The Regulation Mechanism Of Goat MiR-487b-3p On Skeletal Muscle Myogenesis By Targeting IRS1

In today's life science area,one of the most popular categories research is noncoding RNAs,including microRNAs,long non-coding RNAs and circular RNAs.Among them,the research related to microRNAs were most widely.MicroRNAs are small,highly conserved,endogenous non-coding RNAs that can play critical gene-regulatory roles during skeletal muscle development.Here,based on our previous high-throughput of goat skeletal muscle,RT-qPCR analysis of miR-487b-3p levels were performed in various fetal and mature goat tissues(heart,liver,spleen,lung,kidney,small intestine,longissimus dorsi and leg muscle)to investigate the expression profile of miR-487b-3p in goat tissues,and then transfected the constructed pcDNA3.1(+)-miR-487b-3p plasmid,compounded miR-487b-3p oligos into C2C12 myoblasts,to explore the potential regulation functions of goat mi R-487b-3p during myogenesis.Besides,the function of miR-487b-3p target gene IRS1 was been identified and explored.The main results of this study are as follows:(1)Based on the miRbase and TargetScan,mi R-487b-3p sequence and IRS1 mRNA contained a highly conserved binding site were highly conversed in different species,such as goat,human,mouse,cow and sheep.(2)miR-487b-3p was widely expressed in different tissues of Xuhuai goat at different developmental stages(fetal goat and mature goat);A significant difference in miR-487b-3p expression was observed between muscle tissues from fetal and mature Xuhuai goat,implying that miR-487b-3p is differentially expressed in muscle cells and playing potential roles in skeletal muscle myogenesis.(3)The pc DNA3.1(+)-miR-487b-3p,miR-487b-3p mimics,NC,miR-487b-3p inhibitors and anti-NC were respectively transfected into C2C12 myoblasts using Lipofectamine 2000.The cells then were collected for RT-qPCR and Western blot assay to examine the expression of myogenic proliferation and differentiation marker genes after miR-487b-3p overexpression or inhibition.Besides,CCK-8,EdU assay were also used to investigate the effect of miR-487b-3p overexpression or inhibition on C2C12 cells.Taken together,these results indicated that miR-487b-3p plays a negative role in the myogenic proliferation and differentiation of C2C12 myoblasts.(4)Wild-type or mutant 3'UTR of IRS1,ITGA6 and ZYMND8 were successfully constructed into the dual-luciferase reporters with Xho I and Not I sites.Dual-luciferase reporter assays demonstrated mi R-487b-3p could directly target the 3'UTR of IRS1,ITGA6 and ZYMND8.Bioinformatics analysis(David,KEGG),RT-qPCR,Western blot assays indicated that miR-487b-3p induction repressed IRS1 mRNA and protein expression in C2C12 myoblasts.(5)Through the RNAi,Western blot and EdU assays,we found that IRS1 reduction by transfection with siRNA repressed C2C12 myoblast proliferation and differentiation,consistent with the impact of miR-487b-3p overexpression.(6)IRS1 plays a key role in insulin-Ras-MAPK signaling,which transmit signals from IGF-1 receptors to the intracellular MAPK/ERK and PI3K/AKT pathways.These results reveal a miRNA-related regulatory link between miR-487b-3p and the MAPK/ERK and PI3K/AKT pathways during myogenic proliferation and differentiation in which IRS1 plays a vital role,offering key insights into the role of miR-487b-3p in skeletal muscle myogenesis.