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

• Published in: PloS one Vol. 12; no. 1; p. e0169671
• Main Authors: Bermudez, Jaclyn Y, Webber, Hannah C, Brown, Bartley, Braun, Terry A, Clark, Abbot F, Mao, Weiming
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 09-01-2017; Public Library of Science (PLoS)
• Subjects: Animals; Biology and Life Sciences; Biomedical engineering; Care and treatment; Cattle; Computational Biology - methods; Data processing; Dexamethasone; Dosage and administration; Engineering schools; Ethanol; Extracellular matrix; Eye; Eye (anatomy); Eye diseases; Fibronectin; Gene expression; Gene Expression Profiling; Gene Expression Regulation - drug effects; Gene sequencing; Genes; Genomes; Glaucoma; Glucocorticoids; Glucocorticoids - pharmacology; High-Throughput Nucleotide Sequencing; Hypertension; Immunoblotting; Laboratory animals; Medicine and Health Sciences; Pathogenesis; Perfusion; Protein Interaction Mapping; Proteins; Research and analysis methods; Ribonucleic acid; RNA; RNA sequencing; Science; Sequence Analysis, RNA; Signal Transduction; Studies; Trabecular Meshwork - cytology; Trabecular Meshwork - metabolism; Transcriptome; Germany
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0169671

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.