Study On The Mechanism Of Leucine Regulating The Proliferation And Differentiation Of Equine Skeletal Muscle Satellite Cells Based On Multi-omics

The Mongolian horse,one of the unique breeds in China,has excellent endurance quality formed by the half-grazing and half-wild breeding feeding mode on the grassland.Mammalian skeletal muscle satellite cells(SCs)are located between the sarcolemma and the basal lamina of the muscle fiber.When animal muscle is damaged,SCs are activated,which proliferate and differentiate into myoblasts at rest,and then self-renew to supplement the stem cell pool or fuse into new muscle fibers.This will affect the formation,development,and recovery process of skeletal muscle after injury.Leucine(Leu),as an important essential amino acid,plays an important role in the synthesis and metabolism of muscle proteins and the composition of muscle fibers.Therefore,the study on the mechanism of Leu regulating the proliferation and differentiation of equine SCs can not only improve the exercise performance of horses but also reduce muscle injury of horses during exercises.This study systematically studied the molecular mechanism of Leu-induced proliferation and differentiation of equine SCs by using the whole transcriptome and proteome sequencing technology,cell culture technology,molecular biology technology and bioinformatics technology.Combined with the effect on cell proliferation,differentiation and slow muscle fiber expression,the suitable concentration of Leu was screened during the proliferation and differentiation of equine SCs,which provided a theoretical basis for improving the endurance of Mongolian horse and alleviating muscle injury during exercise.The main research results of this thesis were as follows:1.Establish and optimize the isolation and culture method of equine SCs.In this study,two separation methods were established and compared.High purity equine SCs could be obtained by both methods.The experimental period of enzyme digestion was short,and the number of cells is large;the purity of Pax7 positive SCs reached more than96% by immunofluorescence;the isolated primary equine SCs proliferated rapidly and had strong characteristics of myogenic stem cells.After 3 days of differentiation induction,a large number of obvious myotubes can be observed,which can be used for subsequent experiment.2.Leu promoted the proliferation of equine SCs,and 2 m M was the optimal supplemental level.The hub genes were selected from protein-protein interaction network(PPI)network,including ACACB,HMGCR,IDI1,HAO1,SHMT2,PSPH,PSAT1,ASS1,PHGDH,MTHFD2 and DPYD were further identified as candidate biomarkers to regulate the Leu-induced proliferation of equine SCs.These three mi RNAs(Novel699＿star,novel 170＿star and novel 360＿mature)were newly predicted mi RNAs,which will open a new sight in the study of Leu improving exercise performance and post-exercise injury repair in horses.3.We integrated the transcriptome(RNA-Seq)and quantitative proteome profiling analyses on Leu-induced proliferation of equine SCs.Based on the translation relationship between m RNA and protein,110 overlapped genes were validated.In addition,the pathways were compared between differentially expressed genes(DEGs)and differentially expressed proteins(DEPs)enrichment analysis,we found that there were five overlapped pathways,four of which were enriched by down-regulated DEGs and DEPs related to metabolism,including proximal tubule reclamation,alanine,aspartate and glutamate metabolism,glycine,serine and threonine metabolism and mineral absorption.The only one enriched by up-regulated DEGs and DEPs was chemokine signaling pathway.Among them,four genes(CCL26,ASNS,FTL and STEAP1)of them were not only discovered at DEGs and DEPs level in the overlapping signaling pathway,but also in the 110 overlapping DEGs.4.Through immunofluorescence and Western blot,it was found that the number of nuclei in a single myotube and the expression of slow muscle fibers increased significantly with the concentration of Leu(P < 0.01),2 m M was the optimal supplemental level;PPI network analysis identified the central genes related to cholesterol anabolic metabolism and amino acid transport activity,including HAO1,IDI1,LSS,FDFT1,CTH,and SHMT2.Genes related to lipid metabolism were up-regulated,while those related to amino acid transport activity were down-regulated.We also found that four up-regulated mi RNAs(eca-mi R-541,eca-mi R-323-5p,eca-mi R-9112 and eca-mi R-154a)and one down-regulated mi RNA(eca-mi R-150)through the competitive endogenous RNAs(ce RNA)regulatory network.These mi RNAs play an important role in Leu-induced differentiation of equine SCs.5.We integrated RNA-Seq and quantitative proteome profiling analyses on Leu-induced differentiation of equine SCs.According to the translation relationship between gene and protein,133 overlapping DEGs were found.In addition,comparing the pathways for enrichment analysis of DEGs and DEPs,it was found that four up-regulated signal pathways were skeletal muscle fiber development,myofibril assembly,myosin complex,and cholesterol homeostasis,only L-glutamine transmembrane transporter activity was enriched in down-regulated DEGs and DEPs.11 DEGs were not only found in overlapping 133 key DEGs,but also in overlapping GO signaling pathways.These 11 DEGs are presumed to be key genes for Leu to regulate the differentiation of equine SCs,of which 10 are up-regulated(CAV3,KLHL40,ACTA1,TMOD2,LMOD3,MYL1,MYH3,MYH7,LDLR,and LIPG),and one is down-regulated(SLC38A5).6.Based on the above sequencing data,we found that Leu regulated the expression level of related genes and proteins in the AMPK and mTOR signaling pathways.Our validation results of AMPK and mTOR signaling pathways showed that Leu enhanced mitochondrial aerobic respiratory energy supply,AMPK and mTOR signaling pathways mediate Leu to increase the expression of slow muscle fibers in equine SCs,while Leu activated mTOR in an AMPK dependent manner.