Background: Amplified MYCN is observed in about 20% of neuroblastoma (NB) patients and strongly predicts poor prognosis. The transcription factor MYCN targets an exceptionally large number of genes; in addition, indirect regulation occurs by microRNAs and epigenetic mechanisms. In MYCN-amplified neuroblastoma cell lines the knockdown of MYCN expression induces changes in the cell cycle (G1 arrest), apoptosis and differentiation. However, the details of this process are not yet well understood. We argue that regulatory events may occur in a cell cycle phase-dependent manner and are therefore not visible in a bulk population. In this study, we aimed to precisely characterize how MYCN influences expression of its target genes with the help of a time-resolved synchronisation model.
Methods: We used a MYCN-amplified cell line in which the level of MYCN can be regulated by an inducible shRNA vector (IMR5/75 MYCN shRNA). These cells were synchronised with a thymidine block and released into the cell cycle. RNA and miRNA expression was analysed at regular intervals during 22 hours.
Results: RNA sequencing revealed that the number of genes which were up- or downregulated due to MYCN was approximately equal. Clustering algorithms detected five different patterns of regulation, both cell cycle-dependent and independent. Interestingly, regarding expression of MYCN itself, we observed two peaks during G1 and S phase. In our miRNA expression analysis, we found 164 miRNAs to be differentially regulated. Among the most prominent results were several members of the mir-17-92 cluster, whose expression profile recapitulated the MYCN peaks described above. This cluster is known to have oncogenic potential by driving proliferation and angiogenesis while inhibiting differentiation.
Conclusion: This study for the first time describes MYCN-controlled gene regulation as cells pass through the cell cycle. We are currently aiming at completing this analysis with data on histone modifications and protein expression.