title: MYCN-regulated miRNA-92 inhibits secretion of the tumor suppressor DICKKOPF-3 (DKK3) in neuroblastoma.
abstract: The MYCN oncogene is frequently amplified in neuroblastoma. It is one of the most consistent markers of bad prognosis for this disease. Dickkopf-3 (DKK3) is a secreted protein of the DKK family of Wnt regulators. It functions as a tumor suppressor in a range of cancers, including neuroblastoma. MYCN was recently found to downregulate DKK3 mRNA. In this study, we show that MYCN knockdown in MYCN-amplified (MNA) neuroblastoma cell lines increases secretion of endogenous DKK3 to the culture media. MicroRNAs (miRNAs) are ∼20 nt long single-stranded RNA molecules that downregulate messenger RNAs by targeting the 3' untranslated region (3'UTR). Many miRNAs regulate genes involved in the pathogenesis of cancer and are extensively deregulated in different tumors. Using miRNA target prediction software, we found several MYCN-regulated miRNAs that could target the 3'UTR sequence of DKK3, including mir-92a, mir-92b and let-7e. Luciferase expression from a reporter vector containing the DKK3-3'UTR was decreased when this construct was cotransfected with mir-92a, mir-92b or let-7e in HEK293 cells. Mutation of the mir-92 seed sequence in the 3'UTR completely rescued the observed decrease in reporter expression when cotransfected with mir-92a and mir-92b. Antagomir and miRNA-mimic transfections in neuroblastoma cell lines confirmed that DKK3 secretion to the culture media is regulated by mir-92. Consistent with reports from other cancers, we found DKK3 to be expressed in the endothelium of primary neuroblastoma samples and to be absent in tumors with MYCN amplification. Our data demonstrate that MYCN-regulated miRNAs are able to modulate the expression of the tumor suppressor DKK3 in neuroblastoma.
authors.affiliation: Department of Pediatrics, University Hospital of North-Norway, NO-9038 Tromsø, Norway.
authors.forename: Bjørn Helge
authors.initials: BH
authors.lastname: Haug
authors.forename: Jørn R
authors.initials: JR
authors.lastname: Henriksen
authors.forename: Jochen
authors.initials: J
authors.lastname: Buechner
authors.forename: Dirk
authors.initials: D
authors.lastname: Geerts
authors.forename: Ellen
authors.initials: E
authors.lastname: Tømte
authors.forename: Per
authors.initials: P
authors.lastname: Kogner
authors.forename: Tommy
authors.initials: T
authors.lastname: Martinsson
authors.forename: Trond
authors.initials: T
authors.lastname: Flægstad
authors.forename: Baldur
authors.initials: B
authors.lastname: Sveinbjørnsson
authors.forename: Christer
authors.initials: C
authors.lastname: Einvik
mesh_terms: D020413:3' Untranslated Regions; D048868:Adaptor Proteins, Signal Transducing; D001808:Blood Vessels / Q000378:metabolism; D045744:Cell Line, Tumor; D018925:Chemokines; D019175:DNA Methylation; D055785:Gene Knockdown Techniques; D016147:Genes, Tumor Suppressor; D006801:Humans; D007150:Immunohistochemistry; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D035683:MicroRNAs / Q000235:genetics / Q000502:physiology*; D000071447:N-Myc Proto-Oncogene Protein; D009447:Neuroblastoma / Q000098:blood supply / Q000378:metabolism* / Q000473:pathology; D009687:Nuclear Proteins / Q000235:genetics / Q000502:physiology*; D015513:Oncogene Proteins / Q000235:genetics / Q000502:physiology*; D009857:Oncogenes; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D020413:3' Untranslated Regions; D048868:Adaptor Proteins, Signal Transducing; D018925:Chemokines; C498624:DKK3 protein, human; D036341:Intercellular Signaling Peptides and Proteins; C541874:MIRN92 microRNA, human; C459801:MYCN protein, human; D035683:MicroRNAs; D000071447:N-Myc Proto-Oncogene Protein; D009687:Nuclear Proteins; D015513:Oncogene Proteins