Abstract
The worst subtype of neuroblastoma is caused by MYCN oncogene amplification and N-Myc oncoprotein over-expression. Long noncoding RNAs (lncRNAs) are emerging as critical regulators of gene expression and tumourigenesis. While Myc oncoproteins are well-known to exert tumourigenic effects by regulating the expression of protein-coding genes and microRNAs, little is known about which lncRNAs are Myc targets and whether the Myc target lncRNAs play a role in Myc-induced oncogenesis. Here we performed differential gene expression studies using lncRNA microarray in neuroblastoma cells after transfection with control or N-Myc-specific small interfering RNA (siRNA), and identified N-Myc target lncRNAs including the novel lncRNA linc00467, the expression and function of which were completely unknown. RT-PCR, chromatin immunoprecipitation and luciferase assays showed that N-Myc suppressed linc00467 gene expression through direct binding to the linc00467 gene promoter and reducing linc00467 promoter activity. While N-Myc suppressed the expression of RD3, the protein-coding gene immediately down-stream of linc00467 gene, through direct binding to the RD3 gene promoter and reducing RD3 promoter activity, linc00467 reduced RD3 mRNA expression. Moreover, Affymetrix microarray analysis revealed that one of genes significantly up-regulated by linc00467 siRNA was the tumour suppressor gene DKK1. Importantly, knocking-down linc00467 expression with siRNA in neuroblastoma cells reduced the number of viable cells and increased the percentage of apoptotic cells, and co-transfection with DKK1 siRNA blocked the effects. These findings therefore demonstrate that N-Myc-mediated suppression of linc00467 gene transcription counterintuitively blocks N-Myc-mediated reduction in RD3 mRNA expression, and reduces neuroblastoma cell survival by inducing DKK1 expression.
Highlights
Neuroblastoma is a solid extracranial paediatric cancer that arises from neural crest cells, and accounts for 15% of cancerrelated death in children [1]
One of the Long noncoding RNAs (lncRNAs) most significantly upregulated by N-Myc small interfering RNA (siRNA)-1 was linc00467, which was identified by Human Genome Organisation Gene Nomenclature Committee (HGNC) according to published DNA and cDNA sequencing data [16,17,18,19,20]
Discussion lncRNAs are emerging as critical regulators of gene transcription, tumour initiation, progression and metastasis [9,10,26]
Summary
Neuroblastoma is a solid extracranial paediatric cancer that arises from neural crest cells, and accounts for 15% of cancerrelated death in children [1]. Amplification of MYCN oncogene and consequent N-Myc oncoprotein over-expression occur in approximately 40% of high risk neuroblastoma, and is clinically associated with cancer metastasis, resistance to therapies and poor patient outcome [1,2]. Myc oncoproteins, including N-Myc and c-Myc, exert biological effects through modulating gene transcription. After Myc oncoproteins dimerize with Max, the Myc-MAX complex binds to Myc-responsive element E-boxes at target gene promoters, leading to transcriptional activation [3,4]. Myc oncoproteins repress gene transcription by forming transcriptional repressor complexes with histone deacetylases at Sp1-binding sites of target gene promoters [5,6,7,8]. Identifying N-Myc target genes and understanding the function of the N-Myc target genes are important in developing better anticancer therapies
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