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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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Strategy to identify putative targets of SAmiRs.A) The 3’UTR sequences of the 139 mRNAs down-regulated in replicative senescent fibroblasts and reported in Table S1 were analyzed with four different target prediction algorithms. This in silico analysis revealed 30 putative SAmiR targets: 20 of SAmiR-494, 7 of SAmiR-486-5p and 3 common to both SAmiRs. B) List of the 30 predicted target genes of both SAmiR-494 or SAmiR-486-5p. In grey, the three putative targets common to both SAmiRs.
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pone-0098669-g001: Strategy to identify putative targets of SAmiRs.A) The 3’UTR sequences of the 139 mRNAs down-regulated in replicative senescent fibroblasts and reported in Table S1 were analyzed with four different target prediction algorithms. This in silico analysis revealed 30 putative SAmiR targets: 20 of SAmiR-494, 7 of SAmiR-486-5p and 3 common to both SAmiRs. B) List of the 30 predicted target genes of both SAmiR-494 or SAmiR-486-5p. In grey, the three putative targets common to both SAmiRs.

Mentions: To select candidate targets of SAmiR-494 or SAmiR-486-5p, we speculated that SAmiRs induced upon Replicative Senescence (RS) of HDFs could contribute to the suppression of genes that must be kept down-regulated on the induction of RS. Thus, we generated a list of mRNAs down-regulated on HDFs senescence by comparing the normalized data of six different microarray gene expression profiles available on public databases [23]–[28]. This analysis allowed to select 139 mRNAs down-regulated(≥1.5 folds) in at least 3 out of 6 different arrays (Table S1). As summarized in Fig. 1A, we analyzed the 139 mRNAs for the presence of consensus motifs for SAmiR-494 and/or for SAmiR-486-5p, by using four different target prediction algorithms (Target Scan v6.2, miRDB, Diana, miRanda) and focusing on the results common to at least two algorithms. This screening led to the generation of the list of candidate targets shown in Fig. 1B, with 20 putative targets of SAmiR-494, 7 of SAmiR-486-5p and 3 common to both SAmiRs (in grey). Among them, there are many mRNAs encoding proteins involved in cell cycle regulation (e.g. CCNE2, NUSAP1, ZWINT) and DDR (e.g. RAD51, RAD51AP1, DEK), two biological processes modified by ectopic expression of SAmiRs [17] (see also Table S1 for functional annotations). Interestingly, some of the candidates are members of the same protein family (e.g. BUB3 and BUB1b; CDCA2, CDCA4 and CDCA7; RFC2 and RFC3). We excluded BIRC5 (survivin) from further investigation, as it was already validated by others [31].


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)

Strategy to identify putative targets of SAmiRs.A) The 3’UTR sequences of the 139 mRNAs down-regulated in replicative senescent fibroblasts and reported in Table S1 were analyzed with four different target prediction algorithms. This in silico analysis revealed 30 putative SAmiR targets: 20 of SAmiR-494, 7 of SAmiR-486-5p and 3 common to both SAmiRs. B) List of the 30 predicted target genes of both SAmiR-494 or SAmiR-486-5p. In grey, the three putative targets common to both SAmiRs.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098669-g001: Strategy to identify putative targets of SAmiRs.A) The 3’UTR sequences of the 139 mRNAs down-regulated in replicative senescent fibroblasts and reported in Table S1 were analyzed with four different target prediction algorithms. This in silico analysis revealed 30 putative SAmiR targets: 20 of SAmiR-494, 7 of SAmiR-486-5p and 3 common to both SAmiRs. B) List of the 30 predicted target genes of both SAmiR-494 or SAmiR-486-5p. In grey, the three putative targets common to both SAmiRs.
Mentions: To select candidate targets of SAmiR-494 or SAmiR-486-5p, we speculated that SAmiRs induced upon Replicative Senescence (RS) of HDFs could contribute to the suppression of genes that must be kept down-regulated on the induction of RS. Thus, we generated a list of mRNAs down-regulated on HDFs senescence by comparing the normalized data of six different microarray gene expression profiles available on public databases [23]–[28]. This analysis allowed to select 139 mRNAs down-regulated(≥1.5 folds) in at least 3 out of 6 different arrays (Table S1). As summarized in Fig. 1A, we analyzed the 139 mRNAs for the presence of consensus motifs for SAmiR-494 and/or for SAmiR-486-5p, by using four different target prediction algorithms (Target Scan v6.2, miRDB, Diana, miRanda) and focusing on the results common to at least two algorithms. This screening led to the generation of the list of candidate targets shown in Fig. 1B, with 20 putative targets of SAmiR-494, 7 of SAmiR-486-5p and 3 common to both SAmiRs (in grey). Among them, there are many mRNAs encoding proteins involved in cell cycle regulation (e.g. CCNE2, NUSAP1, ZWINT) and DDR (e.g. RAD51, RAD51AP1, DEK), two biological processes modified by ectopic expression of SAmiRs [17] (see also Table S1 for functional annotations). Interestingly, some of the candidates are members of the same protein family (e.g. BUB3 and BUB1b; CDCA2, CDCA4 and CDCA7; RFC2 and RFC3). We excluded BIRC5 (survivin) from further investigation, as it was already validated by others [31].

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