Silencing METTL3 Increases HSP70 Expression and Alleviates Fibrosis in Keratocytes

To explore the potential role of N6-methyladenosine (m6A) and its regulatory factors in corneal fibrosis response using both in vivo and in vitro models.

This study utilized the C57BL/6 mouse corneal alkali burn as an in vivo model and stimulated keratocytes with transforming growth factor beta 1 (TGF-β1) in vitro. Small interfering RNA (siRNA) was employed to downregulate the expression of YTH domain family member 2 (YTHDF2), methyltransferase-like 3 (METTL3), and fat mass and obesity-associated protein (FTO) in keratocytes. The expression of relevant genes was quantified by real-time quantitative reverse-transcription PCR (qRT-PCR), western blotting, and immunohistochemistry.

After an alkali burn, m6A modification in corneas increased, with the most notable increase observed on the fourth day after the injury. The levels of METTL3 and FTO initially decreased and then increased. After 21 days following an alkali burn, the corneal fibrosis was most significant. The levels of METTL3 and FTO were elevated. There were higher levels in m6A modification and the expression of METTL3 and FTO in keratocytes stimulated by TGF-β1. In corneas after alkali burns and in keratocytes stimulated by TGF-β1, the expression of heat shock protein 70 (HSP70) was negatively correlated with fibrotic response markers. Silencing METTL3 and YTHDF2 in keratocytes increased HSP70 expression and reduced the expression of fibrosis-related indicators in keratocytes stimulated by TGF-β1. However, silencing FTO did not significantly affect the expression of HSP70 and fibrosis.

These findings indicate that METTL3 is involved in the modulation of corneal fibrosis through the regulation of HSP70 expression in a manner that is dependent on YTHDF2.