Effect Of Long Noncoding RNA NEAT1 On Proliferation And Differentiation Of Myoblasts And Its Epigenetic Regulatory Mechanism

Skeletal muscle is the most abundant tissue in the mammalian body and plays a pivotal role in regulating body metabolism and homeostasis.The growth and development processes of skeletal muscle are complex and delicate,and regulated by many factors.In recent years,many studies have shown that epigenetic regulation plays an important role in skeletal muscle development.Enhancer of zeste homolog 2(EZH2)is the core component of the polycomb repressive complex 2(PRC2),it contains a typical SET domain,and can make the target gene promoter produce H3K27me3 epigenetic inhibition marker,thus inhibiting gene expression.It is reported that EZH2 has a vital role in the growth and development of skeletal muscle.Therefore,RNA immunoprecipitation combined with high throughput sequencing(RIP-seq)was carried out in pig skeletal muscle tissue in our previous study,and a large number of EZH2-binding RNA transcripts were identified.This provides a theoretical basis for EZH2 to regulate the complex network of skeletal muscle in pigs.In recent years,long noncoding RNA(lncRNA)has been reported to play an important role in myogenesis,and many studies have shown that lncRNA can play a regulatory role through EZH2.In this study,we re-analyzed our previous RIP-seq data,and focused on EZH2-binding intergenic lncRNA(linc RNA)transcripts,and found a linc RNA NEAT1,which is conserved in humans,pigs and mice.We studied the effect of NEAT1 on the proliferation and differentiation of myoblasts in pigs,and mice,explored the effect of NEAT1 on postnatal muscle growth and muscle regeneration,and the mechanism of NEAT1 in mice.The main results are as follows:1.Through combined analysis of RIP-seq data and lncRNA-seq data,a total of 356 EZH2-binding novel intergenic lncRNAs(linc RNAs)transcripts were identified.Some EZH2-binding novel linc RNAs from RIP-seq were verified by RIP-PCR experiment.Then though conservative analysis and literature reports,we selected linc RNA NEAT1 for further study,it is conserved in humans,pigs,and mice.2.Using q PCR to detect the expression pattern of mouse Neat1,we found that Neat1 was up-regulated in the early stage of C2C12 cell differentiation and muscle regeneration then down-regulated;Gain-or loss-of-function studies in C2C12 cells demonstrated that Neat1 accelerates myoblast proliferation but suppresses myoblast differentiation and fusion.3.In vivo knockdown of Neat1 by injecting with lentivirus containing Neat1 si RNA fragment into mice hindlimb muscles showed that the volume and weight of the quadriceps,anterior tibial,and gastrocnemius muscles were increased by Neat1 knockdown,the cross-sectional areas of the above muscles were increased after Neat1 knockdown.These results indicated that Neat1 knockdown could promote the growth of postnatal muscles.To detect the effect of Neat1 on muscle regeneration,mice hind limb muscles were injected with lentivirus containing Neat1 si RNA fragment followed by CTX injection.The results showed that Neat1 knockdown could inhibit the repair process after muscle injury.4.The RIP and RNA pulldown results showed that Neat1 could bind to Ezh2;RNA pulldown result of a series of deletions in full-length Neat1 showed that the 1001-1540 fragment of Neat1 full-length was required for its recruitment of Ezh2;overexpression a series of deletions in full-length Neat1 in C2C12 cells,we found that the 1001-1540 fragment of Neat1 full-length was required for its function on myogenesis.The Ch IP-q PCR and co-transfection experiments results showed that Neat1 can recruit Ezh2 to the promoters of p21,Myog,Myh4 and Tnni2 genes,thus increase H3k27me3 accumulation in these gene promoters,and promote myoblast proliferation and inhibit myoblast differentiation.The Ch IRP-q PCR results showed that Neat1 can directly bind to p21,Myog,Myh4 and Tnni2 promoters.5.The full-length sequence of pig NEAT1(p NEAT1)was obtained by RACE technology.The results of nucleocytoplasmic localization showed that p NEAT1 was specifically located in the nucleus.The q PCR result showed that p NEAT1 expression was increased during porcine skeletal muscle satellite cells differentiation.The RNA pulldown assay was used to further verify the interaction between p NEAT1 and Ezh2.Then p NEAT1 si RNA fragment was designed and p NEAT1 gene was cloned to knockdown or overexpressed p NEAT1 expression.Gain-or loss-of-function studies in porcine skeletal muscle satellite cells showed that p NEAT1 could promote the proliferation of porcine skeletal muscle satellite cells and inhibit the differentiation of porcine skeletal muscle satellite cells.6.Ectopic expression of p NEAT1 in C2C12 cells showed that p NEAT1 can promote C2C12 proliferation but inhibit C2C12 differentiation;moreover,mice hind limb muscles injected with lentivirus containing p NEAT1 expression vector decreased the quadriceps,anterior tibial,and gastrocnemius muscles volume,weight and cross-sectional area of mice.These results showed that NEAT1 has a conserved function on myogenesis.In summary,through RIP-seq combined with lncRNA-seq analysis,we found a conserved linc RNA NEAT1 binds to EZH2.This lncRNA can promote myoblast proliferation and inhibit myoblast differentiation in mice and pigs.In vivo,it can inhibit muscle growth and promote muscle regeneration.Mechanically,NEAT1 plays a regulatory role by interacting with EZH2.Altogether,we uncover a previously unknown function of NEAT1 in muscle development and the molecular mechanism by which NEAT1 regulates myogenesis.It is of great significance to understand the complex regulatory network of muscle development.