Proteomics unveils novel targets of mir-494 involved in senescence

Cellular senescence is a permanent cell cycle arrest triggered by different stimuli such as telomere attrition, activated oncogenes, oxidative stress. We recently identified up-regulation of miR-494 as a component of the genetic program leading to senescence of human diploid IMR90 fibroblasts. Here, we used 2D-DIGE coupled to mass spectrometry analysis to profile protein expression changes induced by adoptive overexpression of miR-494 in IMR90 cells. miR-494 induced robust perturbation of the IMR90 proteome by significantly (p≤0.05) down-regulating a number of proteins. Combination of mass spectrometry-based identification of down-regulated proteins and bioinformatic prediction analysis of the miR-494 binding sites on the relevant mRNAs identified 26 potential targets of miR-494. Among them, computational target prediction analysis identified 7 putative miR-494 targets, the 3’UTRs of which featured evolutionary conservation of binding sites for miR-494. Functional miR-494 binding sites were confirmed in 3’UTRs of four of them (hnRNPA3, PDIA3, RAD23B, and SYNCRIP/hnRNPQ). Accordingly, their reduced expression correlated with miR-494 up-regulation in different models of senescence. RNA interference-mediated knockdown of hnRNPA3 and, to a lesser extent, RAD23B mirrored the senescent phenotype induced by miR-494 overexpression, blunting cell proliferation and causing up-regulation of SA-β-galactosidase and DNA damage. Ectopic expression of hnRNPA3 or RAD23B slowed the appearance of the senescent phenotype induced by miR-494 in IMR90 cells. Overall, these findings identify novel miR-494 direct targets that are involved in cellular senescence.