Mir-15a/b Promote Adipogenesis In Porcine Pre-adipocyte Via Repressing Foxo1

Diabetes and many other metabolism syndromes are closely related to obesity. In orderto reveal the underlying mechanism of fat deposition, an increasing number of studies arefocusing on the functions of miRNAs during adipocytes development. Previous studies haveproved that miR-15a/b play important roles in multiple physiological processes; however,their functions during adipogenesis remain unclear. To reveal this, we detected the expressionprofiles of miR-15a/b during adipogenesis in porcine pre-adipocyte. Then two adenovirusvectors were constructed and infected the pre-adipocyte. After that, the target genes ofmiR-15a/b were filtrated and the dual luciferase reporter assay vectors of target genes wereconstructed. The target gene of miR-15a/b was identified by dual luciferase reporter assay.The results of this study are:1. miR-15a/b are conversed among mammals and highly expressed during the earlystage of adipogenesis. The data showed that the expression levels of miR-15a/b reached thehighest on the4thday, after that the expression levels of miR-15a/b decreased during4-10days. Which indicated that miR-15a/b might play a role in the early stage of adipogenesis.2. Two overexpression adenovirus vectors were constructed. We used adenoviruses totrasfected the cells, and the transfection efficiency in pre-adipocytes achieved almost100%onday0. qRT-PCR showed that the expression levels of miR-15a/b after infection raised2-foldsand4-folds respectively.3. The mRNA expression levels of PPARγ, aP2and SREBP-1C and protein expressionlevels of PPARγ, aP2and Fas were detected and the expression levels in miR-15a/bover-expression group were dramaticly high. And the Oil Red O staining results alsoillustrated that there were more lipid droplets in miR-15a/b groups than GFP and controlgroups.4. To identify the target gene of miR-15a/b, we performed a bioinformatics analysis andover thousands of potential target genes were predicted. The common target genes associatedwith adipogenesis predicted by at least three programs were FoxO1(predicted by TargetScan,RNA22and miRanda) and IRS1(predicted by TargetScan, PITA and miRanda). Theexpression levels of FoxO1and IRS1were detected and we found the expression level ofFoxO1were significantly decreased after the over-expression of miR-15a/b.5. Three dual luciferase reporter assay vectors (psiCHECK-2-FoxO1、psiCHECK-2-FoxO1-M and psiCHECK-2-IRS1) were constructed and identified, the results shown thatFoxO1is the target of miR-15a/b. The inhibition of FoxO1expression level caused by miR-15a/b overexpression had apositive effect on adipogenesis. Thus, we conclude that miR-15a/b promote adipogenesis inporcine pre-adipocyte via repressing FoxO1.