Study On Mir-142 And Mir-144 Down-Regulation Contributions To Differentiation Of Ovine Preadipocytes By Targeting FoxO1 Gene

FoxO1(Forkhead box protein 01)is one of the most deepest researched members of the FoxO subfamily and plays an important role in the regulation of adipocyte differentiation.FoxO1 protein can be modified by acetylation,deacetylation,phosphorylation,dephosphorylation,etc,thus plays its biological function.It is generally considered that the FoxO1 plays the roles in regulating of adipocyte differentiation through binding with its target gene PPARy(Peroxisome proliferator activated receptor γ),which is a key regulator for adipocyte differentiation.The microRNAs(miRNAs)are single-strand small RNAs.Although they do not encode proteins,they can bind to 3’ UTR or 5’ UTR(a few to the CDS)regions of the corresponding target genes,thereby promoting or inhibiting the expression of target genes.The mechanism of miRNAs regulating FoxO1 gene in ovine,is lack of study.This study aims to predict and validate miRNAs that regulate the expression of FoxO1 gene,and elucidate the functional mechanism of these miRNAs in adipocytese differentiation.Four online softwares were used to predict the miRNAs that may target FoxO1.Dual-luciferase reporter system was employed to detect luciferase activity and verify whether the key miRNAs and FoxO1 have targeting relationships.qPCR,western blotting and Oil red O staining were used to detect the effect of these miRNAs on the expression of FoxO1 at mRNA and protein levels,and on the preadipocyte differentiation.Main results were shown as follows:(1)After obtaining the intersection of four target prediction softwares,6 miRNAs were found to have a target relationship with FoxO1,After a further screening,miR-142 and miR-144 were finally selected as the research subjects.Sequences of the miR-142 and miR-144 seed regions are completely complementary to the 2524～2529 bp and 2679～2685 bp of the 3’ UTR of FoxO1.It is demonstrated that miR-142 and miR-144 have a theoretic target relationships with FoxO1 gene.(2)The FoxO 1-3’ UTR vector was successfully constructed.Dual-luciferase reporter assay showed that the fluorescence activity in the over-expression group(co-transfected with pmir-FoxO1 and miR-142 mimic)was significantly lower(P<0.01)than that in the negative control group.Similarly,the fluorescence activity in the overexpressed group that co-transfected with pmir-FoxO1 and miR-144 mimic was also significantly lower than that in the negative control group(P<0.05).It is demonstrated that the binding of miR-142 or miR-144 to the 3’ UTR region of FoxO1 significantly inhibits the fluorescence activity of the dual-luciferase reporter vector.(3)Results of qPCR and western blotting showed that the overexpression of miR-142 mimic(or miR-144 mimic)in ovine preadipocytes down-regulated the expression of FoxO1 mRNA(P<0.01)and its encoded protein(P<0.05).This demonstrates that miR-142 and miR-144 negatively regulate the expression of FoxO1.(4)qPCR detection for PPARy expression showed that miR-142 and miR-144 promote the expression of PPARy mRNA.Both miR-142 and miR-144 promoted the differentiation of ovine preadipocytes and accelerated the formation of lipid droplets.Collectively,miR-142 and miR-144 can down-regulate the expression of FoxO1 and further down-regulate PARγ expression,thus play roles in promoting the differentiation of ovine preadipocytes.