Cai Jingwen, Perkumas Kristin M, Qin Xuejun, Hauser Michael A, Stamer W Daniel, Liu Yutao
Department of Cellular Biology and Anatomy, Georgia Regents University, Augusta, Georgia, United States.
Department of Ophthalmology, Duke University, Durham, North Carolina, United States.
Invest Ophthalmol Vis Sci. 2015 Oct;56(11):6747-53. doi: 10.1167/iovs.15-17720.
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.
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).
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.
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.
高眼压是青光眼的主要危险因素，施莱姆管（SC）内壁内皮细胞参与房水流出阻力的调节。本研究旨在鉴定青光眼患者SC细胞原代培养物中差异表达的基因。
本研究检测了3例青光眼患者和4例对照者的SC样本。从8只不同的人死后眼睛中分离出施莱姆管细胞。提取总RNA，进行标记，并与Illumina HumanWG-6 BeadChips杂交，该芯片包含针对约47,000个人类转录本的探针。使用Illumina GenomeStudio软件提取数据后，使用Bioconductor中的R包limma对数据进行标准化和分析。使用蛋白质进化关系分析（PANTHER）软件，分别在对照组和青光眼组中对高表达基因进行基因本体分析。使用基于网络的基因集分析工具包（WebGestalt）对差异表达基因进行通路分析。使用液滴数字PCR（ddPCR）验证选定的基因。
基因本体分析表明病例组和对照组的功能类别相似。差异分析共鉴定出113个基因，其在病例组中的表达变化至少为2倍。通路分析表明，细胞黏附、肝素结合、糖胺聚糖结合、丝状伪足和细胞外基质重塑相关基因显著富集。使用ddPCR成功验证了18个选定的差异表达基因。
本研究是首次对青光眼患者的人原发性SC细胞进行全基因组表达研究，并提供了一份潜在的靶点清单，这些靶点可调节SC细胞硬度和孔隙形成，最终调节青光眼个体的流出阻力。
