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

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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
StatePublished - Dec 11 2009

Fingerprint

MicroRNAs
Endothelial cells
sulofenur
Endothelial Cells
RNA-Induced Silencing Complex
Cells
Untranslated Regions
Neural Cell Adhesion Molecules
Untranslated RNA
Messenger RNA
Gene Ontology
5' Untranslated Regions
Brain-Derived Neurotrophic Factor
3' Untranslated Regions
Bioinformatics
Computational Biology
Immunoprecipitation
Proteomics
Vascular Endothelial Growth Factor A
Cell Movement

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Fasanaro, P., Greco, S., Lorenzi, M., Pescatori, M., Brioschi, M., Kulshreshtha, R., ... Martelli, F. (2009). An integrated approach for experimental target identification of hypoxia-induced miR-210. Journal of Biological Chemistry, 284(50), 35134-35143. https://doi.org/10.1074/jbc.M109.052779

An integrated approach for experimental target identification of hypoxia-induced miR-210. / Fasanaro, Pasquale; Greco, Simona; Lorenzi, Maria; Pescatori, Mario; Brioschi, Maura; Kulshreshtha, Ritu; Banfi, Cristina; Stubbs, Andrew; Calin, George A.; Ivan, Mircea; Capogrossi, Maurizio C.; Martelli, Fabio.

In: Journal of Biological Chemistry, Vol. 284, No. 50, 11.12.2009, p. 35134-35143.

Research output: Contribution to journalArticle

Fasanaro, P, Greco, S, Lorenzi, M, Pescatori, M, Brioschi, M, Kulshreshtha, R, Banfi, C, Stubbs, A, Calin, GA, Ivan, M, Capogrossi, MC & Martelli, F 2009, 'An integrated approach for experimental target identification of hypoxia-induced miR-210', Journal of Biological Chemistry, vol. 284, no. 50, pp. 35134-35143. https://doi.org/10.1074/jbc.M109.052779
Fasanaro P, Greco S, Lorenzi M, Pescatori M, Brioschi M, Kulshreshtha R et al. An integrated approach for experimental target identification of hypoxia-induced miR-210. Journal of Biological Chemistry. 2009 Dec 11;284(50):35134-35143. https://doi.org/10.1074/jbc.M109.052779
Fasanaro, Pasquale ; Greco, Simona ; Lorenzi, Maria ; Pescatori, Mario ; Brioschi, Maura ; Kulshreshtha, Ritu ; Banfi, Cristina ; Stubbs, Andrew ; Calin, George A. ; Ivan, Mircea ; Capogrossi, Maurizio C. ; Martelli, Fabio. / An integrated approach for experimental target identification of hypoxia-induced miR-210. In: Journal of Biological Chemistry. 2009 ; Vol. 284, No. 50. pp. 35134-35143.
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AU - Banfi, Cristina

AU - Stubbs, Andrew

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