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Elsevier, Molecular and Cellular Proteomics, 8(13), p. 2114-2131, 2014

DOI: 10.1074/mcp.m113.035808

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Early targets of miR-34a in neuroblastoma

This paper is made freely available by the publisher.
This paper is made freely available by the publisher.

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Data provided by SHERPA/RoMEO

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 hours). Proteome analysis using post-metabolic labeling led to the identification of 2,082 proteins, and among these 186 were found regulated (112 proteins down- and 74 up-regulated). Prediction of miR-34a targets by bioinformatics showed that 32 transcripts hold miR-34a seed sequences in their 3 UTR. By combining the proteomics data with the Kaplan Meier gene-expression studies, 7 new gene products were identified (ALG13, TIMM13, TGM2, ABCF2, CTCF, Ki67 and LYAR) that correlated with worse clinical outcome. These were further validated in vitro by 3 UTR seed sequence regulation. In addition, Michigan Molecular Interactions (MiMI) searches indicated that altogether these proteins affect signaling pathways that regulate cell cycle and proliferation, focal adhesions and other cellular properties which 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 neuroblastoma tumorigenesis. Along with previous studies, our data strongly suggest miR-34a as useful tool for a better chance in therapeutic success of NBL.