MYCN-regulated miRNAs Inhibit 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 a bad prognosis for this disease. Dickkopf-3 (DKK3) is a secreted protein of the Dickkopf 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. MiRNAs are ~20-nt-long RNAs encoded by the genome that downregulate mRNAs 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 and 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 these miRNAs. 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. These data demonstrate a previously unknown tumor promoting mechanism for MYCN-regulated miRNAs.