The novel long noncoding RNA lncUSMycN in N-Myc-induced oncogenesis
One of the worst subtypes of neuroblastoma is caused by the amplification of a 130 kb genomic DNA region containing the MYCN oncogene. While MYCN has been extensively investigated, it is unknown whether other genomic elements within the 130 kb amplicon play a role in neuroblastoma. In this study, I experimentally validated the novel long noncoding RNA up-stream of MYCN (lncUSMycN), which was originally annotated by bioinformatics analyses, within the 130 kb amplicon. Using rapid amplification of cDNA ends PCR, 140 extra nucleotides at the 5’-end of lncUSMycN were identified. Gene copy number analysis of human neuroblastoma samples showed that the lncUSMycN gene was co-amplified with MYCN, leading to lncUSMycN RNA over-expression. Repression of lncUSMycN decreased N-Myc mRNA expression and cell proliferation in MYCN amplified neuroblastoma cells. Microarray differential gene expression studies demonstrated that lncUSMycN regulated the expression of N-Myc target genes at genome-wide level. RNA-binding protein pull-down assays identified NonO as a lncUSMycN RNA binding protein, and RNA-immunoprecipitation assays showed that NonO bound to lncUSMycN and N-Myc RNAs. While knocking down NonO and lncUSMycN expression reduced N-Myc mRNA expression, forced over-expression of lncUSMycN resulted in N-Myc mRNA up-regulation by binding to NonO. ChIP assays showed that neither lncUSMycN nor NonO bound to the MYCN gene promoter to alter histone H3 trimethylation at lysine 4, a marker for active gene transcription, indicating that lncUSMycN up-regulated N-Myc mRNA expression through a post-transcriptional mechanism. Pearson correlation and Kaplan-Meier survival analyses demonstrated that high levels of lncUSMycN and NonO RNA expression in human neuroblastoma tissues correlated with high levels of N-Myc mRNA expression and predicted poor prognoses in three independent cohorts of neuroblastoma patients. Multivariable Cox regression analyses showed that high levels of lncUSMycN and NonO expression in neuroblastoma tissues independently predicted poor patient prognoses. Moreover, treatment with antisense oligonucleotides targeting lncUSMycN in neuroblastoma-bearing mice significantly reduced N-Myc expression and blocked tumour progression. In conclusion, this study demonstrates the important roles of lncUSMycN and its binding partner NonO in regulating N-Myc expression, cell proliferation and neuroblastoma progression, and provides the first evidence that amplification of long noncoding RNA genes can contribute to tumourigenesis.
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