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

J Cell Physiol. 2010 Jul;224(1):242-9. doi: 10.1002/jcp.22126.

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-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 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-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.

MeSH terms

  • Binding Sites
  • Breast Neoplasms / genetics
  • Breast Neoplasms / metabolism*
  • Breast Neoplasms / pathology
  • Cell Hypoxia
  • Cell Line, Tumor
  • Cobalt / pharmacology
  • Down-Regulation
  • Female
  • Gene Expression Regulation, Neoplastic
  • Humans
  • Hypoxia-Inducible Factor 1, alpha Subunit / genetics
  • Hypoxia-Inducible Factor 1, alpha Subunit / metabolism*
  • MicroRNAs / metabolism*
  • Promoter Regions, Genetic
  • RNA Interference
  • STAT3 Transcription Factor / genetics
  • STAT3 Transcription Factor / metabolism*
  • Signal Transduction* / drug effects
  • Signal Transduction* / genetics
  • Time Factors
  • Transfection
  • Vascular Endothelial Growth Factor A / genetics
  • Vascular Endothelial Growth Factor A / metabolism*

Substances

  • HIF1A protein, human
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • MIRN20a microRNA, human
  • MicroRNAs
  • STAT3 Transcription Factor
  • STAT3 protein, human
  • VEGFA protein, human
  • Vascular Endothelial Growth Factor A
  • Cobalt
  • cobaltous chloride