Fig. 5

miR-212-3p inhibits osteogenic differentiation of hBMSCs. a Relative expression of miR-212-3p during osteoinduction of hBMSCs (n = 3). GAPDH was used for normalization relative to the day 0 group. b Transfection efficiency of miR-212-3p mimic or inhibitor by qRT-PCR in hBMSCs (n = 3). U6 was used for normalization. c Confocal microscopy of RUNX2 with DAPI counterstaining in the miR-NC-inhibitor, miR-212-3p-inhibitor, miR-NC-mimic, miR-212-3p-mimic groups after osteogenic induction for 7 days (n = 3). Scale bars: 20 μm. d Relative mRNA expression of ALP, RUNX2, and OCN measured via qRT-PCR in PM conditions (n = 3). GAPDH was used for normalization. e Images of ALP staining after 7 days of osteogenic differentiation, and ARS staining after 14 days of osteogenic differentiation in the miR-NC-inhibitor, miR-212-3p-inhibitor, miR-NC-mimic, miR-212-3p-mimic groups (n = 3). Histograms show ALP activity and AZR staining quantification by spectrophotometry. f, g Western blotting analyses of the protein expression of RUNX2, OCN, and β-actin in the miR-NC-inhibitor, miR-212-3p-inhibitor and miR-NC-mimic, miR-212-3p-mimic groups after osteogenic induction for 7 days (n = 3). Histograms show the quantification of band intensities. β-actin was used for normalization. (*p < 0.05; **p < 0.01; ***p < 0.001). ALP, alkaline phosphatase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GDF5, growth differentiation factor 5; hBMSCs, human bone marrow mesenchymal stem cells; PM, proliferation medium; OCN, osteocalcin; OM, osteogenic medium; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; RUNX2, runt-related transcription factor 2