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Age-dependent increase in miRNA-34a expression in the posterior pole of the mouse eye.

Smit-McBride Z, Forward KI, Nguyen AT, Bordbari MH, Oltjen SL, Hjelmeland LM - Mol. Vis. (2014)

Bottom Line: An inverse relationship between the levels of expression of miR-34a and its target Sirt1 mRNA was found at 18 and 24 months of age.Our data showed that miR-34a expression increased in the retina and RPE with age.This suggests that miR-34a may play a role in the senescence and apoptosis of the retina and RPE cells in the aging eye.

View Article: PubMed Central - PubMed

Affiliation: UC Davis School of Medicine, Department of Ophthalmology, Vitreoretinal Research Lab, Davis, CA.

ABSTRACT

Purpose: MicroRNA-34a (miR-34a) has been implicated in neurodegeneration. MiR-34a belongs to a signaling network involving p53 and Sirt-1. This network responds to DNA damage with further downstream signals that induce senescence or apoptosis. Our goal was to measure the expression level of miR-34a in the mouse retina and RPE as a function of age.

Methods: The age-dependent change in miR-34a expression was quantified using a real-time PCR (RT-PCR) assay on microRNA isolates from eye tissue: the retina and RPE/choroid (4, 18, 24, and 32 months of age). Tissue localization of miR-34a was determined by in situ hybridization (ISH) for a series of time points. Expression of the miR-34a target gene Sirt1 was analyzed using RT-PCR and immunohistochemistry.

Results: MiR-34a examined with real-time PCR showed a linear increase in expression with age when compared to that of 4-month-old mice. However, the level of expression between the 24 and 32-month-old animals showed mild downregulation. An age-related increase in miR-34a expression was confirmed in the mouse eye using in situ hybridization. An inverse relationship between the levels of expression of miR-34a and its target Sirt1 mRNA was found at 18 and 24 months of age.

Conclusions: Our data showed that miR-34a expression increased in the retina and RPE with age. The level of DNA damage in mitochondria in the retina and RPE followed a similar time course. This suggests that miR-34a may play a role in the senescence and apoptosis of the retina and RPE cells in the aging eye.

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Real-time PCR (RT-PCR) results for the expression of miR-34a target mRNA Sirt1 in the aging posterior mouse eye. RT–PCR of the retina (A) and RPE/choroid (B) isolated total RNA samples at 4, 18, 24, and 32 months of age. There were three samples (n=3) for each of the four time points, for a total of twelve animals (n=12). Total RNA samples from each animal tissue at each time point were run in triplicate, resulting in each time point representing an average of nine data values. Data were normalized to a geometric mean of the three control genes (Gapdh, B2M, and Hprt1) and calibrated to the 4-month-old sample. The y-axis represents the fold change compared to the 4 month old. * denotes statistically significant differences (p<0.01). Error bars represent standard deviation (SD) values.
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f4: Real-time PCR (RT-PCR) results for the expression of miR-34a target mRNA Sirt1 in the aging posterior mouse eye. RT–PCR of the retina (A) and RPE/choroid (B) isolated total RNA samples at 4, 18, 24, and 32 months of age. There were three samples (n=3) for each of the four time points, for a total of twelve animals (n=12). Total RNA samples from each animal tissue at each time point were run in triplicate, resulting in each time point representing an average of nine data values. Data were normalized to a geometric mean of the three control genes (Gapdh, B2M, and Hprt1) and calibrated to the 4-month-old sample. The y-axis represents the fold change compared to the 4 month old. * denotes statistically significant differences (p<0.01). Error bars represent standard deviation (SD) values.

Mentions: Age-related change in the expression of the miR-34a target mRNA, Sirt1, was analyzed using RT–PCR (Figure 4). The Sirt1 mRNA showed more than a twofold upregulation in the retina (Figure 4A) and more than a fivefold upregulation in RPE (Figure 4B) at 18 months compared to the 4-month-old animals. Differences in the Sirt1 mRNA expression levels between 4 and 18 months were statistically significant for the retina and the RPE (p<0.01; Figure 4A,B, respectively). The level of expression dropped at 24 months in the retina (FC=0.40) and the RPE (FC=–1.84). The downregulation of Sirt1 mRNA expression in the RPE at 24 months was statistically significant compared to 4 months (FC=–1.84; p<0.05). The level of expression of Sirt1 mRNA at 32 months in the retina and the RPE returned to the same level as that of the 4-month-old animals.


Age-dependent increase in miRNA-34a expression in the posterior pole of the mouse eye.

Smit-McBride Z, Forward KI, Nguyen AT, Bordbari MH, Oltjen SL, Hjelmeland LM - Mol. Vis. (2014)

Real-time PCR (RT-PCR) results for the expression of miR-34a target mRNA Sirt1 in the aging posterior mouse eye. RT–PCR of the retina (A) and RPE/choroid (B) isolated total RNA samples at 4, 18, 24, and 32 months of age. There were three samples (n=3) for each of the four time points, for a total of twelve animals (n=12). Total RNA samples from each animal tissue at each time point were run in triplicate, resulting in each time point representing an average of nine data values. Data were normalized to a geometric mean of the three control genes (Gapdh, B2M, and Hprt1) and calibrated to the 4-month-old sample. The y-axis represents the fold change compared to the 4 month old. * denotes statistically significant differences (p<0.01). Error bars represent standard deviation (SD) values.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4225137&req=5

f4: Real-time PCR (RT-PCR) results for the expression of miR-34a target mRNA Sirt1 in the aging posterior mouse eye. RT–PCR of the retina (A) and RPE/choroid (B) isolated total RNA samples at 4, 18, 24, and 32 months of age. There were three samples (n=3) for each of the four time points, for a total of twelve animals (n=12). Total RNA samples from each animal tissue at each time point were run in triplicate, resulting in each time point representing an average of nine data values. Data were normalized to a geometric mean of the three control genes (Gapdh, B2M, and Hprt1) and calibrated to the 4-month-old sample. The y-axis represents the fold change compared to the 4 month old. * denotes statistically significant differences (p<0.01). Error bars represent standard deviation (SD) values.
Mentions: Age-related change in the expression of the miR-34a target mRNA, Sirt1, was analyzed using RT–PCR (Figure 4). The Sirt1 mRNA showed more than a twofold upregulation in the retina (Figure 4A) and more than a fivefold upregulation in RPE (Figure 4B) at 18 months compared to the 4-month-old animals. Differences in the Sirt1 mRNA expression levels between 4 and 18 months were statistically significant for the retina and the RPE (p<0.01; Figure 4A,B, respectively). The level of expression dropped at 24 months in the retina (FC=0.40) and the RPE (FC=–1.84). The downregulation of Sirt1 mRNA expression in the RPE at 24 months was statistically significant compared to 4 months (FC=–1.84; p<0.05). The level of expression of Sirt1 mRNA at 32 months in the retina and the RPE returned to the same level as that of the 4-month-old animals.

Bottom Line: An inverse relationship between the levels of expression of miR-34a and its target Sirt1 mRNA was found at 18 and 24 months of age.Our data showed that miR-34a expression increased in the retina and RPE with age.This suggests that miR-34a may play a role in the senescence and apoptosis of the retina and RPE cells in the aging eye.

View Article: PubMed Central - PubMed

Affiliation: UC Davis School of Medicine, Department of Ophthalmology, Vitreoretinal Research Lab, Davis, CA.

ABSTRACT

Purpose: MicroRNA-34a (miR-34a) has been implicated in neurodegeneration. MiR-34a belongs to a signaling network involving p53 and Sirt-1. This network responds to DNA damage with further downstream signals that induce senescence or apoptosis. Our goal was to measure the expression level of miR-34a in the mouse retina and RPE as a function of age.

Methods: The age-dependent change in miR-34a expression was quantified using a real-time PCR (RT-PCR) assay on microRNA isolates from eye tissue: the retina and RPE/choroid (4, 18, 24, and 32 months of age). Tissue localization of miR-34a was determined by in situ hybridization (ISH) for a series of time points. Expression of the miR-34a target gene Sirt1 was analyzed using RT-PCR and immunohistochemistry.

Results: MiR-34a examined with real-time PCR showed a linear increase in expression with age when compared to that of 4-month-old mice. However, the level of expression between the 24 and 32-month-old animals showed mild downregulation. An age-related increase in miR-34a expression was confirmed in the mouse eye using in situ hybridization. An inverse relationship between the levels of expression of miR-34a and its target Sirt1 mRNA was found at 18 and 24 months of age.

Conclusions: Our data showed that miR-34a expression increased in the retina and RPE with age. The level of DNA damage in mitochondria in the retina and RPE followed a similar time course. This suggests that miR-34a may play a role in the senescence and apoptosis of the retina and RPE cells in the aging eye.

Show MeSH
Related in: MedlinePlus