hsa-miR-30c2 negatively regulates RUNX2, FRZB, and BGLAP in primary human bone cells. (A) Predicted binding sites for hsa-miR-30c2 in RUNX2, FRZB, and BGLAP 3′ UTR by the TargetScan, miRanda, and PicTar softwares. (B) Pearson correlation scatter plots showing a negative correlation between hsa-miR-30c2 RUNX2 (P = 0.0148), FRZB (P = 0.003), and BGLAP (P = 0.0325). (C) RT-PCR analysis of RUNX2, FRZB, and BGLAP mRNA levels normalized to GAPDH. (D) The amount of RUNX2 and FRZB 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.
