The Effect Of Circular Rnas On Chicken Embryonic Skeletal Muscle Development

Embryonic growth and development of chicken skeletal muscle is a major determinant of muscle mass,and muscle mass directly affects the performance of broiler.The growth and development of skeletal muscle involves a series of very complex biological processes which are regulated by many genes and non-coding RNAs.Circular RNA was found as a novel class of functional biomacromolecule with special ring structure in recent years.It plays an important role in many biological processes and diseases.However,its functions on the growth and development of poultry skeletal muscle have not been reported yet.In this study,we used Xinghua chicken skeletal muscle at embryonic age(E)11,E16,and 1-day post hatch(P1)for circular RNA sequencing,and analyzed the expression of circular RNA in chicken muscle tissues.We screened the critical circular RNAs during chicken embryo skeletal muscle growth and differentiation,and analyze their interactions with genes or miRNAs.At the same time,we performed proteomic analysis on the chicken skeletal muscle by iTRAQ.We identified the differentially expressed proteins during skeletal muscle embryonic development,and analyzed their functional networks.Furthermore,these proteome data were performed integrated analysis with transcriptome and circular RNA sequencing data,to construct an integrate interaction network between protein,lncRNA and circular RNA,provide a more comprehensive insight into molecular regulatory pathway of chicken muscle development.The major results in the present study were showed as follows.A total of 13,377 circRNAs were identified in skeletal muscle of Xinghua chickens.Among them,11,064 circRNAs are derived from coding genes(numbers = 3,589),and most of these circRNAs are from exonic sequences,especially from coding sequences(numbers = 8,110).There were 3036 circular RNA expressed abundantly(BSRP > 1)in skeletal muscle.Comparing the three different developmental stages of leg muscle at E11,E16 and P1,a total of 1470 differentially expressed circular RNAs were identified.GO analysis of the parternal genes of these circular RNAs found that they were enriched in multiple muscle related biological processes.The miRNA binding site analysis of exon circular RNA revealed that 946 circular RNAs contained one or more miRNA binding sites,involving a total of 150 known miRNAs.We found that both chicken circRBFOX2.2-3 and circRBFOX2.2-4 interacted with miRNA-206,promoted myoblast proliferation and inhibited myoblast differentiation;circSVIL.6-14 interacted with miRNA-203,promotes myoblast proliferation and differentiation.We identified 3,240 proteins in E11,E16 and P1 of chicken leg muscle by proteomic analysis,and 491 of them were differentially expressed proteins.Protein interaction network analysis indicated that these differentially expressed proteins were mainly involved in the pathway of protein synthesis,muscle contraction and oxidative phosphorylation.A small number of differential proteins may be regulated by lncRNA and circRNA,especially in the process of muscle contraction,such as DMD,MYL3,TNNI2,TNNT3 and TPMs.These differentially expressed proteins which regulated by their corresponding lncRNA and together take part in the regulation of muscle development.We cloned the chicken MEF2 D gene and identified five novel variant transcripts.These transcripts contain highly conserved sequences,including the MADS-Box and MEF2-Domain functional regions.The MEF2D-V4 is specific expressed in muscle tiuesses,and its expression was regulated by RBFOX2 gene.The MEF2D-V4 could promote the differentiation of chicken myoblasts.The SNP g.36195C> T of MEF2 D gene was significantly associated with multiple growth traits in chickens,especially at ages of 0-4 weeks.In conclusion,this study found that circular RNAs are abundant and dynamically expressed during embryoinc skeletal muscle development in chickens.Chicken circular RNA can regulate myoblast proliferation and differentiation through interacted with miRNAs,thus affecting the growth and development of embryonic skeletal muscle.At the same time,we analyzed the functional interaction network between differentially expressed proteins and non-coding RNAs in embryonic muscle,and thus provided a more comprehensive molecular regulation network for growth and development of chicken embryonic skeletal muscle.