Regulating Mechanism Of Non-Coding RNA(microRNA、lncRNA、circRNA) In Qinchuan Cattle Myoblasts Proliferation And Differentiation

Skeletal muscle is the most important component of animal body(constitutes 40%-60%of the animal body),and provides structural support and energy storage.Skeletal muscle is an important object in the study of meat quality.The development and growth of skeletal muscle is a complex process,and the regulatory mechanisms underlying the differences in meat quality are largely unknown.In prenatal and very early postnatal development,muscle growth depends on the increased number of muscle fibers(hyperplasia).Once that growth phase has been completed,the fibre numbers would not increase any more but the volume of fibres increases(hypertrophy).These processes influence meat quality after the slaughter of livestock.In fact,various noncoding RNAs have been identified and suggested to regulate myogenesis such as microRNAs and long non-coding RNAs.Many studies show that the regulatory networks center on the myogenetic transcription factor PAX7 and MyoD,and play a key role in the process of muscle generation and muscle regeneration.Recent researches demonstrate that the physiology and pathology of skeletal muscle are profoundly influenced by miRNAs.The majority of lncRNAs are engaging inepigenetic/transcriptional regulation on chromatins via their ability to interact with chromatin regulators.In addition,lncRNAs can also modulate post-transcriptional regulation in myogenesis,for example through acting as sponges of microRNAs(miRNAs)to titrate them away from their target mRNAs thus named as competing endogenous RNAs(ceRNAs).Circular RNAs(circRNAs)have been identified in various tissues and cell types from human,monkey,porcine and mouse.However,knowledge on circRNAs in bovine muscle development is limited.In this study,Ribo-Zero RNA-Seq method was performed to analyze the embryonic and adult musculus longissimus of Qinchuan cattle in whole transcriptome with an unparalleled depth.These experimental techniques of RACE,RNA interfering,double luciferase gene reporter system,RIP,RNA pull down and EdU incorporation were used to explore the regulatory mechanism of lncRNA MDNCR,circRNA circFUT10 and circFGFR4 in muscle cell proliferation and differentiation.Our main results are as follows:1.Identification of lncRNAs in muscle tissue of Qinchuan cattleIn this study,Ribo-Zero RNA-Seq method was performed to analyze the embryonic and adult musculus longissimus of Qinchuan cattle in whole transcriptome with an unparalleled depth.A total of 13,580 candidates were identified,and 4,343 and 76 lncRNAs were expressed only in the embryo and adult,respectively.According to the cuffcompare classes,we found the most of candidates(12,957)aligned to intergenic regions(u).Compared with mRNA,lncRNA has lower level of expression,fewer exons and shorter length of transcript and ORF.The distribution of detected lncRNAs number is not uniform in the chromosomes,but as a whole,the number of reads located in the chromosome increased with the increase of chromosome length.2,944 lncRNAs were significantly different(P < 0.05)between the embryonic stage and adult stage libraries.There were 826 lncRNAs were up-regulated at least 2 fold-change,while 2,095 lncRNAs were down-regulated when comparing adult to embryonic muscle tissue.lncRNA MDNCR was the most down-regulated lncRNA in the adult sample compared to the embryonic sample.2.MDNCR promotes differentiation of myoblasts via sponging miR-133a5’ and 3’ RACE analyses revealed that the full-length of MDNCR was 1,974 nucleotides,and located in bovine chromosome 29 including 5 exons and 4 introns.MDNCR was a muscle-specific lncRNA and up-regulated during differentiation.MDNCR is expressed in cytoplasm and is rarely expressed in the nucleus,suggesting that it may work through the trans mechanism.Overexpression of MDNCR could promote myoblast differentiation,inhibit myoblast proliferation,and promote myoblast apoptosis.The mRNA-miRNA-lncRNA network revealed that MDNCR has multiple binding sites for muscle development related miRNAs,with 32 potential binding sites of miR-133 a.Via luciferase screening,RIP and RNA pull down assays,MDNCR was observed to directly bind to miR-133 a with thirty-two potential binding sites.It is revealed that MDNCR can sponge miR-133 a as a competitive endogenous RNA.Then Ribo-Zero RNA-Seq method was performed to analyze the embryonic and adult musculus longissimus of Qinchuan cattle transcriptome.We analyzed and screened the differentially expressed mRNAs associated with myocyte proliferation and differentiation.Via the verification of bioinformatic prediction,luciferase screening,q PCR and Western blot,it was indicated that GosB is the downstream target gene of MDNCR and miR-133 a.Mechanistically,MDNCR promoted myoblast differentiation by functioning as a ceRNA for miR-133 a,then augmenting the expression of the miR-133 a target gene,GosB,thereby promoting myoblast differentiation and inhibiting myoblast proliferation.3.circFUT10 promotes differentiation of myoblasts via sponging miR-133aThe full-length of circFUT10 was 295 nt,and was named based on its host gene FUT10 in chromosome 27,as a candidate circRNA to further explore its role in cattle muscle development.The tissue expression assay showed that the expression of circFUT10 was high in longissimus muscle,and to be much higher(7.43 fold)at the embryonic stagecompared with the adult stage,suggesting a potential role in muscle development.We found that circFUT10 was expressed predominantly in muscle and weakly expressed in other tissues.Softwares RNAhybrid and Target Scan used for miRNA recognition sequences on bovine circFUT10 revealed the presence of three putative miR-133 a binding sites.Via luciferase screening,RIP and RNA pull down assays,circFUT10 was observed to directly bind to miR-133 a.The expression of circFUT10 is higher in cytoplasm than in the nucleus,suggesting that circFUT10 may regulate gene expression after transcription.The expression of circFUT10 significantly increased in differentiated myoblasts.Mechanistically,circFUT10 promoted myoblast differentiation by functioning as a ceRNA for miR-133 a,thereby promoting myoblast differentiation and inhibiting myoblast proliferation.4.circFGFR4 promotes differentiation of myoblasts via sponging miR-107The full-length of circFGFR4 was 963 nt,and was named based on its host gene FGFR4 in chromosome 7,as a candidate circRNA to further explore its role in cattle muscle development.The tissue expression assay showed that the expression of circFGFR4 was high in longissimus muscle,suggesting a potential role in muscle development.Softwares RNAhybrid and Target Scan used for miRNA recognition sequences on bovine circFGFR4 revealed the presence of eighteen putative miR-107 binding sites.Via luciferase screening and RNA pull down assays,circFGFR4 was observed to directly bind to miR-107.The expression of circFGFR4 is mainly expressed in cytoplasm,suggesting that circFGFR4 may regulate gene expression after transcription.The expression of circFGFR4 significantly increased in differentiated myoblasts.Mechanistically,circFGFR4 promoted myoblast differentiation by functioning as a ceRNA for miR-107,thereby promoting myoblast differentiation and inhibiting myoblast proliferation.In this study,lncRNA and circRNA were screened by high-throughput sequencing,and detected the expression of lncRNA and circRNA,then through ceRNA theory to reveal their regulatory mechanism of muscle differentiation.It is indicated that non-coding RNA plays an important role in the muscle development.