Identification Of Differentially Expressed MiRNAs During MSCs Adipogenesis And Myogenesis In Cattle And Regulation Mechanism Of MiR-23a In Adipogenesis

Intramuscular fat(IMF)is critical indicator that can reflect meat quality,which is closely related to beef juiciness,tenderness and flavor.Skeletal muscle and IMF are both developed from the mesenchymal stem cells(MSCs)located in mesoderm.In bovine,skeletal muscle development initiates from the early pregnancy while IMF development initiates during mid pregnancy.During embryo and fetal period,MSCs develop into skeletal muscle,bone,adipose tissue,fibroblast and vessel under regulation of molecules including transcription factor,miRNAs etc.The differentiation of MSCs into skeletal muscle and IMF is crucial for postnatal performance.To illuminate the mechanisms of miRNAs involved in MSCs differentiation into myocyte and adipocyte,we isolated MSCs from fetal skeletal muscle at 5-month of gestation and constructed the in vitro myogenesis and adipogenesis model.We aim to identify the differentially expressed miRNAs between undifferentiated and differentiating cells using miRNA sequencing and bioinformatics analyses.Moreover,the mechanism of one differentially expressed miRNA,bta-miR-23 a,which was involved in adipogenic differentiation,was further investigated and validated.The major findings of this study are as follows:1.On the basis of previous collagenase IV digesting method,we used CD140 a as a surface marker to isolate and purify the intramuscular preadipocyte and myoblast.After myogenic induction,CD140 a negative cells display strong myogenesis and emerge a large number of long myotubes,while only a small percentage of CD140 a positive cells form short myotube.qPCR analysis showed that expression of MYOD,MYOG and MYF5 in CD140 a negative cells were significantly higher than those in CD140 a positive cells.After adipogenic induction,CD140 a positive cells display strong adipogenesis and lipid accumulation.However,only a small percentage of CD140 a negative cells exhibited lipid accumulation.qPCR analysis showed that expressions of ZNF423,PPAR?,C/EBP?,FABP4 in CD140 a positive cells were significantly higher than those in CD140 a negative cells.In this study,we constructed an optimal method for isolate intramuscular preadipocyte and myoblast from fetal bovine skeletal muscle.Our findings will provide a powerful method for studying bovine myogenesis and IMF adipogenesis in vitro.2.On the basis of previous adipogenic method using dexamethasone,IBMX,insulin and rosiglitazone,we evaluated the BMP4 effects on the bovine preadipocyte differentiation.After BMP4 treatment and induction,BMP4 group cells exhibit more lipid numbers and larger lipid size.qPCR analysis showed that expressions of ZNF423,PPAR?,C/EBP?,FABP4 in BMP4 treatment group were significantly higher than those of control group.These results suggested BMP4 may enhance bovineadipogensis in vitro.3.Using miRNAs sequencing and bioinformatics analysis,we identified 90 miRNAs differentially expressed between undifferentiated and differentiating myoblast cells and 45 miRNAs were up-regulated and 45 were down-regulated.Additionally,56 miRNAs differentially expressed between undifferentiated and differentiating preadipocytes were identified,and 30 of those were up-regulated and 26 were down-regulated.4.In adipogenesis of fetal bovine skeletal muscle derived preadipocyte,miR-23 a was identified as a differentially expressed miRNA and was down-regulated immediately 1 day after adipogenic induction.After transfection of miR-23 a mimics,the adipogenic differentiation was inhibited and lipid accumulation decreased.qPCR revealed that PPAR?,C/EBP? decreased significantly.Meanwhile,after transfection of miR-23 a inhibitors the expression level of PPAR?,C/EBP?,FABP4 increased.In this study,ZNF423 was predicted as a miR-23 a target according to the Targetscan software.This interaction between miR-23 a and ZNF423 3'UTR was identified using dual luciferase reporter assay system.The protein level of ZNF423 was decreased after miR-23 a mimics transfection,while up-regulated of protein level of ZNF423 was found after inhibitors transfection.These results showed that miR-23 a inhibited fetal bovine skeletal muscle derived preadipocyte differentiation by targeting ZNF423.