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Comparative analysis of gene expression data reveals novel targets of senescence-associated microRNAs.

Napolitano M, Comegna M, Succoio M, Leggiero E, Pastore L, Faraonio R, Cimino F, Passaro F - PLoS ONE (2014)

Bottom Line: Senescence-inducing stimuli are myriad and, recently, we and others have demonstrated the role exerted by microRNAs in the induction and maintenance of senescence, by the identification of a subset of Senescence-Associated microRNAs (SAmiRs) up-regulated during replicative or stress-induced senescence and able to induce a premature senescent phenotype when over-expressed in human primary cells.The expression profiles of selected candidates have been validated on replicative and stress-induced senescence and the targeting of the 3'UTRs was assessed by luciferase assay.Furthermore, we demonstrated that the over-expression of CDCA2 in human primary fibroblasts was able to partially counteract etoposide-induced senescence by mitigating the activation of DNA Damage Response.

View Article: PubMed Central - PubMed

Affiliation: IRCCS SDN Foundation, Naples, Italy.

ABSTRACT
In the last decades, cellular senescence is viewed as a complex mechanism involved in different processes, ranging from tumor suppression to induction of age-related degenerative alterations. Senescence-inducing stimuli are myriad and, recently, we and others have demonstrated the role exerted by microRNAs in the induction and maintenance of senescence, by the identification of a subset of Senescence-Associated microRNAs (SAmiRs) up-regulated during replicative or stress-induced senescence and able to induce a premature senescent phenotype when over-expressed in human primary cells. With the intent to find novel direct targets of two specific SAmiRs, SAmiR-494 and -486-5p, and cellular pathways which they are involved in, we performed a comparative analysis of gene expression profiles available in literature to select genes down-regulated upon replicative senescence of human primary fibroblasts. Among them, we searched for SAmiR's candidate targets by analyzing with different target prediction algorithms their 3'UTR for the presence of SAmiR-binding sites. The expression profiles of selected candidates have been validated on replicative and stress-induced senescence and the targeting of the 3'UTRs was assessed by luciferase assay. Results allowed us to identify Cell Division Cycle Associated 2 (CDCA2) and Inhibitor of DNA binding/differentiation type 4 (ID4) as novel targets of SAmiR-494 and SAmiR-486-5p, respectively. Furthermore, we demonstrated that the over-expression of CDCA2 in human primary fibroblasts was able to partially counteract etoposide-induced senescence by mitigating the activation of DNA Damage Response.

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Expression profile of putative targets upon the induction of replicative or stress-induced senescence.The expression levels of the 26 candidates were measured by Real Time PCR in replicative (RS: IMR90 cells at PDL 58), etoposide- (EIS: PDL 33 IMR90 cells treated with 20 µM etoposide for 24 h and then subcultivated for additional 10 days) and DEM-induced (DIS: PDL 33 IMR90 cells treated with 150 µM DEM on alternate days for 10 days) senescent cells. The mRNA relative expression was calculated by assigning the arbitrary value 1 to the amount found in young or DMSO-treated cells. SD is used to refer to the values obtained in 2 different experiments. Results showed that 7 mRNAs, highlighted by a star, resulted significantly (p<0.05) down-regulated, with a cut-off≥2 folds, in all conditions.
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pone-0098669-g002: Expression profile of putative targets upon the induction of replicative or stress-induced senescence.The expression levels of the 26 candidates were measured by Real Time PCR in replicative (RS: IMR90 cells at PDL 58), etoposide- (EIS: PDL 33 IMR90 cells treated with 20 µM etoposide for 24 h and then subcultivated for additional 10 days) and DEM-induced (DIS: PDL 33 IMR90 cells treated with 150 µM DEM on alternate days for 10 days) senescent cells. The mRNA relative expression was calculated by assigning the arbitrary value 1 to the amount found in young or DMSO-treated cells. SD is used to refer to the values obtained in 2 different experiments. Results showed that 7 mRNAs, highlighted by a star, resulted significantly (p<0.05) down-regulated, with a cut-off≥2 folds, in all conditions.

Mentions: To validate the results of our comparative analysis, we investigated in IMR90 cells the expression profile of putative targets upon induction of RS, EIS and DIS (Fig. 2). With the exception of CDCA7, SOCS2 and ZNF367, whose expression in IMR90 was undetectable (not shown), the results obtained by Real Time PCR demonstrated a common signature of gene expression in replicative and stress-induced senescence, with 17 out of 26 putative target genes that resulted significantly (p<0.05) down-regulated, with a fold variation≥2, upon RS, 14 out of 26 upon EIS and 7 out of 26 upon DIS. These results prompted us to select for further investigation the 7 candidates, highlighted in Fig. 2 by a star, whose expression resulted down-regulated in all the examined conditions (RS, EIS and DIS).


Comparative analysis of gene expression data reveals novel targets of senescence-associated microRNAs.

Napolitano M, Comegna M, Succoio M, Leggiero E, Pastore L, Faraonio R, Cimino F, Passaro F - PLoS ONE (2014)

Expression profile of putative targets upon the induction of replicative or stress-induced senescence.The expression levels of the 26 candidates were measured by Real Time PCR in replicative (RS: IMR90 cells at PDL 58), etoposide- (EIS: PDL 33 IMR90 cells treated with 20 µM etoposide for 24 h and then subcultivated for additional 10 days) and DEM-induced (DIS: PDL 33 IMR90 cells treated with 150 µM DEM on alternate days for 10 days) senescent cells. The mRNA relative expression was calculated by assigning the arbitrary value 1 to the amount found in young or DMSO-treated cells. SD is used to refer to the values obtained in 2 different experiments. Results showed that 7 mRNAs, highlighted by a star, resulted significantly (p<0.05) down-regulated, with a cut-off≥2 folds, in all conditions.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048207&req=5

pone-0098669-g002: Expression profile of putative targets upon the induction of replicative or stress-induced senescence.The expression levels of the 26 candidates were measured by Real Time PCR in replicative (RS: IMR90 cells at PDL 58), etoposide- (EIS: PDL 33 IMR90 cells treated with 20 µM etoposide for 24 h and then subcultivated for additional 10 days) and DEM-induced (DIS: PDL 33 IMR90 cells treated with 150 µM DEM on alternate days for 10 days) senescent cells. The mRNA relative expression was calculated by assigning the arbitrary value 1 to the amount found in young or DMSO-treated cells. SD is used to refer to the values obtained in 2 different experiments. Results showed that 7 mRNAs, highlighted by a star, resulted significantly (p<0.05) down-regulated, with a cut-off≥2 folds, in all conditions.
Mentions: To validate the results of our comparative analysis, we investigated in IMR90 cells the expression profile of putative targets upon induction of RS, EIS and DIS (Fig. 2). With the exception of CDCA7, SOCS2 and ZNF367, whose expression in IMR90 was undetectable (not shown), the results obtained by Real Time PCR demonstrated a common signature of gene expression in replicative and stress-induced senescence, with 17 out of 26 putative target genes that resulted significantly (p<0.05) down-regulated, with a fold variation≥2, upon RS, 14 out of 26 upon EIS and 7 out of 26 upon DIS. These results prompted us to select for further investigation the 7 candidates, highlighted in Fig. 2 by a star, whose expression resulted down-regulated in all the examined conditions (RS, EIS and DIS).

Bottom Line: Senescence-inducing stimuli are myriad and, recently, we and others have demonstrated the role exerted by microRNAs in the induction and maintenance of senescence, by the identification of a subset of Senescence-Associated microRNAs (SAmiRs) up-regulated during replicative or stress-induced senescence and able to induce a premature senescent phenotype when over-expressed in human primary cells.The expression profiles of selected candidates have been validated on replicative and stress-induced senescence and the targeting of the 3'UTRs was assessed by luciferase assay.Furthermore, we demonstrated that the over-expression of CDCA2 in human primary fibroblasts was able to partially counteract etoposide-induced senescence by mitigating the activation of DNA Damage Response.

View Article: PubMed Central - PubMed

Affiliation: IRCCS SDN Foundation, Naples, Italy.

ABSTRACT
In the last decades, cellular senescence is viewed as a complex mechanism involved in different processes, ranging from tumor suppression to induction of age-related degenerative alterations. Senescence-inducing stimuli are myriad and, recently, we and others have demonstrated the role exerted by microRNAs in the induction and maintenance of senescence, by the identification of a subset of Senescence-Associated microRNAs (SAmiRs) up-regulated during replicative or stress-induced senescence and able to induce a premature senescent phenotype when over-expressed in human primary cells. With the intent to find novel direct targets of two specific SAmiRs, SAmiR-494 and -486-5p, and cellular pathways which they are involved in, we performed a comparative analysis of gene expression profiles available in literature to select genes down-regulated upon replicative senescence of human primary fibroblasts. Among them, we searched for SAmiR's candidate targets by analyzing with different target prediction algorithms their 3'UTR for the presence of SAmiR-binding sites. The expression profiles of selected candidates have been validated on replicative and stress-induced senescence and the targeting of the 3'UTRs was assessed by luciferase assay. Results allowed us to identify Cell Division Cycle Associated 2 (CDCA2) and Inhibitor of DNA binding/differentiation type 4 (ID4) as novel targets of SAmiR-494 and SAmiR-486-5p, respectively. Furthermore, we demonstrated that the over-expression of CDCA2 in human primary fibroblasts was able to partially counteract etoposide-induced senescence by mitigating the activation of DNA Damage Response.

Show MeSH
Related in: MedlinePlus