The Role And Molecular Mechanism Of Long Noncoding RNA SYISL In The Regulation Of Skeletal Muscle Growth And Development

Skeletal muscle is an important part of animal organisms,the muscle growth rate and quantity of are one of the economically important traits in animal production.Skeletal muscle development is a complex process,which is regulated by various transcription factors and epigenetic regulators.Therefore,it is of great theoretical and practical value to analyze the regulatory network of muscle growth and development.Long noncoding RNAs(lncRNAs)are defined sequences as more than 200 nt and with no protein-coding capacity,and play an important function in diverse biological processes.While numerous lncRNAs have been identified in muscle,most of their roles in myogenesis remain unclear,and many more lncRNAs have yet to be identified.Based on the previous study,we systematically characterized the expression profiles of lncRNAs during C2C12 cells myogenic differentiation,and identified a muscle-enriched lncRNA-AK004418,that is up-regulated during myogenic differentiation.AK004418 is transcribed from the fourth intron of the SYNPO2 gene,we named it SYISL(SYNPO2 intron sense-overlapping lncRNA).In present study,we investigated the role and molecular mechanism of lncRNA SYISL in the regulation of skeletal muscle growth through the SYISL knockout mice model and cell experiments.The results are as follows:1.qPCR results showed that SYISL was highly expressed in muscle tissue,including leg muscle,longissimus dorsi and tongue.The expression of SYISL was significantly up-regulated during C2C12 cell differentiation.Luciferase reporter assay and ChIP results found that MyoD could specifically bind to the E-box between-200 bp and +200bp of SYISL promoter,suggesting the expression of SYISL is regulated by MyoD.2.To determine the role of SYISL in muscle development at the individual animal level,we used CRISPR-Cas9 mediated genome editing to generate SYISL heterozygous knockout mice.The founder heterozygous mice were randomly hybridized to get SYISL homozygous knockout mice(SYISL KO mice).SYISL KO mice were healthy and manifested no fignificant differences in weight and the growth rate compared with WT mice.SYISL knockout in mice significantly increases muscle mass and muscle fiber density,decrease the cross-sectional areas of muscle fibers.We isolated skeletal muscle stallite cells from the leg muscle of WT and SYISL KO mice,respectively,and found that SYISL KO in myoblasts significantly decreased cell proliferation and fusion,promoted primary myoblasts differentiation.3.The results of RNA pulldown and RIP assays showed that SYISL directly interacts with EZH2 protein,and the full-length sequence of SYISL is required for its physical interaction with EZH2 to exert its function.ChIP-qPCR and cotransfection experiments showed that SYISL inhibits myogenic differentiation through its recruitment of PRC2 to promoter of myogenic genes MyoG,Myh4 and MCK,leading to H3K27me3 and epigentic silencing of myogenic genes.Moreover,SYISL promotes myoblasts proliferation through recruitment PRC2 to promoter of p21 gene,leading to H3K27me3 and epigentic silencing of p21 gene.4.CHIRP-qPCR results showed that SYISL could physically bind to the promoters of target genes.Next,EZH2 gene overexpression experiments were performed in primary myoblasts from WT and KO mice,and the results showed that EZH2 overexpression significantly inhibited p21,MyoG,Myh4 and MCK expression and increased CDK6 expression in WT primary myoblasts,while no significantly differences for the expression levels of these genes were detected in KO primary myoblasts.These results indicated that SYISL is required for EZH2-mediated epigentic silencing of muscle-specific genes and cell cycle genes.5.Conservation analysis of SYISL in pig genome showed that pig lncRNA AK238214 is transcribed from the first intron of the SYNPO2 genes.The full-length cDNA sequence of AK238214 was obtained by RACE assays.AK238214 was highly expressed in muscle tissue,and the expression of AK238214 increased during myogenic differentiation.AK238214 was mianly located in nucleus of proliferating(D0)and differentiated 3 days(D3)primary myoblasts.qPCR,Western blot,EdU staining and immunofluorescence were used to detect the role of AK238214 in proliferation and differentiation of primary myoblasts.The results showed that knockdown of AK238214 significantly inhibits cell proliferation,but promotes myoblasts differentiation.In conclusion,lncRNA SYISL could inhibit myoblasts differentiation and decreased muscle mass through interacting with PRC2.Meanwhile,we identified a homologous lncRNA AK238214 in pig genome,and confirmed the role of AK238214 in pig myoblasts proliferation and differentiation.Our study provides a novel regulator and melocular mechanism for the regulation of muscle growth and development,and is of great significance to deeply analyze the molecular regulatory network of muscle growth and development.