The Regulation Mechanism Of MiR-299a-3p On Ovine Adipocytes Differentiation By Targeting UCP2

Fat accumulation of mammals is mainly determine by the proliferation,differentiation and hypertrophy of adipocytes.And the formation of adipocytes,including proliferation,directional and terminal differentiation of mesenchymal stem cells.This process involving a lot of adipocytes transcription factors cascade control,multiple signaling pathways and a lot of regulatory miRNAs.In the research of our lab before,high through-put sequencing was used to identify a group of potential regulatory miRNAs of fat metabolism.This study selected miR-299a-3p from those miRNAs,to explore its relationship with the target gene in sheep fat metabolism regulation.This research used sheep preadipocytes for in vitro research model.Using bioinformatics analyzed the potential targets of miR-299a-3p and mapping to KEGG pathway.Detected the expression of miR-299a-3p between different breeds of fat tail sheep,and the temporal expression of miR-299a-3p and UCP2 during the differentiation of preadipocytes by qPCR.MiR-299a-3p over-expression vector were constructed and transfected in sheep preadipocytes,tested the facticity of the binding site by luciferase report system,qPCR and western blotting tested the expression of UCP2 after over-expression of miR-299a-3p.The main results were as follows:(1)MiRanda,TargetScan and DIANA-microT these three online target gene prediction softwares were used and analyzed UCP2 as a candidate target of miR-299a-3p,and reverse predicted the regulatory miRNAs by these three prediction softwares which binding with UCP2 3'-UTR,found that and UCP2 is the most appropriate target of miR-299a-3p relationship,and the combination is highly conservative in multiple species.(2)The expression of miR-299a-3p and its potential target genes UCP2 were significantly(P<0.01)negative correlation during sheep preadipocytes differentiation(3)Dual-luciferase report system proved that the reality of binding site between miR-299a-3p UCP23'-UTR region.Results of qPCR and western blotting showed that miR-299a-3p can significantly(P<0.05)inhibited UCP2 mRNA and UCP2 protein expression.(4)Oil red O stain,and the expression of two marker gene PPARy and AP2 were tested,by these results found miR-299a-3p up-regulate the process of sheep preadipocytes differentiation.Combining the results found that UCP2 is a directly target of miR-299a-3p,and miR-299a-3p down-regulate expression of UCP2,further up-regulate the sheep preadipocytes differentiation process.The above results lay the foundation for further study of miR-299a-3p on sheep preadipocytes differentiation mechanism.