Identification And Function Analysis Of Skeletal Muscle Growth Related Long Noncoding RNA In Chicken

The genome of eukaryotes can be transcribed into substantial noncoding RNAs and most of them are long noncoding RNA(lncRNA).Lnc RNAs have large numbers and can play different roles in the regulation of multi levels of protein coding gene expressions.Chicken not only is an important model organism for genetic research,but also a kind of important economic animal.However,currently little is known of lncRNAs regulatory mechanism in chicken,and skeletal muscle growth related lnc RNAs are remaining to be explored.The aim of this study was to identify the skeletal muscle growth related lncRNAs and elucidate theirs mechanism.Firstly,we used RNA sequencing to profile the Xinghua chicken(XH)leg muscle transcriptome(lncRNA and mRNA)at three stages of skeletal muscle development in the chicken: embryonic day 11(E11),embryonic day 16(E16),and 1 day after hatching(D1).To identify the skeletal muscle growth related lncRNA,we identified the cis-and trans-regulatory target genes of differentially expressed lncRNAs,and constructed lncRNA-gene interaction networks.Secondly,we predicted the cis-acting and trans-acting target genes of differentially expressed lncRNAs between fast-growing broilers(white recessive rock,WRR)and slow-growing broilers(XH)for construction of lncRNA-mRNA interaction network.Thirdly,we used our published miRNA-seq data to predict the target lncRNAs and mRNAs of differentially expressed miRNAs,and constructed lncRNA-miRNA-mRNA interaction network with lncRNA as competitive endogenous RNA(ceRNA).We proposed the hypothesis that lncIRS1 targets mi R-15 family and hence regulates the expression of IRS1 which is a key gene in IGF-1 signaling pathway.We further studied the role of lncIRS1-mi R-15-IRS1 interaction in muscle growth.The main results were as follows:1.A total of 1995 new lncRNAs were identified based on RNA-seq results drawn from the leg skeletal muscle of XH chicken in embryo age of 11 days and 16 days,as well as 1-day-old.We predicted the target genes of differentially expressed lncRNA,and constructed lncRNA-mRNA network.The function of LncRNA-mRNA network was involved in cellular development,cellular growth and proliferation,embryonic development,organismal development,tissue development,and developmental disorders.LncRNA-mRNA pair,including lnc00003323-TEAD4,lnc00005738-DMD,lnc00073545-DLK1 and lnc00023378-FGF13,could be candidates for the regulation of skeletal muscle development.2.Based on the prediction for the target genes of differentially expressed lncRNAs between WRR and XH chicken with 7 weeks age,we discovered that 64 DE-lncRNAs targeted with 67 differentially expressed genes(DEGs).We further constructed the lncRNA-mRNA interaction network.The function of network was involved in skeletal and muscular disorders,cellular assembly and organization,and cellular function and maintenance.3.We constructed an lncRNA-miRNA-gene interaction network which is related to skeletal muscle growth.LncRNA-mi RNA-gene interaction network contains 1370 pairs of lncRNA-miRNA-gene,involving 45 DE-miRNAs,155 DE-lncRNAs and 30 DEGs.MiR-15 family(miR-15a;miR-15b-5p;miR-15c-5p;miR-16-5p;miR-16c-5p)is the core component of this interaction network with 792 pairs of lncRNA-miRNA-gene.The network genes,including IRS1,IRS2,FOXO1,FOXO3 and FOS,were significantly enriched in IGF-1 signaling pathway.4.Full-length transcripts of IRS1 and lncIRS1 amplified by RACE PCR.IRS1 and lncIRS1 contain the binding site of miR-15 family.By dual-luciferase reporter assays and RNA-binding protein immunoprecipitation,we proved that IRS1 and lncIRS1 were the targets of miR-15 family.Overexpression of lncIRS1 enhanced its “sponge” ability to miR-15 family,and promoted the expression of IRS1.The opposite effect was observed by knockdown of lncIRS1.LncIRS1 could affect myoblasts proliferation and differentiation by regulating IGF-1 signal pathway.Our study constructed “lncRNA-mRNA” network and lncRNA acts as the ceRNA “lncRNA-miRNA-gene” network.These interaction networks were involved in skeletal muscle growth.LncIRS1 “sponged” miR-15 family to promote IRS1 expression and myoblast cells proliferation and differentiation.These networks not only further complements the molecular regulatory pathways that are involved in chicken skeletal muscle development,but also provides theoretical help for improving the growth rate of chicken muscle.