Abstract
Purpose: To elucidate the expression profile and the potential role of long non-coding ribonucleic acids (RNAs) (lncRNAs) in a dry eye disease (DED) model.Methods: A DED model was established in C57BL/6J mice with 0.2% benzalkonium chloride (BAC) twice a day for 14 days. The differentially expressed lncRNAs were detected by RNA-seq technology (Gene Expression Omnibus, GEO GSE186450) and the aberrantly expressed lncRNAs were further verified by RT-qPCR. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to predicate the related candidate genes and potential pathological pathways. Cells from a human corneal epithelial cell line (HCECs) were cultured under hyperosmolarity. The regulation of inflammatory factors by silencing potential targeted lncRNAs was verified in vitro in HCECs.Results: In our study, a significant increase in corneal fluorescence staining and a reduction in tear production were observed in DED mice at all follow-ups compared with the controls, and the differences were increasing over time. In total, 2,649 upregulated and 704 downregulated lncRNAs were identified in DED mice. We selected six aberrantly expressed and most abundant lncRNAs and performed RT-qPCR using the samples for RNA-seq. Chrnb2, Gabarapl2, and Usp31 were thereby confirmed as the most significantly altered lncRNAs. Pathway analysis revealed that the neuroactive ligand–receptor interaction signaling pathway was the most enriched, followed by the calcium signaling pathway and cytokine–cytokine receptor interaction. Following treatment of Gabarapl2 siRNA and Chrnb2 siRNA, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 were significantly downregulated in the HCECs.Conclusion: Our study suggests that Chrnb2 and Gabarapl2 may be involved in the inflammation response by regulating TNF-α, IL-1β, and IL-6 in DED. These candidate lncRNAs may be both potential biomarkers and therapeutic targets for DED.
Highlights
Hundreds of millions of people throughout the world are affected by dry eye disease (DED), typically suffering symptoms such as blurred vision, ocular discomfort, and a stinging, burning, scratchy, or gritty sensation while reading, driving, or working with computers (1–5)
Seven days after the treatment with 0.2% benzalkonium chloride (BAC), obvious epithelial punctate defects were detected using a fluorescence sodium dye in DED mice compared with the control group (Figures 1A–C)
The results showed that cholinergic receptor nicotinic beta 2 (Chrnb2) and autophagy target factor (gamma-aminobutyric acid (GABA) a receptor-associated protein-like 2, Gabarapl2) were significantly increased following DED compared with the control group (p < 0.01) (Figure 3E)
Summary
Hundreds of millions of people throughout the world are affected by dry eye disease (DED), typically suffering symptoms such as blurred vision, ocular discomfort, and a stinging, burning, scratchy, or gritty sensation while reading, driving, or working with computers (1–5). (6), severe symptoms of DED are associated with decreased work productivity and levels of activity (7). Dry eye is a multifactorial disease of the ocular surface characterized by a loss of homeostasis of the tear film and accompanied by ocular symptoms, in which tear film instability and hyperosmolarity, ocular surface inflammation and damage, and neurosensory abnormalities play etiological roles (5). Numerous cellular and molecular components have been found to contribute to immune-cell activation and associated inflammation in the pathogenesis of dry eye, including inflammatory cytokines, metalloproteinases, and chemokines and their receptors (8–11). Considerable attention is being paid to the role and mechanisms of ocular-surface nerve inflammation in DED pain sensitivity (13). Neuropathic pain is difficult to cure: all existing therapies, such as serotonin– noradrenaline reuptake inhibitors, anticonvulsants acting at calcium channels, and topical lidocaine and opioids, have only variable success in alleviating pain, while failing to tackle the underlying mechanisms (14, 15)
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