Fig. 5

miR-320a-3p antagonised osteogenic differentiation of VSMCs. (a) The heatmap shows the differentially expressed miRNAs (absolute fold change ≥ 1.5, p < 0.05) between CT-Exo and RT-Exo (n = 3 per group). (b) qRT-PCR analysis of miR-320a-3p expression in exosomes from the plasma of the RT or CT mice (n = 6). (c) qRT-PCR analysis of miR-320a-3p expression in vessel s from RT or CT mice (n = 6). (d) qRT-PCR was performed to evaluate the expression of miR-320a-3p in VSMCs transfected with specific miR-320a-3p mimics or inhibitor (n = 4). (e) The ALP activity was evaluated by using specific kits in VSMCs transfected with specific miR-320a-3p mimics or inhibitors (n = 4). (f) Western blotting was performed to determine the protein expression of RUNX2, BMP2, LC3B, ATG5 and p21 in VSMCs transfected with specific miR-320a-3p mimics or inhibitors (n = 4). (g) The data are presented as densitometric ratios normalised to β-actin. (h) qRT-PCR analysis of miR-320a-3p expression in CT-Exo + AntagomiR-320a-3p (n = 6). ARS staining (i, j), ALP staining (k) and ALP activity (l) quantification of SA-β-gal-stained positive cells was shown (m, n). The black scale bar represents 200 μm (n = 5 per group). The PS group represents the positive control group with only β-GP treatment. The data are presented as the mean ± standard deviation. The data were analysed with one-way ANOVA with the Bonferroni post hoc test or the unpaired, two-tailed Student’s t-test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001