miR-20a regulates adipocyte differentiation by targeting lysine-specific demethylase 6b and transforming growth factor-beta signaling

BACKGROUND: Several types of microRNAs (miRNAs) have recently been defined as important regulators in adipocyte differentiation, the role of other miRNAs in the processes and the mechanisms involved remain to be explored. METHODS: miR-20a expression was quantified in primary cultured marrow stromal cells and adipogenic cell lines after adipogenic treatment. Effects of miR-20a on adipocyte differentiation were studied following supplementing or depleting miR-20a in murine 3T3-L1 preadipocytes, ST2 stromal cells and C3H10T1/2 mesenchymal cells. Bioinformatics prediction of miRNA targets was performed, and potential targets of miR-20a were verified by using dual luciferase activity assays. Gain-of-function and loss-of-function studies were performed to examine the effects of the target genes on adipocyte differentiation. RESULTS: miR-20a was induced in primary cultured marrow stromal cells and established adipogenic lines after adipogenic treatment. Supplementing miR-20a activity suppressed the growth of 3T3-L1 preadipocytes and induced 3T3-L1, ST2 and C3H10T1/2 cells to differentiate into mature adipocytes, along with the induction of adipocyte-specific transcription factors peroxisome proliferator-activated receptor gamma (PPAR gamma), CCAAT/enhancer binding protein-alpha (C/EBP alpha), C/EBP beta and the marker gene adipocyte protein 2 (aP2). Conversely, inhibition of the endogenous miR-20a repressed 3T3-L1, ST2 and C3H10T1/2 cells to fully differentiate. Transforming growth factor-beta receptor II (Tgfbr2) and lysine-specific demethylase 6b (Kdm6b) were shown to be direct targets of miR-20a. Supplementing miR-20a activity in ST2 reduced levels of KDM6B and TGFBR2 proteins, while suppression of endogenous miR-20a increased KDM6B and TGFBR2. While TGF-beta signaling is a well-documented inhibitor of adipogenesis, the effects of Kdm6b on adipocyte formation need to be clarified. We demonstrated that overexpression of Kdm6b inhibited, while knockdown of Kdm6b promoted the differentiation of the ST2 cells into mature adipocytes. CONCLUSION: The present work provides evidence that mouse miR-20a promotes adipocyte progenitor cells to differentiate and this function may depend upon its inhibitory effects on Kdm6b and TGF-beta signaling.