RNA m⁵C Modification Modulates Corneal Epithelial Wound Healing

Abstract

Purpose : Corneal epithelial wound healing (CEWH) is essential for restoring corneal integrity and transparency after injury. Post-transcriptional regulations are known to affect this process through controlling gene expression levels. RNA 5-methylcytosine (m⁵C) modification, catalyzed by NOP2/Sun RNA methyltransferase 2 (NSUN2), is one possible regulatory mechanism since it is involved in many biological processes. To address if m⁵C modification actually affects CEWH, we determined here the role of NSUN2 in modulating this process.

Methods : Dot blots determined m⁵C levels during mouse CEWH. Quantitative RT-PCR and Western blot analysis examined the NSUN2 gene and protein expression levels. MTS, flow cytometry, and a scratch wound assay evaluated the effects of NSUN2 knockdown on human corneal epithelial cell (HCEC) proliferation and migration, respectively. An in vivo murine corneal epithelial debridement model evaluated the effects of NSUN2 knockdown on wound healing rate by monitoring mouse corneal fluorescein staining patterns.

Results : The expression level of NSUN2 and the m⁵C modification level were significantly increased during mouse CEWH. In contrast, knockdown of NSUN2 led to a significant decrease of m⁵C level in HCECs. Furthermore, downregulation of NSUN2 significantly inhibited HCEC proliferation and migration. In addition, NSUN2 knockdown markedly delayed in vivo CEWH.

Conclusions : Our study reveals a crucial role of m⁵C RNA modification in regulating CEWH. Therefore, m⁵C can serve as a novel epitranscriptomic marker in this process.

This is a 2020 ARVO Annual Meeting abstract.