Identification of miR‐494 direct targets involved in senescence of human diploid fibroblasts

Abstract

Cellular senescence is a permanent cell cycle arrest triggered by different stimuli. We recently identified up-regulation of microRNA (miR)-494 as a component of the genetic program leading to senescence of human diploid IMR90 fibroblasts. Here, we used 2-dimensional differential gel electrophoresis (2D-DIGE) coupled to mass spectrometry 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 of the miR-494 binding sites on the relevant mRNAs identified 26 potential targets of miR-494. Among them, computational analysis identified 7 potential evolution-conserved miR-494 targets. Functional miR-494 binding sites were confirmed in 3'-untranslated regions (UTRs) of 4 of them [heterogeneous nuclear ribonucleoprotein A3 (hnRNPA3), protein disulfide isomerase A3 (PDIA3), UV excision repair protein RAD23 homolog B (RAD23B), and synaptotagmin-binding cytoplasmic RNA-interacting protein (SYNCRIP)/heterogeneous nuclear ribonucleoprotein Q (hnRNPQ)]. Their reduced expression correlated with miR-494 up-regulation in senescent cells. 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. Overall, these findings identify novel miR-494 direct targets that are involved in cellular senescence.

Translational upregulation of Aurora-A by hnRNP Q1 contributes to cell proliferation and tumorigenesis in colorectal cancer

“…27, 28 It is possible that the increased hnRNP Q mRNA in tumors might be due to mechanisms involving transcriptional upregulation or post-transcriptional regulation, such as miRNA. 29 The content of cancer cells might contribute additional signals in the posttranslational modification of hnRNP Q1 for its retention in the cytoplasm and its function in the translational regulation of mRNAs. 26 …”

Noncoding RNA control of cellular senescence

“…153 miR-494 reduces the levels of hnRNPA3, hnRNPQ, protein disulfide isomerase A3 (PDIA3), and UV excision repair protein RAD23 homolog B (RAD23B), and triggers senescence in lung cancer cells and diploid fibroblasts. 154,155 OncomiRs miR-372, miR-373, and miR-214 prevent cancer cell senescence; 156,157 in light of the fact that a rise in miR-214 reduced the efficacy of radiotherapy, knockdown of microRNA-214 in radioresistant lung cancer cells sensitized them to radiotherapy and stimulated senescence. 157 The lncRNA UCA1 (urothelial cancer-associated 1) enhances tumorigenesis of bladder and breast cancer cells.…”

Proteomic analysis reveals novel common genes modulated in both replicative and stress-induced senescence

“…The population doubling level (PDL) was calculated by using the formula PDL = log(n h /n i )/log2, where n i is the initial number of cells and n h is the final number of cells at each passage. To induce premature senescence, IMR90 at PDL34 or WI38 cells at PDL30 were treated with 100 μM diethylmaleate (DEM) (Sigma-Aldrich) for ten days or challenged with 50 μM etoposide (Sigma-Aldrich) for 24 h, and then subcultured for another 10 days [37,38].…”