YTHDF1-Mediated m6A Modification of NREP Promotes Corneal Fibrosis via TGF-β-Smad Signaling

Corneal fibrosis ranks among the foremost drivers of global vision loss and blindness, yet therapeutic options remain scarce. This study investigated neuronal regeneration-related protein (NREP), which is recognized by the N6-methyladenosine (m6A) modification reader protein YTH domain family member 1 (YTHDF1), in promoting corneal fibrosis in keratocytes.

In vivo, a standardized alkali burn model was created in C57BL/6 mice with corneas harvested on days 7, 14, and 21. Fibrosis, NREP, and m6A modifications were estimated using slit-lamp, real-time quantitative polymerase chain reaction (RT-qPCR), and western blotting. In vitro, isolated keratocytes were treated with TGF-β1. Small interfering RNA (si-RNA) was used to downregulate NREP and YTHDF1 expression in keratocytes. SKLB-Y13 was used to specifically inhibit YTHDF1. Cell proliferation, migration, and alpha-smooth muscle actin (α-SMA) expression were assessed using CCK-8 assays, scratch-wound assays, flow cytometry, immunofluorescence, and liquid chromatography-mass spectrometry. Following keratocyte fibrosis induction using 10% fetal bovine serum, the indicators were evaluated.

Corneal fibrosis peaked on day 14 post-injury, coinciding with increased NREP protein levels versus controls. NREP knockdown reduced keratocyte proliferation and migration and abolished TGF-β1-induced α-SMA expression. YTHDF1 depletion phenocopied these effects and lowered NREP protein levels, suggesting translational control. Sequence-based RNA adenosine methylation site predictor (SRAMP) revealed enriched m6A modifications in NREP mRNA. YTHDF1 deficiency reduced NREP translation and impaired TGF-β-Smad signaling, leading to decreased keratocyte proliferation and motility and impaired fibroblast-to-myofibroblast differentiation.

YTHDF1 promotes corneal fibrosis through m6A-dependent enhancement of NREP translation, potentiating TGF-β-Smad and myofibroblast transdifferentiation, and thus may serve as a therapeutic target for fibrotic corneal disorders.