mRNA and microRNA Expression Profile of Corneal and Conjunctival Impression Cytology Samples

Purpose: To characterize the messenger RNA (mRNA) and microRNA (miRNA) expression profiles of the normal human cornea and conjunctiva using impression cytology (IC) samples and to investigate their molecular characteristics and regulatory networks. Methods: Corneal and conjunctival IC samples were collected from healthy subjects. Whole-transcriptome and miRNA sequencing were performed, followed by differential expression and bioinformatics analyses. Regulatory networks, protein interaction networks, and functional enrichment analyses were constructed. Selected genes and miRNAs were validated by RT-qPCR. Results: A total of 1676 differentially expressed genes and 175 differentially expressed miRNAs were identified between the cornea and conjunctiva. Functional analyses revealed that genes showing higher expression in the cornea were mainly associated with epithelial structure, barrier function, and antiviral immune responses. In contrast, genes showing higher expression in the conjunctiva were primarily involved in immune regulation, secretion, metabolic detoxification, and tissue remodeling. PPI network analysis showed that hub genes in the cornea were predominantly interferon-stimulated genes related to antiviral responses, while those in the conjunctiva were mainly involved in cell cycle regulation and metabolic detoxification. GO and KEGG analyses further supported these functional distinctions. RT-qPCR validation generally supported the expression patterns identified by RNA sequencing. Conclusions: Our findings reveal that the cornea is characterized by gene expression programs supporting epithelial homeostasis, barrier function, and antiviral immunity, whereas the conjunctiva exhibits transcriptional signatures related to immune surveillance, secretion, metabolic processing, and tissue remodeling. The miRNA–mRNA regulatory networks constructed in this study provide new insights into the molecular regulatory mechanisms of the ocular surface and offer a theoretical basis for future research into disease mechanisms and targeted therapeutic strategies.