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Zeljka Smit-McBride, Leonard Hjelmeland; Age-related Upregulation of MicroRNA miR-34a In the Mouse Retina and RPE/choroid. Invest. Ophthalmol. Vis. Sci. 2013;54(15):288.
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Age-related upregulation of miR-34a in blood and in brain has recently been identified as a senescence marker for brain and liver tissue in mice. In order to test the hypothesis that miR-34a can be considered as a senescence marker in retina and RPE/choroid, we conducted a real-time PCR analysis to dissect further 2mo, 18mo, 24mo and 32mo old RPE/choroid and retina of male and female mouse.
Mice were obtained from The JAX and from NIA. Research was conducted in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Total microRNA from the tissue was isolated using the Qiagen miRNeasy kit. Samples of miRNA were then analyzed using Taqman assays using preamp primer mix set A, miR-34a and three control small RNAs - sno-135, sno-202 and U6. MicroRNA assays were run at 1:1000 dilutions, in triplicate, with 3 biological replicas for each time point. Raw data was normalized with the geometric mean of the 3 control small RNAs and calibrated to the average value for young mice.
MiR-34a showed upregulation with age in RPE/choroid at all time points examined, 18mo, 24mo and 32mo in comparison with to 2mo old levels. Differences between 2mo and 24mo old expression levels were statistically significant in males (p<0.02), while 2mo vs. 18mo old was statistically significant in females (p<0.01). In retina, statistical significance was achieved for males at 24mo (p<0.01) and 32mo (p<0.05). The highest upregulation has been observed in RPE at 24mo old males (FC=6.35). The individual variability at age 32mo old males was much greater, which brought the average fold change down (FC=1.94). MiRNA-34a is part of the regulatory network of genes that includes tumor suppressor p53, SIRT1 (a longevity related gene), and transcription factors FOXO3 and PGC1-alpha which are regulators of several oxidative stress genes (NRF1, UCP1, UCP3, ESRRA, PPARG). Ingenuity Pathway Analysis (IPA) of mRNA targets of miR-34a will be presented.
Our data show that miR-34a expression increases with aging of retina and RPE/choroid and thus can be considered as a senescence marker. The extreme variability between samples at the age of 34mo could be the result of age-related epigenetic regulatory changes, i.e. methylation of the CpG islands in the miR-34a promoter.
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