Fig. 3
Hypoxia represses GATA6-AS1 expression in PDAC through ETS1 expression at the transcriptional levels. A Luciferase activity assay analysis of the reporter constructs containing 2 kb of the GATA6-AS1 promoter in 293T cells exposed to 1% or 20% O2. B A schematic diagram illustrating the two putative ETS1 binding sites (P1 and P2) in the GATA6-AS1 promoter. C, Luciferase report assays for the GATA6-AS1 promoter region containing either wild-type (WT) or mutated (MUT1, MUT2, MUT1 + 2) ETS1 binding sites in 293T cells exposed to 1% or 20% O2. The GATA6-AS1 gene promoter was predicted by using JASPAR (http://jaspar.genereg.net/) online tool. D ChIP analysis of ETS1 enrichment at the GATA6-AS1 promoter for lysates from Panc-1 and BxPC 3 cells exposed to 1% or 20% O2. ChIP products were amplified by qPCR. IgG was used as controls. E, RT-qPCR and western blot assays analysis of ETS1 expression in Panc-1 and BxPC 3 cells exposed to 1% or 20% O2. F RT-qPCR assay analysis of GATA6-AS1 expression in Panc-1 and BxPC 3 with transfection of shRNAs targeting ETS1 or shNC under 1% or 20% O2 conditions. G-H, MTT (G) and Edu (H) assays analysis of cell viability in Panc-1 and BxPC 3 cells transfection with the indicated vectors under 1% or 20% O2 conditions. HIF Hypoxia-inducible factor, NC negative control, ChIP chromatin immunoprecipitation, ns not significant. Data represent mean ± S.D. from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001