Research On T2DM Pathogenesis By Transcriptome And Proteome Of Skeletal Muscle In Bama Mini-pig

Type 2 diabetes mellitus(T2DM)is a complex metabolic disease affected by multiple factors.Large-scale and multi-omics methods provide new strategies for T2DM pathogenesis study,diagnosis,and developing of animal T2DM model.In this study,we constructed T2DM model(T2D)and non-T2DM model(NT2D)of Guangxi Bama mini-pigs.RNA-seq and iTRAQ techniques were used to analyze the transcriptomics and proteomics of skeletal muscle from these animals,in order to explore the pathogenesis of T2DM in Bama mini-pigs.Forty Guangxi Bama mini-pigs(18 sows and 22 castrated pigs)with similar age(6-7 months old)and weight(about 20 kg)were selected and 9 and 31 of them were randomly assigned into control group(CON)and experimental group respectively.The control group was fed a basic diet,while the experimental group was fed a high-fat and high-sugar diet(consisting of 60%basic diet,35%sucrose and 5%lard).After fed with high-fat and high-sugar diets for 12 months,the blood glucose levels in fasting state and intravenous glucose tolerance test(IVGTT)were determined,and six pigs with abnormal blood glucose and six pigs with normal blood glucose were selected and assigned into T2D group and NT2D group,respectively.The transcriptome and proteome of longissimus dorsi muscle from Bama mini-pigs were used RNA-seq and iTRAQ technology.The main experimental results are as follows:1.The construction of Guangxi Bama mini-pigs T2DM modelCompared with NT2D individuals,fasting blood glucose increased,lipid metabolism disordered,absolute values of HOMA-IR index and HOMA-ISI index significantly increased(p<0.05),and glucose tolerance of IVGTT test decreased in T2D individuals.The histological analysis of liver,kidney and pancreas were carried out and the pathological changes were found in these organs from T2D individuals.2.The transcriptome of skeletal muscle in Bama mini-pigsThe transcriptome of longissimus dorsi muscle from Bama mini-pigs revealed 290 differentially expressed genes(DEGs)in NT2D compared with CON groups,572 DEGs in T2D compared with CON groups,and 300 DEGs in T2D compared with NT2D groups.The qPCR results of PGC1?,NR4A3,CSRP3,MYH7,MYH2,MYH1,GLUT4,PPARy,LEP,ATP5H,UCP2 and MTOR genes from the transcriptome indicated that the transcriptomic result was reliable.The analysis of KEGG displayed that differentially expressed genes in three groups mainly involved in the signaling pathways related to metabolism,disease,inflammation,immunity,genetic modification,cytoskeleton and signal transduction.The differentially expressed genes in T2D and NT2D groups mainly involved in signaling pathways associated with cardiovascular diseases,infectious disease and cancer-related signaling pathways,inflammatory and immune-related signaling pathways,and the cytoskeleton and signal transduction pathways.A comparative analysis of the transcriptome of T2DM Bama mini-pigs with T2DM patients(GSE29221)revealed 17 differentially co-expressed genes which mainly involved in adipocytokine signaling pathway and hypertrophic cardiomyopathy(HCM).Comparing the annotated signaling pathways,six identical signaling pathways were obtained,e.g.hypertrophic cardiomyopathy,dilated cardiomyopathy,thyroid cancer,cadiac muscle contraction,tight junction and calcium signaling pathway,which indicated that the T2DM pathogenesis is similar between human and Bama mini-pig in these pathways.3.The proteome of skeletal muscle in T2D and NT2D Bama mini-pigsThe proteome of longissimus dorsi muscle from Bama mini-pigs revealed that 13 differentially expressed proteins(DEPs)existed between T2D group and NT2D group,of which one protein was up-regulated and 12 proteins were down-regulated.The protein expression levels of LUM,DCN,OGN,PRELP,BGN,and CSRP3 detected by PRM technology were consistent with iTRAQ results.GO analysis revealed that most DEPs were extracellular matrix proteins.KEGG analysis revealed that DEPs involved inflammation and disease(proteoglycans in cancer,malaria,phagosome,and focal adhesion),metabolism(2-oxocarboxylic acid metabolism;pantothenate and CoA biosynthesis;cysteine and methionine metabolism;valine,leucine and isoleucine degradation;biosynthesis of amino acids;protein digestion and absorption)and signal transduction pathways(TGF-beta signaling pathway,ECM-receptor interaction,and PI3K-Akt signaling pathway).The associated analysis based on proteome and transcriptome revealed that 1641 genes were associated with each other,of which 13 were differentially expressed proteins and 95 were differentially expressed genes.Both proteomic and transcriptomic analysis found that the CSRP3 expression was significantly down-regulated in T2D animals(p<0.05).The genes associated proteome with transcriptome were mainly involved in metabolism,genetic and environmental information processing,cellular and organismal systems and human diseases and other related signal pathways.4.Function analysis of CSRP3 gene in T2D and NT2D Bama mini-pigsThe CDS sequence size of CSRP3 gene from Bama mini-pig was 585 bp.Four synonymous mutations were detected and the homology with CDS sequence released by NCBI was 99.7%.The expression level of CSRP3 gene in T2D group was lower than that of NT2D group.The trend of CSRP3 gene expression was the most similar to MYHC1 gene,positively correlated with MYHC1,MYHC2A and MYHC2X genes,and negatively correlated with MYHC2B gene.The CSRP3 gene positively correlated with TG,TC,HDLC and LDLC,and negatively correlated with ISI index.The relationship of CSRP3 gene expression and T2DM related-index was not significant(p>0.05)from 1 month to 11 month(M),while it was significantly positively correlated with TG in 1M(p<0.05).There was a significantly positive correlation with FBG,INS and IR index(p<0.05),and a significantly negative correlation with ISI index at 12M(p<0.05),suggesting that the gene expression level of CSRP3 may be a marker of T2DM.Overexpression and RNAi experiments of CSRP3 gene in C2C12 cells showed that up-regulated CSRP3 expression could significantly down-regulated the IRS1,AKT1,GLUT4 and LPL expression(p<0.05),and down-regulated CSRP3 expression could significantly up-regulated LPL and down-regulated IRS1 expression(p<0.05).In summary,the signaling pathways related to metabolism,signal transduction,inflammation and disease in skeletal muscle from T2DM Bama mini-pig were dramatically changed in both transcriptome and proteome,which may be the important causes for T2DM developed in Bama mini-pig.The decreased expression of CSRP3 gene may affect the fiber type conversion,fat storage and oxidative metabolism in skeletal muscle,which may be related with the T2DM pathogenesis.