Figure 5

Hypoxia-driven HIF-1α is critical for upregulation of miR-18b-5p in OS. (a) The luciferase reporter assay accessed luciferase activity of MG-63/143b cells transfected with luciferase reporter plasmid carrying miR-18b promoter sequence or scrambled vector under normoxia and hypoxia. (b) RT-qPCR assay showed miR-18b-5p expression in MG-63/143b cells cultured under hypoxia for 0 h, 6 h, 12 h and 24 h. (c) (Up) Schematic diagram showed the predicted HRE site on the promoter of miR-18b. (Down) ChIP assay using antibodies against HIF-1α or IgG were performed to validate the binding of HIF-1α on the promoter of miR-18b. (d) The luciferase reporter assay accessed luciferase activity of MG-63 and 143b cells transfected with luciferase reporter plasmid carrying WT- miR-18b or MUT- miR-18b, siRNA targeting HIF-1α and negative control under normoxia or hypoxia. (e) Western blotting assay accessed protein level of HIF-1α in MG-63/143b cells transfected with siRNA targeting HIF-1α and negative control under normoxia or hypoxia. (f,g) RT-qPCR assay showed the expression of miR-18b-5p and PHF2 mRNA in MG-63/143b cells transfected with siRNA targeting HIF-1α and negative control under normoxia or hypoxia.