An Integrated Approach for Experimental Target Identification of Hypoxia-induced miR-210
Autor: | Cristina Banfi, Maura Brioschi, Maurizio C. Capogrossi, Mircea Ivan, Pasquale Fasanaro, Simona Greco, Fabio Martelli, Ritu Kulshreshtha, Andrew P. Stubbs, George A. Calin, Mario Pescatori, Maria Lorenzi |
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Přispěvatelé: | Fasanaro, P, Greco, S, Lorenzi, M, Pescatori, M, Brioschi, M, Kulshreshtha, R, Banfi, C, Stubbs, A, Calin, G, Ivan, M, Capogrossi, M, Martelli, F |
Rok vydání: | 2009 |
Předmět: |
Proteomics
Untranslated region Proteome Computational biology RNA-Mediated Regulation and Noncoding RNAs Biology Biochemistry microRNA Animals Cluster Analysis Humans Coding region Gene silencing Hypoxia miRNA proteomics endothelial cells Molecular Biology Cells Cultured Genetics Regulation of gene expression Base Sequence Gene Expression Profiling Cell Biology Gene expression profiling MicroRNAs Gene Expression Regulation XIST |
Zdroj: | Journal of Biological Chemistry. 284:35134-35143 |
ISSN: | 0021-9258 |
Popis: | miR-210 is a key player of cell response to hypoxia, modulating cell survival, VEGF-driven endothelial cell migration, and the ability of endothelial cells to form capillary-like structures. A crucial step in understanding microRNA (miRNA) function is the identification of their targets. However, only few miR-210 targets have been identified to date. Here, we describe an integrated strategy for large-scale identification of new miR-210 targets by combining transcriptomics and proteomics with bioinformatic approaches. To experimentally validate candidate targets, the RNA-induced silencing complex (RISC) loaded with miR-210 was purified by immunoprecipitation along with its mRNA targets. The complex was significantly enriched in mRNAs of 31 candidate targets, such as BDNF, GPD1L, ISCU, NCAM, and the non-coding RNA Xist. A subset of the newly identified targets was further confirmed by 3′-untranslated region (UTR) reporter assays, and hypoxia induced down-modulation of their expression was rescued blocking miR-210, providing support for the approach validity. In the case of 9 targets, such as PTPN1 and P4HB, miR-210 seed-pairing sequences localized in the coding sequence or in the 5′-UTR, in line with recent data extending miRNA targeting beyond the “classic” 3′-UTR recognition. Finally, Gene Ontology analysis of the targets highlights known miR-210 impact on cell cycle regulation and differentiation, and predicts a new role of this miRNA in RNA processing, DNA binding, development, membrane trafficking, and amino acid catabolism. Given the complexity of miRNA actions, we view such a multiprong approach as useful to adequately describe the multiple pathways regulated by miR-210 during physiopathological processes. |
Databáze: | OpenAIRE |
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