title: Epigenetic silencing of Kruppel like factor-3 increases expression of pro-metastatic miR-182.
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.
authors: Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710, USA.
authors: Mohit
authors: M
authors: Sachdeva
authors: Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710, USA.
authors: Rebecca D
authors: RD
authors: Dodd
authors: Department of Obstetrics and Gynecology, Duke University Medical Center, Durham, NC 27710, USA.
authors: Zhiqing
authors: Z
authors: Huang
authors: Department of Obstetrics and Gynecology, Duke University Medical Center, Durham, NC 27710, USA.
authors: Carole
authors: C
authors: Grenier
authors: Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710, USA.
authors: Yan
authors: Y
authors: Ma
authors: Department of Cancer Biology, University of Texas, MD Anderson Cancer Center, Houston, TX, USA.
authors: Dina C
authors: DC
authors: Lev
authors: Department of Pathology, Duke University Medical Center, Durham, NC 27710, USA; Department of Pharmacology & Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA.
authors: Diana M
authors: DM
authors: Cardona
authors: Department of Obstetrics and Gynecology, Duke University Medical Center, Durham, NC 27710, USA.
authors: Susan K
authors: SK
authors: Murphy
authors: Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710, USA; Department of Pathology, Duke University Medical Center, Durham, NC 27710, USA; Department of Pharmacology & Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA. Electronic address: david.kirsch@duke.edu.
authors: David G
authors: DG
authors: Kirsch
mesh_terms: D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000235:genetics* / Q000378:metabolism; D008175:Lung Neoplasms / Q000378:metabolism* / Q000556:secondary; D008819:Mice, Nude; D008822:Mice, Transgenic; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D009368:Neoplasm Transplantation; D011401:Promoter Regions, Genetic; D012509:Sarcoma / Q000378:metabolism* / Q000556:secondary
chemical_list: C501168:KLF3 protein, human; D051741:Kruppel-Like Transcription Factors; D035683:MicroRNAs; C538984:Mirn182 microRNA, human
keywords: Epigenetics; Gene regulation; Metastasis; microRNA