Figure 4
Effects of microRNA-18a on hypoxia-inducible factor 1α expression and hypoxic response. (A) MicroRNA-18a (miR-18a) directly targeted the 3′ untranslated region (3′-UTR) of hypoxia-inducible factor 1α (HIF1A) mRNA and ectopic miR-18a expression decreased HIF1A protein levels in MB231RN-LM cells cultured at either normoxia or hypoxia (2% O2 for 16 hours). HIF1A-3′-UTR-Luciferase reporter activities were measured in MDA-MB-231/DROSHA-shRNA cells cotransfected with either pEZX-MR06 (expressing miR-18a) or control vector pEZX-GFP. GFP, Green fluorescent protein; shRNA, Short-hairpin RNA. Luciferase activities were normalized to internal control pSV-β-galactosidase and are presented as mean ± SD. *P < 0.05 by Student’s t-test (n = 4). WT, mutant and deletion represent wild type, miR-18a binding site mutation and miR-18a binding site deletion of HIF1A-3′-UTR-Luciferase reporter, respectively. HIF1A protein levels in MB231RN-LM cells transduced with miR-18a or GFP were measured by a cell-based enzyme-linked immunosorbent assay, normalized to cytochrome c and presented as mean ± SD (n = 4). *P < 0.05 by Student’s t-test. (B) miR-18a attenuated the induction of HIF1A target genes by hypoxia in MB231RN-LM cells. Cells were exposed to hypoxia (2% O2 for 16 hours). mRNA levels were measured by quantitative PCR and normalized to ACTB, and data are presented as mean ± SD (n = 3). The inset shows the expression levels of indicated proteins in nuclear extracts (NEs) or whole-cell lysates (WCEs) detected by immunoblotting. TATA-binding protein (TBP) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as loading controls for NEs and WCEs, respectively. (C) Ectopic miR-18a expression in MB231RN-LM cells reduced viable cell numbers after cells were exposure to hypoxia (2% O2) and lactic acidosis (25 mM lactic acid, pH 6.7) for 48 hours. *P < 0.05 by Student’s t-test. n, the number of experimental repeats.