Abstract
Purpose :
Age-related macular degeneration (AMD), one of the leading causes of blindness worldwide, is a neurodegenerative retinal disease. Non-protein coding RNAs have been implicated in the pathophysiology of various neurodegenerative diseases. In this study, we aimed to investigate the pathophysiology of dry AMD by screening exosomal miRNAs differential expressed in vitreous samples from patients with dry AMD and identify target molecules regulated by the miRNAs.
Methods :
The vitreous samples were collected during vitrectomy and total RNA was extracted from the exosomes. After identifying biologically relevant miRNA expression changes between patients with dry AMD and the control group, target genes were screened for the most differentially expressed miRNA, miR-146a-5p. To validate the function of miR-146a-5p in retinal pigment epithelial (RPE) cells, human induced pluripotent stem cell derived RPE cells (iPSC-RPE) and ARPE-19 cells were transfected with 25nM of miRNA-146a-5p mimic for 24 hours. Those treated with 300μM of H2O2 or 100μM of A2E for 3 hours were used for mimicking RPE cellular stress in dry AMD. The expression of mRNA and protein in each condition was analyzed by real-time PCR and western blotting, respectively. In addition, whether miR-146a-5p directly targets the target gene was confirmed using luciferase assay. We also performed H2-DCF-DA fluorescence staining to measure the oxidative stress by miR-146a-5p mimic.
Results :
The expression level of miR-146a-5p was increased in patients with dry AMD and RPE cells treated with H2O2 or A2E, compared to normal subjects and the control group, respectively. Bioinformatic analysis showed that tumor necrosis factor receptor associated factor 6 (TRAF6) and interleukin-1-receptor-assocated kinase 1 (IRAK1) were targeted molecules for miRNA 146a-5p. The expression levels of these target genes, both mRNA and protein, were reduced by drug treatment or inhibition by miR-146a-5p mimic. Through luciferase assay, it was confirmed that miR-146a-5p directly targets TRAF6 and IRAK1. In addition, higher oxidative stress was measured by transfection of miR-146a-5p mimic.
Conclusions :
Our results identified miR-146a-5p as a potential target for further functional analysis regarding their putative role in the pathogenesis of dry AMD via TRAF6 and IRAK1 regulation.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.