RNA-Seq Analysis of Hydroquinone-Induced Oxidative Stress in Porcine Corneal Endothelial Cells

Abstract

Purpose : To understand the association between cigarette smoke and Fuchs Endothelial Corneal Dystrophy, we examine the effect of hydroquinone, an oxidative component of cigarette smoke, on gene expression in porcine corneal endothelial cell cultures through RNA sequencing.

Methods : Porcine corneal endothelial cells were isolated from four porcine eyes, proliferated to confluency on a 6-well plate, then treated with 100µM HQ or PBS control for 2 hours. Total RNA was extracted using the Qiagen RNeasy Mini Kit. Samples that had OD 260/280 ratio > 2 were sent to UCSD Institute of Genomic Medicine for library prep and Next Generation Sequencing. Quantity of RNA was assessed using a Qubit fluorometer and integrity (RIN > 8) using a TapeStation. Sample libraries were prepared using the Illumina TrueSeq RNA Library Prep Kit v2 and then run on an Illumina NovaSeq S4 to obtain 100 bp paired end reads at a coverage depth of 25 million reads/sample. Differentially expressed genes (DEGs) were selected based upon an adjusted p-value of < 0.05 and a fold change of ≥ 1.5 or ≤ −1.5.

Results : Significantly upregulated DEGs included those involving antioxidant defense (GSR, PRDX1) and oxidative stress (NQO1, SRXN1) (adj. p < 0.05). Downregulated DEGs included those involving cell division, replication, and repair. Functional pathway analysis confirmed DEGs in the pathways involving cellular response to oxidative stress (G6PD, GSR, SRNX1) and antioxidant defense (PRDX1, SRNX1) (adj. p < 0.05). Moreover, a subset of genes involved in inflammatory pathways and oxidative stress, including inflammatory cytokines (IL-6, CXCL8), antioxidant capacity (SOD1, SOD2, GPX1), and inflammatory markers (MAPK14) displayed a trend of upregulation despite the adjusted p values falling slightly short of statistical significance.

Conclusions : We found an association between inflammation, oxidative stress, and endothelial cell division upon exposure to hydroquinone. This finding may provide insight into the potential role of oxidative stress in Fuchs Endothelial Corneal Dystrophy, which may be influenced by smoking. Future studies with human corneal endothelial cells of different genotypes exposed to a variety of hydroquinone concentrations and time points may help further elucidate the combinatory effects of genetics and oxidative stress.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.