Figure 4.

ZEB-1AS1 transcription is regulated by HIF-1α during hypoxia medium. (A) A putative hypoxia-responsive element (HRE) was found in the promoter region of ZEB1-AS1. (B) The expression levels of ZEB1-AS1 (upper) and HIF-1α protein (lower) in BXPC-3/PANC-1 cells were measured after being cultured during normoxia, hypoxia (1%O₂), or CoCl₂ (concentration of 100 µM under 48 h) at the mRNA and protein levels by qRT-PCR and Western blot analysis, respectively. (C, D) After knockdown of HIF-1α with siRNA, the expression of ZEB1-AS1 was evaluated by qRT-PCR in BXPC-3 and PANC-1 cells under normoxia or hypoxia (upper). Lower diagrams indicated HIF-1α protein levels by Western blot analysis. (E) ChIP assays with anti-HIF-1α antibody were performed to affirm the binding between HIF-1α and HRE of ZEB1-AS1 promoter region in PANC-1 cells under normoxia or hypoxia condition. (F) After being cultured in hypoxia or normoxia, ChIP assays with anti-Pol II antibody were performed to ascertain the binding capacity between Pol II and ZEB1-AS1 promoter region in PANC-1 cells. (G) After knockdown of HIF-1α, the expression of ZEB1-AS1 was shown by FISH assays in PANC-1 cells during normoxia and hypoxia condition. (H) Wild-type ZEB1-AS1 promoter-containing pGL3 reporter vector (WT) or mutant-type ZEB1-AS1 promoter-containing pGL3 reporter vector (MUT) of ZEB1-AS1 promoter sequence firefly luciferase reporter activity in PANC-1 cells transfected with siNC or siHIF-1α and cultured under normoxia or hypoxia conditions were assessed by Renilla luciferase reporter assays after 48 h All data were presented as means ± SD of at least three independent experiments. Values are significant at ^* p < 0.05 and ^** p < 0.01 as indicated.