miR-125b inhibits osteoblastic differentiation by down-regulation of cell proliferation
Section snippets
Materials and methods
Cell culture. ST2 cells were obtained from the RIKEN BioResource Center (BRC, Tsukuba, Japan) and cultured according to the protocols supplied by BRC (RPMI1640 supplemented with 10% fetal bovine serum (FBS)).
Osteoblastic differentiation. Osteoblastic differentiation was induced by changing to media containing 10% FBS supplemented with 100 ng/ml BMP-4 (R&D Systems, Minneapolis, MN, USA).
Alkaline phosphatase staining and measurement. Cells were fixed with 10% formalin for 20 min followed by 1 min
Increase in miR-125b expression is attenuated during osteoblastic differentiation
To determine the expression pattern of miR-125b during osteoblastic differentiation in ST2 cells, real-time RT-PCR was performed with the miRNA measurement protocol. As shown in Fig. 1, miR-125b expression was increased within 6 days in the cells that proliferated naturally without osteoblastic induction by BMP-4, suggesting that miR-125b may be involved in proliferation of ST2 cells. As a previous report showed that miR-125b has a negative effect on cell proliferation in cancer-related cells
Discussion
miRNAs have been reported to be involved in several important biological events, such as cell differentiation and proliferation. Although miRNAs are known to act as regulators in the differentiation of skeletal muscle, granulopoiesis and adipogenesis, there have been no reports so far that describe osteoblastogenesis-regulating miRNAs. Here, we reported details of miR-125b, a miRNA that regulates osteoblastic differentiation through the regulation of cell proliferation. We analyzed the function
Acknowledgments
We thank Shino Okumura and Megumi Otsu for technical assistance. We also thank Hiroyoshi Iseki, Hidemasa Bono, Yutaka Nakachi, Itoshi Nikaido, Shigeki Arai, and Riki Kurokawa for helpful discussion. This work was supported by grants of the Genome Network Project from the Ministry of Education, Culture, Sports, Science and Technology of Japan to Y.O., and also in part by the Ministry of Education, Culture, Sports, Science and Technology of Japan, and in particular by a Ministry Grant to Saitama
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- 1
Present address: Antibiotics Laboratory, RIKEN, 2-1 Hirosawa, Wako City, Saitama 351-0198, Japan.
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Present address: Nano Medical Engineering Laboratory, RIKEN, 2-1 Hirosawa, Wako City, Saitama 351-0198, Japan.