Neuroblastoma is a pediatric cancer of the sympathetic nervous system where amplification is a key indicator of poor prognosis. However, mechanisms by which MYCN promotes neuroblastoma tumorigenesis are not fully understood. In this study, we analyzed global miRNA and mRNA expression profiles of tissues at different stages of tumorigenesis from TH-MYCN transgenic mice, a model of MYCN-driven neuroblastoma. On the basis of a Bayesian learning network model in which we compared pretumor ganglia from TH-MYCN mice to age-matched wild-type controls, we devised a predicted miRNA-mRNA interaction network. Among the miRNA-mRNA interactions operating during human neuroblastoma tumorigenesis, we identified miR-204 as a tumor suppressor miRNA that inhibited a subnetwork of oncogenes strongly associated with -amplified neuroblastoma and poor patient outcome. MYCN bound to the miR-204 promoter and repressed miR-204 transcription. Conversely, miR-204 directly bound MYCN mRNA and repressed MYCN expression. miR-204 overexpression significantly inhibited neuroblastoma cell proliferation and tumorigenesis Together, these findings identify novel tumorigenic miRNA gene networks and miR-204 as a tumor suppressor that regulates MYCN expression in neuroblastoma tumorigenesis. Network modeling of miRNA-mRNA regulatory interactions in a mouse model of neuroblastoma identifies miR-204 as a tumor suppressor and negative regulator of MYCN. .

<div>Abstract<p>Neuroblastoma is a pediatric cancer of the sympathetic nervous system where <i>MYCN</i> amplification is a key indicator of poor prognosis. However, mechanisms by which MYCN promotes neuroblastoma tumorigenesis are not fully understood. In this study, we analyzed global miRNA and mRNA expression profiles of tissues at different stages of tumorigenesis from TH-MYCN transgenic mice, a model of MYCN-driven neuroblastoma. On the basis of a Bayesian learning network model in which we compared pretumor ganglia from TH-MYCN^+/+ mice to age-matched wild-type controls, we devised a predicted miRNA–mRNA interaction network. Among the miRNA–mRNA interactions operating during human neuroblastoma tumorigenesis, we identified miR-204 as a tumor suppressor miRNA that inhibited a subnetwork of oncogenes strongly associated with <i>MYCN</i>-amplified neuroblastoma and poor patient outcome. MYCN bound to the miR-204 promoter and repressed miR-204 transcription. Conversely, miR-204 directly bound MYCN mRNA and repressed MYCN expression. miR-204 overexpression significantly inhibited neuroblastoma cell proliferation <i>in vitro</i> and tumorigenesis <i>in vivo</i>. Together, these findings identify novel tumorigenic miRNA gene networks and miR-204 as a tumor suppressor that regulates MYCN expression in neuroblastoma tumorigenesis.</p><p><b>Significance:</b> Network modeling of miRNA–mRNA regulatory interactions in a mouse model of neuroblastoma identifies miR-204 as a tumor suppressor and negative regulator of MYCN. <i>Cancer Res; 78(12); 3122–34. ©2018 AACR</i>.</p></div>

<p>Supplementary Table 2-1 All miRNA-mRNA interactions detected for network presented in Figure 2A, Supplementary Table 2-2 Expression of miRNAs in human adrenal neuroblasts and MYCN-amplified tumours</p>

<p>mRNA and miRNA expression in 1 week, 2 week and 6 week wildtype or TH-MYCN+/+ ganglia or tumors</p>

<p>mRNA and miRNA expression in 1 week, 2 week and 6 week wildtype or TH-MYCN+/+ ganglia or tumors</p>

<p>Supplementary Table 2-1 All miRNA-mRNA interactions detected for network presented in Figure 2A, Supplementary Table 2-2 Expression of miRNAs in human adrenal neuroblasts and MYCN-amplified tumours</p>

<p>This file contains Supplementary Figure legends and Figures Supplementary Figure 1 - Comparisons between time-dependent gene expression quantification models Supplementary Figure 2 - Comparisons of Piscore for miRNA and mRNA members of interaction hubs Supplementary Figure 3 - Survival metrics for miRNA and miRNA signature in human neuroblastoma tumors Supplementary Figure 4 - Gene set enrichment analysis of predicted miR204 target genes upon MYCN siRNA treatment Supplementary Figure 5 - Data to support the creation of dox-inducible miR204 expressing cells and to support miR204 MYCN relationship Supplementary Figure 6 - Data to support miR204 effects on MYCN expression, neuroblastoma proliferation and xenograft experiments.</p>

<p>This file contains Supplementary Figure legends and Figures Supplementary Figure 1 - Comparisons between time-dependent gene expression quantification models Supplementary Figure 2 - Comparisons of Piscore for miRNA and mRNA members of interaction hubs Supplementary Figure 3 - Survival metrics for miRNA and miRNA signature in human neuroblastoma tumors Supplementary Figure 4 - Gene set enrichment analysis of predicted miR204 target genes upon MYCN siRNA treatment Supplementary Figure 5 - Data to support the creation of dox-inducible miR204 expressing cells and to support miR204 MYCN relationship Supplementary Figure 6 - Data to support miR204 effects on MYCN expression, neuroblastoma proliferation and xenograft experiments.</p>