title: MicroRNA-302a stimulates osteoblastic differentiation by repressing COUP-TFII expression.
abstract: 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.
authors.affiliation: 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.
authors.forename: In-Hong
authors.initials: IH
authors.lastname: Kang
authors.forename: Byung-Chul
authors.initials: BC
authors.lastname: Jeong
authors.forename: Sung-Woong
authors.initials: SW
authors.lastname: Hur
authors.forename: Hyuck
authors.initials: H
authors.lastname: Choi
authors.forename: Seung-Ho
authors.initials: SH
authors.lastname: Choi
authors.forename: Je-Hwang
authors.initials: JH
authors.lastname: Ryu
authors.forename: Yun-Chan
authors.initials: YC
authors.lastname: Hwang
authors.forename: Jeong-Tae
authors.initials: JT
authors.lastname: Koh
mesh_terms: D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000378:metabolism; D051837:COUP Transcription Factor II / Q000378:metabolism*; D002454:Cell Differentiation / Q000502:physiology*; D051379:Mice; D035683:MicroRNAs / Q000378:metabolism*; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology
chemical_list: D055396:Bone Morphogenetic Protein 2; D051837:COUP Transcription Factor II; C558265:MIRN302 microRNA, mouse; D035683:MicroRNAs; D012333:RNA, Messenger