U1 RNA Detected by Toll-Like Receptor 3 Plays a Role in the Pathogenesis of Pterygium

Toll-like receptor 3 (TLR3) detects RNA from pterygium epithelial cells (PECs). We previously suggested that pterygium development may be linked to RNA released from abnormally growing PECs: U1 RNA released from ultraviolet B (UVB)-damaged cells may activate TLR3. This study investigated how U1 RNA, polyriboinosinic:polyribocytidylic acid (poly[I:C]), and PEC lysates affect the TLR3 signaling pathway in PECs and conjunctival epithelial cells (CECs).

Human pterygium and ipsilateral pterygium-free conjunctiva from the same patients were used for cell culture and RNA-sequencing analysis. PECs and CECs were cultured, irradiated with UVB, and treated with poly(I:C), PEC lysates, or synthetic U1 RNA. TLR3 and toll/interleukin-1 receptor domain-containing adaptor-inducing interferon-β (TRIF) expression, phosphorylated nuclear factor-kappa B (NF-κB)/NF-κB ratio, IL-6, and IL-8 were evaluated using western blot, quantitative real-time PCR (qPCR), and enzyme-linked immunosorbent assay (ELISA). Cell proliferation was evaluated using water-soluble tetrazolium salt-1 assay.

After UVB irradiation, U1, U2, U4, and U6 RNA increased in PECs and CECs, and TLR3 expression increased in PECs. Western blot and qPCR results indicated an increase in TLR3, TRIF, and NF-κB expression in PECs and CECs treated with poly(I:C), UVB-irradiated PEC lysates, or synthetic U1 RNA compared to controls. However, RNase A inhibited this effect in UVB-irradiated PECs. ELISA showed that IL-6 and IL-8 increased in cell groups treated with poly(I:C), UVB-irradiated PEC lysates, or synthetic U1 RNA. Proliferation of PECs was also increased by poly(I:C).

Several small noncoding RNAs, whose expression was induced by UVB irradiation, may be a possible novel therapeutic target for pterygium treatment through activation of the TLR3 signaling pathway.