MicroRNA-200b Regulates the Proliferation and Differentiation of Ovine Preadipocytes by Targeting p27 and KLF9

Abstract

MicroRNAs (miRNAs) are crucial regulatory molecules in lipid deposition and metabolism. However, the effect of miR-200b on the regulation of proliferation and adipogenesis of ovine preadipocytes is unknown in the sheep (<i>Ovis aries</i>). In this study, the expression profiles of miR-200b were investigated in the seven tissues of Tibetan ewes and differentiated preadipocytes. The effect of miR-200b, as well as its target genes <i>p27</i> and <i>KLF9</i>, on the proliferation of ovine preadipocytes and adipogenesis was also investigated, using cell viability analysis, EdU staining, Oil Red O staining and reverse transcription-quantitative PCR (RT-qRCR). The miR-200b was expressed in all the tissues investigated, and it was highly expressed in lung, liver, subcutaneous adipose and spleen tissues. The expression of miR-200b continuously decreased when the differentiation of ovine preadipocytes initiated. The miR-200b mimic dramatically accelerated the proliferation but inhibited differentiation of ovine preadipocytes. The miR-200b inhibitor resulted in an opposite effect on the proliferation and differentiation of ovine preadipocytes. The dual luciferase reporter assay results showed that miR-200b mimic significantly decreased the luciferase activity of <i>p27</i> and <i>KLF9</i> in HEK293 cells transfected with wild-type dual luciferase reporter vectors. This suggests that <i>p27</i> and <i>KLF9</i> are the target genes of miR-200b. In over-expressed-<i>p27</i> preadipocytes, the number of EdU-labeled preadipocytes and the expression levels of proliferation marker genes <i>CDK2</i>, <i>CDK4</i>, <i>CCND1</i> and <i>PCNA</i> significantly decreased. In addition, the transfection of over-expressed-<i>KLF9</i> vector into adipocytes remarkably increased the accumulation of lipid droplets and the expression levels of differentiation marker genes <i>aP2</i>, <i>PPARγ</i>, <i>LPL</i> and <i>GLUT4</i>. These results suggest that miR-200b accelerated the proliferation but inhibited the adipogenic differentiation of ovine preadipocytes by targeting <i>p27</i> and <i>KLF9</i>, respectively.