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.

Identification of miR-494 direct targets involved in senescence of human diploid fibroblasts / Comegna, Marika; Succoio, Mariangela; Napolitano, M; Vitale, M; D'Ambrosio, C; Scaloni, A; Passaro, Fabiana; Zambrano, Nicola; Cimino, Filiberto; Faraonio, Raffaella. - In: THE FASEB JOURNAL. - ISSN 0892-6638. - 28:8(2014), pp. 3720-3733. [10.1096/fj.13-239129]

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

COMEGNA, Marika;SUCCOIO, MARIANGELA;PASSARO, FABIANA;ZAMBRANO, NICOLA;CIMINO, FILIBERTO;FARAONIO, RAFFAELLA
2014

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.
2014
Identification of miR-494 direct targets involved in senescence of human diploid fibroblasts / Comegna, Marika; Succoio, Mariangela; Napolitano, M; Vitale, M; D'Ambrosio, C; Scaloni, A; Passaro, Fabiana; Zambrano, Nicola; Cimino, Filiberto; Faraonio, Raffaella. - In: THE FASEB JOURNAL. - ISSN 0892-6638. - 28:8(2014), pp. 3720-3733. [10.1096/fj.13-239129]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11588/585863
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