Studies On The Variation Of Transcriptome Of Skeletal Muscle Tissue In Tongcheng And Yorkshire Pigs,and The Regulation Of MiRNA-34a To Functional Gene ENO3

Our study focused on muscle development of Tongcheng(TC)and Yorkshire(YK)pigs,and mainly discussed: transcriptional profiles in TC and YK and miRNA-34 a affecting on functional gene of muscle development.The research’ results are as follows:1.We analysed muscle morphological variances using histochemistry section(H&Estaining)among Tongcheng and Yorkshire pigs.Primary fibers formed 40 dpc(days-post-coitus)until to 63 dpc;while secondary fibers formed 63 dpc until to 90 dpc in the two breeds.The number of myoblasts in TC early embryo was more than YK early embryo.Myogenic precursor cells proliferation and differentiation began earlier in TC.Furthermore,muscle fiber growth in YK was faster than in TC.2.Digital gene expression profiles of TC and YK skeletal muscle were obtained using Solexa/Illumina high-throughput sequencing,and validated by q PCR method.First,differentially expressed genes were screened with NOISeq method in TC and YK pigs(log2Ratio>1and Probability>0.7).4331 DE genes were screened in TC libraries,while2259 DE genes in YK libraries.Cluster analysis showed the differences on genes expression patterns between TC and YK.Second,984 DE genes related to muscle development were identified;of these 851 in TC and 523 in YK.Third,more DE genes were involved in early muscle development;this result indicated genes transcription and expression were more intense in early stage of muscle fiber formation.3.DE genes were aligned to Gene ontology and KEGG databases in order to gain genes functional annotation.The total GO term and pathway list were obtained in each breed.Moreover,GO term and pathway list in each comparison group were screened for significant enrichment(FDR<0.05).These data showed many DE genes were associated with muscle development and growth,cellular signal transduction,cell morphological development and energy metabolism,etc.4.Correlation interaction regulatory networks were constructed for myofibers development and formation.These genes CXCL10,EIF2B5,PSMA6,FBXO32,LOC100622249,NOR.1,LMOD1,IRF1,CPS1 and LOC100518817 were vital roles in muscle regulatory networks in TC pigs,whereas the genes LOC100511980,SGCD,ENG,THBD,CXCL10,TXNIP,AQP4,LOC100519635,MYF6,PSEN2,APOD,GPX1 and BTG2 performed key roles in YK pigs.So molecular regulation mechanism was different in TC and YK skeletal muscle development.5.Muscle fiber type(My HC)expression patterns showed differences in TC and YK pigs.My HC-Ⅰexpression began 40 dpc and higher in TC than YK pigs;while My HC-2a and My HC-2x had no expression in TC and YK early embryo,and started expression from70 dpc.My HC-2b expression increased after birth,and was higher in YK than in TC.6.Our study first detected that micro RNA-34 a had an effect on myoblasts.ENO3 was related to muscle development regulatory and predicted as a targeting gene of miRNA-34 a using Target Scan,Pic Tar and micro RNA software.Dual luciferase report test indicated miRNA-34 a might bind ENO3 3’UTR.Additional,miRNA-34 a had a negative regulatory for ENO3 based on q PCR,western blot and immunofluorescence method.The expression level of ENO3 gene and protein was significantly decreased and increased,respectively(P<0.001),when miRNA-34 a mimics and miRNA-34 a inhibitors transfected C2C12 separately,compared to the control.Furthermore,miRNA-34 a inhibited cell proliferation with CCK-8 test,and flow cytometer test showed that the proportion of S period and G2 period in C2C12 cells was increased when miRNA-34 a was inhibited.Moreover,ENO3 protein interaction network was constructed with STRING 9.0.