Cellular senescence can be induced by various endogenous or exogenous stresses triggering DNA damage and DNA Damage Response (DDR). Cells undergoing senescence have characteristic biochemical features sustained by a reprogrammed gene expression. MicroRNAs (miRNAs) are small non-coding RNAs that act as potent negative regulators at post-transcriptional level. We recently identified 24 miRNAs (SAmiRs) that were either up-or down-regulated in senescent cells . SAmiRs expression was also deregulated in senescence induced by DNA damage (etoposide) or oxidative stress (DEM). Upon adoptive overexpression, 7 up-regulated SAmiRs induced senescence-associated heterochromatin foci (SAHFs) and senescence-associated beta-gal staining (SAbeta-gal) that were accompanied, in the case of miR-210, miR-376a*, miR-486-5p, miR-494 and miR-542-5p, by reduced cell proliferation, induction of double strand DNA breaks, DDR and reactive oxygen species accumulation. Based on these results, our aim is to study regulatory networks induced by SAmiRs in senescence through the identification of their direct targets. To address this point, we used two different approaches. In the first case, we used target-prediction algorithms to generate a list of mRNAs downregulated in replicative senescent fibroblasts, harboring potentially SAmiRs recognition elements within their 3’UTR. Preliminary results indicate that ID4 and CDCA2 are targets of miR-486-5p and miR-494, respectively. In the second case, we performed Two Dimensional Differential- In-Gel Electrophoresis (2D-DIGE) and mass-spectrometry analysis to identify proteins differentially expressed between senescent (replicative or DEM induced) IMR90 fibroblasts and young cells, or after miR-494 overexpression compared to young cells. Among all the protein spots that were differentially detected and identified, we found cytoskeletal, heat shock, metabolic and redox proteins, as well as polypeptide components involved in DDR and others of unknown function. Validation of these results is in progress by integrating in silico computational analysis, luciferase-based and western blot assays
Identification of specific proteins affected by microRNAs in cellular senescence / Comegna, Marika; Napolitano, M; Succoio, Mariangela; Vitale, M; D’Ambrosio, C; Scaloni, A; Zambrano, Nicola; Faraonio, Raffaella; Passaro, Fabiana; Cimino, Filiberto. - (2012). (Intervento presentato al convegno 56° National Meeting of the Italian Society of Biochemistry and Molecular Biology (SIB) tenutosi a Chieti nel 26 th -29 th September, 2012).
Identification of specific proteins affected by microRNAs in cellular senescence
COMEGNA, Marika;SUCCOIO, MARIANGELA;ZAMBRANO, NICOLA;FARAONIO, RAFFAELLA;PASSARO, FABIANA;CIMINO, FILIBERTO
2012
Abstract
Cellular senescence can be induced by various endogenous or exogenous stresses triggering DNA damage and DNA Damage Response (DDR). Cells undergoing senescence have characteristic biochemical features sustained by a reprogrammed gene expression. MicroRNAs (miRNAs) are small non-coding RNAs that act as potent negative regulators at post-transcriptional level. We recently identified 24 miRNAs (SAmiRs) that were either up-or down-regulated in senescent cells . SAmiRs expression was also deregulated in senescence induced by DNA damage (etoposide) or oxidative stress (DEM). Upon adoptive overexpression, 7 up-regulated SAmiRs induced senescence-associated heterochromatin foci (SAHFs) and senescence-associated beta-gal staining (SAbeta-gal) that were accompanied, in the case of miR-210, miR-376a*, miR-486-5p, miR-494 and miR-542-5p, by reduced cell proliferation, induction of double strand DNA breaks, DDR and reactive oxygen species accumulation. Based on these results, our aim is to study regulatory networks induced by SAmiRs in senescence through the identification of their direct targets. To address this point, we used two different approaches. In the first case, we used target-prediction algorithms to generate a list of mRNAs downregulated in replicative senescent fibroblasts, harboring potentially SAmiRs recognition elements within their 3’UTR. Preliminary results indicate that ID4 and CDCA2 are targets of miR-486-5p and miR-494, respectively. In the second case, we performed Two Dimensional Differential- In-Gel Electrophoresis (2D-DIGE) and mass-spectrometry analysis to identify proteins differentially expressed between senescent (replicative or DEM induced) IMR90 fibroblasts and young cells, or after miR-494 overexpression compared to young cells. Among all the protein spots that were differentially detected and identified, we found cytoskeletal, heat shock, metabolic and redox proteins, as well as polypeptide components involved in DDR and others of unknown function. Validation of these results is in progress by integrating in silico computational analysis, luciferase-based and western blot assaysI documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
