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Identification of microRNAs dysregulated in cellular senescence driven by endogenous genotoxic stress.

Nidadavolu LS, Niedernhofer LJ, Khan SA - Aging (Albany NY) (2013)

Bottom Line: Microarray analysis showed three differentially expressed miRNAs in passage 7 (P7) Ercc1-/- MEFs grown at 20% O2 compared to Ercc1-/- MEFs grown at 3% O2.Thirty-six differentially expressed miRNAs were identified in Ercc1-/- MEFs at P7 compared to early passage (P3) in 3% O2.Collectively these results support the conclusion that the miRNAs identified may play an important role in staving off cellular senescence and their altered expression could be indicative of aging.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA.

ABSTRACT
XFE progeroid syndrome, a disease of accelerated aging caused by deficiency in the DNA repair endonuclease XPF-ERCC1, is modeled by Ercc1 knockout and hypomorphic mice. Tissues and primary cells from these mice senesce prematurely, offering a unique opportunity to identify factors that regulate senescence and aging. We compared microRNA (miRNA) expression in Ercc1-/- primary mouse embryonic fibroblasts (MEFs) and wild-type (WT) MEFs in different growth conditions to identify miRNAs that drive cellular senescence. Microarray analysis showed three differentially expressed miRNAs in passage 7 (P7) Ercc1-/- MEFs grown at 20% O2 compared to Ercc1-/- MEFs grown at 3% O2. Thirty-six differentially expressed miRNAs were identified in Ercc1-/- MEFs at P7 compared to early passage (P3) in 3% O2. Eight of these miRNAs (miR-449a, miR-455*, miR-128, miR-497, miR-543, miR-450b-3p, miR-872 and miR-10b) were similarly downregulated in the liver of progeroid Ercc1-/Δ and old WT mice compared to adult WT mice, a tissue that senesces with aging. Three miRNAs (miR-449a, miR-455* and miR-128) were also downregulated in Ercc1-/Δ and WT old mice kidneys compared to young WT mice. We also discovered that the miRNA expression regulator Dicer is significantly downregulated in tissues of old mice and late passage cells compared to young controls. Collectively these results support the conclusion that the miRNAs identified may play an important role in staving off cellular senescence and their altered expression could be indicative of aging.

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QRT-PCR quantification of miRNA identified as down-regulated in the kidney of old WT and progeroid Ercc1−/Δ mice compared to adultWT kidneyQRT-PCR analysis was performed on kidneys of WT young (20 weeks), Ercc1−/Δ (20 weeks), and WT old mice (30 months). (A) miR-449a. (B) miR-455*. (C) miR-128. All three miRNAs identified in the microarray were significantly downregulated in kidney tissue of Ercc1−/Δ progeroid mice and old mice compared to WT young mice. No RT, no reverse transcriptase added. Three mouse kidneys are in each condition. The mean of three experimental replicates for each sample is graphed as relative to WT young samples, which were normalized to a value of -1. The standard deviation is plotted as error bars. P-values were calculated comparing samples to WT Young using Welch's t-tests and are indicated by * (p < .05), ** (p< .01), *** (p< .001) and # (p<.0001).
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Figure 2: QRT-PCR quantification of miRNA identified as down-regulated in the kidney of old WT and progeroid Ercc1−/Δ mice compared to adultWT kidneyQRT-PCR analysis was performed on kidneys of WT young (20 weeks), Ercc1−/Δ (20 weeks), and WT old mice (30 months). (A) miR-449a. (B) miR-455*. (C) miR-128. All three miRNAs identified in the microarray were significantly downregulated in kidney tissue of Ercc1−/Δ progeroid mice and old mice compared to WT young mice. No RT, no reverse transcriptase added. Three mouse kidneys are in each condition. The mean of three experimental replicates for each sample is graphed as relative to WT young samples, which were normalized to a value of -1. The standard deviation is plotted as error bars. P-values were calculated comparing samples to WT Young using Welch's t-tests and are indicated by * (p < .05), ** (p< .01), *** (p< .001) and # (p<.0001).

Mentions: In addition to severe liver abnormalities, ERCC1-deficient mice also develop significant renal dysfunction, as demonstrated by increased proteinuria and creatinine levels [32]. Renal histopathology is evident, including dilated renal tubules, nuclear abnormalities and fibrosis [33]. We examined RNA prepared from kidneys of young (20 weeks) Ercc1−/Δ and WT mice, and old (30 months) WT mice to determine whether any of the aging-associated miRNAs we identified in this study were similarly dysregulated in the kidney tissue. Of the 8 downregulated miRNAs in Ercc1−/Δ and WT old mouse liver compared to WT young mouse liver (Figure 1), three miRNAs (miR-449a, miR-455*, miR-128) were also downregulated in the kidneys of progeroid mice compared to WT young mice (Figure 2). Interestingly, these three miRNAs were also downregulated in the kidneys of old WT mice compared to the young WT mice (Figure 2), further strengthening the conclusion that these miRNAs may be aging-associated.


Identification of microRNAs dysregulated in cellular senescence driven by endogenous genotoxic stress.

Nidadavolu LS, Niedernhofer LJ, Khan SA - Aging (Albany NY) (2013)

QRT-PCR quantification of miRNA identified as down-regulated in the kidney of old WT and progeroid Ercc1−/Δ mice compared to adultWT kidneyQRT-PCR analysis was performed on kidneys of WT young (20 weeks), Ercc1−/Δ (20 weeks), and WT old mice (30 months). (A) miR-449a. (B) miR-455*. (C) miR-128. All three miRNAs identified in the microarray were significantly downregulated in kidney tissue of Ercc1−/Δ progeroid mice and old mice compared to WT young mice. No RT, no reverse transcriptase added. Three mouse kidneys are in each condition. The mean of three experimental replicates for each sample is graphed as relative to WT young samples, which were normalized to a value of -1. The standard deviation is plotted as error bars. P-values were calculated comparing samples to WT Young using Welch's t-tests and are indicated by * (p < .05), ** (p< .01), *** (p< .001) and # (p<.0001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3824412&req=5

Figure 2: QRT-PCR quantification of miRNA identified as down-regulated in the kidney of old WT and progeroid Ercc1−/Δ mice compared to adultWT kidneyQRT-PCR analysis was performed on kidneys of WT young (20 weeks), Ercc1−/Δ (20 weeks), and WT old mice (30 months). (A) miR-449a. (B) miR-455*. (C) miR-128. All three miRNAs identified in the microarray were significantly downregulated in kidney tissue of Ercc1−/Δ progeroid mice and old mice compared to WT young mice. No RT, no reverse transcriptase added. Three mouse kidneys are in each condition. The mean of three experimental replicates for each sample is graphed as relative to WT young samples, which were normalized to a value of -1. The standard deviation is plotted as error bars. P-values were calculated comparing samples to WT Young using Welch's t-tests and are indicated by * (p < .05), ** (p< .01), *** (p< .001) and # (p<.0001).
Mentions: In addition to severe liver abnormalities, ERCC1-deficient mice also develop significant renal dysfunction, as demonstrated by increased proteinuria and creatinine levels [32]. Renal histopathology is evident, including dilated renal tubules, nuclear abnormalities and fibrosis [33]. We examined RNA prepared from kidneys of young (20 weeks) Ercc1−/Δ and WT mice, and old (30 months) WT mice to determine whether any of the aging-associated miRNAs we identified in this study were similarly dysregulated in the kidney tissue. Of the 8 downregulated miRNAs in Ercc1−/Δ and WT old mouse liver compared to WT young mouse liver (Figure 1), three miRNAs (miR-449a, miR-455*, miR-128) were also downregulated in the kidneys of progeroid mice compared to WT young mice (Figure 2). Interestingly, these three miRNAs were also downregulated in the kidneys of old WT mice compared to the young WT mice (Figure 2), further strengthening the conclusion that these miRNAs may be aging-associated.

Bottom Line: Microarray analysis showed three differentially expressed miRNAs in passage 7 (P7) Ercc1-/- MEFs grown at 20% O2 compared to Ercc1-/- MEFs grown at 3% O2.Thirty-six differentially expressed miRNAs were identified in Ercc1-/- MEFs at P7 compared to early passage (P3) in 3% O2.Collectively these results support the conclusion that the miRNAs identified may play an important role in staving off cellular senescence and their altered expression could be indicative of aging.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA.

ABSTRACT
XFE progeroid syndrome, a disease of accelerated aging caused by deficiency in the DNA repair endonuclease XPF-ERCC1, is modeled by Ercc1 knockout and hypomorphic mice. Tissues and primary cells from these mice senesce prematurely, offering a unique opportunity to identify factors that regulate senescence and aging. We compared microRNA (miRNA) expression in Ercc1-/- primary mouse embryonic fibroblasts (MEFs) and wild-type (WT) MEFs in different growth conditions to identify miRNAs that drive cellular senescence. Microarray analysis showed three differentially expressed miRNAs in passage 7 (P7) Ercc1-/- MEFs grown at 20% O2 compared to Ercc1-/- MEFs grown at 3% O2. Thirty-six differentially expressed miRNAs were identified in Ercc1-/- MEFs at P7 compared to early passage (P3) in 3% O2. Eight of these miRNAs (miR-449a, miR-455*, miR-128, miR-497, miR-543, miR-450b-3p, miR-872 and miR-10b) were similarly downregulated in the liver of progeroid Ercc1-/Δ and old WT mice compared to adult WT mice, a tissue that senesces with aging. Three miRNAs (miR-449a, miR-455* and miR-128) were also downregulated in Ercc1-/Δ and WT old mice kidneys compared to young WT mice. We also discovered that the miRNA expression regulator Dicer is significantly downregulated in tissues of old mice and late passage cells compared to young controls. Collectively these results support the conclusion that the miRNAs identified may play an important role in staving off cellular senescence and their altered expression could be indicative of aging.

Show MeSH
Related in: MedlinePlus