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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 SAmiRs ectopic expression and validation of CDCA2 and ID4.A) Expression levels of SAmiR-494 and SAmiR-486-5p putative targets 2 days after the transfection of the cognate SAmiR pre-miR in PDL 33 IMR90 cells. mRNA levels were measured by Real Time PCR and mRNA relative expression was calculated by assigning the arbitrary value 1 to the amount found in control cells transfected with a scramble pre-miR. SD refers to the values obtained in 3 different experiments and the difference was significant (** p<0.01; * p<0.05). The quantification of the expression levels of SAmiRs after their ectopic over-expression is reported in Panel A of Figure S2. B) Luciferase constructs bearing the normal 3’UTRs (N), reverse 3’UTRs (R) or mutated 3’UTRs (M) of CDCA2 and ID4 were transfected in HEK293 cells together with the cognate pre-miR or control pre-miR. The normal and reverse 3’UTR of OLFM4 were used as positive control. Luciferase levels were reported as fold changes compared to the values measured in control pre-miR transfected cells, after normalization with Renilla luciferase activity. SD refers to the values obtained in 3 different experiments (* p<0.01). C) Wild type seed regions of SAmiR-494 and SAmiR-486-5p, respectively present into the 3’UTRs of CDCA2 and ID4, compared to the mutated seed regions used for luciferase assays. D) Western blot analysis of CDCA2 in IMR90 cells over-expressing SAmiR-494 (pre-miR) or a specific SAmiR-494 inhibitor (anti-miR). E) Western blot analysis of ID4 in IMR90 cells over-expressing SAmiR-486-5p (pre-miR) or a specific SAmiR-486-5p inhibitor (anti-miR). In both cases, the proteins resulted suppressed by the SAmiR over-expression, as well as they resulted up-regulated by the anti-miR transfection, if compared to the control scramble transfected cells. The quantification of the expression levels of SAmiRs after their ectopic over-expression in D and E is reported in Panel B of Figure S2.
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pone-0098669-g003: Expression profile of putative targets upon SAmiRs ectopic expression and validation of CDCA2 and ID4.A) Expression levels of SAmiR-494 and SAmiR-486-5p putative targets 2 days after the transfection of the cognate SAmiR pre-miR in PDL 33 IMR90 cells. mRNA levels were measured by Real Time PCR and mRNA relative expression was calculated by assigning the arbitrary value 1 to the amount found in control cells transfected with a scramble pre-miR. SD refers to the values obtained in 3 different experiments and the difference was significant (** p<0.01; * p<0.05). The quantification of the expression levels of SAmiRs after their ectopic over-expression is reported in Panel A of Figure S2. B) Luciferase constructs bearing the normal 3’UTRs (N), reverse 3’UTRs (R) or mutated 3’UTRs (M) of CDCA2 and ID4 were transfected in HEK293 cells together with the cognate pre-miR or control pre-miR. The normal and reverse 3’UTR of OLFM4 were used as positive control. Luciferase levels were reported as fold changes compared to the values measured in control pre-miR transfected cells, after normalization with Renilla luciferase activity. SD refers to the values obtained in 3 different experiments (* p<0.01). C) Wild type seed regions of SAmiR-494 and SAmiR-486-5p, respectively present into the 3’UTRs of CDCA2 and ID4, compared to the mutated seed regions used for luciferase assays. D) Western blot analysis of CDCA2 in IMR90 cells over-expressing SAmiR-494 (pre-miR) or a specific SAmiR-494 inhibitor (anti-miR). E) Western blot analysis of ID4 in IMR90 cells over-expressing SAmiR-486-5p (pre-miR) or a specific SAmiR-486-5p inhibitor (anti-miR). In both cases, the proteins resulted suppressed by the SAmiR over-expression, as well as they resulted up-regulated by the anti-miR transfection, if compared to the control scramble transfected cells. The quantification of the expression levels of SAmiRs after their ectopic over-expression in D and E is reported in Panel B of Figure S2.

Mentions: In order to identify direct targets of SAmiRs, considering that mRNAs targeted by a miR are generally degraded [32]–[33], we analyzed by Real Time PCR the mRNA levels of BUB1b, CDCA2, FOXM1, ID4, MKI67, NUSAP1 and PCOLCE at day 2 after the transfection of the cognate synthetic SAmiR precursor (pre-miRs). As shown in Fig. 3A, CDCA2, FOXM1 and NUSAP1 resulted significantly (p<0.01) down-regulated 2 days after SAmiR-494 pre-miR transfection, whereas, among SAmiR-486-5p putative targets, ID4 and, at a lower extent, BUB1b, showed a significant reduction upon pre-miR transfection.


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 SAmiRs ectopic expression and validation of CDCA2 and ID4.A) Expression levels of SAmiR-494 and SAmiR-486-5p putative targets 2 days after the transfection of the cognate SAmiR pre-miR in PDL 33 IMR90 cells. mRNA levels were measured by Real Time PCR and mRNA relative expression was calculated by assigning the arbitrary value 1 to the amount found in control cells transfected with a scramble pre-miR. SD refers to the values obtained in 3 different experiments and the difference was significant (** p<0.01; * p<0.05). The quantification of the expression levels of SAmiRs after their ectopic over-expression is reported in Panel A of Figure S2. B) Luciferase constructs bearing the normal 3’UTRs (N), reverse 3’UTRs (R) or mutated 3’UTRs (M) of CDCA2 and ID4 were transfected in HEK293 cells together with the cognate pre-miR or control pre-miR. The normal and reverse 3’UTR of OLFM4 were used as positive control. Luciferase levels were reported as fold changes compared to the values measured in control pre-miR transfected cells, after normalization with Renilla luciferase activity. SD refers to the values obtained in 3 different experiments (* p<0.01). C) Wild type seed regions of SAmiR-494 and SAmiR-486-5p, respectively present into the 3’UTRs of CDCA2 and ID4, compared to the mutated seed regions used for luciferase assays. D) Western blot analysis of CDCA2 in IMR90 cells over-expressing SAmiR-494 (pre-miR) or a specific SAmiR-494 inhibitor (anti-miR). E) Western blot analysis of ID4 in IMR90 cells over-expressing SAmiR-486-5p (pre-miR) or a specific SAmiR-486-5p inhibitor (anti-miR). In both cases, the proteins resulted suppressed by the SAmiR over-expression, as well as they resulted up-regulated by the anti-miR transfection, if compared to the control scramble transfected cells. The quantification of the expression levels of SAmiRs after their ectopic over-expression in D and E is reported in Panel B of Figure S2.
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Related In: Results  -  Collection

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

pone-0098669-g003: Expression profile of putative targets upon SAmiRs ectopic expression and validation of CDCA2 and ID4.A) Expression levels of SAmiR-494 and SAmiR-486-5p putative targets 2 days after the transfection of the cognate SAmiR pre-miR in PDL 33 IMR90 cells. mRNA levels were measured by Real Time PCR and mRNA relative expression was calculated by assigning the arbitrary value 1 to the amount found in control cells transfected with a scramble pre-miR. SD refers to the values obtained in 3 different experiments and the difference was significant (** p<0.01; * p<0.05). The quantification of the expression levels of SAmiRs after their ectopic over-expression is reported in Panel A of Figure S2. B) Luciferase constructs bearing the normal 3’UTRs (N), reverse 3’UTRs (R) or mutated 3’UTRs (M) of CDCA2 and ID4 were transfected in HEK293 cells together with the cognate pre-miR or control pre-miR. The normal and reverse 3’UTR of OLFM4 were used as positive control. Luciferase levels were reported as fold changes compared to the values measured in control pre-miR transfected cells, after normalization with Renilla luciferase activity. SD refers to the values obtained in 3 different experiments (* p<0.01). C) Wild type seed regions of SAmiR-494 and SAmiR-486-5p, respectively present into the 3’UTRs of CDCA2 and ID4, compared to the mutated seed regions used for luciferase assays. D) Western blot analysis of CDCA2 in IMR90 cells over-expressing SAmiR-494 (pre-miR) or a specific SAmiR-494 inhibitor (anti-miR). E) Western blot analysis of ID4 in IMR90 cells over-expressing SAmiR-486-5p (pre-miR) or a specific SAmiR-486-5p inhibitor (anti-miR). In both cases, the proteins resulted suppressed by the SAmiR over-expression, as well as they resulted up-regulated by the anti-miR transfection, if compared to the control scramble transfected cells. The quantification of the expression levels of SAmiRs after their ectopic over-expression in D and E is reported in Panel B of Figure S2.
Mentions: In order to identify direct targets of SAmiRs, considering that mRNAs targeted by a miR are generally degraded [32]–[33], we analyzed by Real Time PCR the mRNA levels of BUB1b, CDCA2, FOXM1, ID4, MKI67, NUSAP1 and PCOLCE at day 2 after the transfection of the cognate synthetic SAmiR precursor (pre-miRs). As shown in Fig. 3A, CDCA2, FOXM1 and NUSAP1 resulted significantly (p<0.01) down-regulated 2 days after SAmiR-494 pre-miR transfection, whereas, among SAmiR-486-5p putative targets, ID4 and, at a lower extent, BUB1b, showed a significant reduction upon pre-miR transfection.

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