MicroRNA-10a Influences Osteoblast Differentiation and Angiogenesis by Regulating β-Catenin Expression

Jun Li, Yongqing Zhang, Qingxia Zhao, Jianghua Wang, Xijing He
2015 Cellular Physiology and Biochemistry  
Background/Aims: Elucidation of the molecular mechanisms governing osteoblast differentiation and angiogenesis are of great importance for improving the treatment of bone-related diseases. In this study, we examined the role of microRNA (miR)-10a in the differentiation of MC3T3-E1 cells and pro angiogenic activity of mouse umbilical vein endothelial cells (MUVECs). Methods: The murine pre-osteoblast cell line MC3T3-E1 and MUVECs were used in the experiment. After transfected with miR-10a mimics
more » ... with miR-10a mimics or inhibitors, with or without LiCl pretreatment, the miR-10a, ALP, Runx2, Osx, OC and Dlx5 expression were assessed by RT-PCR. MC3T3-E1 cells were cultured with BMP2 to differentiate into bone cells, osteogenic differentiation of MC3T3-E1 cells were detected by ALP and ARS staining. MMP2, and VEGF were detected by Western blotting; VEGF and VE-cadherin release were assessed by ELISA, and the migration of MUVECs, as well as tube formation were also detected. Results: MiR-10a expression was obviously down-regulated during osteogenic differentiation. Overexpression of miR-10a inhibited osteogenic differentiation of MC3T3-E1 cells, effectively decreasing MUVECs proliferation, migration, VEGF expression, VE-cadherin concentrations, and tube formation in vitro, whereas miR-10a silence enhanced those processes. Further protein and transcription level, while pretreatment with Wnt signaling activator Licl partially attenuated the suppression effects of miR-10a overexpression on osteoblast differentiation and angiogenesis. Conclusion: in osteoblast differentiation of MC3T3-E1 cells and pro angiogenic activity of MUVECs by the treatment of bone regeneration-related diseases. PCR products were electrophoresed on 1.5% agarose gels, and PCR fragments were visualized by ethidium bromide staining. Real-time PCR experiments for each gene were performed on three separate occasions. Western blotting using commercially available antibodies. Cells were lysed in cell lysis buffer (Beyotime, Haimen, China) and overnight. After incubation with HRP-conjugated secondary antibody for 1 h at room temperature, protein 95% ethanol for 10 min, washed with distilled water, and then were stained by alizarin red solution (1 g Tris and 0.1 g alizarin red (Bio Basic Inc., Canada) in 100 ml ultrapure water, adjusting the Ph to 8.3 by Hcl for 30 min at 37°C. After being washed with distilled water twice, the cells were photographed. To assess the ability of MUVECs to integrate in the vascular structure, a Matrigel tube formation cultured with the test compounds were seeded in the Matrigel-coated plate at concentrations of 5 × 10 ® was assessed under an inverted light microscope.
doi:10.1159/000438576 pmid:26610149 fatcat:jt6vx55ynbeelmp6wv4izfyq2i