Figure 5.

HIF‐2α directly activates the BCRP gene in ovarian cancer cells. (A) The evolutionarily conserved hypoxia‐response element (HRE) sequence (CACGTG), located between −483 and −478 nucleotides upstream of the transcription start site of the human BCRP gene. (B) Luciferase reporter activity was measured in transient co‐transfections with EPAS1‐cDNA or NC‐cDNA plasmids and BCRP promoter vectors (GV238‐BCRP‐WT) or vectors containing mutated HRE sequence binding sites (GV238‐BCRP‐mut) in 293T and OVCAR‐3 cells. The luciferase activities in the cells co‐transfected with NC‐cDNA and GV238‐BCRP‐WT or GV238‐BCRP‐mut plasmids were set as 1. Data are presented as the mean ± SD from three independent experiments. (C) ChIP assays were performed to verify HIF‐2α binding to the BCRP gene in OVCAR‐3 and OVCAR‐3 S cells cultured under hypoxic conditions (1% O₂) for 48 h. (D) ChIP‐qPCR shows the enhanced binding HIF‐2α on BCRP promoter in OVCAR‐3 S cells. Antibody enrichment was quantified relative to the amount of input DNA. Antibody directed against IgG was used as a negative control. (E) The mRNA expression of BCRP was analyzed by qRT‐PCR in the OVCAR‐3 and CAOV‐3 cells transduced with EPAS1‐cDNA or NC‐cDNA lentivirus, and in the OVCAR‐3 S and CAOV‐3 S cells transduced with sh‐EPAS1 or sh‐NC lentivirus under hypoxic conditions (1% O₂) for 48 h. BCRP expression levels in OVCAR‐3 cells or CAOV‐3 cells transduced with EPAS1‐cDNA lentivirus and in OVCAR‐3 S cells or CAOV‐3 S cells transduced with sh‐NC lentivirus were set as 1. Data are presented as the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01. Statistical significance was evaluated by Student's t‐test.