Abstract
Purpose :
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 microRNA modulation 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 age-matched normal cybrids, and that inhibition of these microRNA can alter gene expression in our cybrids. We hypothesize that in addition to gene expression changes, modulation of these microRNAs can potentially altar cellular health.
Methods :
Cybrids 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, differing responses between AMD and age-matched normal cybrids can be attributed to mitochondrial influence. MicroRNA inhibition was achieved through transfection of antisense miRNA inhibitors. Caspase 3/7 assays used the IncuCyte live cell imager. Statistical analyses were by paired t-test.
Results :
Inhibition of mir135b-5p in AMD cybrids decreased expression of pro-apoptotic genes, BAX,BCL2L13 and CASP3 (0.85*, 0.7*, 0.82* fold) and decreased Casp3/7 staining by 40%*. In contrast, inhibition of mir148a-3p increased expression of pro-apoptotic CASP9 gene (1.3* fold) ER Stress gene DDIT3 (4.6* fold), autophagy genes ATG5 and ATG12 (1.75*, 1.73* fold) and inflammatory genes IL6 and IL1B. (12.3*, 4.4* fold). Inhibition of mir148a-3p also increased expression of mitochondrial biogenesis genes TFAM, POLG, TFB2M. (1.5*, 1.98*, 2.0* fold), and decreased ROS levels by 15%*. (*=p<0.05)
Conclusions :
This study shows that inhibition of mir135b-5p decreases expression of apoptotic genes, and decreased Caspase 3/7 staining in AMD cybrids, supporting our hypothesis that modulation of this microRNA may improve ARPE cybrid cell health. In contrast, inhibition of mir148a-3p was associated with increased expression of cell death, inflammation, ER stress, and interestingly an increase in mitochondrial biogenesis genes as well as decreased celluar ROS levels. This data demonstrates that manipulation of specific microRNA can have dramatic effects on many pathways relevant to AMD.
This is a 2021 ARVO Annual Meeting abstract.