Purchase this article with an account.
Colin E Willoughby, Karen Lester, Kasia Whysall, Anshoo Choudhary, Kevin J Hamill, Carl Sheridan; Genome-wide transcriptome profiling of human trabecular meshwork cells treated with TGFβ-2.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4916.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Transforming growth factor beta 2 (TGFβ-2) is elevated in the aqueous humour of primary open angle glaucoma (POAG) patients and results in structural and molecular changes in the human trabecular meshwork (TM). Identifying specific effectors of the TGF-ß2 response in the TM will facilitate the identification of new molecular targets for therapeutic manipulation. RNA-Seq was performed on cultured normal human TM cells treated with TGFβ-2 in order to profile genome-wide transcriptional alterations and to identify the genes and gene-gene networks dysregulated by a known glaucoma stimulus.
Primary normal human TM cells (n=5) were grown to confluence and treated with 5ng/mL TGFβ-2 in serum free low glucose DMEM for 24 hours. Total RNA was extracted using Qiagen AllPrep and RNA quality checked on the Agilent Tapestation. RNA-Seq was performed with 50 bp paired end reads (60 million reads/sample) using the Illumina NextSeq500 by Exiqon. Bioinformatics analysis used a combination of analysis components (Tuxedo, Bowtie2, Tophat and Cufflinks). Gene ontology enrichment analysis was performed with a standard Fisher’s test and the ‘Elim’ method. Realtime-qPCR was performed on Lightcycler480 system (Roche) to validate 10 of the most significant differentially expressed genes.
The read quality was high: 99.9% of reads had a Q score >30. 46.3 million reads were obtained from each sample and the average genome mapping was 82%. Differentially expressed genes (DEGs) were ranked by logFC and p values. A number of key regulators of the TGFβ and Wnt signalling pathways were in the significantly altered DEGs and were validated by qPCR. Gene ontology enrichment analysis of the differentially expressed genes identified extracellular matrix (ECM) organization (GO:0030198) as a key pathway altered by TGFβ-2 in human TM cells.
RNA-Seq is a powerful tool to investigate genome-wide alterations in gene expression in TM treated with a known glaucoma stimulus (TGFβ-2) as a hypothesis-independent approach and implicates alterations in ECM organization in POAG pathogenesis. RNA-Seq can identify therapeutic targets for future molecular therapies to treat the TM in POAG and/or prevent TGF-β2 induced scarring following glaucoma filtration surgery.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
This PDF is available to Subscribers Only