Identification And Differential Expression Analysis Of MicrorRNAs During Porcine Different Muscle Development Stage

Skeletal muscle makes up approximately 40% of the body’s mass. The principal function of skeletal muscleis contraction-related in order to control movement and posture. In response to increased use throughregular exercise training, skeletal muscle can increase its size and capacity to produce force. In contrast, ageing, sedentary lifestyles and immobilization, as well as neuromuscular disorders and chronic disease conditions are associated with the loss of skeletal muscle mass and function. Growth and development of muscle tissue are regulated by epigenetic, such as:DNA methylation, histone modifications and RNA interference (RNAi). DNA methylation and histone modifications mainly affect the level of gene transcription, while RNA interference mainly involved in post-transcriptional regulation. MicroRNAs (miRNAs), which is a class of non-coding RNA, play critical roles in many important biological processes such as growth and development in mammals. The pig (Sus scrofa) has tremendous biomedical importance as a model organism because it has closer phylogenic proximity to humans than mouse or other animals. Here, to identify miRNA and determine their role in skeletal muscle development, we evaluated miRNA transcriptome profiles at five stages of muscle development(90-day post-gestation fetuses, E90; 0,30,180 days and 7 year after birth). We analyzed the difference of the muscle fibersdiameter during the muscle development, and identified new miRNAs transcripts. Meanwhile, we identified the differentially expressed miRNAs using IDEG6 during the muscle development. Furthermore, we aslo identified the expression patterns of differentially expressed miRNAs using STEM (short time-series expression miner). In this study, we investigate the muscle fiber diameter and body weight change during the postnatal muscle development stage and found that muscle fiber diameter and body weight were significantly increased (P< 0.001, one-way ANOVA, n= 100) from Od to 7y.Notably, we found that muscle fiber diameter growth rate significantly slowed down with age (P< 0.05),Interestingly, during the period of 30d-180d, we found that the growth rate of body weight sustainable increased, but that of muscle fiber diameter was decreased (P< 0.05).The results showed that serum biochemical parameters showed significant differences during porcine muscle development stage (P< 0.05). Interestingly, we found that cholesterol (Cholesterol) and lipoprotein a (Lipoprotein α)were significantly increased with age, and the highest in the 180d.We identified 629 pre-miRNAs encoding 936 mature miRNAs in nine small RNA libraries using high throughput sequ encing technology. Of the 936 mature miRNAs,404 known porcine miRNAs,118 novel miRNA*s, and 101 miRNAs that are conserved to other mammals as well as 431 porcine putative new miRNAs.In the muscle miRNA transcriptome, we found that the majority of abundant miRNAs are from few miRNAs. the top 10 unique miRNAs with the highest expression level account for more than 50.58%, by total counts, of all the 903 unique miRNAs.we identified 505 miRNAs (505 of 903,55.92%) that were differentially expressed during the muscle development stages using IDEG6. Of the 505 differentially expression (DE) miRNAs,167 (167 of 505, 33.07%) miRNAs were retained for subsequent analysis (>1000 read counts).We performed a pairwise comparison analysis and identified 17 specifically enriched miRNAs in the early growth stage (E90, Od and 30d) and the later growth stage (180 dand 7y). Of the 17 miRNAs,10 differentially expressed miRNAs were up-regulated in the early growth stage, while 7 differentially expressed miRNAs were up-regulated in the the later growth stage.A short time-series expression miner (STEM) analysis indicated significant variations in miRNA expression across the distinct muscle development, and miRNA target prediction analysis revealed the down-regulated miRNAs were associated mainly with macromolecule biosynthetic process, while up-regulated miRNAs miRNAs were associated mainly with cell apoptosis and death.Furthermore, nine representative differentially expressed miRNAs were chosen for validation by the stem-loop RT-PCR based real-time PCR method using three independent samples. The results were consistent with our sequencing result (Person’s r=0.875), which indicates that deep-sequencing does allow for the successful discovery of miRNAs from pig with high accuracy and efficiencyIn summary, we not only predicted many novel candidate miRNAsbut aslo analyzed specific function to different stages of skeletal muscle development. Our findings will promote the further development of the pig model for human age-related muscle diseases research and improvement of pork quality.