Reciprocal antagonistic regulation of N-myc mRNA by miR-17 and the neuronal-specific RNA-binding protein HuD

Abstract

Neuroblastoma is a childhood cancer originating from embryonic neural crest cells. Amplification of the proto-oncogene N-myc, seen in ~30% of neuroblastoma tumors, is a marker for poor prognosis. Recently discovered small regulatory RNAs, microRNAs (miRNAs), are implicated in cancers, including neuroblastoma. miRNAs downregulate the expression of genes by binding to the 3'-untranslated regions (3'-UTRs), thereby inhibiting translation or inducing degradation of cognate mRNAs. Our study sought to identify miRNAs that regulate N-myc expression and thereby malignancy in neuroblastoma. 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.