Identification Of Genes And MiRNA Associated With Skeletal Muscle Development And Network Interaction In Porcine

In our study, pig oligo array and miRCURYTMLNA miR Array were employed toexplore gene mRNA expression and miRNA expression during prenatal skeletal muscledevelopment (33,65and90days post conception) among Landrace (a leaner western breed),Tongcheng (a typical indigenous Chinese breed) and Wuzhishan (a typical miniature Chinesebreed) pigs.12differentially expressed genes and3differentially expressed miRNAs werevalidated by quantitative PCR. Systematic cluster analysis was performed using Cluster3.0software; Gene Ontology (GO) annotation and pathway analysis were performed usingDAVID database. Subsequently, the expression profiles of the differentially expressed genesin the three pig breeds were analyzed with the Series Test Cluster (STC) algorithm and thegene-gene network were constructed based on the function of the differentially expressedgenes in the profiles. With the expression correlation of the differentially expressed genes andmiRNAs, the miRNA-gene networks were constructed, and then, the miRNA-GO networkswere constructed according to the GO annotation of the target genes in the miRNA-genenetwork. With TargetScan and PicTar databases,4miRNA and their partial target genes werevalidated by luciferase assay, real-time PCR and western blot. Moreover, we detected theporcine OLFML3gene and miR-155tissues distribution in adult Tongcheng pigs and theirdynamical expressions in developmental skeletal muscle (12prenatal and10postnatal stages)from Landrace (lean-type) and Tongcheng (obese-type) pigs. The results of our study arefollowing:1. A total of1402,912and1910transcripts were differentially expressed during prenatalskeletal muscle development in Landrace, Tongcheng and Wuzhishan pigs, respectively.There were190common differentially expressed genes among the three pig breeds. InLandrace and Tongcheng pigs, there were233common differentially expressed genes, inLandrace and Wuzhishan pigs, there were230common differentially expressed genes, andwhile in Tongcheng and Wuzhishan pigs, there were83common differentially expressedgenes (p<0.01, FDR<0.05).2.33,18and67differentially expressed miRNAs were identified during prenatalskeletal muscle development in Landrace, Tongcheng and Wuzhishan pigs, respectively. There were9common miRNAs differentially expressed among the three pig breeds. InLandrace and Tongcheng pigs, there were2common miRNAs differentially expressed; inLandrace and Wuzhishan pigs, there were20common miRNAs differentially expressed; andin Tongcheng and Wuzhishan pigs, there were4common miRNAs differentially expressed(p<0.05, FDR<0.5).3. The real-time PCR results of12genes and3miRNAs were in good agreement withthe array experiments, the correlation coefficients were0.7511(p<0.01) and0.7937(p<0.01),respectively.4. The systematic cluster analysis showed that the Landrace and Tongcheng pigs had asimilar gene expression pattern and clustered into one group, whereas Wuzhishan pigsexhibited a different expression pattern and clustered into another group.5. GO and pathway analysis showed that the differentially expressed genes in Landrace,Tongcheng and Wuzhishan pigs were involved in the common biological processes of mitoticcell cycle, nuclear division, muscle system process, monosaccharide metabolic process,generation of precursor metabolites and energy, muscle contraction, cytoskeleton organization,muscle organ development, glucose metabolic process, cell cycle and so on. The differentiallyexpressed genes in both Landrace and Tongcheng pigs participated in the common pathwaysof muscle contraction, Parkinson's disease, oxidative phosphorylation, viral myocarditis, typeI diabetes mellitus, dilated cardiomyopathy and so on. The differentially expressed genes inboth Landrace and Wuzhishan pigs participated in the pathway of cell cycle and thedifferentially expressed genes in both Landrace and Wuzhishan pigs participated in thepathway of Glutathione metabolism.6. STC analysis indicated that the differentially expressed genes in profiles15inLandrace and Tongcheng pigs and profile11in Wuzhishan pigs were up-regulated; the genesin profile0in Landrace pigs, profile3in Tongcheng pigs and profile4in Wuzhishan pigswere down-regulated. The gene-gene networks indicated that one gene was regulated bymultiple other mRNAs and may take part in the regulation of multiple genes.7. The miRNA-gene interaction networks for differentially expressed genes showed thatthere were290targets for28miRNAs in Landrace pigs,91targets for15miRNAs inTongcheng pigs, and502targets for57miRNAs in Wuzhishan pigs. GO annotation showedthat the target genes in Landrace and Toncheng pigs were involved in15functional categories,while the target genes in Wuzhishan pigs were involved in19functional categories.8. TargetScan and PicTar predicted that OLFML3gene was regulated by miR-155, CNN3gene was regulated by miR-200c, miR-7, miR-1, miR-206, miR-124, miR-153and so on,RAP2C gene was regulated by miR-19ab, miR-130ab, miR-133ab and so on, MAP1A gene was regulated by miR-1, miR-206, miR-613, miR-9, miR-181abc, miR-26ab, miR-34ac andso on, SMARCD1gene was regulated by miR-21, miR-150, miR-122, miR-133b and so on,and SFRS3gene was regulated by miR-21, miR-1, miR-206and so on.9. The validated results indicated that miR-206regulate CNN3, MAP1A and SFRS3genes expression, miR-133b regulate RAP2C and SMARCD1genes expression, miR-155regulate OLFML3gene at mRNA level and miR-1a regulate CNN3gene at protein level.10. Real-time PCR results showed that OLFML3mRNA was highly expressed in liverand pancreas, moderately in lung, small intestines and placenta, and weakly in other tissues.MiR-155was highly expressed in small intestines and lung, moderately in heart and placenta,and weakly in other tissues. Furthermore, the OLFML3gene and miR-155exhibited differentdynamical expression patterns during prenatal skeletal muscle development in Landrace andTongcheng pigs, respectively, indicated that they may be involved in the prenatal skeletalmuscle development of the different pig breeds.