Hypomethylation at non-CpG/CpG sites in the promoter of HIF-1α gene combined with enhanced H3K9Ac modification contribute to maintain higher HIF-1α expression in breast cancer

a Boxplots on the upper row showing relative expression of HIF-1α in basal, HER2 positive, luminal A, luminal B, normal-like, and nonclassified breast cancer patients. Boxplots in the lower-left corner showing relative expression of HIF-1α in ERα positive and negative status. Boxplots in the lower-right corner showing relative expression of HIF-1α in breast cancer patients with grade 1, 2, and 3. b Western blotting analysis of HIF-1α expression in various breast cancer cell lines. c MSP analysis of the methylation status of the HIF-1α promoter. “U” indicates unmethylated amplification, and “M” indicates methylated amplification. d Western blotting shows changes in the expression levels of HIF-1α and HIF-2α after the cells were treated with RG-108 or 5-Aza of different concentrations. e qRT-PCR shows changes in the expression levels of HIF-1α and HIF-2α after the cells were treated with RG-108 of different concentrations. f qRT-PCR shows changes in the expression levels of HIF-1α and HIF-2α after the cells were treated with 5-Aza of different concentrations. g HIF-1α activities in MCF-7 and MDA-MB-231 cells were tested respectively by co-transfecting 6XHRE reporter vector and a Renilla luciferase plasmid. Data are presented as the means？±？SD of three independent experiments. *p？<？.05; **p？<？.01; and ***p？<？.001 (Student’s t-test) as compared to control cell