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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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Adoptive expression of CDCA2 promotes cell cycle progression in Etoposide-Induced Senescence.A) CDCA2 and ID4 coding sequences were transfected in PDL 33 IMR90 cells. Cells transfected with CMV-NEO plasmid were used as control. After 24 h, transfected cells were treated with 20 µM etoposide or 150 µM DEM for 24 h and then incubated with BrdU for 4 h. Coverslips were then fixed, incubated with primary anti-BrdU and secondary fluorescein-conjugated antibodies, counterstained with Hoechst-33258 and counted by immunofluorescence. Counts of at least 800 cells were averaged and expressed as fold changes±SD, with respect to control transfected cells (***p<0.01). B) and C) PDL 33 IMR90 cells were transfected with the coding sequence of CDCA2. After 24 h, transfected cells were treated with 20 µM etoposide for 24 h and then were collected to obtain protein extracts. Cell extracts from CMV-neo over-expressing cells served as control. Western Blot analysis was used to detect the levels of phosphorylated ATM (p-ATM Ser1981), ATM, phosphorylated p53 (p-p53 Ser15), p53, p21Cip1 and CDCA2 in the cell lysates. β-actin was used as a loading control.
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pone-0098669-g005: Adoptive expression of CDCA2 promotes cell cycle progression in Etoposide-Induced Senescence.A) CDCA2 and ID4 coding sequences were transfected in PDL 33 IMR90 cells. Cells transfected with CMV-NEO plasmid were used as control. After 24 h, transfected cells were treated with 20 µM etoposide or 150 µM DEM for 24 h and then incubated with BrdU for 4 h. Coverslips were then fixed, incubated with primary anti-BrdU and secondary fluorescein-conjugated antibodies, counterstained with Hoechst-33258 and counted by immunofluorescence. Counts of at least 800 cells were averaged and expressed as fold changes±SD, with respect to control transfected cells (***p<0.01). B) and C) PDL 33 IMR90 cells were transfected with the coding sequence of CDCA2. After 24 h, transfected cells were treated with 20 µM etoposide for 24 h and then were collected to obtain protein extracts. Cell extracts from CMV-neo over-expressing cells served as control. Western Blot analysis was used to detect the levels of phosphorylated ATM (p-ATM Ser1981), ATM, phosphorylated p53 (p-p53 Ser15), p53, p21Cip1 and CDCA2 in the cell lysates. β-actin was used as a loading control.

Mentions: While DIS program seemed to be unaffected, the transient over-expression of CDCA2, but not ID4, was able to counteract the EIS program, promoting cell cycle progression (Fig. 5A), despite the DNA damage induced by etoposide (Fig. S4A). However, this is a temporary effect, as CDCA2 over-expressing cells finally arrested their growth and showed SA-β-gal accumulation, just like control cells (Fig. S4B).


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)

Adoptive expression of CDCA2 promotes cell cycle progression in Etoposide-Induced Senescence.A) CDCA2 and ID4 coding sequences were transfected in PDL 33 IMR90 cells. Cells transfected with CMV-NEO plasmid were used as control. After 24 h, transfected cells were treated with 20 µM etoposide or 150 µM DEM for 24 h and then incubated with BrdU for 4 h. Coverslips were then fixed, incubated with primary anti-BrdU and secondary fluorescein-conjugated antibodies, counterstained with Hoechst-33258 and counted by immunofluorescence. Counts of at least 800 cells were averaged and expressed as fold changes±SD, with respect to control transfected cells (***p<0.01). B) and C) PDL 33 IMR90 cells were transfected with the coding sequence of CDCA2. After 24 h, transfected cells were treated with 20 µM etoposide for 24 h and then were collected to obtain protein extracts. Cell extracts from CMV-neo over-expressing cells served as control. Western Blot analysis was used to detect the levels of phosphorylated ATM (p-ATM Ser1981), ATM, phosphorylated p53 (p-p53 Ser15), p53, p21Cip1 and CDCA2 in the cell lysates. β-actin was used as a loading control.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098669-g005: Adoptive expression of CDCA2 promotes cell cycle progression in Etoposide-Induced Senescence.A) CDCA2 and ID4 coding sequences were transfected in PDL 33 IMR90 cells. Cells transfected with CMV-NEO plasmid were used as control. After 24 h, transfected cells were treated with 20 µM etoposide or 150 µM DEM for 24 h and then incubated with BrdU for 4 h. Coverslips were then fixed, incubated with primary anti-BrdU and secondary fluorescein-conjugated antibodies, counterstained with Hoechst-33258 and counted by immunofluorescence. Counts of at least 800 cells were averaged and expressed as fold changes±SD, with respect to control transfected cells (***p<0.01). B) and C) PDL 33 IMR90 cells were transfected with the coding sequence of CDCA2. After 24 h, transfected cells were treated with 20 µM etoposide for 24 h and then were collected to obtain protein extracts. Cell extracts from CMV-neo over-expressing cells served as control. Western Blot analysis was used to detect the levels of phosphorylated ATM (p-ATM Ser1981), ATM, phosphorylated p53 (p-p53 Ser15), p53, p21Cip1 and CDCA2 in the cell lysates. β-actin was used as a loading control.
Mentions: While DIS program seemed to be unaffected, the transient over-expression of CDCA2, but not ID4, was able to counteract the EIS program, promoting cell cycle progression (Fig. 5A), despite the DNA damage induced by etoposide (Fig. S4A). However, this is a temporary effect, as CDCA2 over-expressing cells finally arrested their growth and showed SA-β-gal accumulation, just like control cells (Fig. S4B).

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