MicroRNA-27a inhibits porcine type I muscle fibre gene expression by directly targeting peroxisome proliferator-activated receptor-γ coactivator-1α.

MicroRNAs are one of the key determinants of muscle fibre development and phenotype in mammals. The preliminary experiment implied that microRNA-27a (miR-27a) might involve in regulation of muscle fibre type composition of pigs. Thereby, the present study aimed to confirm the regulatory effect of miR-27a on porcine type I muscle fibre-encoding gene (myosin heavy chain gene 7, MYH7) expression and its related mechanism. We firstly observed opposite expression patterns between miR-27a and MYH7 as well as between miR-27a and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) during differentiation of porcine skeletal muscle satellite cells. Through the subsequent transfection analysis in porcine myotubes, we found that miR-27a suppressed the expression of MYH7 and PGC-1α. Besides, miR-27a induced inhibition of PGC-1α downstream targets, namely myocyte enhancer factor-2C (MEF2C) along with mitochondrial biogenesis and oxidative metabolism-related factors such as nuclear respiratory factor 1 (NRF-1), mitochondrial transcription factor A (mtTFA), cytochrome c (Cytc) and cytochrome oxidase IV (COX Ⅳ) and succinodehydrogenase (SDH). Dual-luciferase reporter analysis revealed that miR-27a could bind to the predicted target site in the 3'-untranslated regions of PGC-1α mRNA, confirming a direct targeting of PGC-1α by miR-27a. Moreover, PGC-1α silencing abolished the promotive effects of miR-27a inhibitor on MYH7, PGC-1α and its downstream targets (MEF2C, NRF-1, mtTFA, COX Ⅳ, Cytc and SDH) in porcine myotubes. Collectively, miR-27a inhibits porcine MYH7 expression by negatively regulating PGC-1α and PGC-1α-controlled MEF2C expression as well as mitochondrial biogenesis and oxidative metabolism. Our findings may provide a molecular target for genetic or nutritional control of muscle fibre phenotype of pigs, probably having an important implication for regulating pork quality.

MicroRNA Transcriptome Profile Analysis in Porcine Muscle and the Effect of miR-143 on the MYH7 Gene and Protein.

Porcine skeletal muscle fibres are classified based on their different physiological and biochemical properties. Muscle fibre phenotype is regulated by several independent signalling pathways, including the mitogen-activated protein kinase (MAPK), nuclear factor of activated T cells (NFAT), myocyte enhancer factor 2 (MEF2) and peroxisome proliferator-activated receptor (PPAR) signalling pathways. MicroRNAs are non-coding small RNAs that regulate many biological processes. However, their function in muscle fibre type regulation remains unclear. The aim of our study was to identify miRNAs that regulate muscle fibre type during porcine growth to help understand the miRNA regulation mechanism of fibre differentiation. We performed Solexa/Illumina deep sequencing for the microRNAome during 3 muscle growth stages (63, 98 and 161 d). In this study, 271 mature miRNAs and 243 pre-miRNAs were identified. We detected 472 novel miRNAs in the muscle samples. Among the mature miRNAs, there are 23 highest expression miRNAs (over 10,000 RPM), account for 85.3% of the total counts of mature miRNAs., including 10 (43.5%) muscle-related miRNAs (ssc-miR-133a-3p, ssc-miR-486, ssc-miR-1, ssc-miR-143-3p, ssc-miR-30a-5p, ssc-miR-181a, ssc-miR-148a-3p, ssc-miR-92a, ssc-miR-21, ssc-miR-126-5p). Particularly, both ssc-miR-1 and ssc-miR-133 belong to the MyomiRs, which control muscle myosin content, myofibre identity and muscle performance. The involvement of these miRNAs in muscle fibre phenotype provides new insight into the mechanism of muscle fibre regulation underlying muscle development. Furthermore, we performed cell transfection experiment. Overexpression/inhibition of ssc-miR-143-3p in porcine skeletal muscle satellite cell induced an/a increase/reduction of the slow muscle fibre gene and protein (MYH7), indicating that miR-143 activity regulated muscle fibre differentiate in skeletal muscle. And it regulate MYH7 through the HDAC4-MEF2 pathway.