hsa-miR-125b negatively regulates BGLAP and FRZB in primary human bone cells. (A) Predicted binding sites for hsa-miR-125b in BGLAP and FRZB 3′ UTR by the TargetScan, miRanda, and PicTar softwares. (B) Pearson correlation scatter plots showing a negative correlation between hsa-miR-125b and BGLAP (P = 0.0450) and FRZB (P = 0.0117). (C) RT-PCR analysis of BGLAP and FRZB mRNA levels normalized to GAPDH. (D) The amount of FRZB protein was assessed by Western blot analysis with β-actin as a loading control (left panel) and BGLAP protein secreted into conditioned media was quantified by Western blot. Mean ± SD * is significantly different from negative control (NC) (right panel). (E) ALP staining was visualized at 10× magnification. (F) Deposition of calcium was visualized (left panel) and quantified (right panel), by Alizarin red stain in HOBs after they were treated in osteogenic medium but for 12 d.
