The Molecular Mechanism Of MiR-33a Regulates Preadipocyte Differentiation In Sheep

To adapt to cold and long winters,sheep form a rich deposit of subcutaneous adipose tissue,which is the consequence of long-term adaptive selection.However,the excessive storage of subcutaneous fat can seriously reduce the feed efficiency of domestic animals;further,it is not conducive to the further improvement of production and meat quality.Micro RNAs(miRNAs)comprise a type of endogenous short non-coding RNA.These molecules can degrade or inhibit the translation of m RNA through complementary binding.miR-33 loss-of-function in adipose tissue can increase preadipocyte proliferation,enhance lipid uptake,and impair lipolysis.However,the mechanism that miR-33 a affects the differentiation of ovine preadipocytes and adipogenesis is not fully understood.The aim of this study was to reveal the biological function of miR-33 a during the differentiation of ovine preadipocytes.Bioinformatics software was used to predict the target genes of miR-33 a.The potential target genes were verified by double luciferase report systems.In order to elucidate the regulatory mechanism of miR-33 a during the differentiation of ovine preadipocytes,miR-33 a was overexpressed and inhibited in ovine preadipocytes.In order to elucidate the regulatory mechanism of SIRT6 during the differentiation of ovine preadipocytes,SIRT6 was overexpressed or interfered by lentivirus-mediated method.The main results of this study are shown as follows:(1)miR-33 a targets Lipin1,IRS2 and SIRT6The binding sites of miR-33 a with Lipin1 and IRS2 were found by bioinformatics prediction,the expression levels of miR-33 a and Lipin1,IRS2 exhibited opposite trends during ovine preadipocyte differentiation.miR-33 a significantly reduced the luciferase activity of reporters containing the 3’-UTR of Lipin1,IRS2 and SIRT6,whereas no significant difference was observed in mutant or blank vectors.Lipin1,IRS2 and SIRT6 were identified as target genes of miR-33 a.(2)miR-33 a negatively regulates the differentiation of ovine preadipocytes and inhibits adipogenesisOverexpressing miR-33 a significantly down-regulated the expression of adipogenesis markers including PPARγ,C/EBPα,Adiponectin,FABP4,SREBP1,and SREBP2.In contrast,these markers were significantly up-regulated upon inhibiting miR-33 a expression.Overexpressing miR-33 a reduced lipid droplet deposition,and inhibiting of miR-33 a promoted lipid droplet deposition.These results indicate that miR-33 a affected the expression of adipogenesis markers genes,and inhibited the differentiation of ovine preadipocytes.(3)miR-33 a inhibits the differentiation of ovine preadipocytes and the deposition of lipid droplets by targeting the 3′-UTR of Lipin1,IRS2 and SIRT6.The overexpression of miR-33 a significantly down-regulated the expressions of Lipin1,IRS2 and their encoded proteins.After the miR-33 a was inhibited,the expressions of these genes and proteins were significantly up-regulated.miR-33 a had no significant effect on the expression of SIRT6 m RNA in both cases.However,the expression of SIRT6 protein was down-regulated by overexpressing miR-33 a and up-regulated by inhibiting miR-33 a.These results indicated that miR-33 a regulates SIRT6 expression at the translational level.(4)SIRT6 promotes the differentiation of ovine preadipocytes and the deposition of lipid dropletsThe m RNA expression of these six adipogenesis markers significantly increased after overexpressing SIRT6,but were significantly decreased after interfering with SIRT6.In addition,overexpressing SIRT6 formed more lipid droplets in ovine preadipocytes,whereas interfering with SIRT6 resulted in fewer lipid droplets.Based on pervious experiments,our study shows that miR-33 a play important role in the regulation of ovine preadipocytes differentiation by targeting the 3′-UTR of Lipin1,IRS2 and SIRT6.Our result provides a reference for further exploring the functions of miRNAs in the development of subcutaneous adipose tissue.