Fig. 5.

Targeting relationship between HIF1A-AS2 and miR-665 and their effects on ASCs. a qRT-PCR: expression of miR-665 in normal/induced ASCs over time. b Predicted binding site between miR-556 and wt/mut HIF1A-AS2 3′UTR. c Dual-luciferase assay: luciferase activity in wt HIF1A-AS2 3′UTR + agomiR-665 group was significantly lower than that in wt HIF1A-AS2 3′UTR + agomiR-NC group. d qRT-PCR: expression of miR-665 in induced ASCs after agomiR-665, antagomiR-665, pcDNA3.1-HIF1A-AS2, or sh-HIF1A-AS2 transfection. e qRT-PCR: expression of Runx2, Osterix, and Osteocalcin after transfection. f Western blot analysis of Runx2, Osterix, and Osteocalcin after transfection. g Alizarin red staining (21 days) and ALP staining (14 days): changes of calcium nodule formation and ALP activity after transfection. ^*P < 0.05, ^**P < 0.01, compared with NC group; ^#P < 0.05, ^##P < 0.01, compared with sh-NC group