Figure 4.
miR-130a activates the VEGFR2/STAT3/HIF1 signaling axis in ECFCs under hypoxia
(A) qRT-PCR assessment of angiogenesis-related genes in ECFCs from six biological replicates. ECFCs transfected with miR-130a mimics or control mimics were exposed to hypoxia for 48 h prior to RNA extraction, and their gene expression was compared. Data are presented as dot plots with 95% CI, ∗∗∗p < 0.001 (ANOVA). (B) Immunofluorescent staining for VEGFR2 in green, and nuclei are stained in blue with DAPI. Scale bar, 100 μm. (C) Flow cytometry analysis of VEGFR2 cell surface expression. Gates highlight the positivity threshold established using unstained controls. Percentage of positivity shown on the top right corner. (D) Western blot analysis of VEGFR2, HIF1α, STAT3, and phosphorylated STAT3 in the protein extract of miR-130a and control mimic transfected ECFCs exposed to hypoxia. ImageJ-based densitometry was used for quantification and statistical analysis. ∗∗p < 0.01; ∗p < 0.05 (Paired t test). See also Figure S6. (E) Graph showing cell counts after plating 1 × 105 of ECFCs and culturing for 3 days in hypoxia in basal media supplemented with either FGF or VEGF. Data are presented as bar plots. ∗∗p < 0.01; ns, not significant (Student’s t test). (F) Levels of nuclear STAT3 bound to its DNA target oligo sequence measured by ELISA-based TransAM STAT3 kit as absorbance. Data are presented as boxplots, ∗∗∗p < 0.001 (Student’s t test). (G) Immunofluorescent staining for STAT3 in green and nuclei stained with DAPI in red to highlight increased nuclear localization in the miR-130a mimic-transfected ECFCs. Colocalization shown in white. (H) Quantification of Ki67-positive cells as percentage of the total cell population. ECFCs transfected with miR-130a mimics or controls were treated with STAT3 inhibitor S31-201(stat3i). Data shown as boxplots. ∗∗p < 0.01; ∗p < 0.05 (ANOVA). (I) Western blot analysis of HIF1α in miR-130a mimics-treated ECFCs cultured with stat3 inhibitor. ***p < 0.001; **p < 0.01 (ANOVA).