Epigenetic regulation of leucine rich repeat containing G protein coupled receptor 5 (LGR5) in corneal endothelial cells

Abstract

Human corneal endothelial cells (hCECs) are vital for maintaining corneal transparency. However, their limited capacity for proliferation can result in vision loss, requiring corneal transplantation. The Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) plays a critical role in maintaining various fetal and adult stem cell types by promoting the Wnt/β-catenin signaling. Although LGR5 is present in corneal endothelial progenitors, its expression decreases as these cells mature, coinciding with the loss of replicative properties. In this study, we explore strategies to reactivate LGR5 expression in cultured hCECs through epigenetic modulation. We found low levels of DNA methylation and hydroxymethylation at LGR5 promoter in cultured hCECs. Our findings reveal that high-dose HDAC inhibitors, trichostatin A, and valproic acid enhanced LGR5 expression in cultured hCECs. In addition, combining low-dose valproic acid with small molecules including Wnt3A, R-Spondin1, and BMP inhibitors also promoted LGR5 expression in these cells. Furthermore, we generated CRISPRa targeting at LGR5 promoter which could activate LGR5 expression in HEK293 cells, especially with multiple sgRNAs. Applying this method to cultured hCECs can also specifically enhance LGR5 expression. Our results suggest that epigenome modification is a viable strategy for promoting corneal endothelium regeneration.