Transcriptome Analysis Of Porcine Skeletal Muscle MicroRNA And Primary Study Of MiR-486 Function

MicroRNAs are the length of 22nt small molecule RNAs that regulate genes expression in post-transcriptional levels.It has been proved that miRNAs were involved in the regulation of a series of biological processes which is related to muscle tissue growth and development,such as muscle cell proliferation,differentiation,hypertrophy,muscle fiber type conversion and occurrence of muscle disease.Guangxi Bama mini-pig was characterized with its mini-body size,slower growth rate,lower lean meat percentage and better meat quality compared to Landrace,a modern pig breed from Denmark.So far,there is a few research on the differential expression of miRNAs associated with muscle tissue development between small size and large size pig breeds.Therefore,to explore the differentially expressed miRNAs of skeletal muscle in both Bama mini-pig and Landraces will be helpful to understand the molecular mechanisms of miRNAs involving in the regulation of skeletal muscle development and meat quality.In this study,the expression profiles of miRNAs and muscle development related miRNAs were determined by Solexa sequencing in longissimus dorsi muscle tissue samples from Bama mini-pig and Landrace at two different development stage(Day1 and Day 180).The target genes of differentially expressed miRNAs were enriched to the GO terms and KEGG signaling pathway,which could comprehensively analyze the potential functions of miRNAs.Ten differentially expressed miRNAs were validated further by QRT-PCR to confirm the sequencing results.Meanwhile,their tissues expression distribution and pattern among longissimus dorsi muscle development were detected to analyze their function in muscle development.The expression levels and function of miR-486 in pigs have not been reported.In current study,the full-length cDNA of miR-486 host gene sANK1 was cloned by RACE(Rapid-amplification of cDNA end),the relationship of miR-486 and sANK1 gene and the promoter function were analyzed.For understanding the biological function of miR-486 in pigs,the targets of miR-486 were predicted and their functions were analyzed.The expression of genes from IGF-1-PI3K/AKT-mTOR signaling pathway in longissimus dorsi muscle between Bama mini-pigs and Landrace pigs were detected.The effects and possible mechanisms of miR-486 on muscle development were researched in C2C12 myoblasts cell line.The main results obtained in this study are summarized as follows:1.Small RNA library sequencing in longissimus dorsi muscle tissues both Bama mini-pigs and LandraceFour small RNA library in porcine longissimus dorsi muscle tisseus containing dayl Bama miniature pigs(NB)and day180 Bama mini-pigs(AB),dayl Landrace(NL)and day 180 Landrace(AL)were constructed and sequenced by Solexa.The results showed that 11,955,887;11,975,536;11,955,924 and 11,975,035 high-quality reads were obtained from NB,AB,NL and AL library,respectively.The majority sequence reads were 22nt in length which were consistent with the common size of mammalian miRNAs.After removing the redundant sequence,11,925,255;11,934,400;11,934,796 and 11,931,683 clean reads of 18 to 30 nucleotides in length were obtained from NB,AB,NL and AL library,which corresponding to the unique sequences were 125,347;121,867;152,944 and 136,903 respectively.About 85%of total reads were mapped to the pig genome and more than 50%of unique reads could not be mapped to the pig genome.There were 30,588;39,840;31,861 and 33,533 common sequences among libraries of NB and NL,NL and AL,AB and AL,respectively.The results of annotation showed that the number of sequences in NB,AB,NL and AL libraries mapped to known miRNAs were 85.52%,87.05%,81.94%and 87.13%,however,13.11%,11.79%,16.08%and 11.76%did not mapped to known miRNAs,respectively.2.Longissimus dorsi muscle small RNA expression profiling and variation analysis both Bama mini-pig and LandraceIn four libraries,292 known miRNAs were identified by bioinformatics analysis.86 potentially novel miRNA were identified by Mireap software and 9 candidates were successful validated by RT-PCR and sequencing.Differential method of expression analysis showed that:compared with dayl Bama mini-pig,there were 151 significantly different miRNAs in day 180 Bama minni-pig,18 miRNAs were significantly up-regulated and 133 miRNAs down-regulated.Compared with dayl Landrace,there were 161 significantly different miRNAs in day180 Landrace,21 miRNAs were significantly up-regulated and 140 miRNAs down-regulated.Compare with dayl Landrace,20 miRNAs were significantly up-regulated and 9 miRNAs were down-regulated in dayl Bama mini-pig,compared with dayl 80 Landrace,26 miRNAs up-regulated and 4 miRNAs down-regulated.10 differentially expressed miRNAs were confirmed by QRT-PCR,the results indicated the expression patterns of miRNAs were consistent with sequencing results.3.Functional analysis of differential expressed miRNAsGO annotation of differentially expressed miRNAs showed that the target genes of differentially expressed miRNAs between NB and NL were significantly enriched in cell components;the target genes of differentially expressed miRNAs between AB and AL were significantly enriched in biological processes and molecular functions.The results of KEGG pathway analysis showed that the target genes of differentially expressed miRNAs between NB and NL were significantly enriched in 12 pathways,including Notch signaling pathway,dilated cardiomyopathy and hypertrophic cardiomyopathy,etc.14 pathways were significantly enriched in the target genes of differentially expressed miRNAs between AB and AL,including phosphatidylinositol signaling system,glyoxylic acid and dicarboxylic acid metabolism,lysine degradation,vascular smooth muscle contraction,etc.4.Ten high abundance and differentially expressed miRNAs expression profiling,expression pattern and functional analysisQRT-PCR method was used to detect the expression profile of miR-1,miR-133a-3p,miR-148,miR-206,miR-486,miR-378,miR-181a/b,miR-103/107 in different tissues of day 180 and 5 different developmental stages in longissimus dorsi muscle(postnatal Dayl,Day30,Day60,Day90 and Day 180)in Bama mini-pig and Landrace pigs.The results showed that miR-103/107,miR-148,miR-181a/b were widely expressed in different tissues,miR-1/133/378 belonged to the muscle-specific miRNA,miR-486 was enriched in muscle tissues and miR-206 belonged to the skeletal muscle-specific miRNA.Our results documented that there were significant age-and breed-differences at these miRNAs expression levels during postnatal development of porcine longissimus dorsi muscle muscles.Functional analysis of 10 miRNAs(miR181a/b、miR-103/107、miR-206、miR-378、miR-486、miR-499-5p、miR-423-5p and miR-432-5p)showed that their target genes were significantly enriched in 5 pathways including muscle developmental pathways,muscle disease pathways,cellular processes pathways,metabolic pathways,and neurodevelopmental pathways.The results indicated that these miRNAs played very important roles in porcine muscle development and might affect the differences of muscle growth and meat quality both Bama mini-pig and Landrace.5.Identification of a novel transcript of miR-486 host gene ANK1 in porcine muscleWe first used RACE method to verify that there was a novel alternative exon 1(exon 39a)located in intron 39 of porcine ANK1 gene.It formed a muscle-specific truncated transcript sANK1,which indicated that miR-486 might select the near intron as its promoter.By using full-length cDNA PCR strategy,we cloned and characterized the transcript.The results showed that there were three kinds of ORF(open reading frame)sequences in coding region which can form three kinds of alternative splicing isoforms including sANK1-1,sANK1-2 and sANK1-3.The results of bioinformatics analysis showed that three isoforms of porcine sANK1 gene had function activity and might be involved in signal transduction and the regulation of cell growth and a series of biological processes.6.The correlative analysis of miR-486 expression and its host gene sANK1PCR results showed that miR-486 could be obtained from DNA and cDNA by PCR amplification and the exons of ANK1 gene exhibited a normal splicing,which indicated the primary transcription of miR-486 and the transcription of it host gene ANK1 co-expressed.Semiquantitative RT-PCR showed that miR-486 was highly expressed in muscle tissue,sANK1 was only specifically expressed in myocardium,longissimus dorsi muscle and biceps femoris,which indicated that they were important in muscle development.QRT-PCR results showed that a gradually up-regulated trend of miR-486 and sANK1 was detected during postnatal longissimus dorsi muscle development as well as in the differentiation of C2C12 myoblast cell line.The results of correlative analysis showed that there was a positive correlation between miR-486 and sANKl expression in longissimus dorsi muscle development both Bama mini-pig(adjusted-r2=0.773,P=0.125)and Landrace(adjusted-r2=0.698,P=0.19),as well as in the differentiation of C2C12 myoblast cell line(adjusted-r2=0.999,P<0.05).The results indicated that miR-486 shared the same promoter with sANKl gene in porcine muscle.7.The differentially expressed mechanism of miR-486/sANKl gene in longissimus muscle between Bama-mini-pigs and LandraceQRT-PCR results showed that miR-486 was higher expression in Bama mini-pig at the age of Day1,Day60,Day90 and Day 180 as compared to Landrace,sANK1 was higher expression at the age of Day60,Day90 and Day 180 in Bama mini-pig compared to Landrace.In order to resolve the expression differences of miR-486/sANK1 gene in Bama mini-pig and Landrace,we cloned the promoter of porcine miR-486/sANKl and analyzed the SNPs and transcription factor binding sites in the promoter region.The results showed that there were 18 SNPs in promoter and 14 SNPs among of them were regulatory SNPs that created or abolished putative transcription factor binding motifs.The miR-486/sANK1 promoter expression vectors from Bama mini-pig and Landrace(pEGFP-BP1,pEGFP-BP2,pEGFP-LP1,pEGFP-LP2)were constructed and the functions were verified in different cells lines.The result showed that the promoters had strongest function only in C2C12 cell line,which indicated that the promoter was muscle-specific.Moreover,the activity of P1 promoter was significantly higher than P2 promoter(P<0.01).BP1 and BP2 promoter was significantly higher than LP1 and LP2 promoter respectively(P<0.01),which indicated the activity of promoter in Bama mini-pig was higher than that of in Landrace promoter.Further analysis results showed that a mutation from A to G located at-401 of Bama mini-pig promoter,which could increase a new transcription factor binding sites of MyoD.This may lead to the difference activity of promoter between Bama mini-pigs and Landrace.This difference may be the one of the reasons that the difference expression levels of miR-486/sANK1 gene in longissimus dorsi muscle between Bama mini-pigs and Landrace.8.The expression patterns of IGF-1-PI3K/AKT-mTOR signaling pathway genes in longissimus dorsi muscle between Bama mini-pigs and LandraceIn this study,the complete CDS sequence of pig p85a gene was cloned and the results showed that the CDS full length of p85a were 2175bp in Bama mini-pig.Bioinformatic analysis showed that there were SH3、SH2 domains and RhoGAP domains in p85α gene,which domains made it had a capability to interact with IRS1,PTEN,IRS2 and other proteins.QRT-PCR was used to detected the expression levels of IGF-1,INSR,IRS1,PI3K,p85a,PDK1,AKT1,PTEN,FoxO1,mTOR and 4EBP1 genes in five different developmental stages of longissimus dorsi muscle(postnatal Dayl,Day60,Day90 and Day 180)both Bama mini-pig and Landrace.The results showed that the expression levels of PTEN,FoxO1 and 4EBP1 gene were significantly higher than other genes in early stages of two breed pigs,which suggested that they might play important roles in the regulation of muscle growth and development.In addition,a significant age-and breed-different expression of IGF-1-PI3K/AKT-mTOR related genes at postnatal development of porcine longissimus dorsi muscle,which indicated the difference of gene expression level may affect different muscle performance between Bama mini-pig and Landrace.9.The function of miR-486 in IGF-1-PI3K/AKT-mTOR signaling pathwayIn normal physiological conditions,correlation analysis results showed that there was a negative correlation between miR-486 and IGF-1,p85a,PTEN and FoxO1 genes expression in longissimus dorsi muscle of porcine postnatal development.QRT-PCR was used to detect the expression of IGF-1-PI3K-mTOR relative genes in overexpressed or inhibited the expression of miR-486 in C2C12 cells line.The results showed that overexpression of miR-486 inhibited the mRNA expression levels of PTEN and FoxO1 gene and further a down-regulation of INSR,IRS1,AKT1,PDK1,mTOR and 4EBP1 mRNA expression levels was observed.However,when miR-486 was inhibited,the mRNA expression levels of p85a and PTEN gene were inhibited and further an up-regulation of PDK1 and 4EBP1 mRNA expression levels was detected in 24 hours.The mRNA expression of IGF-1 was increased,while expression level of 4EBP1 was down-regulated in 48 hours.All results indicated that miR-486 regulated IGF-1-PI3K-mTOR signaling pathway by inhibiting target genes in porcine skeletal muscle.