A Comparison of Gene Expression Profiles between Glucocorticoid Responder and Non-Responder Bovine Trabecular Meshwork Cells Using RNA Sequencing

Abstract

The most common ocular side effect of glucocorticoid (GC) therapy is GC-induced ocular hypertension (OHT) and GC-induced glaucoma (GIG). GC-induced OHT occurs in about 40% of the general population, while the other 60% are resistant. This study aims to determine the genes and pathways involved in differential GC responsiveness in the trabecular meshwork (TM). Using paired bovine eyes, one eye was perfusion-cultured with 100nM dexamethasone (DEX), while the fellow eye was used to establish a bovine TM (BTM) cell strain. Based on maximum IOP change in the perfused eye, the BTM cell strain was identified as a DEX-responder or non-responder strain. Three responder and three non-responder BTM cell strains were cultured, treated with 0.1% ethanol or 100nM DEX for 7 days. RNA and proteins were extracted for RNA sequencing (RNAseq), qPCR, and Western immunoblotting (WB), respectively. Data were analyzed using the human and bovine genome databases as well as Tophat2 software. Genes were grouped and compared using Student’s t-test. We found that DEX induced fibronectin expression in responder BTM cells but not in non-responder cells using WB. RNAseq showed between 93 and 606 differentially expressed genes in different expression groups between responder and non-responder BTM cells. The data generated by RNAseq were validated using qPCR. Pathway analyses showed 35 pathways associated with differentially expressed genes. These genes and pathways may play important roles in GC-induced OHT and will help us to better understand differential ocular responsiveness to GCs.

The Molecular Processes in the Trabecular Meshwork After Exposure to Corticosteroids and in Corticosteroid-Induced Ocular Hypertension

“…Bermudez et al 14 kindly provided the complete statistical results of their RNA-sequencing experiment: the averaged Fragments Per Kilobase Million (FPKM) for each tested gene after exposure to dexamethasone and a control medium in both responders and nonresponders.…”

“…After the systematic search, the datasets of five different human microarray studies were selected for further analyses: GSE6298 (Fan et al 6 ), GSE16643 (Nehmé et al 10 ), GSE37474 (Kwon et al, 8 concept article received after contacting the authors), GSE65240 (Matsuda et al 9 ), and GSE124114 (Faralli et al 31 ) In addition, the statistical results of the bovine RNA-sequencing experiment by Bermudez et al 14 were used in the analysis.…”

“…Within this study, we integrated the publicly available gene expression data investigating the effect of dexamethasone on the TM. In addition, Bermudez et al 14 kindly provided a bovine RNA-sequencing dataset in which a distinction between eyes with and without a corticosteroid response was made. Bioinformatics analyses on these datasets identified which processes are significantly changed in the TM after exposure to corticosteroids, and in the TM of bovine responders.…”

Intraocular Pressure Changes Following the Administration of Intravitreal Dexamethasone Implant: A Mini -Review

“…Clinical trials have shown that more than 90% of POAG patients are steroid responders. It has been suggested that steroid responders are at greater risk for development of POAG [12][13][14][15] .…”

Glucocorticoid receptor signaling in the eye

“…Many of GC-induced changes in TM of human eyes can be mimicked in cultured TM cells, indicating a direct action of GCs on TM [48]. Transcriptomic and proteomic analysis of DEX-treated cultured human and bovine TM cells revealed alteration in hundreds of genes and proteins involved in cell metabolism, signal transduction, cell structure/motility, intracellular traffic, and immunity [49–51]. Moreover, DEX induced the upregulation of noncanonical Wnt ligand Wnt5a , which in turn induced cross-linked actin network (CLAN) formation in cultured TM cells through RhoA/ Rho-associated protein kinases (ROCK) [52–54].…”