An Integrated Analysis Revealed Different MicroRNA-mRNA Profiles During Skeletal Muscle Development Between Landrace And Lantang Pigs

Landrace(LR)and Lantang(LT)pigs have significantly differences in muscle production and growth rate.Simultaneously,there were significant differences in muscle fiber development and hypertrophy between LR and LT pig at the same stage.In this study,we performed an integrated analysis of the mi RNA-m RNA expression profiles in LR and LT pigs during 8 stages of skeletal muscle development,including four embryonic stages(35,49,63,and 77 dpc)and four postnatal stages(2,28,90,and 180 dpn)using Solexa sequencing.We compared significantly DE-mi RNAs between breeds at the same stages,together with target gene prediction using transcriptome data,and selected candidate mi RNAs to conduct in vitro validation to investigate the mechanisms of distinct skeletal muscle development between breeds differing in muscle production.1,Embryonic and postnatal stages had distinct mi RNA expression patterns in both breeds257 mi RNAs were found expressed in longissimus dorsi muscle during all stages,of which 152 significantly DE-mi RNAs were identified between LR and LT.DE-mi RNA expression profiles can be well classified into embryonic and postnatal stages by PCA analysis,therefore,we grouped the embryonic stages as G1 and the postnatal stages as G2.To understand the expression characteristics of abundant mi RNAs in LR and LT at G1 and G2,heatmaps of the most abundant mi RNAs at G1(95 mi RNAs)and G2(96 mi RNAs)were generated.More than two-thirds of the mi RNAs with distinct expression patterns between breeds were observed across several stages.In the G1 group,35 and 49 dpc showed notable differences in the mi RNA expression profiles between breeds,of which the LT had more highly expressed mi RNAs.In the G2 group,however,the LR had higher mi RNA expression level,though the expression profiles of most mi RNAs were similar between breeds in this group.Interestingly,the expression patterns of myogenic mi RNAs(mi R-1,206 and 133)also showed distinct between-breed differences in G1 and G2.2.Comparison of significantly DE-mi RNAs between breeds at the same stagesIn the embryonic stages,more down-regulated mi RNAs than up-regulated mi RNAs were found in LR.In the postnatal stages,however,more up-regulated mi RNAs than down-regulated mi RNAs were observed in LR.The top 10 most abundant DE-mi RNAs across eight stages were divided into G1A(up),G1A(down),G2A(up)and G2A(down).From only these most abundant mi RNAs,obvious differences were found between breeds in both the prenatal(35,49 and 63 dpc)and postnatal(2 dpn)stages.From these abundantly expressed DE-mi RNAs,mi R-30 a showed higher expression in LR than in LT,especially at 49 dpc.In a comparison between the pig breeds,49 dpc showed the most significant differences in G1A(up),in which mi R-378,mi R-30 a,mi R-148 a,and mi R-127 showed drastic changes.Moreover,the myogenic mi RNA mi R-206 also showed a higher expression level in LR and distinctly different expression patterns between breeds from 49 to 77 dpc.In G1A(down),the stages from 35 to 63 dpc showed the most significant differences between breeds,in which mi R-20,mi R-499,mi R-451 and mi R-335 had drastic changes.In G2A(up),mi R-148 a was markedly the most different between breeds at 2dpn.In G2A(down),no obviously distinct mi RNA was found between breeds.3.Interaction network analysis of mi RNAs and targets predicted by GO to be involved in myogenic-related BPsWe classified all the DE-mi RNAs into the following four subgroups: G1T(down),G1T(up),G2T(down),and G2T(up).A Venn diagram showed that G1T(down)and G2T(up)had the largest number of DE-mi RNAs as well as the largest number of shared DE-mi RNAs.Thus,we conducted a GO analysis to find out which biological progresses(BPs)these DE-mi RNAs participated in.Notably,in G1T(down)group,most enriched BPs were involved in muscle development,followed by muscle differentiation,whereas in G2T(up),muscle hypertrophy was the second most enriched BP following muscle system process.Therefore,we predicted the target genes of these myogenic mi RNAs by Targetscan.To further validate the interaction mechanisms of genes predicted by GO to have myogenic-related BPs,we performed a network analysis of mi RNAs and their 138 target genes involved in all the significantly enriched profiles resulting from STEM analysis in G1 and G2 of both breeds.The STRING website was used to predict the interaction score of candidate genes.Then,Cytoscape software was performed to visualize the data.The interaction of 22 targets and 35 mi RNAs were observed in the network.Some of these interactions included well-known,crucial myogenic mi RNAs and genes(mi R-206,mi R-133 b,mi R135-5p,MEF2 A,MEF2C,etc.)and myofibrillar genes(MYH7,TPM2,DES,etc.).These findings not only revealed a number of candidate mi RNAs with potential roles in regulating muscle development but also identified critical stages at which these mi RNAs function.4.In vitro validation of predicted myogenic mi RNAs(mi R-127,mi R-30a)In order to validate the predicted mi RNAs by bioinformatic analysis in muscle development,we selected mi R-127 and mi R-30 a for functional analysis in C2C12 cell line.After transfecting mi R-127 mimic,the number of cells in S phase increased while the number of cells in G2/M decreased.Moreover,cells transfecting mimic show decreased cell cycle related factors expression level,such as cyclin A2,cyclin E2 and CDK4,while the level of cyclin-CDK inhibitor P21 increased,indicating after trancfecting mi R-127 mimic,the cell division potential of C2C12 cell decreased.Overexpression of mi R-30 a promoted C2C12 cell proliferation.FACS analysis showed that after overexpression of mi R-30 a,the number of cells in G1 phase decreased and the number of cells in S and G2/M increased.Accordingly,expression level of cell cycle protein cyclin A2 and protein kinase CDK4 increased,while expression level of cell cycle inhibitor tumor inhibited gene Rb decreased,indicating after transfecting mi R-30 a mimic,the proliferation potential of C2C12 cell increased.