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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 liver of old WT mice and progeroid Ercc1−/Δ mice compared to adult WT miceQRT-PCR analysis was performed on livers of WT young (20 weeks), Ercc1−/Δ (20 weeks), and WT old mice (30 months). (A) miR-449a. (B) miR-455*. (C) miR-128. (D) miR-497. (E) miR-543. (F) miR-450b-3p. (G) miR-872. (H) miR-10b. All eight miRNAs were downregulated, most significantly, in Ercc1−1Δ progeroid mice and WT old mice compared to WT young mice. No RT, no reverse transcriptase added. Three mouse livers 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 using Welch's t-tests and are indicated by * (p < .05), ** (p< .01), *** (p< .001) and # (p< .0001).
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Figure 1: QRT-PCR quantification of miRNA identified as down-regulated in the liver of old WT mice and progeroid Ercc1−/Δ mice compared to adult WT miceQRT-PCR analysis was performed on livers of WT young (20 weeks), Ercc1−/Δ (20 weeks), and WT old mice (30 months). (A) miR-449a. (B) miR-455*. (C) miR-128. (D) miR-497. (E) miR-543. (F) miR-450b-3p. (G) miR-872. (H) miR-10b. All eight miRNAs were downregulated, most significantly, in Ercc1−1Δ progeroid mice and WT old mice compared to WT young mice. No RT, no reverse transcriptase added. Three mouse livers 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 using Welch's t-tests and are indicated by * (p < .05), ** (p< .01), *** (p< .001) and # (p< .0001).

Mentions: The liver of 20 week-old progeroid Ercc1−/Δ mice and 26 month-old WT mice show signs of profound cellular senescence [35]. This offers a unique opportunity to determine if the miRNA identified to correlate with senescence in vitro might play a role in senescence and aging in vivo. We analyzed the levels of 13 miRNAs confirmed to be dysregulated in P7 Ercc1−/− MEFs compared to P3 Ercc1−/− MEFs (miR-680, miR-320, miR-22, miR-449a, miR-455*, miR-675-3p, miR-128, miR-497, miR-543, miR-450b-3p, miR-872, miR-369-5p and miR-10b) in RNA samples prepared from the livers of WT young (20 weeks), the progeroid Ercc1−/Δ mice, and WT old mice (30 months). Of the ten miRNAs downregulated in Ercc1−/− MEFs, eight (miR-449a, miR-455*, miR-128, miR-497, miR-543, miR-450b-3p, miR-872 and miR-10b) were also down-regulated in both the progeroid and old WT mouse livers compared to the WT young (20 week) control mouse livers (Figure 1). The two remaining miRNAs downregulated in Ercc1−/− MEFs, miR-369-5p and miR-675-3p, showed no expression changes in Ercc1−/Δ mouse livers (data not shown). Three miRNAs (miR-680, miR-320, and miR-22) which were upregulated in P7 compared to P3 Ercc1−/− MEFs (Table 4) as measured by microarray did not show upregulation in livers from progeroid and WT old mice compared to young WT controls as measured by qRT-PCR (data not shown).


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 liver of old WT mice and progeroid Ercc1−/Δ mice compared to adult WT miceQRT-PCR analysis was performed on livers of WT young (20 weeks), Ercc1−/Δ (20 weeks), and WT old mice (30 months). (A) miR-449a. (B) miR-455*. (C) miR-128. (D) miR-497. (E) miR-543. (F) miR-450b-3p. (G) miR-872. (H) miR-10b. All eight miRNAs were downregulated, most significantly, in Ercc1−1Δ progeroid mice and WT old mice compared to WT young mice. No RT, no reverse transcriptase added. Three mouse livers 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 using Welch's t-tests and are indicated by * (p < .05), ** (p< .01), *** (p< .001) and # (p< .0001).
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Related In: Results  -  Collection

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Figure 1: QRT-PCR quantification of miRNA identified as down-regulated in the liver of old WT mice and progeroid Ercc1−/Δ mice compared to adult WT miceQRT-PCR analysis was performed on livers of WT young (20 weeks), Ercc1−/Δ (20 weeks), and WT old mice (30 months). (A) miR-449a. (B) miR-455*. (C) miR-128. (D) miR-497. (E) miR-543. (F) miR-450b-3p. (G) miR-872. (H) miR-10b. All eight miRNAs were downregulated, most significantly, in Ercc1−1Δ progeroid mice and WT old mice compared to WT young mice. No RT, no reverse transcriptase added. Three mouse livers 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 using Welch's t-tests and are indicated by * (p < .05), ** (p< .01), *** (p< .001) and # (p< .0001).
Mentions: The liver of 20 week-old progeroid Ercc1−/Δ mice and 26 month-old WT mice show signs of profound cellular senescence [35]. This offers a unique opportunity to determine if the miRNA identified to correlate with senescence in vitro might play a role in senescence and aging in vivo. We analyzed the levels of 13 miRNAs confirmed to be dysregulated in P7 Ercc1−/− MEFs compared to P3 Ercc1−/− MEFs (miR-680, miR-320, miR-22, miR-449a, miR-455*, miR-675-3p, miR-128, miR-497, miR-543, miR-450b-3p, miR-872, miR-369-5p and miR-10b) in RNA samples prepared from the livers of WT young (20 weeks), the progeroid Ercc1−/Δ mice, and WT old mice (30 months). Of the ten miRNAs downregulated in Ercc1−/− MEFs, eight (miR-449a, miR-455*, miR-128, miR-497, miR-543, miR-450b-3p, miR-872 and miR-10b) were also down-regulated in both the progeroid and old WT mouse livers compared to the WT young (20 week) control mouse livers (Figure 1). The two remaining miRNAs downregulated in Ercc1−/− MEFs, miR-369-5p and miR-675-3p, showed no expression changes in Ercc1−/Δ mouse livers (data not shown). Three miRNAs (miR-680, miR-320, and miR-22) which were upregulated in P7 compared to P3 Ercc1−/− MEFs (Table 4) as measured by microarray did not show upregulation in livers from progeroid and WT old mice compared to young WT controls as measured by qRT-PCR (data not shown).

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