Differentiating the functional role of hypoxia‐inducible factor (HIF)‐1α and HIF‐2α (EPAS‐1) by the use of RNA interference: erythropoietin is a HIF‐2α target gene in Hep3B and Kelly cells

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Abstract
SPECIFIC AIMSCellular adaptation to hypoxia is achieved by a transcriptional response system mediated by the hypoxia-inducible factor (HIF). Since the individual roles of its two alternative oxygen-regulated α subunits, HIF-1α and HIF-2α, are not fully understood, our aim was to determine functional differences between HIF-1α and HIF-2α in different human cell lines by using RNA interference (RNAi) and to compare the results with HIFα overexpression experiments.PRINCIPAL FINDINGS1. Specificity and efficiency of HIF-1α and HIF-2α siRNAsThe transfection of HIF-1α and HIF-2α siRNAs in HeLa and Hep3B cells selectively and reproducibly reduced mRNA expression of the respective HIFα isoform by >80%, whereas a luciferase control siRNA had no effect. Concordantly, knockdown of HIFα mRNA levels led to substantially diminished HIFα protein expression under inducing conditions.2. Differential effects of HIF-1α and HIF-2α siRNAs on HIF target gene mRNA inductionRNase protection assays revealed that HIF-1α knockdown i...