The Effects And Molecular Mechanism Of 5-azaeytidine On Porcine Preadipocyte Differentiation

Adipose tissue is an important constituent of living organism that regulates and coordinates energy homeostasis. In recent years, adipose tissue has also been identified as a major endocrine organ that regulates diverse activities, such as immunological responses, insulin sensitivity, lipid metabolism and appetite regulation. The maintenance of adipose tissue function depends on white adipocytes, the main cellular component of white adipose tissue. Adipocytes are derived from mesenchymal stem cells(MSCs), which differentiate first into lipoblasts, then into preadipocytes and eventually into mature adipocytes. The differentiation of preadipocytes is regulated by a variety of differentiation-related transcription factors and is accompanied by the temporal expression of a series of adipogenic genes. A number of recent studies showed that DNA methylation/demethylation regulation plays a critical role in several differentiation processes and possibly in adipocyte differentiation, however, the associated molecular pathways and gene regulatory networks is still unclear. 5-azacytidine is a typical DNA methylation inhibitors, belongs to the cytosine nucleoside analogues, can be achieved whole-genome DNA demethylation. In order to further understand the role of DNA demethylation in the adipogenesis, in this study we elucidated the effects and molecular mechanism of 5-Azaeytidine on porcine preadipocyte differentiation.Through isolating and characterizing subcutaneous porcine preadipocytes, we successfully established primary porcine preadipocytes differentiation system ex vivo. After 5-Azacytidine treatment, the long fusiform porcine preadipocytes became elliptical gradually. Comparing to the control group, cell morphology is more similar to the differentiation state. Preadipocytes treated with 5-Azacytidine were then induced for differentiation, the results showed that the number and volume of lipid droplets were increased. Moreover, the m RNA expression levels of PPARγ and C/EBPα were significantly increased, whereas the expression level of KLF2 m RNA was significantly decreased compared to the control group. These data suggested that 5-Azacytidine promote the differentiation of porcine preadipocytes.To clarify the molecular mechanism of the effect of 5-Azaeytidine on porcine preadipocyte differentiation, we use high-throughput sequencing to detect the differential gene expression of porcine preadipocyte treated either with 5-Azaeytidine or control. Porcine preadipocyte with 5-Azaeytidine treatment had 4030 differentially expressed genes compared with the control, including 3332 up-regulated genes, 698 down-regulated genes. We screened out 5 high expression abundance, large fold difference and adipocyte differentiation-related genes: TXNIP, TXNRD1, IGFBP2, IGFBP4 and IGFBP5. Bisulfite sequencing analysis found that 5-Azaeytidine reduces the methylation level of the TXNRD1 gene promoter region, demonstrating its regulatory role of gene expression through DNA demethylation.To analyze the effect of genes on the differentiation of porcine preadipocytes, TXNIP, TXNRD1, IGFBP2, IGFBP4 and IGFBP5 genes were silenced in porcine preadipocytes respectively. Silencing of TXNRD1 decreased the number and volume of lipid droplets, and decreased the m RNA expression levels of PPARγ and C/EBPα, whereas the expression level of KLF2 m RNA was significantly increased, which suggested that silencing of TXNRD1 inhibited the differentiation of porcine preadipocytes. In contrast, silencing of TXNIP, IGFBP2, IGFBP4 or IGFBP5 increased the m RNA expression of the PPARγ, C/EBPα and inhibited KLF2 in adipocytes, and inducing the formation of lipid droplets, which suggested that silencing of TXNIP, IGFBP2, IGFBP4 or IGFBP5 promoted the differentiation of preadipocytes. These results indicated that TXNRD1 is a positive regulator, while TXNIP, IGFBP2, IGFBP4 and IGFBP5 are the negative regulator of porcine preadipocytes differentiation.In summary, we found that 5-Azaeytidine promoted the differentiation of porcine preadipocytes through upregulating the expression of positive regulators, i.e. TXNRD1, and downregulating the expression of negative regulator i.e. TXNIP, IGFBP2, IGFBP4 and IGFBP5. The results of the present study not only provided an insight for understanding physiological regulation of adipogenesis, but also provided a basic knowledge for elucidating the causes of obesity.