The Biological Function Analysis Of Transcriptome Differentially Expressed MiRNAs In Porcine Muscle Fibre Types

MicroRNAs (miRNAs) are 21-23 nt small RNAs that post-transcriptionally regulate the expression their target mRNA genes, mainly through its 5’seed region (2-7 nt) complementary with 5’ non-coding region of target genes to inhibit gene transcript or degradation. Skeletal muscle is a very heterogeneous tissue that comprises different fiber types, and that can be broadly classified as red, intermediate and white fiber type, which combined with the feature of its function, shape, form and proportion of myosin heavy chain. Recently, several studies have identified that several differentially expressed miRNAs between red and white myofibers, and these miRNAs mainly associate with skeletal muscle development, metabolic pathways. Nonetheless, an integral explanation of the miRNA transcriptome differences in three fiber types remains to be further investigated. The results showed that:A total of 15 skeletal muscles in porcine different anatomic locations were collected, and their fiber type were determined by qRT-PCR for MHC isoforms, in which the specificity and reliability of qRT-PCR primers were confirmed by Sanger sequencing. Results showed that these muscles were divided into red, white and intermediate fiber type based on the hierarchical clustering analysis for the ratios of myosin heavy chain (MHC) isoforms. Meanwhile, the mitochondrial contents and relative expression levels of LDHA were detected to distinguish the differences of metabolic capacity for distinct muscle fibers. We further illustrated three muscles, which typically represented each muscle fiber type (i.e. red:peroneal longus (PL), intermediate:psoas major muscle (PMM), white: longissimus dorsi muscle (LDM)), which have distinct metabolic patterns of mitochondrial and glycolytic enzyme expression levels.In PL, PMM and LDM libraries, length distribution analysis showed that the majority of reads ranged from 21 to 23 nt in length, and 80.77% of the mappable reads were mapped to porcine known pre-miRNAs. In addition, we randomly selected 20 miRNAs for validation by qRT-PCR. The small RNA-sequencing data showed a significant positive correlation with qRT-PCR results (r= 0.780, P< 10-6)。Meanwhile, we performed the small RNA library for PL, PMM and LDM using deep sequencing. Results showed that, of the ten universally abundant miRNAs that accounted for more than 70% of the total unique miRNAs reads, four miRNAs (miR-133a, miR-143, miR-27b and miR-lOb) were co-expressed in three libraries. MiR-133a, a muscle-specific miRNA that involved in myogenesis showed little difference (< 1.5-fold) among the three libraries, while miR-143 was differentially expressed (> 1.5-fold) among three libraries, compared with PMM and LDM, both miR-27b and miR-10b were up-regulated (> 1.5-fold) in PL. These miRNAs were mainly associated with metabolic pathways (e.g. mitochondrion, pyruvate metabolism and glycolysis/gluconeogenesis), myogenesis and angiogenesis.In pairwise comparison, we identified 44 differentially expressed miRNAs (P< 0.001, fold-change>1.5 and reads> 10,000). For the functional enrichment analysis, we gathered the target information of the up-regulated DE miRNAs that had been experimentally validated in muscle tissues and/or cells from previous reports. The target genes of the rest miRNAs, whose functions were not reported before, were predicted using the specific algorithms of MiRanda and TargetScan software based on our in-house dataset of the porcine skeletal muscle 3’untranslated region (UTR).Based on previous reports, we found that there were 18 miRNAs involved in myogenesis via functional enrichment analysis of their target genes, of which 12 miRNAs were specifically enriched in PL. Night miRNAs, mainly associated with energy metabolism, were enriched in PL.This study confirmed that skeletal muscles were mainly classified as red, intermediate and white fiber type, and demonstrates that miRNAs play essential roles in myogenesis and metabolic pathways (e.g. energy metabolism) in three fiber types. Overall, this comprehensive analysis will contribute to better understanding the miRNA regulatory mechanism for the phenotypic diversity of skeletal muscles.