The Mechanism Of DNMT3A In Proliferation And Differentiation Of Porcine Intramuscular Preadipocytes

In animal husbandry,intramuscular fat(IMF)content has been demonstrated as one of the crucial factors that affect meat quality characteristics,such as tenderness,juiciness and flavor level.The proliferation and differentiation of adipocytes determine the cell number and the accumulation of lipid droplets,and finally affect the fat deposition.The proliferation and differentiation of adipocyte is a highly coordinated process,which is regulated by a large number of transcription factors and various cytokines.And DNA methylation plays a vital role in this process.DNA methylation is a process by which methyl groups are added to the DNA molecule through DNMTs.It's results from the transfer by DNA methyltransferase of a methyl group from SAM to the cytosine residue within CpG dinucleotides.As a member of DNMTs,DNMT3 A can increases the methylation of microRNA promoter to regulate the progression of gastric cancer and promotes obesity-related inflammatory responses.However,the effect of DNMT3 A in proliferation and differentiation of porcine intramuscular preadipocytes remain unclear.DNA methylation inhibitor were used to test whether DNA methylation participate in the differentiation of porcine intramuscular preadipocytes.To investigate the DNMT3 A biological effect,recombinant adenovirus that can overexpress DNMT3 A were constructed to infect porcine intramuscular preadipocytes.EdU staining,Flow cytometry and CCK-8 were used to evaluate the proliferation,and BODIPY staining were used to evaluate the differentiation.Meanwhile,Real-time PCR and WB were used to detect the expression of related gene.And Bisulfite sequencing were used to clarify the mechanism.The main results of this study are shown as follows:1.5-Aza-DC inhibited the differentiation of porcine intramuscular preadipocytes and repressed the promoter methylation of PPARg.2.Overexpression of DNMT3 A promoted the the proliferation.The percentage of S-phage cells and total cell number was increased in Ad-DNMT3 A treated group compared with Ad-NC.At the same time,overexpression of DNMT3 A promoted cell cycle-related genes(such as cyclin B,cyclin D and PCNA)expression and repressed the cell cycle inhibitor p21.It was related with the increased methylation of p21 promoter.3.Overexpression of DNMT3 A increased the methylation of PPARg promoter,and led to lower expression of PPARg and aP2,finally inhibited the differentiation of porcine intramuscular preadipocytes.In conclusion,the study showed that overexpression of DNMT3 A promoted the the proliferation which was related with methylation of p21 promoter while inhibited the differentiation of porcine intramuscular preadipocytes through the increase of methylation in PPARg promoter.This study reveals the mechanism of fat deposition in intramuscular fat from the perspective of DNA methylation and provides a scientific basis for meat quality traits in breeding.