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

<div><p>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 (<i>MAPK</i>), nuclear factor of activated T cells (<i>NFAT</i>), myocyte enhancer factor 2 (<i>MEF2</i>) 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 10000 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 (<i>MYH7</i>), indicating that miR-143 activity regulated muscle fibre differentiate in skeletal muscle. And it regulate <i>MYH7</i> through the <i>HDAC4-MEF2</i> pathway.</p></div

Potential regulation pathways of muscle fibres by ssc-miR-143-3p.

<p>Potential regulation pathways of muscle fibres by ssc-miR-143-3p.</p

Summary of differential expression miRNAs between the three stages.

<p>DE: differential expression, fold change>2 or fold change<0.5, p<0.05.</p><p>Summary of differential expression miRNAs between the three stages.</p

KEGG pathway analysis of most abundant miRNAs.

<p>KEGG pathway analysis of most abundant miRNAs.</p

MyHC gene expression in the psoas major muscle at 63, 98 and 161 d.

<p>MyHC gene expression in the psoas major muscle at 63, 98 and 161 d.</p

The expression levels of <i>MYH7</i> gene and protein in porcine skeletal muscle satellite cells after transferred ssc-miR-143 mimics and inhibitor.

<p>(A) The expression level of slow fibre gene <i>MYH7</i> after transferred ssc-miR-143-3p mimics; (B) The expression level of slow-twitch muscle myosin heavy chain protein after transfection with ssc-miR-143-3p mimics; (C) The expression level of slow fibre gene <i>MYH7</i> after transferred ssc-miR-143-3p inhibitor; (D) The expression level of slow-twitch muscle myosin heavy chain protein after transferred ssc-miR-143-3p inhibitor. NC is the control group for the miR-143 mimics transferred experiment. And INC is the control group for the miR-143 inhibitor transferred experiment. (*p<0.05, n = 5).</p

The expression levels of key genes in the potential regulation pathways after transferred ssc-miR-143 mimics and inhibitor.

<p>(A) The expression levels of key genes in the potential regulation pathways after transferred ssc-miR-143 mimics; (B) The expression levels of key genes in the potential regulation pathways after transferred ssc-miR-143 inhibitor. (*p<0.05, n = 5).</p

Basic information from the sequencing data.

<p>(A) Length distribution of the total reads; (B) Length distribution of the unique reads; (C) Length distribution of the unique miRNAs; (D) The numbers of detected miRNAs in the three libraries. (p<0.05).</p

RT-PCR verification of miRNAs detected by solexa sequencing.

<p>(<b>A</b>) Fold change of differentially expressed miRNAs at 63, 98 and 161 d as determined by sequencing. (<b>B</b>) Fold change of differentially expressed miRNAs at 63, 98 and 161 d as determined by RT-qPCR. (<b>C</b>) Correlation coefficients (R) between miRNA expression profiles as determined by Illumina sequencing and RT-qPCR. The miRNAs selected for analysis are PC-5p-813_1295, ssc-miR-140-3p, ssc-miR-450c-5p, ssc-miR-143-3p, ssc-miR-195, ssc-miR-1, ssc-miR-92a, ssc-miR-127 and ssc-miR-145-5p.</p

The function of highly expressed muscle-related miRNAs in muscle development.

<p>The function of highly expressed muscle-related miRNAs in muscle development.</p