LncRNA DANCR Regulates PARP1-Mediated Oxidative Stress Response in Corneas by Encoding a Novel Micropeptide miPEPDR

Purpose: Oxidative stress, as a pathogenic factor of multiple corneal diseases, regularly threatens the genome stability in corneal cells. However, the potential significance of long noncoding RNAs (lncRNAs) in this process remains largely unknown. This study aims to elucidate the role and mechanisms of lncRNAs in the oxidative stress response of human corneal epithelial (HCE) cells. Methods: Published RNA sequencing data of human normal cornea (HNC) and keratoconus (HKC) samples were used to investigate differentially expressed lncRNAs, which were further validated in independent clinical corneal epithelial samples. Hydrogen peroxide (H2O2)-induced oxidative stress was used to evaluate the effect of candidate lncRNAs on the cell viability and genome stability through the cell counting kit-8 (CCK-8), micronucleus formation, and alkaline comet assay. LncRNA-encoded micropeptides were investigated through epitope tagging, frameshift mutagenesis, custom antibody, and mass spectrum-based detection. Comprehensive analyses, including immunofluorescence, immunoblotting, and quantitative real-time PCR were used to assess DNA damage repair, protein, and mRNA expression, respectively. Results: LncRNA differentiating antagonistic non-protein-coding RNA (DANCR) was found to be downregulated in keratoconus and to confer resistance to oxidative stress in HCE cells. Knockdown of DANCR reduced oxidative stress-induced DNA damage, as evidenced by decreased micronucleus formation rate and DNA strand breaks. Mechanistically, downregulation of DANCR not only inhibits poly(ADP-ribosyl)ation (PARylation) but also promotes the nucleolar-nucleoplasmic shuttling of poly(ADP-ribose) polymerase 1 (PARP1), both of which could be rescued by miPEPDR, a previously unrecognized 56-amino acid micropeptide encoded by DANCR. Conclusions: LncRNA DANCR functions as a critical regulator of corneal epithelial oxidative stress response through modulating PARP1 activity and nucleolar-nucleoplasmic translocation via its derived micropeptide miPEPDR.