Abstract
Neuroblastoma (NB), a tumor of the sympathetic nervous system, is the most common extracranial solid tumor of childhood. We and others have identified distinct patterns of genomic change that underlie diverse clinical behaviors, from spontaneous regression to relentless progression. We first identified CHD5 as a tumor suppressor gene that is frequently deleted in NBs. Mutation of the remaining CHD5 allele is rare in these tumors, yet expression is very low or absent, so expression is likely regulated by epigenetic mechanisms. In order to understand the potential role of miRNA regulation of CHD5 protein expression in NBs, we examined all miRNAs that are predicted to target the 3′-UTR using miRanda, TargetScan and other algorithms. We identified 18 miRNAs that were predicted by 2 or more programs: miR-204, -211, -216b, -17, -19ab, -20ab, -93, -106ab, -130ab, -301ab, -454, -519d, -3666. We then performed transient transfections in two NB cell lines, NLF (MYCN amplified) and SY5Y (MYCN non-amplified), with the reporter plasmid and miRNA mimic, as well as appropriate controls. We found seven miRNAs that significantly downregulated CHD5 expression in NB: miR-211, 17, -93, -20b, -106b, -204, and -3666. Interestingly, MYCN upregulates several of the candidates we identified: miR-17, -93, -106b & -20b. This suggests that miRNAs driven by MYCN and other genes represent a potential epigenetic mechanism to regulate CHD5 expression.
Highlights
Neuroblastoma (NB) is the most common extracranial solid tumor of childhood
We analyzed 23 genes mapping to the maximal smallest region of consistent deletion (SRD) on 1p36.31, and we identified CHD5 as the most likely tumor suppressor gene (TSG) within this region [11, 17, 18]
We investigated the role of miRNAs on downregulation of CHD5 in NB cell lines in vitro
Summary
Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. NBs show clinical heterogeneity, from spontaneous regression to relentless progression, and NBs account for a disproportionate number of childhood cancer deaths. We analyzed 23 genes mapping to the maximal SRD on 1p36.31, and we identified CHD5 as the most likely TSG within this region [11, 17, 18]. The imperfect pairing between a miRNA and its target allows a given miRNA to bind one or more sites within a 3’-UTR, or to multiple mRNA targets throughout the genome. This allows miRNAs to have a robust influence on gene expression, and it allows simultaneous regulation of multiple components of the signaling network in development and in cancer [32]. We performed functional assays by transfecting miRNA mimics into a cell line with endogenous CHD5 expression to determine if the miRNAs identified by our reporter assay were able to decrease CHD5 protein expression
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