Abstract
Purpose: This work aimed to identify differentially expressed circular RNAs (circRNAs) and elucidate their potential function in aquaporin 5 (AQP5) knockout (AQP5–/–) mice with the primary dry eye phenotype.Methods: A slit lamp examination was performed on AQP5–/– mice to assess corneal epithelial defects using fluorescein sodium staining. Hematoxylin–eosin staining and transmission electron microscopy analysis were performed to identify structural changes in lacrimal gland epithelial cells due to AQP5 deficiency. The expression profiles of circRNA and messenger RNA (mRNA) were determined by a microarray analysis. The selected circRNA was verified by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to predict the biological functions and the potential pathways of parental genes involved in lacrimal gland epithelial cell changes. According to the bioinformatics analysis of identified circRNAs, we predicted a circRNA–miRNA–mRNA network of phagosomes.Results: The AQP5–/– mice spontaneously exhibit dry eye symptoms, wherein the AQP5 deficiency changes the structure of lacrimal gland epithelial cells. The analysis revealed that, compared to AQP5+/+ mice, 30 circRNAs in the lacrimal glands of AQP5–/– mice were differentially expressed (fold change ≥ 2.0, p < 0.05). Nine upregulated circRNAs were identified using qRT-PCR, and nine upregulated validated circRNAs, 40 altered microRNAs (miRNAs), and nine upregulated mRNAs were identified through a network analysis. The KEGG analysis showed that these nine target genes were expressed in phagosomes.Conclusion: The AQP5–/– mice have primary and stable dry eye phenotypes from birth. We identified differently expressed circRNAs in the lacrimal glands of AQP5–/– and AQP5+/+ mice, predicting a circRNA–miRNA–mRNA network of phagosomes. CircRNA likely plays an important role in lacrimal gland epithelial cell pathogenesis. Therefore, it is reasonable to use circRNA as a potential therapeutic agent for the treatment of dry eyes.
Highlights
Dry eye is currently the most common eye disease except for ametropia, with a worldwide incidence of 5–34% (Messmer, 2015)
The miRNA binding sites and target messenger RNA (mRNA) were predicted using proprietary software based on TargetScan and MiRanda
We found that Aquaporin 5 (AQP5)−/− mice naturally develop dry eye symptoms from birth, which makes them a stable, simple, and effective dry eye research animal model that can be used to study the pathogenesis of this condition
Summary
Dry eye is currently the most common eye disease except for ametropia, with a worldwide incidence of 5–34% (Messmer, 2015). The incidence of dry eye is still rising, emphasizing the urgent need to determine its pathogenesis and to develop effective treatment measures. Aquaporin 5 (AQP5), a kind of aquaporin, is highly expressed in the corneal epithelium and lacrimal glands and is closely related to eye diseases (Raina et al, 1995; Takata et al, 2004). AQP5 has been shown to reduce saliva and airway mucus secretions and increase corneal thickness in experimental animals (Ma et al, 1999; Thiagarajah and Verkman, 2002). Lack of AQP5 affects the migration and the proliferation of cells, leading to slow corneal healing (Kumari et al, 2018). Whether AQP5 deficiency can cause dry eye has not been proved yet
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