Integration Of Micrornaome,Transcriptome And Proteomein Prenatal Longissimus Muscles Between Tongcheng And Yorkshire Pigs

Tongcheng(TC)and Yorkshire(YK)are two pig breeds with distinctive muscle morphology.Firstly,porcine miRNA of the longissimus muscle during five developmental stages(40,55,63,70,and 90 days post coitum(dpc))was explored by Solexa sequencing in the present study to find miRNAs involved in the different regulation of skeletal muscle development between the two breeds.Secondly,combined with the transcriptome and proteome data,the miRNAome were analyzed and the differentially expressed miRNAdifferentiated proteins were identified.The result were verficated by dual luciferase reporter assay.Finally,a study of the function of miR-499-5p in C2C12 was performed.The main findings are as follows:1.A total of 320 known porcine miRNAs,64 miRNAs corresponding to other mammals,and 224 potentially novel miRNAs were identified.Principal component analysis and hierarchical cluster analysis suggested that the factor “pig breed” affected the miRNA expression profiles to a lesser extent than the factor “developmental stage”.Fiftyseven miRNAs were differentially expressed(DE)between the neighbor developmental stages in TC and 45 such miRNAs were found in YK,34 in common;there were more down-regulated stage-DE miRNAs than up-regulated.And a total of 23,30,12,6,and 30 breed-DE miRNAs between TC and YK were identified at 40,55,63,70,and 90 dpc,respectively,which were mainly involved in cellular protein modification process,protein transport,and metabolic process.MiR-499-5p was the only highly expressed breed-DE miRNA found in no less than four developmental stages,and also a stage-DE miRNA found both in TC and YK.2.An interaction network between DE miRNAs and DE mRNAs was drawn.The protein whose expression abundance was lower than that of mRNA was characterized by the enriched functional categories.It was found to be related to metabolic process and mRNA spliceosome.Combined DE miRNA and DE proteins,52 pairs of DE miRNA-DE proteins were screened from different development time points in TC,34 pairs in YK and 38 pairs from the same development time points between TC and YK.3.The targeting relationship between miR-499-5p and actin depolymerization factors(DSTN),miR-15 b and PDLIM5,miR-374b-5p and PDLIM5 was verified in pigs and mice using the dual luciferase reporter assay.4.MiR-499-5p inhibits the expression of DSTN protein at the post-transcriptional level and does not affect the mRNA expression of DSTN.MiR-499-5p promoted cell migration,making more cells to remain in the G0 and G1 phases and was essential for myocyte differentiation.Inhibition of DSTN may be one of the mechanisms by which miR-499-5p promoted cell fusion and achieved the functions above.The expression level of miR-499-5p should be kept at a certain level.Too high expression of miR-499-5p and too low expression of miR-499-5p will increase apoptosis.