Limits...
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.

Show MeSH

Related in: MedlinePlus

Knock-down of CDCA2, ID4 or both does not induce premature senescence in PDL 33 IMR90 cells.A) siRNAs designed to target the coding sequence of CDCA2 or ID4 were transfected, individually or as a mix, in PDL 33 IMR90 cells to knock-down the expression levels of target genes. After 72 h, transfected cells were incubated with BrdU for 4 h, then coverslips were fixed, incubated with a primary anti-BrdU antibody, washed and incubated with a secondary fluorescein-conjugated antibody, counterstained with Hoechst-33258 and counted by immunofluorescence. Counts of at least 1,000 cells were averaged and expressed as fold changes±SD, with respect to scrambled transfected cells (*** p<0.001). B) siRNA transfected cells were subcultivated for 10 days and stained for SA-β-gal. Counts of at least 300 cells were averaged and expressed as fold changes±SD, with respect to scrambled transfected cells.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4048207&req=5

pone-0098669-g004: Knock-down of CDCA2, ID4 or both does not induce premature senescence in PDL 33 IMR90 cells.A) siRNAs designed to target the coding sequence of CDCA2 or ID4 were transfected, individually or as a mix, in PDL 33 IMR90 cells to knock-down the expression levels of target genes. After 72 h, transfected cells were incubated with BrdU for 4 h, then coverslips were fixed, incubated with a primary anti-BrdU antibody, washed and incubated with a secondary fluorescein-conjugated antibody, counterstained with Hoechst-33258 and counted by immunofluorescence. Counts of at least 1,000 cells were averaged and expressed as fold changes±SD, with respect to scrambled transfected cells (*** p<0.001). B) siRNA transfected cells were subcultivated for 10 days and stained for SA-β-gal. Counts of at least 300 cells were averaged and expressed as fold changes±SD, with respect to scrambled transfected cells.

Mentions: To analyze the role of CDCA2 and ID4 in senescence, we asked whether their knock-down by RNAi in young cells was able to induce premature senescence, as the up-regulation of the cognate SAmiRs does. To this aim, we transfected siRNAs designed to silence CDCA2 or ID4 in young IMR90 at PDL 33 individually or as a mixture (Fig. S3B), and then we analyzed the cells until ten days after transfection, in order to detect any signs of senescence, as the decreasing of cell proliferation by BrdU incorporation, the change in cell morphology or the appearance of SA-β-gal. As showed in Fig. 4A, the knock-down of these genes seemed to be unable to affect cell proliferation, although a weak but significant decrease in BrdU incorporation was detected in siCDCA2 cells. Accordingly, knock-down cells did not senesce prematurely, as demonstrated by the absence of SA-β-gal staining 10 days after siRNA transfection (Fig. 4B). Probably, neither the transient down-regulation of individual SAmiR target, nor the knock-down of both targets have the “strength” to switching-on the senescence program, as the contemporaneous reduction of many targets exerted by SAmiR’s over-expression do.


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)

Knock-down of CDCA2, ID4 or both does not induce premature senescence in PDL 33 IMR90 cells.A) siRNAs designed to target the coding sequence of CDCA2 or ID4 were transfected, individually or as a mix, in PDL 33 IMR90 cells to knock-down the expression levels of target genes. After 72 h, transfected cells were incubated with BrdU for 4 h, then coverslips were fixed, incubated with a primary anti-BrdU antibody, washed and incubated with a secondary fluorescein-conjugated antibody, counterstained with Hoechst-33258 and counted by immunofluorescence. Counts of at least 1,000 cells were averaged and expressed as fold changes±SD, with respect to scrambled transfected cells (*** p<0.001). B) siRNA transfected cells were subcultivated for 10 days and stained for SA-β-gal. Counts of at least 300 cells were averaged and expressed as fold changes±SD, with respect to scrambled transfected cells.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098669-g004: Knock-down of CDCA2, ID4 or both does not induce premature senescence in PDL 33 IMR90 cells.A) siRNAs designed to target the coding sequence of CDCA2 or ID4 were transfected, individually or as a mix, in PDL 33 IMR90 cells to knock-down the expression levels of target genes. After 72 h, transfected cells were incubated with BrdU for 4 h, then coverslips were fixed, incubated with a primary anti-BrdU antibody, washed and incubated with a secondary fluorescein-conjugated antibody, counterstained with Hoechst-33258 and counted by immunofluorescence. Counts of at least 1,000 cells were averaged and expressed as fold changes±SD, with respect to scrambled transfected cells (*** p<0.001). B) siRNA transfected cells were subcultivated for 10 days and stained for SA-β-gal. Counts of at least 300 cells were averaged and expressed as fold changes±SD, with respect to scrambled transfected cells.
Mentions: To analyze the role of CDCA2 and ID4 in senescence, we asked whether their knock-down by RNAi in young cells was able to induce premature senescence, as the up-regulation of the cognate SAmiRs does. To this aim, we transfected siRNAs designed to silence CDCA2 or ID4 in young IMR90 at PDL 33 individually or as a mixture (Fig. S3B), and then we analyzed the cells until ten days after transfection, in order to detect any signs of senescence, as the decreasing of cell proliferation by BrdU incorporation, the change in cell morphology or the appearance of SA-β-gal. As showed in Fig. 4A, the knock-down of these genes seemed to be unable to affect cell proliferation, although a weak but significant decrease in BrdU incorporation was detected in siCDCA2 cells. Accordingly, knock-down cells did not senesce prematurely, as demonstrated by the absence of SA-β-gal staining 10 days after siRNA transfection (Fig. 4B). Probably, neither the transient down-regulation of individual SAmiR target, nor the knock-down of both targets have the “strength” to switching-on the senescence program, as the contemporaneous reduction of many targets exerted by SAmiR’s over-expression do.

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