Identification of miR-4483 target gene and study on its mechanism of anti fiver fibrosis

Abstract

Objective To analyze and identify the target regulation effect of miR-4483 on TIMP-1 expression levels,and study the anti liver fibrosis mechanism of miR-4483.Methods The target gene of miR-4483 was TIMP-1,predicted by bioinformatics method.The wild-type and mutant recombinant plasmids(pGL3-TIMP-1-wt and pGL3-TIMP-1-mut) were constructed by overlap extension PCR of target gene 3 ' UTR fragment cloning into the vector pGL3-M,which were co-transfected into HSC-T6 cells with the miRNA-4483 mimics and pRL-TK for detection of luciferase expession.Real-time RT-PCR and Western blot were performed to examine the mRNA and protein expression levels of TIMP-1 after miRNA-4483 mimics transfected into HSC-T6 cells.Results The recombinant plasmid was confirmed by restriction enzyme digestion and sequencing.The co-transfected miRNA-4483 mimics can down-regulate the luciferase activity of the wild-type plasmids(P ＜0.05),but can not influence the luciferase activity of the mutant-type plasmids.There was mo significant difference in the luciferase activity between the wild-type plasmids and mutant plasmids co-transfected with miR-NC (P ＞ 0.05).Up-regulation of miR-4483 levels can dramatically suppress the TIMP-1 mRNA and protein expression levels in HSC-T6 cells(P ＜ 0.05).Conclusions TIMP-1 is the target gene of miR-4483,amd miR-4483 has anti liver fibrosis effect by down-regulation of TIMP-1 expession levels. Key words: miR-4483 ; Hepatic fibrosis; TIMP-1 ; Luciferase report