Transcriptional suppression of mir-29b-1/mir-29a promoter by c-Myc, hedgehog, and NF-kappaB.
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.
Mott
Justin L
JL
Division of Gastroenterology and Hepatology, College of Medicine, Mayo Clinic, Rochester, Minnesota 55905, USA. mott.justin@mayo.edu
Kurita
Satoshi
S
Cazanave
Sophie C
SC
Bronk
Steven F
SF
Werneburg
Nathan W
NW
Fernandez-Zapico
Martin E
ME
D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D045744:Cell Line, Tumor; D005786:Gene Expression Regulation; D053823:Hedgehog Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D016328:NF-kappa B / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D014157:Transcription Factors / Q000378:metabolism; D024363:Transcription Initiation Site; D014158:Transcription, Genetic; D014704:Veratrum Alkaloids / Q000494:pharmacology; D000071676:Zinc Finger Protein GLI1
C400551:GLI1 protein, human; D053823:Hedgehog Proteins; C532045:MIRN29a microRNA, human; C489427:MYC protein, human; D035683:MicroRNAs; D016328:NF-kappa B; D016271:Proto-Oncogene Proteins c-myc; D014157:Transcription Factors; D014704:Veratrum Alkaloids; D000071676:Zinc Finger Protein GLI1; C000541:cyclopamine