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MIR152, MIR200B, and MIR338, human positional and functional neuroblastoma candidates, are involved in neuroblast differentiation and apoptosis

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Abstract

MicroRNAs (MIRs) perform critical regulatory functions within cell networks, both in physiology as well as in pathology. Through the positional gene candidate approach, we have identified three MIRs (MIR152, MIR200B, and MIR338) that are located in regions frequently altered in neuroblastoma (NB) and target mRNAs encoding proteins involved in cell proliferation, neuroblast differentiation, neuroblast migration, and apoptosis. Expression analysis in NB biopsies and NB cell lines showed that these MIRs are dysregulated. We have characterized a CpG island, close to the gene encoding MIR200B and hypermethylated in NB samples, that explains its negative regulation. Expression of MIR152, MIR200B, and MIR338 is specifically modulated in NB cell lines during differentiation and apoptosis. Functional genomic experiments through enforced expression of MIR200B and knockdown of MIR152 resulted in a significant decrease of the invasion activity of SH-SY5Y cells. Reconstruction of a NB network comprising MIR152, MIR200B, and MIR338 allowed us to confirm their role in the control of NB cell stemness and apoptosis: This suggests that altered regulation of these MIRs could have a role in NB pathogenesis by interfering with the molecular mechanisms, which physiologically control differentiation and death of neuroblasts. Accordingly, they could be considered as new NB biomarkers and potential targets of antagomirs or epigenetic therapies.

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Abbreviations

ATRA:

All-trans-retinoic acid

FBS:

Fetal bovine serum

GI:

Genome imbalance

GTFs:

General transcription factors

LOH:

Loss of heterozygosity

MIR:

MicroRNA

NB:

Neuroblastoma

NEAA:

Non-essential amino acids

TFBS:

Transcription factor binding site

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Acknowledgments

This project was financed with funds from Ministero dell’Università e della Ricerca Scientifica e Tecnologica to Prof. Michele Purrello (FIRB 2007: Dalla Proteomica alla Biologia Cellulare; FAR 2007: Generation of a technological platform to study the effects of antineoplastic drugs and to investigate their potential efficacy as neuroprotective agents; PRA 2007: Caratterizzazione delle Omiche del Macchinario Apoptotico e dell’Apparato di Trascrizione: ruolo biologico dei microRNA e loro coinvolgimento in Patologia) and to Prof. Massimo Romani (AIRC, Regione Liguria and Ministero della Salute). Primary neuroblastoma tumors were a kind gift of Dr. Giampaolo Tonini (Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy). SH-SY5Y cells were a gift of Prof. Vittoria Spina (Dipartimento di Scienze Chimiche, Sezione di Biochimica e Biologia Molecolare, Università di Catania, Catania, Italy). Fenretinide was kindly provided by Dr. Lizzia Raffaghello (Laboratory of Oncology, G. Gaslini Children’s Hospital, Genova, Italy). Dr. Barbara Banelli is a fellow of Fondazione Italiana per la Lotta al Neuroblastoma.

Authors’ contributions

MP conceived and coordinated the project. MP, MR (Genova), MR (Catania), CDP, AM, BB, and IC designed the experiments, and the other researchers performed them; all contributed to the critical revision of the data. MP, MR (Genova), MR (Catania), and AM wrote the paper. All authors contributed to its revision.

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The authors report no conflict of interest related to this study.

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Correspondence to Michele Purrello.

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Ragusa, M., Majorana, A., Banelli, B. et al. MIR152, MIR200B, and MIR338, human positional and functional neuroblastoma candidates, are involved in neuroblast differentiation and apoptosis. J Mol Med 88, 1041–1053 (2010). https://doi.org/10.1007/s00109-010-0643-0

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