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Kevin Schneider, Marilyn Chwa, Mehrnoosh Saghizadeh, Cristina M Kenney; Modulation of microRNA: Implications for macular degeneration. Invest. Ophthalmol. Vis. Sci. 2018;59(9):353.
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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 and the effects of microRNA modulation on gene expression in our in vitro AMD model. The “personalized” transmitochondrial cybrids are cell lines that have identical nuclei, but mitochondria from different individuals. Previously we showed that AMD cybrids express higher mir135b-5p and mir148a-3p compared to cybrids with age-matched normal mitochondria. We hypothesize that modulation of these microRNAs can alter expression of downstream genes in AMD cybrids, and potentially improve cellular health.
Cybrid cell lines were generated from AMD (n=5) and aged-matched normal (n=5) subjects by fusing platelets from clinically well-characterized patients 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 influence of the mitochondria. MicroRNA inhibition was achieved through transfection of antisense miRNA inhibitors. Statistical analyses were by unpaired t-test.
Inhibition of mir135b-5p in AMD cybrids resulted in a decreased expression of pro-apoptotic genes, BAX (0.85 fold, p=0.04), BCL2L13 (0.7 fold, p=0.002), and CASP3 (0.82 fold, p=0.05). In contrast, inhibition of mir148a-3p resulted in increased expression of pro-apoptotic CASP9 gene (1.3 fold p=0.009) and ER Stress gene DDIT3 (4.6 fold, p=0.0001), as well as increased autophagy genes ATG5 and ATG12 (1.75 and 1.73 fold, p=0.0001).
Previous data has shown that AMD cybrids express higher levels of pro-apoptotic, ER stress and autophagy genes. This study shows that inhibition of mir135b-5p leads to lower expression of apoptotic genes in AMD cybrids, supporting our hypothesis that modulation of this microRNA may improve cellular health and provide therapeutic benefits in RPE cells. Furthermore, mir135b-5p may serve as a marker of AMD. In contrast, inhibition of mir148a-3p was associated with elevated cell death markers and would not likely provide therapeutic benefit. This data demonstrates that manipulation of specific microRNA can have dramatic effects on gene expression relevant to AMD.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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