Fig. 4

RASSF8 is a direct target of miR-224 in gastric cancer cells. a Sequence alignment of miR-224 and RASSF8 3′UTR. There were four putative binding sites on the RASSF8 3′UTR. The red bases represented the mutations. b RASSF8 protein levels were analyzed by Western blot in SGC-7901 and MGC-803 cells exposed to normoxia, 5% O2 and 1% O2. c SGC-7901 and MGC-803 cells were transfected with miR-224 or miR-224 ASO under normoxia or hypoxia, and then subjected to Western blot to examine RASSF8 protein levels. GAPDH was used as a loading control. d Luciferase assay: RASSF8 3′UTR containing miR-224 binding sites or mutant binding sites were cloned downstream of a luciferase reporter gene. The cells were co-transfected with miR-224 and wild-type or mutant luciferase constructs, together with controls, and then subjected to luciferase assay. e The cells transfected with wild-type or mutant luciferase constructs were exposed to hypoxia or normoxia, and then subjected to luciferase assay. *P < 0.05