Department of Pharmacology and Dental Therapeutics, School of Dentistry, Chonnam National University, Gwangju, Republic of Korea; Research Center for Biomineralization Disorders and Dental Science Research Institute, School of Dentistry, Chonnam National University, Gwangju, Republic of Korea.
J Cell Physiol. 2015 Apr;230(4):911-21. doi: 10.1002/jcp.24822.
Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) is a potent transcription factor that represses osteoblast differentiation and bone formation. Previously, we observed that stimuli for osteoblast differentiation, such as bone morphogenetic protein 2 (BMP2), inhibits COUP-TFII expression. This study was undertaken to identify BMP2-regulated and COUP-TFII-targeting microRNAs (miRNAs), and to explore their regulatory roles in osteoblast differentiation. Based on in silico analysis, 12 miRNAs were selected and their expression in BMP2-treated MC3T3-E1 cells was examined. BMP2 induced miR-302a expression in dose- and time-dependent manners with the decrease in COUP-TFII expression. Runx2, a BMP2-downstream transcription factor, specifically regulated miR-302a expression and its promoter activity. A computer-based prediction algorithm led to the identification of two miR-302a binding sites on the 3'-untranslational region of COUP-TFII mRNA (S1: 620-626 bp, S2: 1,016-1,022 bp), and a luciferase assay showed that miR-302a directly targeted S1 and S2. Transfection of miR-302a precursor significantly enhanced expression of osteogenic marker genes with decreasing COUP-TFII mRNA and protein level, alkaline phosphatase activity and matrix mineralization. On the other hand, inhibition of miR-302a significantly attenuated BMP2-induced osteoblast specific gene expression, alkaline phosphatase activity, and matrix mineralization with increasing COUP-TFII mRNA and protein level. These results indicate that miR-302a is induced by osteogenic stimuli and promotes osteoblast differentiation by targeting COUP-TFII. MiR-302a could be a positive regulator for osteoblast differentiation.
鸡卵清蛋白上游启动子转录因子 II（COUP-TFII）是一种有效的转录因子，它抑制成骨细胞分化和骨形成。先前，我们观察到成骨细胞分化的刺激物，如骨形态发生蛋白 2（BMP2），抑制 COUP-TFII 的表达。本研究旨在鉴定 BMP2 调节和 COUP-TFII 靶向的 microRNAs（miRNAs），并探讨它们在成骨细胞分化中的调节作用。基于计算机分析，选择了 12 个 miRNA，并检测了它们在 BMP2 处理的 MC3T3-E1 细胞中的表达。BMP2 以剂量和时间依赖的方式诱导 miR-302a 的表达，同时降低 COUP-TFII 的表达。BMP2 下游转录因子 Runx2 特异性调节 miR-302a 的表达及其启动子活性。基于计算机的预测算法确定了 COUP-TFII mRNA 3'-非翻译区（UTR）上的两个 miR-302a 结合位点（S1：620-626 bp，S2：1016-1022 bp），荧光素酶报告基因检测显示 miR-302a 直接靶向 S1 和 S2。miR-302a 前体的转染显著增强成骨标志物基因的表达，同时降低 COUP-TFII mRNA 和蛋白水平、碱性磷酸酶活性和基质矿化。另一方面，抑制 miR-302a 显著减弱 BMP2 诱导的成骨细胞特异性基因表达、碱性磷酸酶活性和基质矿化，同时增加 COUP-TFII mRNA 和蛋白水平。这些结果表明，miR-302a 是由成骨刺激诱导的，并通过靶向 COUP-TFII 促进成骨细胞分化。miR-302a 可能是成骨细胞分化的正调节剂。
