Figure 2

Overexpression of miR-494 increased HIF-1α and HO-1 expression under both normoxia and hypoxia. (A) Expression of miR-494 in transfected L02 cells. L02 cells were transfected with either miR-494 mimic or miR-negative control at 200 nM under normoxia. MiR-494 levels were analyzed by real-time qRT-PCR at 24 hours for transfection and 48 hours for transfection, respectively. (B-C) Expression of HIF-1α and HO-1 in transfected L02 cells under normoxia. L02 cells were transfected with either miR-494 mimic or miR-negative control at 200 nM under normoxia. After transfection for 48 hours, total RNAs and proteins were extracted and subjected to real-time qRT-PCR (B) or western blot assay (C. Left: Western blot analysis for HIF-1α, HO-1 and β-Tubulin. The sample loading of protein for normoxic condition was 80 μg. The signal indicated by white arrow is HIF-1α. Right: Densitometric analysis using Quantity one software in left). (D-E) Expression of HIF-1αand HO-1 in transfected L02 cells under hypoxia. After transfection for 16 hours, cells were exposed to hypoxia (1%O₂, 5%CO₂ in a 37°C incubator) for 8 hours. Total RNAs and proteins were extracted, and then real-time qRT-PCR (D) and western blot (E. Left: Western blot analysis for HIF-1α, HO-1 and β-Tubulin. The sample loading of protein for hypoxic condition.was 40 μg. Right: Densitometric analysis using Quantity one software in left) were done. Control indicated miR-negative control. The quantitative data for western blot were normalized by the level of β-Tubulin expression. The data were presented as the means ± SD. Columns, mean of three independent experiments; bars, SD; *p < 0.05.