Abstract 3174: Genome-wide analysis of epigenetically silenced miRNAs reveals miR-340 as a methylation repressed potential tumour suppressor miRNA in neuroblastoma

Abstract

Abstract The dysregulation of miRNA contributes to the pathogeneisis of many cancers, including the childhood cancer neuroblastoma (NB). The goal of this study was to identify epigenetically silenced miRNAs that have functional relevance as NB tumor suppressors. MeDIP-ChIP was performed on 15 primary NB tumors and 3 NB cell lines using a custom designed tiling array, allowing the identification of significant peaks of methylation within 10 Kb upstream of pre-miRNAs. Expression profiling data (TaqMan qPCR) was then used to identify up-stream methylation peaks that were inversely correlated with miRNA expression, i.e. high methylation, low expression. In total, 58 miRNA loci had a significant (p < 0.05) inverse correlation between an upstream site of methylation versus expression in the tumor cohort. Twenty-five of these loci exhibited significant re-expression following treatment of NB cell lines with the de-methylating agent 5-aza-cytidine. Remarkably, 56% (n = 14) of the re-activated loci were significantly correlated with poor event free or overall patient survival when under-expressed in tumors. In addition, several loci formed parts of polycistronic clusters, where a common methylation peak appears to co-ordinately regulate the cluster. Genome-wide DNA de-methylation of many protein coding gene promoters occurs during all-trans retinoic acid (ATRA) induced differentiation of NB cell lines (Das et al Can Res 2010;70 7874). Here, this initial finding is extended to include DNA methylation alterations that affect the expression of miRNA loci. MiR-340 was consistently de-methylated and re-expressed following ATRA treatment of both SK-N-BE and SHSY-5Y cells, and also was significantly re-activated in response to 5-aza-cytidine. Moreover, there was a significant inverse correlation between miR-340 methylation and expression levels in NB tumors. Ectopic over-expression of miR-340 mimics in three NB cell lines showed significant reduction in cell viability and colony forming efficiency, further indicating a tumor suppressor function. The transcription factor SOX2 is a predicted target of miR-340, and using luciferase reporter constructs we have experimentally validated direct targeting of the SOX2 mRNA by this miRNA. Over-expression of SOX2 in NB is associated with poor patient survival in NB, and this gene is significantly down-regulated during ATRA induced differentiation. Our results demonstrate that SOX2 down-regulation is mediated post-transcriptionally following de-methylation and re-expression of miR-340, which directly targets SOX2 mRNA. In conclusion, we have determined that DNA methylation decreases the expression of a large set of clinically relevant miRNAs in NB tumors and that the in vitro differentiating effects of ATRA can be attributable, in part, to de-methylation and reactivation of tumor suppressive miRNAs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3174. doi:1538-7445.AM2012-3174