miR-20b Modulates VEGF Expression by Targeting HIF-1α and STAT3 in MCF-7 Breast Cancer Cells

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the expression of different genes, including genes involved in cancer progression. A functional link between hypoxia, a key feature of the tumor microenvironment, and miRNA expression has been documented. We investigated whether and how miR-20b can regulate the expression of vascular endothelial growth factor (VEGF) in MCF-7 breast cancer cells under normoxic and hypoxia-mimicking conditions (CoCl2 exposure). Using immunoblotting, ELISA, and quantitative real-time PCR, we demonstrated that miR-20b decreased VEGF protein levels at 4 and 24 h following CoCl2 treatment, and VEGF mRNA at 4 h of treatment. In addition, miR-20b reduced VEGF protein expression in untreated cells. Next, we investigated the molecular mechanism by which pre-miR-20b can affect VEGF transcription, focusing on hypoxia inducible factor 1 (HIF-1) and signal transducer and activator of transcription 3 (STAT3), transcriptional inducers of VEGF and putative targets of miR-20b. Downregulation of VEGF mRNA by miR-20b under a 4 h of CoCl2 treatment was associated with reduced levels of nuclear HIF-1 alpha subunit and STAT3. Chromatin immunoprecipitation (ChIP) assays revealed that HIF-1 alpha, but not STAT3, was recruited to the VEGF promoter following the 4 h of CoCl2 treatment. This effect was inhibited by transfection of cells with pre-miR-20b. In addition, using siRNA knockdown, we demonstrated that the presence of STAT3 is necessary for CoCl2-mediated HIF-1 alpha nuclear accumulation and recruitment on VEGF promoter. In summary, this report demonstrates, for the first time, that the VEGF expression in breast cancer cells is mediated by HIF-1 and STAT3 in a miR-20b-dependent manner. J. Cell. Physiol. 224: 242-249, 2010. (C) 2010 Wiley-Liss, Inc.