Identification of miR-494 direct targets involved in senescence of human diploid fibroblasts.
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.
Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli Federico II, Naples, Italy; Center of Genetics Engineering (CEINGE) Biotecnologie Avanzate s.c. a r.l, Naples, Italy; and.
Marika
M
Comegna
Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli Federico II, Naples, Italy; Center of Genetics Engineering (CEINGE) Biotecnologie Avanzate s.c. a r.l, Naples, Italy; and.
Mariangela
M
Succoio
Fondazione SDN, Istituto di Ricerca Diagnostica e Nucleare, Naples, Italy;
Marco
M
Napolitano
Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli Federico II, Naples, Italy; Center of Genetics Engineering (CEINGE) Biotecnologie Avanzate s.c. a r.l, Naples, Italy; and.
Monica
M
Vitale
Proteomics and Mass Spectrometry Laboratory, National Research Council, Naples, Italy.
Chiara
C
D'Ambrosio
Proteomics and Mass Spectrometry Laboratory, National Research Council, Naples, Italy.
Andrea
A
Scaloni
Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli Federico II, Naples, Italy; Fondazione SDN, Istituto di Ricerca Diagnostica e Nucleare, Naples, Italy; Center of Genetics Engineering (CEINGE) Biotecnologie Avanzate s.c. a r.l, Naples, Italy; and.
Fabiana
F
Passaro
Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli Federico II, Naples, Italy; Center of Genetics Engineering (CEINGE) Biotecnologie Avanzate s.c. a r.l, Naples, Italy; and.
Nicola
N
Zambrano
Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli Federico II, Naples, Italy; Fondazione SDN, Istituto di Ricerca Diagnostica e Nucleare, Naples, Italy; Center of Genetics Engineering (CEINGE) Biotecnologie Avanzate s.c. a r.l, Naples, Italy; and faraonio@dbbm.unina.it cimino@dbbm.unina.it.
Filiberto
F
Cimino
Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli Federico II, Naples, Italy; Center of Genetics Engineering (CEINGE) Biotecnologie Avanzate s.c. a r.l, Naples, Italy; and faraonio@dbbm.unina.it cimino@dbbm.unina.it.
Raffaella
R
Faraonio
D002460:Cell Line; D016922:Cellular Senescence / Q000235:genetics* / Q000502:physiology; D045643:DNA Repair Enzymes / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D015180:Electrophoresis, Gel, Two-Dimensional; D005347:Fibroblasts / Q000166:cytology*; D020869:Gene Expression Profiling; D057809:HEK293 Cells; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D006801:Humans; D013058:Mass Spectrometry; D035683:MicroRNAs / Q000502:physiology*; D019704:Protein Disulfide-Isomerases / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D020543:Proteome; D034622:RNA Interference; D060888:Real-Time Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D015854:Up-Regulation
D004268:DNA-Binding Proteins; C502034:HNRNPA3 protein, human; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; C547168:MIRN494 microRNA, human; D035683:MicroRNAs; D020543:Proteome; C513287:RAD23B protein, human; D011993:Recombinant Fusion Proteins; C425312:SYNCRIP protein, human; D019704:Protein Disulfide-Isomerases; C085868:PDIA3 protein, human; D045643:DNA Repair Enzymes
DNA damage; PCNA; SA-β-gal; microRNA; p21/WAF1; p53; proteomics