Identification And Functional Verification Of Differentially Expressed Genes And Mirnas In Different Stages Of Duck Embryo Leg Muscle Development

Leg muscles and pectoral muscles are important components of waterfowl skeletal muscle.Previous studies showed that leg muscles and pectoral muscles are significantly different in their development and muscle fiber composition.Previous studies have focused on the development characteristics and regulatory mechanisms of the pectoral muscles.However,systematic studies on the molecular mechanism of leg muscle development have not been reported until now.Therefore,in this study,the cherry duck leg muscles of different development stages were used as research materials.Firstly,we analyzed the development of embryonic leg muscles from histological and gene expression levels.Secondly,RNA-seq and miRNA-seq techniques were used to screen the differentially expressed genes(DEGs),differentially expressed miRNAs,and different signaling pathways in three stages of duck leg muscles.Thirdly,the molecular mechanism of miR-33 a which regulates the proliferation of duck myoblasts at different development stages was explored at the cellular level.Finally,the molecular mechanism of Follistatin(FST)which regulates duck myoblast proliferation was explored at the cellular level.The specific results of this study are as follows:(1)The development process of duck leg muscle was divided into myoblast proliferation stage,myotube stage and myofiber stage,and the corresponding development time points of the three stages were E11,E19 and E27.(2)By using the RNA-seq technique,we found that E11 has 1182 DEGs(FDR≤0.01)compared to E19,875 DEGs were significantly up-regulated and 307 DEGs were significantly down-regulated;E19 has 1967 DEGs(FDR≤0.01)compared to E27,1052 DEGs were significantly up-regulated,915 DEGs significantly down-regulated;E11 has 1484 DEGs(FDR≤ 0.01)compared to E27,654 DEGs were significantly up-regulated and 830 DEGs were significantly down-regulated.The results showed that the stage from myotubes to muscle fibers has more DEGs than the stage from myoblasts to myotubes.(3)By using the miRNA-seq technique,we found that E11 has 194 known differentially expressed miRNAs(FDR≤0.01)compared to E19,105 miRNAs were significantly up-regulated and 89 miRNAs were significantly down-regulated;E19 has 550 known differentially expressed miRNAs(FDR≤0.01)compared to E27,156 miRNAs significantly up-regulated and 394 miRNAs significant down-regulated;E11 has 369 known differentially expressed miRNAs(FDR≤0.01)compared to E27,143 miRNAs were significantly up-regulated,226 miRNAs were significantly down-regulated.The results showed that the stage which from myotubes to muscle fibers has more differentially expressed miRNAs than the stage from myoblasts to myotubes.(4)By using the miRNA-seq technique,we also found that a large number of miRNAs were expressed differentially.E11 has 7 novel differentially expressed miRNAs(FDR≤0.01)compared to E19,5 miRNAs were significantly up-regulated,2 miRNAs were significantly down-regulated;E19 has 25 novel differentially expressed miRNAs(FDR≤0.01)compared to E27,3 miRNAs were significantly up-regulated and 22 miRNAs were significantly down-regulated;E11 has 9 novel differentially expressed miRNAs(FDR≤0.01)compared to E27,2 miRNAs were significantly up-regulated and 7 miRNAs were significantly down-regulated.The results showed that the stage of myotubes developed into myofiber had more novel miRNAs than the stage of myoblasts developed into myotubes.The differential miRNAs played an important regulatory role in the development of myotubes into myofibers.(5)From the KEGG pathway analysis results of RNA-seq and miRNA-seq sequencing,we found that the DEGs mainly enriched in MAPK signaling,cell cycle signaling and p53 signaling;the mRNA expression levels of nine genes including Zinc finger protein RFP-like 1,DNAJC6,ZSWIM2,CDKL2,CDK14,CCNB3,RRM2,CCNB2 and cytochrome c-like on p53 signaling pathway were verified by real-time PCR,the results are consistent with transcriptome sequencing results.The results implying that these three signaling pathways play an important role in regulating the development of leg muscle.(6)The results of miRNA-seq revealed that miR-33 a was significantly expressed at the three time points of myofiber development,and was highest expressed in the proliferation phase of myoblasts.In order to study the mechanism of miR-33 a in myoblast proliferation,miR-33 a was over-expressed and interference.Results shown that miR-33 a can inhibit the proliferation of duck myoblasts through targeted IGF-1,Follistatin(FST)and CCND1 by regulating PI3K/Akt/mTOR signaling pathway.(7)From the result of overexpressing FST,we found that overexpression of FST has the ability to increase myoblast activity and promote myoblast proliferation.The mechanism of FST promoting the proliferation of duck myoblasts is mainly through activation of PI3K/Akt/mTOR signaling pathway.In summary,from the level of tissue and cells,this study investigated the differences of gene expression and miRNAs expression at different stages of duck leg muscle development,and discussed the key regulatory genes,miRNAs signaling pathway which affects the development of duck leg muscles.The above studies will lay a theoretical foundation for systematically elaborating the molecular mechanism of skeletal muscle development.