Dickkopf-3 is Regulated by the MYCN-induced MiR-17-92 Cluster in Neuroblastoma

Neuroblastoma (NB) is a paediatric tumour with a remarkable diverse clinical behaviour. Approximately half of the high stage aggressive tumours are characterized by MYCN gene amplification but our understanding of the role of MYCN in NB oncogenesis is incomplete. Previous studies have shown that MYCN expression is inversely correlated with expression of Dickkopf-3 (DKK3), a gene encoding an extracellular protein with presumed tumour suppressor activity, but direct MYCN regulation of DKK3 was excluded leaving the mechanism of regulation unexplained. Given the recently established role of MYCN-regulated miRNAs in downregulation of protein-coding genes and predicted seeds for miR-17-92 cluster members within the DKK3 3'UTR, we hypothesized that this mechanism would act in MYCN regulation of DKK3. To investigate this, we used a validated miR-17-92-inducible cellular system and could demonstrate robust downregulation of DKK3 mRNA and protein levels upon miR-17-92 overexpression. Next, two of the three predicted miRNAs, miR-19b and miR-92a, were shown to lower DKK3 protein levels, in addition to measurable DKK3 mRNA knock-down by miR-92a. Direct interaction between miR-19b or miR-92a and the 3'UTR of DKK3 was validated using luciferase reporter assays. In conclusion, this study demonstrates that the MYCN-induced downregulation of DKK3 results from direct upregulation of miR-17-92 components effecting both DKK3 mRNA stability and translation which further contributes to the pleiotropic oncogenic effect of elevated MYCN levels. The strict MYCN-mediated regulation of DKK3 is suggestive for an important downstream function of the MYCN protein and thus warrants further investigations to unravel the role of DKK3 in NB.