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Early targets of miR-34a in neuroblastoma.

Molecular & cellular proteomics : MCP (2014-06-11)
Pasqualino De Antonellis, Marianeve Carotenuto, Jonathan Vandenbussche, Gennaro De Vita, Veronica Ferrucci, Chiara Medaglia, Iolanda Boffa, Alessandra Galiero, Sarah Di Somma, Daniela Magliulo, Nadia Aiese, Alessandro Alonzi, Daniela Spano, Lucia Liguori, Cristina Chiarolla, Antonio Verrico, Johannes H Schulte, Pieter Mestdagh, Jo Vandesompele, Kris Gevaert, Massimo Zollo
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-ALG13 antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution
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Anti-LYAR antibody produced in rabbit, affinity isolated antibody
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4-tert-Octylphenol monoethoxylate solution, 1 μg/mL in acetone, analytical standard
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Anti-TGM2 (AB2) antibody produced in rabbit, affinity isolated antibody
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Anti-LYAR antibody produced in mouse, IgG fraction of antiserum, buffered aqueous solution
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Anti-LYAR antibody produced in mouse, IgG fraction of antiserum, buffered aqueous solution
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Anti-ALG13 antibody produced in mouse, purified immunoglobulin, buffered aqueous solution
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Anti-TGM2 antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution
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Anti-TGM2 antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution
Supelco
4-tert-Octylphenol monoethoxylate solution, 10 μg/mL in acetone, analytical standard
Sigma-Aldrich
Anti-TGM2 antibody produced in mouse, purified immunoglobulin, buffered aqueous solution
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Anti-LYAR antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution
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ANTI-ALG13 (CENTER) antibody produced in rabbit, IgG fraction of antiserum, buffered aqueous solution
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Anti-TGM2 (AB1) antibody produced in rabbit, affinity isolated antibody
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Anti-TGM2 antibody produced in goat, affinity isolated antibody, buffered aqueous solution
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Anti-TGM2 antibody produced in goat, affinity isolated antibody, buffered aqueous solution
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Anti-TIMM13 antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution