Abstract
Purpose :
Glucocorticoids-induced ocular hypertension (GC-OHT) is a serious side effect of prolonged GC therapy that can lead to secondary glaucoma and blindness in ~35% of treated patients. Apart from its anti-inflammatory effect, GC action in the trabecular meshwork (TM) involves activation of disease-associated pathology. Our goal is to identify the regulatory mechanisms underlying GC response in the TM. LncRNAs are epigenetic modulators of the chromatin network that regulate gene expression by altering epigenetic marks or acting as molecular RNA switches for transcription regulation. Despite its ability to affect large scale transcriptomic changes, not much is known about LncRNAs role in the TM. Our current study identifies and validates the select group of GC-responsive LncRNAs that is potentially involved in GC-induced pathology in the TM.
Methods :
Primary human TM cells (n=3 biological replicates) were isolated, cultured, and characterized as recommended. TM cells were transduced with Ad5-Null (50 MOI), Ad5-GRβ (50 MOI), or left untreated for 24 hrs before treatment with potent GC Dexamethasone (DEX; 100 nM) or vehicle for 7 days. Cells were then lysed for RNA (Qiagen RNAeasy Micro Kit; RIN>8) and stranded total RNA library preparation and sequencing using Illumina Kit and Platform was performed. Trimmed reads were mapped and count tables were generated using Qiagen CLC Genomics Workbench. Differential expression (DE) analysis was performed to identify significantly altered LncRNAs with respect to DEX. Fold-change (FC)>2 and False Discovery Rate (FDR)<5% was considered significant. Technical validation performed using qPCR.
Results :
We identified 15 differentially expressed LncRNA in response to DEX treatment. Among these, 13 were significantly upregulated and 2 were downregulated. LINC1088 (FC=15.99; FDR=<0.001), LINC2884 (FC=15.67; FDR<0.001), and LINC0008 (FC=34.37; FDR<0.001) were the top 3 upregulated LncRNAs. ENSG00000235872 (FC=–43.30; FDR=0.006) and ENSG00000267512 (FC=–40.10; FDR=0.006) were the only two downregulated LncRNAs. Some of these LncRNAs can modulate genes differentially expressed by DEX treatment. The expression of these LncRNAs is normalized by transduction with Ad5-GRβ.
Conclusions :
The LncRNAs and genes identified in this study represent multiple networks that may influence GC-induced pathology in the TM.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.