Abstract
Purpose :
We previously described polarized proteomes of high-quality exosomes isolated from the basal and apical sides of differentiated primary porcine retinal pigmented epithelial cells (pRPE) grown on Transwell cell culture inserts. Using this same pRPE system we analyzed the miRNA cargo in RPE-derived exosomes isolated from the basal and apical media to determine the role of these miRNAs in RPE function.
Methods :
We used two different exosome isolation methods: differential ultracentrifugation (DUC), and cushioned iodixanol buoyant density gradient ultracentrifugation (C-DGUC) to compare RPE-specific miRNAs isolated from pRPE exosomes. Exosomes were isolated from apical and basal conditioned media collected from established pRPE cell cultures grown on Transwell inserts. miRNAs were isolated from these exosomes with a Qiagen miRNeasy micro kit and miRNA libraries were prepared using a QIAseq miRNA library kit. miRNAs were sequenced at the Duke Sequencing and Genomic Technologies center using Illumina MiSeq v3 kits. The resulting miRNA datasets were curated using the Qiagen GeneGlobe RNA Data Analysis program.
Results :
The yield of exosomes and resulting miRNA from apical and basal secreted exosomes isolated by C-DGUC was lower than those isolated by DUC, as expected since this method yields higher purity exosomes. A total of 250 miRNAs were identified from the RNA-Seq data from exosomes isolated by each isolation method, including RPE-specific miRNAs important for development, the visual cycle, and barrier function. We identified miR-204, miR-125b, and miR-9, which are important for RPE development and present in both apical and basal datasets. There was a 10-fold increase in apical compared to basal secreted exosomal miRNAs involved in oxidative stress and neovascularization. These differences were consistent when using DUC or C-DUGC. Exosomes isolated by the C-DUGC method had a 3-fold increase in basal secreted miRNAs associated with RPE development compared to DUC.
Conclusions :
We have identified a set of miRNAs associated with RPE cellular health and are currently repeating these studies in pRPE cells under chronic oxidative stress. Our findings support that miRNAs in exosomes contribute to RPE homeostasis and function in a polarized manner.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.