Figure 3

miR-133a strands -3p and -5p attenuate endothelial cell proliferation. HUVEC were transfected with mimics for miR negative control (miR-NC) or mimics for miR-133a strands -3p (miR-133a-3p) or -5p (mir-133a-5p) and were left unstimulated (0 h) or stimulated with VEGF-A₁₆₅ as indicated. (A) Proliferation rate of transfected cells assayed by MTT. Proliferation rate was calculated as absorbance in MTT assay after 3 days divided by absorbance at 0 days. Data are shown as mean ± SE, n = 8. ***, P ≤ 0.001 miR-133a-3p versus miR-NC at 3 Days analysed by two-way ANOVA with post hoc Tukey’s comparison test. (B) Volcano plot representation showing differential gene expression analysis of a RT2 Profiler PCR Array Human Cell Cycle Kit (Ref PAHS-020Z, Qiagen) using RNA isolated from HUVEC transfected with miR-133a-3p or negative control (miR-NC) and stimulated for 1 h with VEGF-A₁₆₅ (50 ng/ml). Results of three independent experiments were analysed using GeneGlobe Data Analysis Center (Qiagen). Genes showing a Log2(miR-133a-3p/miR-NC) > 1 and − Log10(P value) P ≤ 0.05 were selected as upregulated (red). Those with Log2(miR-133a-3p/miR-NC) < − 1, and − Log10(P value) P ≤ 0.05 were selected as downregulated (green). (C–I) HUVEC transfected with mimics for negative control (NC, grey bars), miR-133a-3p (3p, blue bars) or miR-133a-5p (5p, red bars) were left unstimulated (0 h) or stimulated with VEGF (50 ng/ml) for the indicated times. RNA isolated from these cells were used to determine changes in gene expression using qPCR TaqMan Gene Expression Assays specific for each gene. Data are shown as mean ± SE, n = 9. Data were analysed for statistical differences by two-way ANOVA with post hoc Tukey’s comparison test. ns = non-significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (J) Flow cytometry cell cycle analysis of HUVEC transfected with negative control (miR-NC) or miR-133a-3p mimics. The proportion of HUVECs in each phase of the cell cycle was determined by quantification of DNA content using propidium iodide. Representative cell count/PI plots are shown. Data are shown as mean ± SE, n = 6. ****, P ≤ 0.0001 two-way ANOVA with post hoc Tukey’s comparison test for the indicated groups.