Differential Expression Analysis And Molecular Network Reconstruction For Genes Associated With Muscle Growth And Adipose Deposition In Obese And Lean Pigs

During the growth and development of skeletal muscle cells and adipose cells,the regulatory mechanism of micro-effect polygenes determines porcine meat quality,carcass characteristics and other relative quantitative traits.Obese and lean type pig breeds show obvious differences in muscle growth and adipose deposition;however,the molecular mechanism underlying this phenotypic variation remains unknown.We used pathway-focused oligo microarray studies to examine the expression changes of 140 genes associated with muscle growth and adipose deposition in longissimus dorsi muscle at six growth stages(birth,1,2,3,4 and 5 months)and in backfat at five growth stages(1,2,3,4 and 5 months)of Landrace(a leaner,Western breed)and Taihu pigs(a fatty,indigenous,Chinese breed).(ⅰ)Variance analysis(ANOVA)revealed that differences in the expression of 18 genes in Landrace pigs and 3 genes in Taihu pigs were very significant(P_ANOVA＜0.01)and differences for 22 genes in Landrace pigs and 7 genes in Taihu pigs were significant(P_ANOVA＜0.05)in longissimus dorsi muscle among six growth stages.The differences in the expression of 25 genes in Landrace pigs were significant(P_ANOVA＜0.05)in backfat among five growth stages.Gene class test(GCT)indicated that a gene-group was very significant in longissimus dorsi muscle of Landrace pigs across six growth stages(P_ErmineJ＜0.01),which consisted of 10 genes for enzymes related to fatty acid biosynthesis.A gene-group was very significant in backfat between two pig breeds across five growth stages(P_ErmineJ＜0.01),which consisted of 23 genes encoding enzymes and regulatory proteins associated with lipid and steroid metabolism.These findings suggest that the distinct differences in fat-deposition ability,muscle-fiber number and growth rate between Landrace and Taihu pigs may closely correlate with the expression changes of these genes.(ⅱ)STEM(Short Time-series Expression Miner)clustering analysis revealed a very high level of significance(P_STEM＜0.01)for 4 gene expression patterns in longissimus dorsi muscle,in which genes that showed strong up-regulation were mainly associated with the positive regulation of myofiber formation and fatty acid biogenesis and genes that showed strong down-regulation were mainly associated with the inhibition of cell proliferation and positive regulation of fatty acidβ-oxidation.A very high level of significance(P_STEM＜0.01)for 2 gene expression patterns in Landrace pigs and a high level of significance(P_STEM＜0.05)for 2 gene expression patterns in Taihu pigs for backfat.Also,expression patterns of genes were more diversified in Taihu pigs than in Landrace pigs,which suggests that the regulatory mechanism of micro-effect polygenes in adipocytes may be more complex in Taihu pigs than in Landrace pigs.(ⅲ)Principal component analysis(PCA)revealed that 7 and 16 genes of Landrace,6 and 13 genes of Taihu pigs in longissimus dorsi muscle and backfat displayed distinctive expression pattern from other genes,which suggested that these genes maybe came under special regulation during the growth and development of skeletal muscle cells and adipose cells.(ⅳ)ANOVA revealed that differences in the expression of 42 genes in Landrace pigs and 51 genes in Taihu pigs were very significant(P_ANOVA＜0.01)and differences for 27 genes in Landrace pigs and 20 genes in Taihu pigs were significant(P_ANOVA＜0.05)between longissimus dorsi muscle and backfat across five growth stages.The resluts of two-way hierarchical clustering could be segmented to three clusters,and mapped to the two- and three- dimensional map of PCA.We can highlight some genes which have tissue specific expression patterns on which to base further study of the differences in molecular mechanism between the depostation of subcutaneous fat and intramuscular fat(IMF).(ⅴ)Based on a dynamic Bayesian network(DBN)model,gene regulatory networks(GRNs) were reconstructed from time-series data for each pig breed and tissue.These four GRNs initially revealed the distinct differences in physiological and biochemical aspects of muscle growth and adipose deposition between the two pig breeds;from these results,some potential key genes could been identified.(ⅵ)Pathway mapping revealed that the biological functions of four pathways maybe changed during the process of growth and development between two pig breeds,in which the pathway of fat acid metabolism and biosynthesis involved the competitive equilibrium of fat depostation,and the pathway of PPAR(peroxisome proliferator-activated receptor)signaling and adipocytokine signaling involved the rugulation of fat depostation.These results could be explained the phenotypic variation of fat traits between obese and lean type pig breeds to some extent.(ⅶ)Based on a natural language processing(NLP)approach,literature co-citation networks of genes that belong to predominant temporal expression patterns using a unique STEM algorithm were reconstructed.We can choose seed genes from sub-networks that have higher rank scores as potential key genes for porcine meat quality and carcass traits.(ⅷ)Based on human homologous genes,transcriptional regulatory networks(TRNs)of differentially expressed genes(P_ANOVA＜0.05 and 0.01)in longissimus dorsi muscle were inferred. We found 27 transcription factors(TFs)have significantly overabundant binding sites in the promoters of 26 genes(P_TF＜0.05).These findings suggest that these TFs maybe indirectly influence muscle and fat traits via directly stimulate target genes.(ⅸ)Repeatability analysis indicated that average coefficient of variation((?))within-arrays and average correlation coefficient((?))between-arrays were 5.95%±3.15%and 0.894±0.038 for expression profiling of backfat,and 5.19%±2.87%and 0.917±0.041 for expression profiling of longissimus dorsi muscle.Quantitative,real-time RT-PCR(QRT-PCR)was used to verify the microarray data for five modulated genes within each tissue,and a good correlation between the two measures of expression was observed for both two pig breeds at different growth stages (backfat:r=0.874±0.071 and longissimus dorsi muscle:r=0.876±0.095).These resultes demonstrate that the microarray technique used in this study is accurate and reproducible.These results highlight some possible candidate genes for porcine meat quality and carcass traits and provide some data on which to base further study of the molecular mechanism of muscle growth and fat deposition.