Corneal diseases and injuries leading to visual impairment have significant impact on the quality of life of patients and constitute a major problem for health care system. Corneal diseases are often manifested through limbal epithelial stem cell (LESC) dysfunction and loss, leading to corneal opacity, visual impairment, and blindness. LESC located in the limbal niche are the source of constant corneal epithelial renewal and are required to maintain corneal epithelial homeostasis by adapting properly to internal and external stimuli. However, persistent exposure to pathological stress disrupts the adaptive mechanisms of LESC, leading to serious corneal problems such as altered wound healing, limbal stem cell deficiency (LSCD) and diabetic keratopathy. Therefore, understanding the precise spatial and temporal regulatory pathways and how each LESC/limbal epithelial cells (LECs) responds to various stimuli at the single-cell level governing corneal epithelial regeneration could be key to elucidating the pathogenesis of various corneal diseases and ultimately developing new and effective treatment strategies. Previously, we have shown the important regulatory role of miR-146a in corneal epithelial homeostasis and wound healing via its different targeted pathways such as EGFR, Notch and NF-κB signaling. However, due to the heterogeneity of limbal basal cells and their proximity to limbal stromal cells, it remains to be determined how miRNAs (miRs) regulate maintenance, differentiation, and the active stage of limbal progenitor cells in various corneal cell populations in wounded and unwounded normal and diseased corneas. Recently several studies have shown the heterogeneity and complexity of limbal niche in relation to regeneration and wound healing and identified new putative LESC markers using newly emerged empowering scRNA-seq technology. We propose to investigate the regulatory role and contribution of each miR-146 main targets (EGFR, TRAF6, IRAK1, NUMB and NOTCH-2) in corneal epithelial regeneration in wounded and unwounded states in normal and diabetic corneas using scRNA-seq, proteomics, loss and gain of functional studies. We hypothesize that miR-146a contribute to the molecular regulation of cellular heterogeneity in the limbal niche steady state. We predict that differential miR-146a expression in limbus vs. central corneal epithelium impacts its regulation of limbal niche cells, LECs and limbal stromal cells, and mediates corneal regeneration and wound healing in normal and diseased states. Our Specific Aims are: Specific Aim 1: To identify and characterize downstream targets including EGFR, Notch and inflammatory mediators of miR-146a in distinct populations of heterogeneous limbal stem cells that contribute to homeostatic maintenance and regeneration in human normal unwounded and wounded cornea. Specific Aim 2: To reveal the differential and converged regulatory role of EGFR, inflammatory, and Notch signaling by miR-146a in corneal epithe...