Figure 5.

BMP2 is the direct target gene of miR‐204 and miR‐211. A, Schematic representation of miR‐204/211 putative target sites in BMP2 3′‐UTR and alignment of miR‐204/211 with WT and MUT BMP2 3′‐UTR showing pairing. B, Luciferase reporter assays were performed using luciferase constructs carrying a WT or mutant BMP2 3′‐UTR cotransfected into VSMCs with miR‐204/211 mimics compared with an empty vector control. Firefly luciferase activity was normalised to Renilla luciferase activity. C, miR‐204/211 mimics or miR‐204/211 inhibitor regulated BMP2, RUNX2 and p21 protein expression as indicated by Western blotting. D, Knock‐down of BMP2 by siRNA decreased the protein level of BMP2, RUNX2 and p21 as indicated by Western blotting. BMP2 siRNA attenuated the effect of exosomes from MT‐treated VSMCs (MT exo) on RUNX2 and p21 expression detected by Western blotting. E, ALP activity was measured with an ALP measurement kit in the VSMCs with BMP2 siRNA group, BMP2 siRNA plus exosomes from MT‐treated VSMC group (MT exo) and the siRNA group. F, CCK8 assay was measured in the VSMCs with BMP2 siRNA group, BMP2 siRNA plus exosomes from MT‐treated VSMCs (MT exo) and the siRNA group. G and H, VSMCs treat with BMP2 siRNA group, BMP2 siRNA plus exosomes from MT‐treated VSMCs (MT exo) and siRNA group then subjected to SA‐β‐gal staining. Semi‐quantitative analyses of SA‐β‐gal–positive cells were performed. Representative microscopic views are shown. Scale bar represents 200 µm. Three independent experiments were performed, and representative data are shown. The data represent the mean ± SD. NC, normal control. *P < .05, **P < .01