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Cynthia Yu-Wai-Man, Nicholas Owen, Jonathan Lees, Aristides D. Tagalakis, Stephen L. Hart, Andrew R Webster, Christine A Orengo, Peng Tee Khaw; Genome-wide RNA-Sequencing analysis reveals a distinct fibrosis gene signature in the conjunctiva after glaucoma surgery. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2140.
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Fibrosis is a complex multifactorial disease but little is known about the mechanisms and molecular pathways driving the fibrotic process. We have performed the first genome-wide RNA-Sequencing study in fibrosis to compare the conjunctival gene expression profiles between patients with and without previous glaucoma surgery.
We prospectively collected conjunctival tissues from patients at the time of glaucoma filtration surgery at the Moorfields Eye Hospital (London, UK). We established 10 primary fibrotic (FFs) and 7 non-fibrotic (NFs) human conjunctival fibroblast cell lines from patients with and without previous glaucoma surgery, respectively. The patients were matched for ethnicity and age. The RNA sequencing was performed using TruSeq RNA Library Prep kit v2 and paired-end sequence reads were aligned to the human hg38 reference genome using STAR 2.4.2a. DESeq2 (1.12.3) analysis was used to identify differentially expressed genes between FFs and NFs. We also carried out gene ontology and protein network enrichment analyses on the list of differentially expressed genes. We further validated genes that were highly upregulated or downregulated using real-time quantitative PCR.
A total of 246 genes (46 upregulated, 200 downregulated) were differentially expressed in FFs compared to NFs with more than a two-fold change and p < 0.05 (Fig 1). Principal component analysis revealed tight clustering of FFs whereas there was more variability in NFs. We identified distinct pathways linked to smooth muscle contraction, inflammatory cytokines, immune mediators, extracellular matrix proteins, and oncogene expression. Smooth muscle contraction, proteinaceous extracellular matrix, and growth factor binding were enriched ontology groups in the biological processes, cellular, and molecular function components, respectively. There was also a very high correlation between the RNA-Seq results and validation of differentially expressed genes by real-time quantitative PCR (Spearman’s r > 0.9).
There is a distinct fibrosis gene signature in the conjunctiva after glaucoma surgery. Our study provides new insights into the mechanistic pathways driving the fibrotic process, as well as potential novel therapeutic targets and biomarkers of disease severity in conjunctival fibrosis and other contractile scarring conditions in the eye.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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