Fig. 2

HIF-1α upregulates miR-224 expression at the transcriptional level. a There was a putative hypoxia response element (HRE) in the region of miR-224 promoter. MiR-224 promoter containing the HRE was cloned upstream of a luciferase reporter gene. SGC-7901 and MGC-803 cells were transfected with the above luciferase construct under hypoxia or normoxic condition, and the luciferase intensity of miR-224 promoter was determined. In addition, the cells were co-transfected the above construct and HIF-1α siRNA or si-control under hypoxia, and subjected to luciferase assay. b SGC-7901 and MGC-803 cells were exposed to hypoxia or normoxic condition and harvested for ChIP assay. Chromatin-bound DNA was precipitated with the antibody against HIF-1α. Anti-IgG antibody was used as a negative control. c Schematic representation of HIF-1α binding sites in miR-224 promoter, together with a mutation generated within the binding sites. The mutated binding sites were cloned upstream of the luciferase reporter gene. The cells were transfected with the mutant constructs under hypoxia or normoxia and subjected to luciferase assay. In addition, the cells were co-transfected the mutant construct and HIF-1α siRNA or si-control under hypoxia, and subjected to luciferase assay. *P < 0.05