MiR-194-5p Targets MAP3K3 To Promote Rabbit Preadipocyte Differentiation

MicroRNAs(miRNAs)are a kind of endogenous non-coding small RNAs with a length of 22~24nt,and a lot of researches has found that miRNAs play important roles in fat metabolism and the differentiation of preadipocytes.It has been confirmed that COUP-TFⅡ is a target gene of miR-194-5p and overexpression of COUP-TFⅡ can suppress the differentiation of 3T3-L1 cells,indicating that miR-194-5p can take part in the regulation of preadipocyte differentiation.According to the prediction analysis,MAP3K3 is another target gene of miR-194-5p.MAP3K3 can activate MEK5,and the latter can activate ERK5,which can phosphorylate PPARγ and then suppress the differentiation of preadipocytes.In order to explore the influence and function of miR-194-5p on rabbit preadipocyte differentiation,this study isolated and cultured rabbit preadipocytes,and utilized miR-194-5p mimic to transfected the rabbit preadipocytes which were then induced into differentiation and we detected the changes of intracellular lipid droplets as well as the mRNA levels of adipogenesis-related genes during differentiation.For identifying the potential target relationship between MAP3K3 gene and miR-194-5p,we constructed pmir-GLO-MAP3K3-3’UTR and pmir-GLO-Mut-MAP3K3-3’UTR recombinant plasmids and detected the luciferase activity via dual-luciferase system reflecting the combination of MAP3K3 and miR-195-5p.For further exploring the effect of MAP3K3 gene on differentiation of rabbit preadipocytes,we inhibited the expression of MAP3K3 using siRNA and then detected the changes of lipid droplets and the mRNA levels of related genes.Besides,we detected the changes in protein levels of PPARγ and MAP3K3 after overexpression of miR-194-5p or inhibiton of MAP3K3,respectively.All the results are as follows:(1)During the induction of rabbit preadipocyte differentiation,the expression levels of miR-194-5p increased gradually and the expression levels on 5 d and 9 d were significantly higher than that on 0 d(P<0.01)and it showed the highest on 9 d.(2)Transfection with miR-194-5p mimic significantly increased the expression level of miR-194-5p in cells(P<0.01).Overexpression of miR-194-5p has increased intracellular lipid droplets formation and promoted triglyceride(TG)content in cells(P<0.05).In addition,overexpression of miR-194-5p significantly promoted the mRNA levels of C/EBPα and PPARγ(P<0.05),while it reduced the mRNA levels of MAP3K3,MEK5 and ERK5.(3)Target gene prediction indicated that MAP3K3 gene was a potential target gene of miR-194-5p,and the binding sites were conserved in many species.According to the dualluciferase system analysis,we found that miR-194-5p could directly target MAP3K3 and extremely significantly down-regulated firefly luciferase activity(P<0.01).(4)Using siRNA to inhibit the expression of MAP3K3 in cells,the expression of MAP3K3 was reduced by 60% and the expression of the downstream genes,MEK5 and ERK5,were significantly down-regulated(P<0.05).Inhibiting the expression of MAP3K3 gene significantly promoted the intracellular lipid droplets formation(P<0.01),and the expression levels of C/EBPα and PPARγ genes were significantly increased(P<0.01).Besides,the results of Western-blotting showed that overexpression of miR-194-5p and inhibition of MAP3K3 had similar results,which reduced the protein level of MAP3K3 while increased that of PPARγ.All above results have indicated that during the differentiation of rabbit preadipocyte,miR-194-5p could promote rabbit preadipocyte differentiation via targeting MAP3K3.