Fig. 4

MiR-203‐3p inhibited osteogenic differentiation of hBMSCs. (A) The binding sites between miR‐203‐3p and RUNX2, predicted by bioinformatics analysis. (B) RUNX2-wt and RUNX2-mut luciferase activity in 293T cells treated with miR‐203‐3p mimic or NC mimic, determined by dual‐luciferase reporter gene assay. *P < 0.05, as comparison to the NC mimic group. The luciferase activity of RUNX2-wt with NC mimic group was normalized. (C) Transfection efficiency after coculturing miR‐203‐3p mimic or miR‐203‐3p inhibitor, validated by qRT-PCR. ****P < 0.0001, as comparison to the NC mimic group; **P < 0.01, as comparison to the NC inhibitor group. The miR-203-3p expression of NC mimic group was normalized. (D-E) Relative protein expression of RUNX2 in hBMSCs after transfection with miR‐203‐3p mimic or miR‐203‐3p inhibitor, measured by western blot. *P < 0.05, as comparison to the NC mimic group; *P < 0.05, as comparison to the NC inhibitor group. The RUNX2 expression of NC mimic group was normalized. (F) Relative ALP activity was examined in hBMSCs after transfecting miR‐203‐3p mimic or miR‐203‐3p inhibitor. *P < 0.05, as comparison to the NC mimic group; **P < 0.01, as comparison to the NC inhibitor group. The ALP activity of NC mimic group was normalized. (G) The mineralization levels of hBMSCs after transfecting miR‐203‐3p mimic or miR‐203‐3p inhibitor, employed by Alizarin red staining