An integrated approach for experimental target identification of hypoxia-induced miR-210

Pasquale Fasanaro, Simona Greco, Maria Lorenzi, Mario Pescatori, Maura Brioschi, Ritu Kulshreshtha, Cristina Banfi, Andrew Stubbs, George A. Calin, Mircea Ivan, Maurizio C. Capogrossi, Fabio Martelli

Research output: Contribution to journalArticle

Abstract

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 3b-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.

Original languageEnglish
Pages (from-to)35134-35143
Number of pages10
JournalJournal of Biological Chemistry
Volume284
Issue number50
DOIs
Publication statusPublished - Dec 11 2009

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

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