Discovery of GLO1 New Related Genes and Pathways by RNA-Seq on A2E-Stressed Retinal Epithelial Cells Could Improve Knowledge on Retinitis Pigmentosa

Abstract

Endogenous antioxidants protect cells from reactive oxygen species (ROS)-related deleterious effects, and an imbalance in the oxidant/antioxidant systems generates oxidative stress. Glyoxalase 1 (GLO1) is a ubiquitous cellular enzyme involved in detoxification of methylglyoxal (MG), a cytotoxic byproduct of glycolysis whose excess can produce oxidative stress. In retinitis pigmentosa, one of the most diffuse cause of blindness, oxidative damage leads to photoreceptor death. To clarify the role of GLO1 in retinitis pigmentosa onset and progression, we treated human retinal pigment epithelium cells by the oxidant agent A2E. Transcriptome profiles between treated and untreated cells were performed by RNA-Seq, considering two time points (3 and 6 h), after the basal one. The exposure to A2E highlighted significant expression differences and splicing events in 370 GLO1 first-neighbor genes, and 23 of them emerged from pathway clustered analysis as main candidates to be associated with retinitis pigmentosa. Such a hypothesis was corroborated by the involvement of previously analyzed genes in specific cellular activities related to oxidative stress, such as glyoxylate and dicarboxylate metabolism, glycolysis, axo-dendritic transport, lipoprotein activity and metabolism, SUMOylation and retrograde transport at the trans-Golgi network. Our findings could be the starting point to explore unclear molecular mechanisms involved in retinitis pigmentosa etiopathogenesis.

Keywords: A2E; GLO1; RNA-Seq; oxidative stress; retinitis pigmentosa.

Figure 1

Cell viability from methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Retinal pigment epithelium (RPE) cell viability percentage is expressed as mean of replicates ± standard error of mean, considering 3 replicates for each independent experiment (n = 3). Multiple t-tests were performed for statistical comparisons (p-value < 0.05). Results were estimated at both treatment considered time points (3 and 6 h) compared to the time zero untreated group.

Figure 2

Pathway analysis of GLO1 and its best neighbors. The represented network highlights GLO1 and its best neighbors, emerged from GENEMANIA pathway analysis. Green edges indicate genetic interactions. Pink edges indicate physical interactions.

Figure 3

qRT-PCR validation of the ten most differentially expressed genes. (a) Histograms show the mean expression values (n° of replicate for each group = 6) of nine chosen differentially expressed genes produced by qRT-PCR experiments, resulting after application of the 2^−ΔΔCt method, normalized to the best stable gene RASGRP3 (not shown) and control group. Computed results were statistically significant (ANOVA Bonferroni-corrected p-values < 0.01). (b) Correlation plot between RNA-Seq log₂FC and qRT-PCR log₂FC data, as mean of all considered replicates, confirmed the RNA-Seq result validity. Empty Diamond = RNA-Seq log₂FC value for that gene at 3 h (3 h vs. basal time). Full circle = qRT-PCR log₂FC value for that gene at 6 h (6 h vs. basal time).