miRNAs whose expression negatively correlates with N-myc expression were identified from a miRNA microarray of 4 N-myc-amplified neuroblastoma cell lines. Three of these miRNAs (miR-17, miR-20a and miR-18a) belong to the miR-17-92 cluster, previously shown to be upregulated by N-myc. qPCR validation of these miRNAs in a larger panel of cell lines revealed that levels of miR-17 were inversely proportional to N-myc mRNA amounts in the N-myc-amplified cell lines. Notably, miR-17 also downregulated N-myc protein synthesis in the N-myc-amplified cells, thereby generating a negative feedback regulatory loop between the proto-oncogene and this miRNA. Moreover, the neuronal-specific RNA-binding protein HuD (ELAVL4), which regulates the processing/stability of N-myc mRNA, competes with miR-17 for a binding site in the 3′-UTR of N-myc.
Thus, N-myc levels appear to be modulated by the antagonistic interactions of both miR-17, as a negative regulator, and HuD, as a positive regulator, providing further evidence of the complex cellular control mechanisms of this oncogene in N-myc-amplified neuroblastoma cells.