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Kevin Schneider, Cristina M Kenney, Mehrnoosh Saghizadeh Ghiam; Mitochondrial regulation of microRNA and implications for macular degeneration. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5263.
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The role of microRNA in age-related macular degeneration (AMD) is of great interest for understanding disease progression, and the development of possible treatment(s). This study investigates the role that mitochondria play in regulation of microRNA. The transmitochondrial cybrid model has allowed us to take a “personalized” approach because while all our cybrids have identical nuclei, each cell line has mitochondria from different individuals. We hypothesize that cybrids containing mitochondria from AMD subjects will differentially express microRNAs compared to cybrids with age-matched normal mitochondria.
Our experiments utilize a transmitochondrial cybrid model in which platelets, containing numerous mitochondria but no nuclei, from clinically well-characterized patients, are isolated and fused with human retinal pigmented epithelial cells (ARPE-19) lacking mitochondria (Rho0). Since all cell lines have identical nuclei, changes in microRNA patterns between AMD cybrids and age-matched normal cybrids can be attributed to the influence of the mitochondria. Cybrid cell lines were generated from AMD (n=5) and aged-matched normal (n=5) subjects. Normal and AMD cybrid cells were cultured and total RNA was extracted, specifically enriching for small RNA utilizing the mirVana RNA Isolation kit. Isolated microRNA were amplified, sequenced and then compared with microRNA database MIRbase. MicroRNA expression levels were then calculated and normalized to total read number. Statistical analyses were by unpaired t-test.
Cybrids containing AMD mitochondria exhibited a statistically significant downregulation of 30-40% in microRNA mir125a, mir155, mir146a, mir30d, mir204, mir95 and mir222 (p<0.05). Decreased expression of the above microRNA have been associated with increased inflammation, oxidative stress, increased expression of vascular endothelial growth factor (VEGF) and increased cell autophagy.
Our results support our hypothesis that there are a subset of microRNA whose expression is regulated by the mitochondria. Mitochondria from patients with AMD induce expression of microRNA consistent with the inflammatory and angiogenic characteristics of wet AMD. These microRNA could serve as potential biomarkers for AMD, and modulation of their levels could provide therapeutic benefits.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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