miR-20b modulates VEGF expression by targeting HIF-1α and STAT3 in MCF-7 breast cancer cells

✍ Scribed by Sandra Cascio; Aleco D'Andrea; Rita Ferla; Eva Surmacz; Eliana Gulotta; Valeria Amodeo; Viviana Bazan; Nicola Gebbia; Antonio Russo

• Publisher: John Wiley and Sons
• Year: 2010
• Tongue: English
• Weight: 316 KB
• Volume: 224
• Category: Article
• ISSN: 0021-9541
• DOI: 10.1002/jcp.22126

✦ Synopsis

Abstract

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 (CoCl~2~ exposure). Using immunoblotting, ELISA, and quantitative real‐time PCR, we demonstrated that miR‐20b decreased VEGF protein levels at 4 and 24 h following CoCl~2~ 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 CoCl~2~ treatment was associated with reduced levels of nuclear HIF‐1α subunit and STAT3. Chromatin immunoprecipitation (ChIP) assays revealed that HIF‐1α, but not STAT3, was recruited to the VEGF promoter following the 4 h of CoCl~2~ 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 CoCl~2~‐mediated HIF‐1α 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 © 2010 Wiley‐Liss, Inc.