Transcriptional suppression of mir-29b-1/mir-29a promoter by c-Myc, hedgehog, and NF-kappaB

J Cell Biochem. 2010 Aug 1;110(5):1155-64. doi: 10.1002/jcb.22630.

Abstract

MicroRNAs regulate pathways contributing to oncogenesis, and thus the mechanisms causing dysregulation of microRNA expression in cancer are of significant interest. Mature mir-29b levels are decreased in malignant cells, and this alteration promotes the malignant phenotype, including apoptosis resistance. However, the mechanism responsible for mir-29b suppression is unknown. Here, we examined mir-29 expression from chromosome 7q32 using cholangiocarcinoma cells as a model for mir-29b downregulation. Using 5' rapid amplification of cDNA ends, the transcriptional start site was identified for this microRNA locus. Computational analysis revealed the presence of two putative E-box (Myc-binding) sites, a Gli-binding site, and four NF-kappaB-binding sites in the region flanking the transcriptional start site. Promoter activity in cholangiocarcinoma cells was repressed by transfection with c-Myc, consistent with reports in other cell types. Treatment with the hedgehog inhibitor cyclopamine, which blocks smoothened signaling, increased the activity of the promoter and expression of mature mir-29b. Mutagenesis analysis and gel shift data are consistent with a direct binding of Gli to the mir-29 promoter. Finally, activation of NF-kappaB signaling, via ligation of Toll-like receptors, also repressed mir-29b expression and promoter function. Of note, activation of hedgehog, Toll-like receptor, and c-Myc signaling protected cholangiocytes from TRAIL-induced apoptosis. Thus, in addition to c-Myc, mir-29 expression can be suppressed by hedgehog signaling and inflammatory pathways, both commonly activated in the genesis of human malignancies.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites / genetics
  • Cell Line
  • Cell Line, Tumor
  • Gene Expression Regulation
  • Hedgehog Proteins / antagonists & inhibitors
  • Hedgehog Proteins / metabolism*
  • Humans
  • MicroRNAs / genetics*
  • NF-kappa B / metabolism*
  • Promoter Regions, Genetic / genetics*
  • Protein Binding
  • Proto-Oncogene Proteins c-myc / genetics
  • Proto-Oncogene Proteins c-myc / metabolism*
  • Signal Transduction / drug effects
  • Transcription Factors / metabolism
  • Transcription Initiation Site
  • Transcription, Genetic
  • Veratrum Alkaloids / pharmacology
  • Zinc Finger Protein GLI1

Substances

  • GLI1 protein, human
  • Hedgehog Proteins
  • MIRN29 microRNA, human
  • MYC protein, human
  • MicroRNAs
  • NF-kappa B
  • Proto-Oncogene Proteins c-myc
  • Transcription Factors
  • Veratrum Alkaloids
  • Zinc Finger Protein GLI1
  • cyclopamine