Dataset Information

Differential expression of genes in cells cultured from juxtacanalicular trabecular meshwork and Schlemm's canal.

ABSTRACT: The purpose of this study was to distinguish differences in gene expression between cells cultured from the juxtacanalicular trabecular meshwork (JCTM) and those from Schlemm's canal (SC), to gain clues to differences between those cell types, and to add to our baseline knowledge of gene expression differences in these cell types for later comparison between cells from nonprimary open-angle glaucoma (POAG) and POAG outflow tissues.A set of JCTM and SC cells was cultured from each of 2 donor eyes by an explant method, grown to passage 3, and frozen in liquid nitrogen. The cells were thawed, total RNA was extracted, and the probes made from total RNAs were hybridized to MICROMAX human cDNA microarray slides in 2 separate trials. Differentially expressed genes were analyzed using PubMed, Prosite, and IPA software, and the expression of several of the genes including intercellular adhesion molecule-1 (ICAM-1), tenascin, and ?-spectrin was assessed by immunofluorescence.Schlemm's canal cells differentially expressed ICAM-1, spectrin, complement, fibulin-1, and several genes consistent with an endothelial origin in both arrays, while the JCTM cells more often overexpressed genes consistent with contractile, matrix function, and neural character. At the same time, many genes highly expressed in the first array were not highly overexpressed in the second. One highly overexpressed gene in the JCTM in both arrays, that for heparan sulfate 3-O-sulfotransferase-1 precursor, is thought to be somewhat unique, and could affect the glycosaminoglycan functionality in the extracellular matrix (ECM).We found generally good agreement between the 2 array trials, but some contradictions as well. Many of the genes overexpressed in each cell type had been described in earlier work, but several were new. Tables of genes, grouped by cellular function, and the complete datasets are provided for the development of new hypotheses.

SUBMITTER: O'Brien ET

PROVIDER: S-EPMC3991976 | biostudies-literature | 2014 Mar-Apr

REPOSITORIES: biostudies-literature

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Differential expression of genes in cells cultured from juxtacanalicular trabecular meshwork and Schlemm's canal.

O'Brien E Timothy ET Wang Yanhong Y Ying Hongyu H Yue Beatrice Y J T BY

Journal of ocular pharmacology and therapeutics : the official journal of the Association for Ocular Pharmacology and Therapeutics 20140310 2-3

<h4>Purpose</h4>The purpose of this study was to distinguish differences in gene expression between cells cultured from the juxtacanalicular trabecular meshwork (JCTM) and those from Schlemm's canal (SC), to gain clues to differences between those cell types, and to add to our baseline knowledge of gene expression differences in these cell types for later comparison between cells from nonprimary open-angle glaucoma (POAG) and POAG outflow tissues.<h4>Methods</h4>A set of JCTM and SC cells was cu ...[more]

PMID: 24611521

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Project description:PurposeSchlemm's canal (SC) endothelial cells derived from donors with or without glaucoma showed different mechanical properties and gene expression. As an important contributor to the regulation of intraocular pressure (IOP) and pathogenesis of primary open-angle glaucoma (POAG), the heritable key epigenetic changes, methylation may play an important role in the physiologic function of SC cells. This study aims to identify differentially methylated CpG sites (DMSs) in primary cultures of human SC cells with or without glaucoma.MethodsWe examined the methylation pattern of seven strains of primary human cells (two glaucoma and five normal SC cell samples), which were isolated and characterized using established protocols. DNA methylation was profiled using Illumina Human Methylation 450 BeadChip. Raw data were extracted and exported using Illumina GenomeStudio software. After quantile normalization, DNA methylation data were analyzed using R package RnBeads in Bioconductor. DMSs were filtered with p ≤ 1E-5, methylation change ≥ 0.1, and false discovery rate ≤ 0.05. The closest genes and the location of each CpG site were annotated using R package FDb.InfiniumMethylation.hg19. Gene Ontology and pathway analysis was performed using WebGestalt. Selected DMSs were validated using the Zymo qMethyl kit.ResultsWe used five non-glaucoma and two glaucomatous SC cell samples to profile genome-wide DNA methylation using Illumina Infinium Methylation BeadChips. Principle component analysis showed the separation between the glaucoma and control samples. After quality control and differential analysis, we identified 298 highly significant DMSs (p ≤ 1E-5). Among them, 221 DMSs were within 1 kb of a nearby gene. Gene Ontology analysis demonstrated significant enrichment in positive regulation of cell migration, negative regulation of endothelial cell proliferation, and stress fiber and actin filament bundles. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed enrichment in cell adhesion and gap junctions. Several glaucoma-related genes were identified, including TGFBR3, THBS1, PITX2, DAXX, TBX3, TNXB, ANGPT1, and PLEKHA7. We also examined differentially methylated regions (DMRs) near these CpG sites and identified significant DMRs in TBX3, TNXB1, DAXX, and PITX2.ConclusionsThis study represents the first genome-wide DNA methylation profiling in cultured human primary SC cells. The DMSs were enriched in the pathways related to outflow resistance. Several DMRs were validated in glaucoma-associated genes, further suggesting the role of DNA methylation in glaucoma development. This study could provide comprehensive understanding of DNA methylation in glaucoma and its effect on aqueous humor outflow.

Dataset Information

Differential expression of genes in cells cultured from juxtacanalicular trabecular meshwork and Schlemm's canal.

Publications

Differential expression of genes in cells cultured from juxtacanalicular trabecular meshwork and Schlemm's canal.

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