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miR‐20b modulates VEGF expression by targeting HIF‐1α and STAT3 in MCF‐7 breast cancer cells
Author(s) -
Cascio Sandra,
D'Andrea Aleco,
Ferla Rita,
Surmacz Eva,
Gulotta Eliana,
Amodeo Valeria,
Bazan Viviana,
Gebbia Nicola,
Russo Antonio
Publication year - 2010
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.22126
Subject(s) - chromatin immunoprecipitation , stat3 , gene knockdown , microrna , cancer research , vascular endothelial growth factor , transfection , stat protein , transcription factor , hypoxia inducible factors , hif1a , microbiology and biotechnology , vascular endothelial growth factor a , chemistry , biology , gene expression , promoter , angiogenesis , signal transduction , gene , vegf receptors , biochemistry
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.