Abstract
Neuroblastoma is the most common human extracranial solid tumor during infancy. Involvement of several miRNAs in its pathogenesis has been ascertained. Interestingly, most of their encoding genes reside in hypermethylated genomic regions: thus, their tumor suppressor function is normally disallowed in these tumors. To date, the therapeutic role of the demethylating agent 5′-Aza-2 deoxycytidine (5'-AZA) and its effects on miRNAome modulation in neuroblastoma have not been satisfactorily explored. Starting from a high-throughput expression profiling of 754 miRNAs and based on a proper selection, we focused on miR-29a-3p, miR-34b-3p, miR-181c-5p and miR-517a-3p as candidate miRNAs for our analysis. They resulted downregulated in four neuroblastoma cell lines with respect to normal adrenal gland. MiRNAs 29a-3p and 34b-3p also resulted downregulated in vivo in a murine neuroblastoma progression model. Unlike the amount of methylation of their encoding gene promoters, all these miRNAs were significantly overexpressed following treatment with 5′-AZA. Transfection with candidate miRNAs mimics significantly decreased neuroblastoma cells proliferation rate. A lower expression of miR-181c was significantly associated to a worse overall survival in a public dataset of 498 neuroblastoma samples (http://r2.amc.nl). Our data strongly suggest that CDK6, DNMT3A, DNMT3B are targets of miR-29a-3p, while CCNE2 and E2F3 are targets of miR-34b-3p. Based on all these data, we propose that miR-29a-3p, miR-34b-3p, miR-181c-5p and miR-517a-3p are disallowed tumor suppressor genes in neuroblastoma and suggest them as new therapeutic targets in neuroblastoma.
Highlights
Neuroblastoma is a neuroectodermal tumor that originates from precursor cells of the sympathetic nervous system
We investigated the involvement of miRNAs encoding genes and the role of their promoter methylation in neuroblastoma
MiR-34a is a known tumor suppressor in neuroblastoma; it has been suggested as an epigenetic target for treatment of diffuse large B-cell lymphoma by 5’-Aza-2 deoxycytidine (5’-AZA) [10, 24, 25]
Summary
Neuroblastoma is a neuroectodermal tumor that originates from precursor cells of the sympathetic nervous system. The most frequently characterized cytogenetic alterations in neuroblastoma include: (i) amplification of the gene encoding MYCN (a protooncogenic transcription factor), localized at 2p24; (ii) loss of heterozygosity (LOH) or rearrangements of the distal portion of 1p31-ter, 3p22, 11q23; (iii) gain of chromosome arms 1q or 17q. Besides these abnormalities, gain of chromosomes 4q, 6p, 7q, 11q and 18q, amplification of MDM2 and MYC genes, and LOH at 14q, 10q, 19q13 have been described [3, 4]. To clarify the role of miRNAs in neuroblastoma, we sought to: (1) investigate the effects of 5’-AZA on the global expression of miRNAs in five commonly used neuroblastoma cell line models; (2) analyze the relationship between the expression of four prioritized miRNAs and the methylation status of their encoding gene promoters; (3) identify and characterize the expression of the most interesting among their targets; (4) assay their involvement in neuroblastoma cell viability; (5) analyze the prognostic value of altered expression of miRNAs and their targets
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