Abstract 191: The role of microRNAs in the epigenetic silencing of CHD5, a tumor suppressor in neuroblastoma

Abstract

Abstract Background: Neuroblastoma (NB) is a tumor of the sympathetic nervous system that is the most common extracranial solid tumor of childhood. NBs show remarkable clinical heterogeneity, and we, along with others, have identified different patterns of genomic change that underlie these diverse clinical behaviors. We first identified 1p deletion as a characteristic change in advanced stage NBs. CHD5, a tumor suppressor gene, was first identified because of its location on 1p36. However, mutation of the remaining allele of CHD5 is rare in these tumors. Therefore, it is likely that epigenetic mechanisms play important roles in CHD5 downregulation. MicroRNAs (miRNAs) are small RNAs that bind to the 3′ UTR to mediate downregulation of target gene expression by translational repression or cleavage of the target gene message. Dysregulation of CHD5 via miR-211 has been reported in colon cancer. In order to understand the role of miRNA regulation of CHD5 in NBs, we wanted to assess the function of all miRNAs that are predicted to target CHD5. Method/approach: We used TargetScan, miRanda, MirTarget2 and other prediction algorithms to identify miRNAs that were predicted to bind to the CHD5-3′UTR. We identified 18 miRNAs that were predicted by 2 or more programs: miR-204-5p, -211, -216b, -17, -19ab, -20ab, -93, -106ab, -130ab, -301ab, -454, -519d, -3666. We used a renilla-luciferase reporter plasmid that contains 103 bp of the 3′UTR of CHD5 targeted by all miRNAs except miR-204, -211, - 219b, and -3666. We also created control plasmids with no CHD5 3′UTR insert and a mutated CHD5 3′UTR with mutated miRNA binding sites. Allstar siRNA served as an additional negative control. We performed transient transfections in two NB cell lines, NLF (a MYCN amplified cell line) and SY5Y (a MYCN nonamplified cell line), with the reporter plasmid and miRNA mimic. We measured luciferase (internal control) and renilla (reporter) activity. Results: Our results from the luciferase reporter assay indicate that four miRNAs are strong candidates for CHD5 downregulation in NB: miR-17, -93, -20b, and miR-106b. Each exhibited a significant decrease of fluorescence activity in cells transfected with CHD5 3′UTR in comparison to cells transfected with either of the control plasmids. Interestingly, MYCN upregulates three of the four miRNA candidates: miR-17, -93, & -20b. We further confirmed consistent downregulation of the CHD5 by all four miRNAs. Conclusion: CHD5 expression is downregulated by miR-17, -93, -2b, and 106b. MYCN amplification and overexpression leads to upregulation of miR-17, -93, and -20b, and this may lead to down regulation of CHD5. Citation Format: Koumudi Naraparaju, Venkatadri Kolla, Tiangang Zhuang, Mayumi Higashi, Gerd A. Blobel, Garrett M. Brodeur. The role of microRNAs in the epigenetic silencing of CHD5, a tumor suppressor in neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 191. doi:10.1158/1538-7445.AM2015-191