An integrated approach for experimental target identification of hypoxia-induced miR-210.
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.
Fasanaro
Pasquale
P
IRCCS-Policlinico San Donato, San Donato Milanese, 20097 Milan, Italy.
Greco
Simona
S
Lorenzi
Maria
M
Pescatori
Mario
M
Brioschi
Maura
M
Kulshreshtha
Ritu
R
Banfi
Cristina
C
Stubbs
Andrew
A
Calin
George A
GA
Ivan
Mircea
M
Capogrossi
Maurizio C
MC
Martelli
Fabio
F
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
C543800:MIRN210 microRNA, human; D035683:MicroRNAs; D020543:Proteome