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.miRNAs are 21-23-nucleotide non-protein coding RNA molecules that regulate the stability and/or the translational efficiency of target messenger RNAs (1-3). Mature miRNAs are loaded into the RNA-induced silencing complex (RISC) 3 and mediate the translational inhibition of target mRNA, albeit a few opposing examples have been described as well (4 -6). The rules that guide miRNA-mRNA interaction are very complex and still under investigation. However, the current paradigm states that a Watson-Crick pairing between the mRNA and the 5Ј-region of the miRNA centered on nucleotides 2-7, termed "seed sequence," is required for miRNA-mediated inhibition (7). RISC-miRNA complexes can move the mRNAs they bind to the P-bodies, which are specialized cytoplasmic compartments where translational repression and mRNA turnover is thought to occur (8). Because P-bodies contain many enzymes involved in mRNA exonucleolitic degradation, miRNAs may also have a secondary quantitative inhibitory effect on mRNAs. A role for miRNAs in mRNA destabilization is also suggested by studies reporting robust correlations between the levels of miRNAs and the message of multiple predicted or validated targets (9 -11).miR-210 is currently regarded as "master miRNA" of hypoxic response, because it was found up-regulated by hypoxia in all the ce...

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