Histological and microRNA Signatures of Corneal Epithelium in Keratoconus

PURPOSE: To illustrate the histopathology of kerato-conic corneal epithelia and its micro-ribonucleic acid (miRNA) regulation. METHODS: Corneal epithelia were collected from 27 patients with keratoconus and 26 normal patients after surgery or by impression cytology. The miRNA profile was determined using miRNA microarray. The biological roles of miRNA target genes were delineated by gene ontology and pathway analyses. The expressions of significant miRNAs were validated using TaqMan polymerase chain reaction (PCR), whereas protein localization and expression of the miRNA target genes were examined by immunofluorescence and immunoblotting analyses. RESULTS: Histological assessment showed that corneal epithelia in patients with keratoconus were thinner with loosely packed cells compared to normal patients. Microarray analysis revealed that 12 miRNAs were significantly downregulated in keratoconic corneal epithelia. Gene ontology analysis demonstrated that the predicted miRNA target genes participated in cell junction, cell division, and motor activity, whereas pathway analysis highlighted the involvement of syndecan-mediated signaling pathway. TaqMan PCR validated the altered expression of six miRNAs in corneal epithelia from surgery (hsa-miR-151a-3p, hsa-miR-138-5p, hsa-miR-146b-5p, hsa-miR-194-5p, hsa-miR-28-5p, and hsa-miR-181a-2-3p) and four miRNAs in squamous corneal epithelial samples collected from impression cytology (hsa-miR-151a-3p, hsa-miR-195-5p, hsa-miR-185-5p, and hsa-miR-194-5p). In addition, higher S100A2 expression was found in the epithelial basal cell layer of keratoconic corneal epithelia. CONCLUSIONS: The miRNA and histological analyses in this study demonstrated structural and biological changes in keratoconic corneal epithelia, broadening the understanding of keratoconus pathology. In addition, impression cytology is useful to collect corneal epithelial tissues for gene expression analysis.