Figure 3.

Effects of miR-1281 on VEGFA expression and cell migration. (A) VEGFA expression in HUVEC cells exposed to increasing amounts (0, 100, 250 μL) of conditioned medium from ARPE-19 cells, which underwent high glucose-induced miR-1281. VEGFA mRNA and protein levels were measured 48 h later by RT-qPCR and WB, respectively. VEGFA mRNA data are expressed as means ± SEM of three independent measurements, each performed in triplicate. *p < 0.05 vs. control (white bar). A representative WB of VEGFA out of three independent experiments is shown in the autoradiogram. β-actin, control of protein loading. (B) VEGFA mRNA and protein levels were measured as in (A), in HUVEC cells untreated or pretreated with 150 ng miR-1281 mimic for 48 h. Data for RT-qPCR and WB are representative of at least 3 independent experiments. *p < 0.05 vs. control (white bar). (C) Human VEGFA-Luc reporter vector (2 μg) was transfected into HUVEC cells, in the absence or presence of miR-1281 mimic (150 ng), and Luc activity was measured 48 h later. Data are means ± SEM for three separate transfections. Values are expressed as the factor of increase above the level of Luc activity obtained in cells transfected with VEGFA-Luc reporter vector in the absence of miR-1281 mimic (control), which is assigned an arbitrary value of 1. White bar, mock (no DNA); black bar, pGL3-basic (effector vector without an insert). *p < 0.05 vs. control. A representative WB of endogenous VEGFA in untreated and miR-1281 mimic-treated cells is shown in the autoradiogram.