Fig. 3

Hypoxia downregulates miR-4521 via ETS1. a qRT-PCR analysis of miR-4521 expression under hypoxic conditions (1% oxygen, 36 h) in various tumor cell lines. b miR-4521 expression in HIF1α-silenced cells after hypoxia stimulation. CA9 serves as a control for HIF1α defect during hypoxia. Meanwhile, Western blotting was performed to detect HIF1α and CA9 protein levels. c A schematic diagram illustrating the two putative ETS1 binding sites (S1 and S2) in the miR-4521 promoter. Bioinformatic analysis of potential ETS1 binding sites in the miR-4521 promoter. d Luciferase report assays for the miR-4521 promoter region containing either wild-type (WT) or mutated (Mut1, Mut2, Mut1 + 2) ETS1 binding sites under hypoxic conditions. Hypoxia reduced the luciferase activities in WT cells but not in Mut1, Mut2 and Mut1 + 2 cells. e qRT-PCR and Western blot assays of ETS1 expression in SGC7901-L and BGC823-L cells exposed to 1% O₂ for 16 or 36 h. f miR-4521 expression was examined by qRT-PCR analysis in SGC7901-L and BGC-L cells transfected with ETS1 siRNAs during hypoxia (upper). Western blotting was performed to assess the inhibition efficiency in the same cells (lower). g ChIP analysis of ETS1 enrichment at the miR-4521 promoter during normoxia or hypoxia. IgG and anti-GAPDH antibodies were used as controls. h SGC7901-L and BGC-L cells during hypoxia were infected with miR-4521 lentiviruses and analyzed in cell invasion assays. Scale bar, 150 μm. i-j 5 × 10⁵ SGC7901-H cells stably expressing ETS1 shRNA or control shRNA were intraperitoneally into BALB/c nude mice to generate peritoneal dissemination (n = 5 mice/group). Representative images of bioluminescent view and bright view are shown. Arrows or circles indicate metastatic nodules. The number of metastatic nodules was quantified. In all cases, error bars indicate SD. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant