MicroRNA-302a stimulates osteoblastic differentiation by repressing COUP-TFII expression.

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.