ABSTRACT: Background: Damage due to ultraviolet (UV) light from the sun is implicated in the development of two proliferative lesions of the ocular surface: pterygium and pinguecula. In this study, RNA-sequencing technology was used to investigate underlying molecular mechanisms. Methods: Pterygium and pinguecula specimens were collected at the time of surgical removal, along with adjacent specimens of healthy conjunctiva. RNA was extracted and sequenced. Data from individual samples were grouped as 1) healthy conjunctiva, 2) pterygia-highly exposed to UV (pterygia-E), 3) pterygia-normally exposed (pterygia-NE) and 4) pinguecula. Pairwise comparison was made of differentially expressed genes (DEGs). Randomly selected DEGs were validated in independent tissue specimens by RT-PCR. Gene sets were further analyzed by Ingenuity Pathway Analysis (IPA), and by assembly of cell type, tissue differentiation and pathological process markers. Results: Transcripts from 18,630 genes were identified. Pterygium exhibited 69% similarity, and pinguecula exhibited 73% similarity to healthy conjunctiva. Pinguecula and pterygia exhibited 95% similarity to each other and pterygia-E and pterygia-NE exhibited 98% similarity to each other. Among the most interesting DEGs were four tumor suppressor genes downregulated in pterygia-E and pterygia-NE: C10orf90, RARRES1, DMBT1, and SCGB3A1. C10orf90 and RARRES1 were also downregulated in pinguecula. Comparison of pterygia-E and pterygia-NE uncovered evidence of genomic instability due to failure to repair DNA damage, and associated with inflammation and the immune response; these changes were also observed in pinguecula, but to a lesser extent, and are consistent with atypia and dysplasia documented pathologically. IPA overwhelmingly linked DEGs to cancer and predicted activation of 12 different upstream regulators known to mediate epithelial cell proliferation. Cell type markers were present for all epithelial and stroma cell types quiescent stem cells of both epithelia and stroma were lacking. Cell-type and differentiation marker expression indicated well-differentiated epithelial components of mixed conjunctival, limbal, and corneal cell-types with mucosal differentiation. Upregulation of MUC6 in all three lesions suggested the presence of a glandular cell type. In all three lesions, genes characteristic of palmoplantar epidermis: KRT9 and TRPV3, were upregulated, suggesting transdifferentiation due to stress. Markers of cornification characteristic of inflammation were also upregulated. Matrix metalloproteinase expression was only slightly changed, suggesting the lesions were not invasive, however fibrovascular proliferation was evidenced. Conclusions: A hallmark of cancer, genomic instability begins in the precancerous stage, and can be caused by failure to repair DNA damage due to UV light exposure. This can be exacerbated by inflammation due to products of DNA damage. Our findings suggest that pinguecula and pterygia, while benign lesions, are both on the pathological path towards cancer.
