title: Early targets of miR-34a in neuroblastoma.
abstract: Several genes encoding for proteins involved in proliferation, invasion, and apoptosis are known to be direct miR-34a targets. Here, we used proteomics to screen for targets of miR-34a in neuroblastoma (NBL), a childhood cancer that originates from precursor cells of the sympathetic nervous system. We examined the effect of miR-34a overexpression using a tetracycline inducible system in two NBL cell lines (SHEP and SH-SY5Y) at early time points of expression (6, 12, and 24 h). Proteome analysis using post-metabolic labeling led to the identification of 2,082 proteins, and among these 186 were regulated (112 proteins down-regulated and 74 up-regulated). Prediction of miR-34a targets via bioinformatics showed that 32 transcripts held miR-34a seed sequences in their 3'-UTR. By combining the proteomics data with Kaplan Meier gene-expression studies, we identified seven new gene products (ALG13, TIMM13, TGM2, ABCF2, CTCF, Ki67, and LYAR) that were correlated with worse clinical outcomes. These were further validated in vitro by 3'-UTR seed sequence regulation. In addition, Michigan Molecular Interactions searches indicated that together these proteins affect signaling pathways that regulate cell cycle and proliferation, focal adhesions, and other cellular properties that overall enhance tumor progression (including signaling pathways such as TGF-β, WNT, MAPK, and FAK). In conclusion, proteome analysis has here identified early targets of miR-34a with relevance to NBL tumorigenesis. Along with the results of previous studies, our data strongly suggest miR-34a as a useful tool for improving the chance of therapeutic success with NBL.
authors.affiliation: From the ‡Centro di Ingegneria Genetica e Biotecnologie Avanzate (CEINGE), 80145 Naples, Italy; §Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università Federico II, 80131 Naples, Italy;
authors.forename: Pasqualino
authors.initials: P
authors.lastname: De Antonellis
authors.affiliation: From the ‡Centro di Ingegneria Genetica e Biotecnologie Avanzate (CEINGE), 80145 Naples, Italy; §Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università Federico II, 80131 Naples, Italy;
authors.forename: Marianeve
authors.initials: M
authors.lastname: Carotenuto
authors.affiliation: ‖Department of Medical Protein Research, VIB, B-9000 Ghent, Belgium; **Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium;
authors.forename: Jonathan
authors.initials: J
authors.lastname: Vandenbussche
authors.affiliation: From the ‡Centro di Ingegneria Genetica e Biotecnologie Avanzate (CEINGE), 80145 Naples, Italy; §Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università Federico II, 80131 Naples, Italy;
authors.forename: Gennaro
authors.initials: G
authors.lastname: De Vita
authors.affiliation: From the ‡Centro di Ingegneria Genetica e Biotecnologie Avanzate (CEINGE), 80145 Naples, Italy; §Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università Federico II, 80131 Naples, Italy;
authors.forename: Veronica
authors.initials: V
authors.lastname: Ferrucci
authors.affiliation: From the ‡Centro di Ingegneria Genetica e Biotecnologie Avanzate (CEINGE), 80145 Naples, Italy; §Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università Federico II, 80131 Naples, Italy;
authors.forename: Chiara
authors.initials: C
authors.lastname: Medaglia
authors.affiliation: From the ‡Centro di Ingegneria Genetica e Biotecnologie Avanzate (CEINGE), 80145 Naples, Italy; §Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università Federico II, 80131 Naples, Italy;
authors.forename: Iolanda
authors.initials: I
authors.lastname: Boffa
authors.affiliation: From the ‡Centro di Ingegneria Genetica e Biotecnologie Avanzate (CEINGE), 80145 Naples, Italy; §Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università Federico II, 80131 Naples, Italy;
authors.forename: Alessandra
authors.initials: A
authors.lastname: Galiero
authors.affiliation: From the ‡Centro di Ingegneria Genetica e Biotecnologie Avanzate (CEINGE), 80145 Naples, Italy; §Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università Federico II, 80131 Naples, Italy;
authors.forename: Sarah
authors.initials: S
authors.lastname: Di Somma
authors.affiliation: From the ‡Centro di Ingegneria Genetica e Biotecnologie Avanzate (CEINGE), 80145 Naples, Italy; §Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università Federico II, 80131 Naples, Italy;
authors.forename: Daniela
authors.initials: D
authors.lastname: Magliulo
authors.affiliation: From the ‡Centro di Ingegneria Genetica e Biotecnologie Avanzate (CEINGE), 80145 Naples, Italy; §Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università Federico II, 80131 Naples, Italy;
authors.forename: Nadia
authors.initials: N
authors.lastname: Aiese
authors.affiliation: From the ‡Centro di Ingegneria Genetica e Biotecnologie Avanzate (CEINGE), 80145 Naples, Italy; §Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università Federico II, 80131 Naples, Italy;
authors.forename: Alessandro
authors.initials: A
authors.lastname: Alonzi
authors.affiliation: From the ‡Centro di Ingegneria Genetica e Biotecnologie Avanzate (CEINGE), 80145 Naples, Italy; §Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università Federico II, 80131 Naples, Italy;
authors.forename: Daniela
authors.initials: D
authors.lastname: Spano
authors.affiliation: From the ‡Centro di Ingegneria Genetica e Biotecnologie Avanzate (CEINGE), 80145 Naples, Italy; §Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università Federico II, 80131 Naples, Italy;
authors.forename: Lucia
authors.initials: L
authors.lastname: Liguori
authors.affiliation: From the ‡Centro di Ingegneria Genetica e Biotecnologie Avanzate (CEINGE), 80145 Naples, Italy; §Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università Federico II, 80131 Naples, Italy;
authors.forename: Cristina
authors.initials: C
authors.lastname: Chiarolla
authors.affiliation: From the ‡Centro di Ingegneria Genetica e Biotecnologie Avanzate (CEINGE), 80145 Naples, Italy; §Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università Federico II, 80131 Naples, Italy; ‡‡Department of Translational Medical Science, Section of Pediatrics, University of Naples Federico II, 80131 Naples, Italy;
authors.forename: Antonio
authors.initials: A
authors.lastname: Verrico
authors.affiliation: §§University Children's Hospital Essen, Essen, Germany;
authors.forename: Johannes H
authors.initials: JH
authors.lastname: Schulte
authors.affiliation: ¶¶Center for Medical Genetics, Ghent University Hospital, B-9000 Ghent, Belgium;
authors.forename: Pieter
authors.initials: P
authors.lastname: Mestdagh
authors.affiliation: ¶¶Center for Medical Genetics, Ghent University Hospital, B-9000 Ghent, Belgium;
authors.forename: Jo
authors.initials: J
authors.lastname: Vandesompele
authors.affiliation: ‖Department of Medical Protein Research, VIB, B-9000 Ghent, Belgium; **Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium;
authors.forename: Kris
authors.initials: K
authors.lastname: Gevaert
authors.affiliation: From the ‡Centro di Ingegneria Genetica e Biotecnologie Avanzate (CEINGE), 80145 Naples, Italy; §Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università Federico II, 80131 Naples, Italy; ‖‖Centro di Medicina Trasfusionale, Azienda Ospedaliera Federico II, 80131 Naples, Italy massimo.zollo@unina.it.
authors.forename: Massimo
authors.initials: M
authors.lastname: Zollo
mesh_terms: D020413:3' Untranslated Regions; D045744:Cell Line, Tumor; D003609:Dactinomycin / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D053858:Metabolic Networks and Pathways; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D009447:Neuroblastoma / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D013752:Tetracycline / Q000494:pharmacology
chemical_list: D020413:3' Untranslated Regions; C522425:MIRN34 microRNA, human; D035683:MicroRNAs; D003609:Dactinomycin; D013752:Tetracycline