Single-cell RNA-seq analysis revealed that BHLHE40 and its binding protein THBS1 are involved in diabetic corneal epitheliopathy.

BACKGROUND: Diabetic corneal epitheliopathy is a significant ocular complication of diabetes whose pathogenic mechanisms remain incompletely understood. This study aimed to elucidate the cellular and molecular mechanisms underlying diabetic corneal epitheliopathy and to identify potential therapeutic targets using single-cell RNA sequencing (scRNA-seq). METHODS: Corneal epithelial tissues were processed into single cells and clustered based on marker gene expression. Integrated bioinformatic analyses were performed, including pseudotime trajectory, cell cycle, gene set enrichment, hdWGCNA, and cell communication analyses. This approach identified BHLHE40 and its interacting partner THBS1 as key candidate genes. Their functional significance was confirmed through knockdown experiments in high glucose-induced human corneal epithelial cells, supporting their potential role in diabetic corneal epitheliopathy pathogenesis. RESULTS: We analyzed 40,976 single cells from human corneal epithelium, classifying them into seven distinct clusters. The most pronounced differences were observed in the basal cells of the limbal stem cell niche between diabetic and control groups. Diabetic epithelia exhibited upregulation of genes associated with glycolysis, circadian rhythm, and hypoxia, alongside downregulation of genes involved in corneal differentiation and oxidative phosphorylation. Knockdown of BHLHE40 reversed high glucose-induced oxidative stress and concurrently reduced THBS1 expression, while knockdown of THBS1 itself lowered the expression of inflammatory factors. Direct binding between BHLHE40 and THBS1 was confirmed by CUT&Tag-qPCR analysis. CONCLUSION: Our findings demonstrate that BHLHE40 and THBS1 are implicated in the pathogenesis of diabetic corneal epitheliopathy, highlighting a promising novel target for therapeutic intervention.