Tear MicroRNA-Based Molecular Signature for Keratoconus Stratification: A Cross-Cohort Integration Study

Keratoconus (KC) is a progressive corneal ectatic disorder characterized by structural remodeling and immune-associated molecular alterations. However, whether tear-derived microRNAs reflect corneal molecular changes remains unclear. We analyzed three publicly available corneal transcriptomic datasets (N = 112) to identify immune-associated gene expression patterns using differential expression analysis and Gene Set Enrichment Analysis. In a prospective tear cohort (N = 20), tear miRNA profiling was performed, and candidate miRNAs were selected using LASSO regression. miRNA patterns were integrated with immune-related transcriptomic alterations, and an exploratory molecular-structural index was constructed using standardized hsa-miR-326 expression and keratometry (K2). We identified immune-associated transcriptional alterations in KC corneas and derived a five-miRNA tear signature. The signature showed discriminatory performance in an independent corneal epithelial miRNA dataset (AUC = 0.89, 95% CI: 0.69-1.00; N = 16) and was further evaluated in an additional tear cohort using RT-qPCR (N = 52). Among these candidates, hsa-miR-326 showed consistent downregulation across datasets. Network analysis identified associations between candidate miRNAs and immune-related genes, including IL1B, CD4, CXCL10, and TGFB1. The exploratory molecular-structural index demonstrated an inverse association with keratometric steepening (Spearman ρ = -0.86, p < 0.001). These findings suggest that tear-derived miRNAs may provide complementary molecular information associated with immune-related corneal alterations and warrant further investigation in KC characterization.