Genome-wide Transcriptome Profiling of Human Trabecular Meshwork Cells treated with Dexamethasone

Abstract

Purpose : The purpose of the present study was to profile genome-wide transcriptional alterations in cultured normal human trabecular meshwork (TM) cells treated with dexamethasone and also to identify the genes and gene-gene networks dysregulated by a known glaucoma stimulus.

Methods : Primary normal human TM cells (n=6) were grown to confluence and treated with 100nM dexamethasone for 16 hours. Total RNA was extracted after treatment and 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, Cufflinks and Bioconductor). Gene ontology enrichment analysis was performed with a standard Fisher’s test and the ‘Elim’ method. Ingenuity Pathway Analysis (IPA) was used for further functional analyses. Nine candidate genes were selected and validated by real time-qPCR.

Results : The read quality was high: 99.9% of reads had a Q score >30. 40-57 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. 4793 genes were differentially expressed. Zinc Finger and BTB Domain Containing 16 (ZBTB16) and FK506 binding protein 5 (FKBP5) were the most significantly up-regulated (ZBTB16: logFC=8.3; FKBP5: logFC= 2.68) genes. Plasminogen Activator Inhibitor Type 2 (PAI) and Matrix metallopeptidase 1 (MMP-1) were the most significantly down-regulated genes (PAI: logFC=-3.59; MMP1: logFC=-1.81). IPA analysis highlighted angiogenesis, axonal guidance signalling and p38 MAPK signalling as key enriched pathways.

Conclusions : RNA-Seq is a powerful tool to investigate genome-wide alterations in gene expression in TM treated with a known glaucoma stimulus (steroid) as a hypothesis-independent approach. A number of key DEGs and pathways were identified. RNA-Seq can identify therapeutic targets for future molecular therapies to treat the TM in steroid induced glaucoma

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.