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Identification of miR‐494 direct targets involved in senescence of human diploid fibroblasts
Author(s) -
Comegna Marika,
Succoio Mariangela,
Napolitano Marco,
Vitale Monica,
D'Ambrosio Chiara,
Scaloni Andrea,
Passaro Fabiana,
Zambrano Nicola,
Cimino Filiberto,
Faraonio Raffaella
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.13-239129
Subject(s) - senescence , ploidy , identification (biology) , wi 38 , biology , cellular senescence , microbiology and biotechnology , genetics , phenotype , gene , botany
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.—Comegna, M., Succoio, M., Napolitano, M., Vitale, M., D'Ambrosio, C., Scaloni, A., Passaro, F., Zambrano, N., Cimino, F., Faraonio, R. Identification of miR‐494 direct targets involved in senescence of human diploid fibroblasts. FASEB J. 28, 3720–3733 (2014). www.fasebj.org