关键词:  , 过敏性结膜炎；可变剪接事件；CDKN1A 相互作用锌指蛋白1；紧密连接；生物标志物

Abstract: Objective Analyse the expression changes of the CDKN1A-interacting zinc finger protein 1 gene (CIZ1) in the serum of patients with allergic conjunctivitis, clarify its alternative splicing patterns, and explore its correlation with clinical indicators. Design Case-control study. Participants Thirty-two patients with active allergic conjunctivitis were recruited from the Ophthalmology Department of Inner Mongolia Baotou Second Affiliated Hospital from July 2020 to December 2020, along with 32 age- and gender-matched healthy volunteers. Methods The transcriptome of serum RNA from healthy volunteers and patients with allergic conjunctivitis was sequenced. Alternative splicing events were identified and quantified using rMATS. The mRNA levels of CIZ1 in clinical samples were quantified by real-time fluorescence quantitative PCR (qPCR), and serum total immunoglobulin E (IgE) levels were measured by ELISA. Human conjunctival epithelial cells (HConEpiCs) were cultured in vitro, and treated with IL-4 and IL-13 for 24 hours, and transfected with CIZ1 siRNA to knockdown its expression. The expression changes of CIZ1, occludin, and claudin-1 were detected by qPCR and Western blot. Main Outcome Measures CIZ1 alternative splicing patterns, serum CIZ1 mRNA levels, serum IgE levels, total ocular symptom score (TOSS) values, and tight junction protein expression levels at the cellular level. Results There were 262 differentially expressed long non-coding RNAs and 1425 differentially expressed mRNAs. A total of 128 genes had significant differential splicing events. CIZ1 exhibited mutually exclusive exons (MXE) alternative splicing events. The total serum CIZ1 mRNA level in patients with allergic conjunctivitis during the acute phase was significantly higher than that in the healthy control group (P<0.0001). Correlation analysis revealed significant positive correlations between serum CIZ1 levels and both total IgE (r=0.7907, P<0.0001) and TOSS scores (r=0.6955, P<0.0001). The serum total mRNA level of patients with TOSS score > 5 was significantly higher than that of patients with TOSS score ≤5 (P<0.001). In vitro experiments showed that IL-4 and IL-13 treatment significantly upregulated CIZ1 expression in HConEpiCs cells and concomitantly downregulated occludin and claudin-1 expression; furthermore, CIZ1 knock down reversed the downregulation of tight junction proteins induced by IL-4 and IL-13. Conclusion In the serum of patients with allergic conjunctivitis, CIZ1 undergoes aberrant MXE alternative splicing. Its expression is significantly upregulated in patient serum and correlates positively with disease severity. CIZ1 may disrupt ocular surface epithelial barrier function by downregulating tight junction proteins.

Key words: Allergic conjunctivitis, Alternative splicing events, CIZ1, Tight junction, Biomarker