Jingwen Cai1, Kristin M Perkumas2, Xuejun Qin3, Michael A Hauser4, W Daniel Stamer5, Yutao Liu6. 1. Department of Cellular Biology and Anatomy, Georgia Regents University, Augusta, Georgia, United States. 2. Department of Ophthalmology, Duke University, Durham, North Carolina, United States. 3. Duke Molecular Physiology Institute, Duke University, Durham, North Carolina, United States 4Department of Medicine, Duke University, Durham, North Carolina, United States. 4. Department of Ophthalmology, Duke University, Durham, North Carolina, United States 3Duke Molecular Physiology Institute, Duke University, Durham, North Carolina, United States 4Department of Medicine, Duke University, Durham, North Carolina, United State. 5. Department of Ophthalmology, Duke University, Durham, North Carolina, United States 5Department of Biomedical Engineering, Duke University, Durham, North Carolina, United States. 6. Department of Cellular Biology and Anatomy, Georgia Regents University, Augusta, Georgia, United States 6James & Jean Culver Vision Discovery Institute, Georgia Regents University, Augusta, Georgia, United States 7Center for Biotechnology and Genomic Medi.
Abstract
PURPOSE: Ocular hypertension is a major risk factor for glaucoma and the inner wall of Schlemm's canal (SC) endothelia participates in the regulation of aqueous humor outflow resistance. This study aimed to identify differentially expressed genes in primary cultures of SC cells from glaucoma patients. METHODS: This study examined SC samples from three glaucoma cases and four controls. Schlemm's canal cells were isolated from eight different postmortem human eyes. Total RNA was extracted, labeled, and hybridized to Illumina HumanWG-6 BeadChips containing probes for approximately 47,000 human transcripts. After extracting the data using Illumina GenomeStudio software, the data were normalized and analyzed using the R package limma in Bioconductor. Using Protein ANalysis THrough Evolutionary Relationships (PANTHER) software, gene ontology analysis of highly expressed genes was executed in controls and glaucoma groups separately. Pathway analysis was performed with differentially expressed genes using WebGestalt (WEB-based GEne SeT AnaLysis Toolkit). Selected genes were validated using droplet digital PCR (ddPCR). RESULTS: Gene ontology analysis indicated similar functional categories in cases and controls. Differential analysis identified a total of 113 genes with at least 2-fold expression changes in cases. Pathway analysis indicated significant enrichment of genes in cell adhesion, heparin binding, glycosaminoglycan binding, filopodium, and extracellular matrix remodeling. Eighteen selected genes with differential expression were successfully validated using ddPCR. CONCLUSIONS: This study represents the first genome-wide expression study of human primary SC cells from glaucoma patients and provides a potential list of targets regulating SC cell stiffness and pore formation, eventually the outflow resistance in glaucoma individuals.
PURPOSE:Ocular hypertension is a major risk factor for glaucoma and the inner wall of Schlemm's canal (SC) endothelia participates in the regulation of aqueous humor outflow resistance. This study aimed to identify differentially expressed genes in primary cultures of SC cells from glaucomapatients. METHODS: This study examined SC samples from three glaucoma cases and four controls. Schlemm's canal cells were isolated from eight different postmortem human eyes. Total RNA was extracted, labeled, and hybridized to Illumina HumanWG-6 BeadChips containing probes for approximately 47,000 human transcripts. After extracting the data using Illumina GenomeStudio software, the data were normalized and analyzed using the R package limma in Bioconductor. Using Protein ANalysis THrough Evolutionary Relationships (PANTHER) software, gene ontology analysis of highly expressed genes was executed in controls and glaucoma groups separately. Pathway analysis was performed with differentially expressed genes using WebGestalt (WEB-based GEne SeT AnaLysis Toolkit). Selected genes were validated using droplet digital PCR (ddPCR). RESULTS: Gene ontology analysis indicated similar functional categories in cases and controls. Differential analysis identified a total of 113 genes with at least 2-fold expression changes in cases. Pathway analysis indicated significant enrichment of genes in cell adhesion, heparin binding, glycosaminoglycan binding, filopodium, and extracellular matrix remodeling. Eighteen selected genes with differential expression were successfully validated using ddPCR. CONCLUSIONS: This study represents the first genome-wide expression study of human primary SC cells from glaucomapatients and provides a potential list of targets regulating SC cell stiffness and pore formation, eventually the outflow resistance in glaucoma individuals.
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