The Effect Of MicroRNA-204-5p On 3T3-L1 Pre-adipocytes Proliferation,Apoptosis And Differentiation

Excessive accumulation of lipids lead to metabolic abnormalities related to diseases such as cardiovascular disease,type 2 diabetes,and cancers.Body fat is also an important factor affecting meat quality of livestock and poultry products.It is helpfull to understand obesity or other related diseases and conducive to guide the work of animal genetic breeding that further study on the molecular mechanism of adipogenesis.Adipogenesis is a complex and tightly regulated process regulated by a network of lipid-related regulatory factors.Micro RNAs(miRNAs)are a class of 18-22 nt single-stranded RNA molecules,which have been proved to be a widely used mechanism for post transcriptional regulation of important cellular pathways,for instance,adipocytes proliferation,differentiation and apoptosis.Numerous studies suggested that miRNAs are significantly involved in adipose development,and mediated the adipocyte cells proliferation or differentiation,and participate in the genetic regulation of fat cells.In this study,obesity model of mice induced by high-fat diet were used to investigate the relationship between miR-204-5p and obesity.Meanwhile,3T3-L1 preadipocytes was transfected miR-204-5p mimics,inhibitors and negative control to investigate the regulatory mechanism and the effect of miR-204-5p on adipocyte proliferation,apoptosis and differentiation.The results are as follows:1.Mice induced by High fat diet(HFD)about which the total body weight,body mass index,serum levels of total triglyceride and total cholesterol were higher than mice feeding with normal chow(p < 0.05).The expression of key genes associated adipogenesis and miRNAs markers up-regulated significantly(p < 0.01),The expression levels of miR-204-5p were significantly negative correlation with mice weight,those clues show that miR-204-5-p may participate in the obesity genetic regulation.2..Experiment with 3T3-L1 preadipocytes proved that:miR-204-5p repressed CDK4,CDK2 and CDK5(p < 0.05),promoted p21 expression(p < 0.05).miR-204-5p inhibits3T3-L1 preadipocytes proliferation.3.Overexpression or inhibition of miR-204-5p in 3T3-L1 preadipocytes increased or decreased the expression levels of p53(p < 0.05),Luciferase reporter assays showed that miR-204-5p may induce adipocytes apoptosis by inhibiting Bcl-2.4.The results of qRT-PCRT indicated that: overexpression or inhibition of miR-204-5p increased or decreased the expression levels of genes(PPARγ、C/EBPα、Fabp4)associated with adipogenesis(p < 0.05).Accordingly,oil red staining and immunofluorescence analysis showed that overexpression or inhibition miR-204-5p could obviously increase the accumulation of triglyceride and adiponectin in cell.5.Overexpression or inhibition of miR-204-5p increased or decreased the expression levels of KLF3 in 3T3-L1 preadipocytes(p< 0.05).Fluorescence activity assay confirmed that miR-204-5p enhanced adipocytes differentiation by directly targeting the 3’-UTR of KLF3.6.The results of qRT-PCRT indicated that: miR-204-5p promote the expression of these genes related to fatty acid synthesis,lipogenic transcription,fatty acid transportation(P < 0.05),but diminish the expression of these genes related fatty acid β-oxidation(P <0.05).Those results indicated that miR-204-5p take part in the fatty acid metabolism.In conclusion,miR-204-5p has a variety of functions to regulate adipocyte:such as inhibited 3T3-L1 pre-adipocytes proliferation,induced apoptosis by directly targeting Bcl-2,promoted 3T3-L1 pre-adipocytes differentiation via negatively regulating KLF3.These data enrich the knowledge about that miRNA mediated the process of adipocyte development.