De Antonellis Pasqualino, Carotenuto Marianeve, Vandenbussche Jonathan, De Vita Gennaro, Ferrucci Veronica, Medaglia Chiara, Boffa Iolanda, Galiero Alessandra, Di Somma Sarah, Magliulo Daniela, Aiese Nadia, Alonzi Alessandro, Spano Daniela, Liguori Lucia, Chiarolla Cristina, Verrico Antonio, Schulte Johannes H, Mestdagh Pieter, Vandesompele Jo, Gevaert Kris, Zollo Massimo
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 Medical Protein Research, VIB, B-9000 Ghent, Belgium; **Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium;
Mol Cell Proteomics. 2014 Aug;13(8):2114-31. doi: 10.1074/mcp.M113.035808. Epub 2014 Jun 9.
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.
已知几个编码参与增殖、侵袭和凋亡的蛋白质的基因是miR-34a的直接靶标。在这里，我们使用蛋白质组学来筛选神经母细胞瘤（NBL）中miR-34a的靶标，NBL是一种起源于交感神经系统前体细胞的儿童癌症。我们在两个NBL细胞系（SHEP和SH-SY5Y）中使用四环素诱导系统在表达的早期时间点（6、12和24小时）检测了miR-34a过表达的影响。使用代谢后标记的蛋白质组分析鉴定出2082种蛋白质，其中186种受到调控（112种蛋白质下调，74种蛋白质上调）。通过生物信息学预测miR-34a靶标显示，32个转录本在其3'-UTR中含有miR-34a种子序列。通过将蛋白质组学数据与卡普兰-迈耶基因表达研究相结合，我们鉴定出七种与较差临床结果相关的新基因产物（ALG13、TIMM13、TGM2、ABCF2、CTCF、Ki67和LYAR）。这些通过3'-UTR种子序列调控在体外进一步得到验证。此外，密歇根分子相互作用搜索表明，这些蛋白质共同影响调节细胞周期和增殖、粘着斑以及其他总体上促进肿瘤进展的细胞特性的信号通路（包括TGF-β、WNT、MAPK和FAK等信号通路）。总之，蛋白质组分析在此鉴定出了与NBL肿瘤发生相关的miR-34a的早期靶标。连同先前研究的结果，我们的数据强烈表明miR-34a是提高NBL治疗成功几率的有用工具。
