Epigenetic silencing of Kruppel like factor-3 increases expression of pro-metastatic miR-182

Cancer Lett. 2015 Dec 1;369(1):202-11. doi: 10.1016/j.canlet.2015.08.016. Epub 2015 Aug 24.

Abstract

Accumulating evidence indicates that microRNAs (miRs) regulate cancer metastasis. We have shown that miR-182 drives sarcoma metastasis in vivo by coordinated regulation of multiple genes. Recently, we also demonstrated that in a subset of primary sarcomas that metastasize to the lung, miR-182 expression is elevated through binding of MyoD1 to the miR-182 promoter. However, it is not known if there are also transcription factors that inhibit miR-182 expression. Defining negative regulators of miR-182 expression may help explain why some sarcomas do not metastasize and may also identify pathways that can modulate miR-182 for therapeutic benefit. Here, we use an in silico screen, chromatin-immunoprecipitation, and luciferase reporter assays to discover that Kruppel like factor-3 (Klf-3) is a novel transcriptional repressor of miR-182. Knockdown of Klf-3 increases miR-182 expression, and stable overexpression of Klf-3, but not a DNA-binding mutant Klf-3, decreases miR-182 levels. Klf-3 expression is downregulated in both primary mouse and human metastatic sarcomas, and Klf-3 levels negatively correlate with miR-182 expression. Interestingly, Klf-3 also negatively regulates MyoD1, suggesting an alternative mechanism for Klf-3 to repress miR-182 expression in addition to direct binding of the miR-182 promoter. Using Methylation Specific PCR (MSP) and pyrosequencing assays, we found that Klf-3 is epigenetically silenced by DNA hypermethylation both in mouse and human sarcoma cells. Finally, we show the DNA methylation inhibitor 5'Azacytidine (Aza) restores Klf-3 expression while reducing miR-182 levels. Thus, our findings suggest that demethylating agents could potentially be used to modulate miR-182 levels as a therapeutic strategy.

Keywords: Epigenetics; Gene regulation; Metastasis; microRNA.

Publication types

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

MeSH terms

  • Animals
  • Base Sequence
  • Cell Line, Tumor
  • DNA Methylation
  • Epigenesis, Genetic*
  • Gene Expression Regulation, Neoplastic*
  • Humans
  • Kruppel-Like Transcription Factors / genetics*
  • Kruppel-Like Transcription Factors / metabolism
  • Lung Neoplasms / metabolism*
  • Lung Neoplasms / secondary
  • Mice, Nude
  • Mice, Transgenic
  • MicroRNAs / genetics
  • MicroRNAs / metabolism*
  • Neoplasm Transplantation
  • Promoter Regions, Genetic
  • Sarcoma / metabolism*
  • Sarcoma / secondary

Substances

  • KLF3 protein, human
  • Kruppel-Like Transcription Factors
  • MicroRNAs
  • Mirn182 microRNA, human