Screening Of Key MRNAs,lncRNAs And MiRNAs Related To Myogenic Differentiation Of Jinghai Yellow Chicken

The meat production potential is closely related to muscle fibers number of livestock and poultry,but during postembryonic period and after birth,the number of muscle fibers were almost fixed.Embryonic myogenesis depends on the proliferation and differentiation of myoblasts.During myogenesis,the degree of myoblast proliferation and differentiation largely determined how many muscle fibers are formed.In order to study the regulation of skeletal muscle development in poultry,especially the process of myoblast differentiated into myotubes,RNA sequencing(RNA-seq)was used to profile chicken primary myoblasts and fused myotubes transcriptomes,messenger RNAs(mRNAs),long non-coding RNAs(lncRNAs),and microRNAs(miRNAs)at four stages of myoblast differentiation.Three main parts of studies were conducted in this thesis:Part one:the primary myoblasts were isolated,cultured and induced to differentiation.RNA-seq was utilized to analyze the expression level of IncRNAs and mRNAs at day 0,1,3,and 5 of differentiation.LncRNAs were identified and their functions during differentiation were predicted using bioinformatics.Moreover,differentially-expressed IncRNAs and mRNAs during differentiation of primary myoblasts were screened and subjected to K-means analysis.1.Of a total of 2,484 IncRNA transcripts,2,285 were long intergenic non-coding RNAs(lincRNAs)and 199 were antisence IncRNAs.The distribution of theses IncRNA transcripts among chicken chromosomes had obvious preference.Notably,the proportion of IncRNAs located in chromosome 1 was the highest compared with other chromosomes.Moreover,the candidate IncRNAs in this study were shorter in both transcript length and ORF length,fewer in exon number than chicken known mRNAs.2.Additionally,1,530 lncRNAs were neighboring 2,041 protein-coding gens(<10 kb upstream and downstream)and the functionally enriched in several pathways related to skeletal muscle development that have been extensively studied,including regulation of actin cytoskeleton,insulin signaling pathway,MAPK signaling pathway,FoxO signaling pathway,and focal adhesion.3.In addition,Pearson' s correlation coefficients demonstrated that 990 lncRNAs and 7,436 mRNAs were possibly in trans-regulatory relationships.These co-expressed mRNAs were enriched in various developmentally-related to myoblast proliferation,differentiation and fusion via cell cycle,focal adhesion,the p53 signaling pathway and extracellular matrix(ECM)-receptor interactions.4.906 differentially-expressed lncRNAs and 4,422 differentially-expressed mRNAs were screened during the differentiation of primary myoblast differentiation.Function annotation showed that differentially-expressed genes were mainly enriched in several developmentally-related biological process,such as skeletal muscle development,mitosis,cell migration and adhesion.And dozens of pathways mainly involved in the process of myogenesis,including ECM-receptor interactions,focal adhesion,DNA replication,cell adhesion molecules(CAM),cell cycle,regulation of actin cytoskeleton,and PPAR signaling pathway.5.Then,4,422 differentially-expressed genes were assigned to four clusters according to K-means analysis.Genes in the K1 cluster likely play important roles in myoblast proliferation or the shift from myoblasts proliferation to differentiation,and those in k4 cluster were likely associated with the initiation of myoblast differentiation,while genes in the K2 and K3 clusters were likely related to myoblast migration,adhesion and fusion.Part two:Differentially-expressed lncRNAs and their corresponding differentially-expressed cis-target genes were used to constructed IncRNA-genes interaction networks and then screen the potential candidate factors that involved in the regulation of chicken skeletal muscle development;Furthermore,we chose the differentially-expressed lncRNAs and mRNAs during myoblasts differentiation as the data input for weight gene co-expressed analysis(WGCNA).Important candidate factors regulating myoblast differentiation were identified from stage-specific modules.1.Differentially-expressed lncRNAs and their corresponding differentially-expressed cis-target genes were used to constructed IncRNA-genes interaction networks.The co-expressed cis-target genes in the network were enriched in several myogenesis-related pathways,including regulation of actin cytoskeleton,ErbB signaling pathway and insulin signaling pathway.SIX2.PAK3?HACDI?SULF1?SOCSl?MFN2?SIK1?CFL2?ACTC1?TNNI2/etal.and their corresponding lncRNAs could be regarded as important candidate factors that involved in regulation of skeletal muscle development.2.Additionally,differentially-expressed lncRNAs and mRNAs were chosen as the data input for WGCNA,and we have identified six gene modules related to mitosis,muscle cell differentiation,dynamic regulation of actin cytoskeleton,energy metabolism,cell migration and adhesion and so on.Dozens of highly connected genes were identified in six stage-specific modules by visualizing them in Cytoscape,including TRIM55,DES,FAM65B,ACTCl,TNNI2,FENl,AIF1L,ALDBGALT0000002581,NEK6,FAR-1 and so on.Part three:RNA-seq was utilized to obtained the profiles of miRNAs at day 0,1,3,and 5 of myoblast differentiation.miRNAs were identified and their functions during myoblasts differentiation were predicted using bioinformatics.Moreover,differentially-expressed miRNAs and their corresponding negative-correlated target genes were used to constructed the interaction network and then screen the potential candidate factors that involved in the regulation of chicken skeletal muscle development.1.A total of 712 known miRNAs were identified in the chicken myoblasts and myotubes,corresponding to 584 pre-miRNAs,belonging to 123 miRNAs family.Furthermore,244 novel miRNAs were identified.2.The expression level of miRNAs in chicken myoblast and myotubes were generally low.Of a total of 956 miRNAs,85.32%were low expression level with TPM within 1-10 and only 1.4%were abundant expressed miRNAs with TPM over 10,000.3.Expression analysis of the 15 most abundant miRNAs in every library indicated that functional miRNAome that involved in regulation of myogenesis may be smaller and tend to be highly expressed.4.A total of 298 differentially-expressed miRNAs(DEMs)between different differentiative stages.Functional analysis indicated that DEMs were mainly enriched in myogenesis-related pathways that associated with the biological process of skeletal muscle development,mitosis,cell migration and adhesion.5.Differentially-expressed miRNAs and their corresponding negative-correlated target genes were used to constructed the interaction network and then screen the potential candidate factors that involved in the regulation of chicken skeletal muscle development.Dozens of potential candidate factors involved in skeletal muscle development were identified in miRNA-mRNA interaction network,including miR-206?miR-133b?miR-204?miR-214?miR-7?miR-7b.miR-146b-5p?miR-222b-5p?miR-10a-5p?miR-203a?miR-10b-5p?miR-34a-5p?miR-218-5p?CDK6?AOX1?FSTL1?SESNI?TMP2 and so on.