Zhou J, Guo F, Wang G, Wang J, Zheng F, Guan X, Chang A, Zhang X, Dai C, Li S, Li X, Wang B
Key Laboratory of Hormones and Development (Ministry of Health), 2011 Collaborative Innovation Center of Tianjin for Medical Epigenetics, Metabolic Diseases Hospital and Institute of Endocrinology, Tianjin Medical University, Tianjin, China.
1] Division of Endocrinology, Tianjin Medical University General Hospital, Tianjin, China [2] Division of Endocrinology, No. 3 Hospital of Xingtai, Hebei Province, China.
Int J Obes (Lond). 2015 Aug;39(8):1282-91. doi: 10.1038/ijo.2015.43. Epub 2015 Mar 30.
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.
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.
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 γ (PPARγ), CCAAT/enhancer binding protein-α (C/EBPα), C/EBPβ 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-β 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-β 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.
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-β signaling.
最近几种类型的微小RNA（miRNA）已被确定为脂肪细胞分化中的重要调节因子，其他miRNA在这些过程中的作用及其涉及的机制仍有待探索。
在成脂处理后的原代培养骨髓基质细胞和成脂细胞系中对miR-20a表达进行定量。在小鼠3T3-L1前脂肪细胞、ST2基质细胞和C3H10T1/2间充质细胞中补充或去除miR-20a后，研究miR-20a对脂肪细胞分化的影响。进行miRNA靶标的生物信息学预测，并通过双荧光素酶活性测定验证miR-20a的潜在靶标。进行功能获得和功能缺失研究以检查靶基因对脂肪细胞分化的影响。
在原代培养的骨髓基质细胞和成脂处理后的已建立的成脂细胞系中诱导了miR-20a。补充miR-20a活性抑制了3T3-L1前脂肪细胞的生长，并诱导3T3-L1、ST2和C3H10T1/2细胞分化为成熟脂肪细胞，同时诱导脂肪细胞特异性转录因子过氧化物酶体增殖物激活受体γ（PPARγ）、CCAAT/增强子结合蛋白-α（C/EBPα）、C/EBPβ和标记基因脂肪细胞蛋白2（aP2）。相反，抑制内源性miR-20a可抑制3T3-L1、ST2和C3H10T1/2细胞完全分化。转化生长因子-β受体II（Tgfbr2）和赖氨酸特异性去甲基化酶6b（Kdm6b）被证明是miR-20a的直接靶标。在ST2中补充miR-20a活性可降低KDM6B和TGFBR2蛋白水平，而抑制内源性miR-20a则增加KDM6B和TGFBR2。虽然TGF-β信号是一种已充分记录的脂肪生成抑制剂，但Kdm6b对脂肪细胞形成的影响尚需阐明。我们证明，Kdm6b的过表达抑制，而Kdm6b的敲低促进ST2细胞分化为成熟脂肪细胞。
本研究提供了证据表明小鼠miR-20a促进脂肪细胞祖细胞分化，且该功能可能取决于其对Kdm6b和TGF-β信号的抑制作用。
