Fig. 7

PDCD4 was a direct target gene of miR-320a-3p and regulated VSMCs calcification. (a) A Venn diagram showing bioinformatics analysis of miR-320a-3p target genes. (b) Schematic representation of miR-320a-3p putative target sites in the PDCD4 3′-UTR and the alignment of miR-320a-3p with wild type and mutant PDCD4 3′-UTR showing pairing. (c) Luciferase reporter assays were performed using luciferase constructs carrying a wild type or mutant PDCD4 3′-UTR co-transfected into VSMCs with miR-320a-3p mimics compared with empty vector control. Firefly luciferase activity was normalised to Renilla luciferase activity. (d, f) PDCD4 protein expression in VSMCs transfected with miR-320a-3p mimics or miR-320a-3p inhibitor was determined by western blot (n = 4). (e and g) The efficiency of PDCD4 knockdown in VSMCs by siRNA was measured by western blot (n = 4). (h-i) RUNX2 expression was measured in the VSMCs treated with siPDCD4#3 or siRNA control (n = 4). (j) ARS staining in β-GP-treated VSMCs transfected with inhibitors of miR-320a-3p in the presence or absence of PDCD4 siRNA for 28 days; representative micrographs are shown. (K) SA-β-gal staining was measured in VSMCs incubated with β-GP for 10 days. n = 4, the data are presented as the ratio of positive ARS (j) and SA-β-gal (m) staining area. The scale bar is 200 μm. The data are presented as the mean ± standard deviation. The data were analysed with one or two-way ANOVA with the Bonferroni post hoc test. ns > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001 and ****p < 0.0001