title: An integrated approach for experimental target identification of hypoxia-induced miR-210.
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 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.
authors.affiliation: IRCCS-Policlinico San Donato, San Donato Milanese, 20097 Milan, Italy.
authors.forename: Pasquale
authors.initials: P
authors.lastname: Fasanaro
authors.forename: Simona
authors.initials: S
authors.lastname: Greco
authors.forename: Maria
authors.initials: M
authors.lastname: Lorenzi
authors.forename: Mario
authors.initials: M
authors.lastname: Pescatori
authors.forename: Maura
authors.initials: M
authors.lastname: Brioschi
authors.forename: Ritu
authors.initials: R
authors.lastname: Kulshreshtha
authors.forename: Cristina
authors.initials: C
authors.lastname: Banfi
authors.forename: Andrew
authors.initials: A
authors.lastname: Stubbs
authors.forename: George A
authors.initials: GA
authors.lastname: Calin
authors.forename: Mircea
authors.initials: M
authors.lastname: Ivan
authors.forename: Maurizio C
authors.initials: MC
authors.lastname: Capogrossi
authors.forename: Fabio
authors.initials: F
authors.lastname: Martelli
mesh_terms: D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D016000:Cluster Analysis; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D000860:Hypoxia / Q000378:metabolism*; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D020543:Proteome / Q000032:analysis; D040901:Proteomics / Q000379:methods
chemical_list: C543800:MIRN210 microRNA, human; D035683:MicroRNAs; D020543:Proteome