The Regulatory Mechanism Study Of MiR-122 On Preadipocytes 3T3-L1 Differentiation And Maturation

MiR-122 is highly expressed in liver.It has been shown that C/EBPα is a transcriptional activator of miR-122 and AP-2α could negatively regulate C/EBPα.However,it is still unclear whether miR-122 could feedback regulate C/EBPα.C/EBPα is well-known as a key marker gene in the differentiation and maturation process from preadipocytes to adipocytes.In our studies,western blot detected decreased expression of C/EBPα when AP-2α was overexpressed.Semi-quantitative PCR and western blot assay showed decreased AP-2α expression and increased C/EBPα expression after overexpression of miR-122 in preadipocytes.Oil red O staining were taken in order to intuitively observe the influence of miR-122 on morphological changes and lipid droplet forming of adipocytes.Data showed that overexpression of miR-122 and silencing of AP-2α by RNA interference could enhance the lipid droplet forming in preadipocytes,but overexpression of AP-2α could decrease the lipid droplet accumulation.In most cases,miRNAs could negatively regulate gene expression by binding to the 3’untranslated region(3’UTR)of target mRNA and inhibit its translation.Using a computational program-TargetScan,we searched for MREs in the 3’UTR of the AP-2α gene,but we didn’t find any MRE in the 3’UTR of AP-2α mRNA.Besides targeting 3’UTR of mRNA,microRNA is also involved in DNA methylation.In order to verify the impact of miR-122 on DNA methylation in the promoter region of AP-2a,we did methylation-specific PCR analysis.The results showed that overexpression of miR-122 could lead to DNA methylation of AP-2a.In brief,this study revealed a new mechanism for the differentiation and maturation of preadipocytes:through inducing DNA methylation in the promoter region of AP-2α,miR-122 negatively regulate the transcription of AP-2a,thus positively regulate C/EBPα and ultimately promote preadipocytes differentiation and maturation.As C/EBPa is a transcription activator of miR-122,the increased C/EBPα will further promote the expression of miR-122.There exists a positive feedback regulatory loop.The excessive accumulation of lipids will lead to lipid metabolism disorders and organ pathophysiological changes,such as obesity and fatty liver.It is significant to investigate the regulatory mechanisms of miR-122 on 3T3-L1 preadipocytes differentiation and maturation.