Figure 6.

FZD4 is a direct target of miR-1292. (A) Bioinformatics analysis was performed to predict the miR-1292-binding seed sequence in the 3′ UTR of FZD4. Wild-type (WT) or mutant-type (MUT) constructs were inserted into the psiCHECK-2 reporter vector. Dual luciferase activity was measured in the lysates of different groups. (B and C) qRT-PCR and western blot were performed to analyze FZD4 expression in hADSCs. (D) The knockdown efficiency of three FZD4-specific siRNAs was confirmed by qRT-PCR and western blot. (E) SA-β-gal staining was performed after FZD4 knockdown. (F and G) The expression of critical regulators of cellular senescence and osteogenesis was analyzed by qRT-PCR and western blot. (H) ALP staining was performed on day 4 after FZD4 knockdown. (I) Mineral deposition was detected by alizarin red staining on day 15 of osteogenic differentiation. GAPDH was used as an internal control for western blot analysis and qRT-PCR data were normalized to PPIA or GAPDH expression; results are presented as the mean ± SD, n = 3. ^*P < 0.05, ^**P < 0.01, ^***P<0.001 compared to the control. Scale bars: 200 μm.