Abstract
Purpose :
Limited data exist on how Non-Pigmented Ciliary Epithelium (NPCE) Extracellular Vesicles (EVs) affect trabecular meshwork (TM) under oxidative stress. This study employs microRNA/mRNA microarray assays and R-based bioinformatic tools to: 1) comprehensively explore miRNA profiles in NPCE EVs during oxidative stress (OS); 2) identify OS-associated microRNAs in NPCE cell exosomes; 3) validate associations between these microRNAs in NPCE EVs and their respective targets in TM cells.
Methods :
NPCE cells underwent AAPH (1.5mm, 24h) exposure for free radical generation. Extracellular vesicles (EVs), analyzed via GeneChip® miRNA 4.0 Array and Flashtag™ Bundle–ThermoFisher, were isolated with non-oxidized NPCE-derived EVs as controls. HTM cells were treated with OS-NPCE-EVs or controls for 24h, followed by total mRNA extraction and Affymetrix GeneChip Transcriptome Profiling with human Clariom™ S assay. Using R, differential gene (DEG) and microRNA (DEmicroRNA) datasets were established (p-value < 0.05, Log2FC ≥ 2/-2). ClusterProfile conducted GO/KEGG analysis of up/down DEGs, and R packages multiMiR and miRInterBase predicted DEmicroRNA targets.
Results :
In HTM cells, 73 genes were downregulated, and 135 were upregulated, particularly focusing on ECM maintenance genes like DPT, ECM2, TNC, LYPD3, JAM2, CDCP1, SCIN, and PCDH9. Upregulated pathways involve PI3K-Akt, MAPK, RAS, Focal adhesion, Fluid shear stress, and ECM signaling, while downregulated pathways include ROS signaling. Analysis identified 29 genes affected by downregulation and validated targets influenced by upregulated microRNAs; a separate analysis revealed 100 targets. Pathway enrichment emphasized ECM remodeling pathways in upregulated genes and associated microRNAs. Under OS, NPCE cells signal TM cells, upregulating OS response genes and activating protective pathways related to cell adhesion, collagen binding, structural organization, and cell adhesion. Simultaneously, cellular responses downregulate oxidative phosphorylation and non-canonical Wnt signaling pathways.
Conclusions :
Our study in HTM cells unveiled a significant impact on gene expression, particularly in genes associated with ECM organization. Upregulated KEGG pathways indicated the involvement of key cellular processes, while downregulated pathways
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.