered Hes1 decoy ODN into the nucleus of HSC-T6 and primary HSCs which were separated and extracted from elder SD rats,TGF-β1 was used to stimulate their activation.(4)It was confirmed that Hes1 decoy ODN effectively downregulated the expression of Hes1,Smad 2/3,COLIα1,COLIα2,ITGα5,TIMP1 and TGF-β2,and up-regulated the expression of MMP2 and MMP9 in HSC-T6,and effectively downregulated the expression of COLIα1,COLIα2 and ITGα5 in HSC-T6 which activated by TGF-β1.(5)Hes1 decoy ODN effectively downregulated the expression of COLIα1 and ITGα5 in primary HSCs which activated by TGF-β1.(6)Hes1 decoy ODN induced the expression of endogenous miR-27 b in HSC-T6.Conclusion(1)Hes1 down-regulates the activity of miR-23b/27b/24-1 promoter;miR-23b/27b/24-1 down-regulates the expression of Hes1;and there is a negative feedback regulatory loop between the Hes1 and the miR-23b/27b/24-1.(2)Aptamer AC4 WJF delivers Hes1 decoy ODN into the nucleus of HSCs,Hes1 decoy ODN induces the expression of endogenous miR-27 b by interfering with the regulatory loop between the Hes1 and the miR-23b/27b/24-1,and then inhibits the activation of HSC.