Regulatory Mechanism Of CircUBE3A And DNA Methylation On Skeletal Muscle Development Of Mutton Sheep

Skeletal muscle development of livestock is a key process in individual growth and development,which has an economic impact on animals and it is the focus of continuous attention and research in the breeding industry.The growth and development of skeletal muscle is the process of myoblast proliferation,differentiation and fusion into muscle fibers.These processes are not only regulated by a network of known myogenic regulatory factors,but also complexly regulated by epigenetic factors.However,the molecular mechanism remains unclear.Therefore,it is very important to study the epigenetic regulation mechanism of skeletal muscle development to accelerate the breeding process of mutton sheep.In recent years,with the rapid development of high-throughput sequencing technology and bioinformatics,the mechanism of circular RNA(circRNA)and DNA methylation modification involved in the regulation of skeletal muscle growth and development has attracted increasing attention.Therefore,first,this study took the longissimus dorsi muscle of Haimen goat as the research object.using RNA-seq technology,we studied the genomic characteristics and expression patterns of circRNAs in the longissimus dorsi muscle of 75-day-old fetal and 1-day-old lambs,and screened out the differentially and highly expressed circRNA circUBE3 A in muscle tissue.The role and molecular mechanism of circUBE3 A regulating goat myoblast proliferation and differentiation were verified by bioinformatics analysis,dual-luciferase reporter vector etc.Second,genome-wide DNA methylation analysis was performed on the longissimus dorsi of 110-day-old sheep and 2-year-old Hu sheep by WGBS.This project aims at the frontier of the subject and explores the regulation mechanism of goat muscle development,which has important practical significance for accelerating the breeding and growth regulation of goat.The main content includes the following five parts:1.Differential expression and bioinformatics analysis of circRNAs in goat skeletal muscle at different developmental stagesIn this study,high-throughput sequencing technology and bioinformatics methods were used to analyze the expression patterns and changes of circRNAs in the longissimus dorsi muscle of two key developmental stages(fetal and lamb)of goats.The results showed that a total of 25,500 circRNAs were identified in the longissimus dorsi of fetal and newborn lambs;more than 89% of circRNAs contained exon sequences,and the transcription level of circRNAs in fetal longissimus dorsi musclewas higher than that of the lamb group.Compared with fetal,a total of 831 differentially expressed circRNAs were identified in lamb skeletal muscles,including 486 up-regulated circRNAs and 345down-regulated circRNAs.GO and KEGG functional enrichment analysis found that these differential circRNAs were enriched in 97 GO terms and 99 KEGG pathways,respectively.q RT-PCR verified the authenticity of the sequencing results,and the highly expressed and differentially expressed circUBE3 A in skeletal muscle was selected as a candidate circRNA.Then,its role and mechanism in goat skeletal muscle development were further explored.In conclusion,this study mapped the expression pattern of circRNAs during goat muscle development,laying a foundation for the subsequent in-depth exploration of the molecular mechanism of circRNAs regulating goat skeletal muscle development.2.Effects of circUBE3 A on the Proliferation and differentiation of goat myoblastsIn this experiment,goat myoblasts were used as the research object,and circUBE3 A and its localization in myoblasts were identified for the first time.Second,circUBE3 A siRNAs were transfected into goat myoblasts to explore their effects on the proliferation and differentiation of goat myoblasts.The results showed that circUBE3 A was generated by circularization of exons 2-4 of the UBE3 A gene,with a full length of 229 bp.The authenticity of circUBE3 A was confirmed by RNase digestion and Sanger sequencing.TheRNA-FISH assay found that circUBE3 A was present in the nucleus and cytoplasm.The results of CCK-8,Ed U,cell cycle analysis,etc.,showed that si-circUBE3 A inhibited the proliferation activity of goat myoblasts and the expression of proliferation marker gene PCNA and inhibited the expression of myoblast myotubes and the differentiation marker genes Myo D,Myo G and My HC.In conclusion,interfering with circUBE3 A inhibited the proliferation and differentiation of goat myoblasts.3.circUBE3 A as a molecular sponge of miR-28-5p regulates the proliferation and differentiation of goat myoblastsThis part mainly explores the molecular mechanism of circUBE3 A promoting the proliferation and differentiation of goat myoblasts.Given that circUBE3 A is formed by the circularization of exons and expressed in the cytoplasm of goat myoblasts,it is suggested that circUBE3 A may act as a molecular sponge of miRNA to regulate the proliferation and differentiation of goat myoblasts.Analysis using Target Scan and miRanda software found that circUBE3 A has potential binding sites with eight miRNAs,and the dual-luciferase reporter system found that only miR-28-5p had a targeted binding relationship with circUBE3 A.The expression of miR-28-5p in the longissimus dorsi of lamb was higher than that in fetal,and it was highly conserved among different species.miR-28-5p mimics inhibited the expression level of circUBE3 A,interfered with miR-28-5p to promote the expression of circUBE3 A,and the expression of miR-28-5p was negatively correlated with circUBE3 A.miR-28-5p mimics can inhibit the viability of myoblasts and the expression of the mRNA and protein of PCNA,a marker gene of proliferation.The proportion of cells in G1 phase increased,and the proportion of cells in S phase decreased,indicating that miR-28-5p mimics inhibited cell viability and suppressed cell viability and cycle progression;miR-28-5p inhibitor promotes cell viability and the expression of genes related to proliferation.miR-28-5p mimics inhibited myotube formation and the expression of myoblast differentiation marker genes Myo D,Myo G and My HC at mRNA and protein levels;miR-28-5p inhibitor promoted myotube formation and the expression of myoblast differentiation marker genes,indicating that miR-28-5p inhibits goat myoblast differentiation.In addition,in goat myoblasts,transfection of miR-28-5p mimics and induction of differentiation for three days,increased the mRNA levels of Bax and Beclin 1 and the ratio of Bax/Bcl2,but decreased the expression of Caspase9,Bcl2 and P62,indicating that miR-28-5p promotes apoptosis and inhibits autophagy during goat myoblast differentiation.In the co-transfection experiment of miR-28-5p inhibitor and si-circUBE3 A,it was found that interfering with miR-28-5p could promote the proliferation and differentiation of goat myoblasts,but this effect could be alleviated when circUBE3 A was simultaneously interfered.The above results showed that circUBE3 A promoted the proliferation and differentiation of goat myoblasts by competitively binding to miR-28-5p.4.circUBE3 A regulates HADHB by adsorbing miR-28-5pmiRNAs typically target the 3’ untranslated region(UTR)of a specific mRNA and regulate its stability or translation.This experiment aimed to further explore the mechanism of circUBE3 A promoting the proliferation and differentiation of goat myoblasts by adsorbing miR-28-5p.First,using RNAhybird and Target Scan databases,combined with mRNA sequencing data of two key stages of muscle development in goats for comprehensive analysis,the downstream targets of miR-28-5p were screened out,and it was confirmed by dual luciferase reporter assay that miR-28-5p could target and bind to the3’UTR of HADHB.Then,in goat myoblasts,overexpression of miR-28-5p significantly inhibited the expression of HADHB,while knockdown of miR-28-5p could induce high expression of HADHB,further indicating that miR-28-5p can target and regulate the expression of HADHB in myoblasts.In addition,knockdown of circUBE3 A could significantly inhibit the expression levels of HADHB mRNA and protein;co-transfection of circUBE3 A siRNA and miR-28-5p inhibitor into goat myoblasts showed that interfering with circUBE3 A could inhibit the expression of HADHB induced by miR-28-5p inhibitor,indicating that miR-28-5p could mediate the regulation of HADHB by circUBE3 A.Finally,interfering with HADHB in goat myoblasts attenuated the promoting effect of miR-28-5p inhibitor on the number of Ed U positive cells,cell proliferation coefficient and PCNA expression level;moreover,transfection of miR-28-5p inhibitor or co-transfection of miR-28-5p inhibitor and si-HADHB in goat myoblasts,and induction of myogenic differentiation for three days,it was found that interfering with HADHB can effectively restore the increased mRNA and protein expression levels of Myo D,Myo G and My HC and myotube formation induced by miR-28-5p inhibitor.The above results indicated that circUBE3 A could regulate the expression level of HADHB by targeting miR-28-5p,thereby promoting the proliferation and differentiation of goat myoblasts.5.Analysis of DNA Methylation Profiles During Sheep Skeletal Muscle Development Using Whole-Genome Bisulfite SequencingDNA methylation is an epigenetic regulatory form that plays an important role in regulating the gene expression and the tissues development.However,DNA methylation regulators involved in sheep muscle development remain unclear.To explore the functional importance of genome-scale DNA methylation during sheep muscle growth,this study systematically investigated the genome-wide DNA methylation profiles at key stages of Hu sheep developmental(fetus and adult)using deep whole-genome bisulfite sequencing(WGBS).Our study found that the expression levels of DNA methyltransferase(DNMT)-related genes were lower in fetal muscle than in the muscle of adults.The methylation levels in the CG context were higher than those in the CHG and CHH contexts,and methylation levels were highest in introns,followed by exons and downstream regions.Subsequently,we identified 48491,17,and 135 differentially methylated regions(DMRs)in the CG,CHG,and CHH sequence contexts and 11522 differentially methylated genes(DMGs).The results of bisulfite sequencing PCR(BSP)correlated well with the WGBS-Seq data.Moreover,Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)functional annotation analysis revealed that some DMGs were involved in regulating skeletal muscle development and fatty acid metabolism.By combining the WGBS-Seq and previous RNA-Seq data,a total of 159 overlap genes were obtained between differentially expressed genes(DEGs)and DMGs(FPKM >10 and fold change >4).Finally,we found that 9 DMGs were likely to be involved in muscle growth and metabolism of Hu sheep.We systemically studied the global DNA methylation patterns of fetal and adult muscle development in Hu sheep,which provided new insights into a better understanding of the epigenetic regulation of sheep muscle development.