Role of m⁶A methyltransferase METTL3 in keratoconus pathogenesis

Keratoconus (KC) is the most common ectatic corneal disease with unknown pathogenesis. This study aimed to investigate the role of methyltransferase-like enzyme 3 (METTL3) in KC pathogenesis. In the present study, we examined the levels of METTL3 and other N6-methyladenosine (m⁶A) modification-related proteins in KC samples and human stromal keratocyte (HTK) cells stimulated by mechanical stretch (MS) using Western blotting and immunohistochemistry. The level of m⁶A RNA methylation was quantified using the m⁶A RNA methylation assay kit. Genetic (Mettl3 knockdown mice) and pharmacological (STM2457) approaches were employed to investigate the effect of METTL3 on the expression of metalloproteinases (MMPs) in MS-treated corneal stromal cells (CSCs) via Western blotting and real-time polymerase chain reaction. Moreover, YAP signaling activity was assessed to explore the relationship between METTL3 and MMPs in MS-treated CSCs. Increased expression of METTL3 and decreased expression of METTL14, WTAP, and YTHDF2 were detected in KC samples and MS-stimulated HTK cells. Correspondingly, the m⁶A levels in KC specimens and MS-stimulated CSCs were significantly higher than those in controls but were significantly reduced when METTL3 activity was genetically and pharmacologically blocked. Inhibition of METTL3 significantly reduced the expression of MMP1 and MMP3 in mechanically stretched CSCs and reduced YAP activity. Furthermore, pharmacologically inhibiting YAP signaling in MS-stimulated HTK cells significantly reduced MMP1 and MMP3 expression. Our findings highlight the pathogenic role of METTL3 in KC. Further investigation is required to investigate the underlying mechanism.