Refractive error stands as a primary cause of reversible visual impairment. In recent decades, myopia has emerged as a global health issue of great concern (Dolgin, 2015; Kim et al., 2019). Projections indicate that by 2050, nearly half of the global population will be affected by myopia (Holden et al., 2016). With the improvement in the quality of life, an increasing number of patients are turning to surgical correction, such as photorefractive keratectomy (PRK), laser in situ keratomileusis (LASIK), and small incision lenticule extraction (SMILE) (Nair et al., 2023) for refractive errors. Although these surgeries are generally successful, they are accompanied by complications. A particularly notable concern is corneal epithelial hyperplasia (Chen et al., 2015; Kanellopoulos and Asimellis, 2014; Luft et al., 2016). Corneal epithelial hyperplasia can give rise to refractive regression, thereby undermining the long-term success of the surgery (Kang and Kim, 2019; Yan et al., 2018).

Fluorometholone (FML, C22H29FO4), a synthetic glucocorticoid, is widely prescribed for the management of corneal epithelial hyperplasia following refractive surgery. Previous studies have demonstrated that FML can reduce corneal epithelial thickening and prevent refractive regression (Li et al., 2022). However, despite its established efficacy, not all patients respond adequately to FML treatment, indicating that its effectiveness is not universal. This phenomenon arises from two primary factors: the heterogeneity in corneal epithelial wound healing pathways, and pharmacogenomic variations affecting FML metabolism. Corneal epithelial wound healing represents a highly dynamic and personalized biological process governed by intricate molecular networks. Central to this process is the epidermal growth factor receptor (EGFR) signaling axis, which orchestrates epithelial cell migration, proliferation, and differentiation during wound repair (Ljubimov and Saghizadeh, 2015). Functional single-nucleotide polymorphisms (SNPs) within the EGF and EGFR genes have been associated with altered cancer treatment responses (Marinović et al., 2022), suggesting potential impacts on wound healing outcomes through modulation of receptor-ligand interactions. As a synthetic glucocorticoid (GC), FML's pharmacokinetic profile is significantly influenced by genetic variations in drug-metabolizing enzymes. Specifically, polymorphisms in the CYP3A5 gene encoding cytochrome P450 enzyme have been linked to differential glucocorticoid metabolism, contributing to variable therapeutic responses in asthma (Nkoy et al., 2024). Additionally, genetic variants in the glucocorticoid receptor gene (NR3C1) modulate receptor-ligand binding affinity and downstream transcriptional activity, thereby affecting treatment outcomes in childhood acute lymphoblastic leukemia (Xue et al., 2015; Gasic et al., 2018). To address this therapeutic inconsistency, mechanistic studies are needed to characterize FML's effects on corneal epithelial cells (CECs). Identification of differentially expressed genes and regulatory pathways will provide critical insights into treatment resistance mechanisms. Identifying its target genes is crucial for elucidating the potential reasons for its therapeutic failure in certain individuals.

Rho guanosine triphosphatases (GTPases) belong to a family of small GTPases within the broader Ras GTPase superfamily. These proteins govern crucial cellular processes, including cytoskeletal dynamics, cell morphology, motility, the cell cycle, and cellular differentiation (Crosas-Molist et al., 2022). In the human genome, 20 distinct Rho GTPases are encoded. Among them, RhoA, Rac1, and Cdc42 have been the most intensively investigated. These three prototypical Rho GTPases play a central part in regulating cell migration and proliferation in diseases such as cancer, cardiovascular disorders, and neurological conditions through their impact on cytoskeletal reorganization and intracellular signaling pathways (Svensmark and Brakebusch, 2019; Voena and Chiarle, 2019; Kalpachidou et al., 2019; Dandamudi et al., 2023). Prior research has demonstrated that knockdown of RhoA impairs CEC proliferation and migration, disrupting actin dynamics and the integrity of focal adhesions, suggesting its essential role in actin cytoskeleton remodeling (Yin et al., 2008). Rac1 signaling, which is involved in regulating fibronectin (a key extracellular matrix protein) influences cell adhesion and motility, both of which are essential for corneal epithelial repair (Kimura et al., 2006). Simultaneously, Cdc42 has been shown to promote cell migration and polarization, thereby facilitating corneal epithelial wound healing (Hou et al., 2013; Pothula et al., 2013). Studies have revealed that GCs target distinct Rho GTPases, thereby resulting in different therapeutic effects. In mammary epithelial tumor cells, GCs regulate the formation of the junctional complex and tight junctions by down-regulating RhoA (Rubenstein et al., 2003). During the treatment of nephrotic syndrome, GC treatment decreases the activity of the pro-migratory small GTPase regulator Rac1, consequently reducing podocyte motility (McCaffrey et al., 2017). Additionally, in human ovarian cancer cells, GCs induce the expression of RhoB (a member of the Rho GTPases family, which has been implicated as a negative regulator of cell proliferation), while having no impact on RhoA and RhoC (Chen et al., 2006).

In this study, our findings unequivocally demonstrate that FML treatment significantly inhibits the proliferation, migration, and wound healing capacity of human corneal epithelial cells (HCECs). At the molecular level, FML treatment induced down-regulation of RhoA, Rac1, and Cdc42. Concurrently, corresponding decreases in the activation of the crucial signaling pathways, namely the Erk and NF-κB pathways, were observed in both HCECs and the corneal epithelia of mice. Significantly, these molecular alterations were also detected in tear samples from clinical patients undergoing FML treatment. Furthermore, inhibition of Rac1 and Cdc42 attenuated the inhibitory effect of FML on CECs. These results suggest that FML inhibits the proliferation and migration of CECs by modulating the RhoA/Rac1/Cdc42, thereby suppressing the Erk/NF-κB pathways.