Transcriptome Sequencing Analysis Of Porcine Intramuscular Adipocyte Exosomes And Its Effect On Intramyocellular Lipids

Pork is one of the most important meat foods in daily life.It is rich in protein,fat,carbohydrates,calcium,phosphorus and other nutrients.The fat content in muscle is an important factor affecting its juiciness,flavor and tenderness.The fat in muscle mainly exists in three forms: intramuscular fat,intermuscular fat and intramyocellular lipids.The content of intramuscular fat is mainly determined by the number and size of intramuscular adipocytes,which is affected by various factors such as obesity,diet,aging and gender.The intramyocellular lipids are related to adipose tissue hypertrophy and increased fatty acid intake.In addition to storing energy and releasing free fatty acids,adipose tissue also acts as an endocrine organ to secrete leptin,adiponectin and other biologically active adipokines.In recent years,exosomes secreted by adipose tissue are regarded as a new type of adipokines.Exosomes are extracellular vesicles with a diameter of 40-160 nm secreted by a variety of cells,which can be absorbed by target cells to regulate various biological processes.Adipose-derived exosomes mediate cell-to-cell communication between adipocytes and macrophages in adipose tissue,and also enter the blood circulation to affect the insulin sensitivity of organs such as liver and skeletal muscle.However,intramuscular adipocytes as an important cell cluster that regulates intramuscular fat,the composition and function of exosomes secreted by them are still unclear.In this study,intramuscular preadipocytes were cultured and induced to differentiate in vitro,and exosomes were separated by ultracentrifugation and polyethylene glycol precipitation(PEG).Transcriptome sequencing was used to construct a c DNA library of porcine intramuscular preadipocytes and intramuscular mature adipocytes exosomes.After filtering and quality control of the sequencing data,the differentially expressed non-coding RNA(sRNA and lnc RNA)and messenger RNA(m RNA)were screened.Combined with database comparison and bioinformatics analysis,the adsorption relationship of non-coding RNA in exosomes,the GO and KEGG enrichment analysis of target genes,and the pathway enrichment status and function of m RNA were predicted.In addition,through the co-culture of exosomes and muscle cells,the effect of exosomes secreted by intramuscular preadipocytes and intramuscular mature adipocytes on intramyocellular lipids was explored.The main research results obtained are as follows:1.Exosomes were separated from the culture supernatant of intramuscular preadipocytes and intramuscular mature adipocytes using ultracentrifugation and PEG precipitation.Transmission electron microscopy analysis,nanometer particle size analysis and exosomal labeling protein WB were used to identify exosomes.In addition,the purity of exosomes separated by ultracentrifugation is better,and protein contamination is less.2.After sRNA annotation and classification,exosomal miRNAs of intramuscular preadipocytes accounted for 0.06% of sRNA,and intramuscular mature adipocytes exosomes miRNAs accounted for 0.11% of sRNA.A total of 99 miRNAs were detected,13 of which were differentially expressed,including 5 up-regulated and 8 down-regulated miRNAs.Differential miRNAs target genes may regulate RNA transcription and protein phosphorylation,and participate in the regulation of growth,development and metabolism.3.After sequencing analysis,a total of 32 differential lnc RNAs were detected in exosomes.Compared with intramuscular preadipocytes,28 exosomal lnc RNAs of the intramuscular mature adipocytes were up-regulated and 4 were down-regulated,including23 known and 9 newly identified lnc RNAs.A network interaction of lnc RNA and miRNA that may be adsorbed was constructed.The function of lnc RNAs target genes may be involved in the regulation of cell growth and development,glucose and lipid metabolism.4.A total of 3249 m RNAs were detected in intramuscular preadipocytes and intramuscular mature adipocyte exosomes,including 860 differentially expressed m RNAs.Compared with the former,the latter had 787 up-regulated and 73 down-regulated m RNAs.KEGG and GO enrichment analysis found that they are mainly involved in the process of protein translation,and are related to pathways such as autophagy and lipid metabolism.5.Exosomes secreted by intramuscular mature adipocytes significantly promoted lipid deposition and the expression of lipid synthesis genes in muscle cells.In summary,this study has isolated and identified the exosomes of porine intramuscular preadipocytes and intramuscular mature adipocytes,and detected the RNA expression profile of two cells types exosomes by transcriptome sequencing.It was found that the differentially expressed exosomal RNA may be involved in processes such as transcription and protein translation,and is related to lipid metabolism.In addition,intramuscular mature adipocytes significantly promoted intramyocellular lipids deposition.The study is of great significance for exploring the functions of porine intramuscular adipocytes exosomes and their intercellular communication with muscle cells.