MicroRNA-148a Regulates the Proliferation and Differentiation of Ovine Preadipocytes by Targeting PTEN

SIMPLE SUMMARY: The miR-148a has been shown to play an important role in preadipocyte differentiation. Herein, we explored the role of miR-148a in ovine adipocyte development, using Oil Red O staining, CCK-8, EdU, flow cytometry and RT-qPCR. The results showed that miR-148a suppressed proliferation and facilitated the differentiation of preadipocytes. The dual fluorescent reporter vector experiments showed that miR-148a directly targeted PTEN. Meanwhile, we demonstrated that PTEN significantly inhibited the differentiation of preadipocytes. In conclusion, our research provides new insights that miR-148a inhibits ovine preadipocyte proliferation and accelerates differentiation by the negative regulation of PTEN. ABSTRACT: MicroRNAs (miRNAs) have been found to be involved in lipid deposition and metabolism. However, there have been no reports on the roles of miR-148a in the proliferation and adipogenesis of preadipocytes in sheep. In this study, the expression of miR-148a was profiled in the eight tissues of Tibetan ewes and differentiated preadipocytes, and the role of miR-148a in differentiation and proliferation of ovine preadipocytes was investigated using Oil Red O staining, CCK-8, EdU staining, cell cycle detection, and RT-qPCR. The effect of PTEN on the differentiation of ovine preadipocytes was also investigated. The miR-148a was widely expressed in the eight tissues investigated and had significantly increased expression in liver, spleen and subcutaneous adipose tissues, and the heart. The expression of miR-148a continued to increase with the differentiation of ovine preadipocytes. The over-expression of miR-148a significantly promoted differentiation but inhibited the proliferation of ovine preadipocytes. The inhibition of miR-148a had the opposite effect on the differentiation and proliferation of ovine preadipocytes with over-expressed miR-148a. The results from the dual luciferase reporter assays showed that miR-148a mimic significantly decreased the luciferase activity of PTEN-3′UTR dual luciferase reporter vector, suggesting that PTEN is a target gene of miR-148a. In over-expressed-PTEN preadipocytes, the number of lipid droplets remarkably decreased, and the expression levels of adipogenesis marker genes PPARγ, FASN, FATP4, GLUT4, C/EBPβ and LPL were also significantly down-regulated. These results suggest that miR-148a accelerated the adipogenic differentiation of ovine preadipocytes by inhibiting PTEN expression, and also inhibited the proliferation of ovine preadipocytes.