| Summary: | <i>Background and Objectives:</i> Atropine is a nonselective muscarinic antagonist which has been used to prevent worsening of myopia in children. Different concentrations of atropine were used for myopia, ranging from 0.01% to 1.0%. However, there are still potential toxicity of different doses of atropine to the cornea. Here, we present a study of investigating novel genes potentially involved in the effects of very low dose atropine treatment (0.003%) on corneal epithelial cells using next-generation sequencing (NGS) and bioinformatics approaches. <i>Materials and Methods:</i> Human corneal epithelial cells were treated with 0.003% atropine, cultured until confluence, and RNA extracted for differential expression profiling of mRNA and microRNA (miRNA) between control and atropine-treated corneal epithelial cells. The functional enrichment analysis for differentially expressed genes was performed using two bioinformatics databases, including Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity<sup>®</sup> Pathway Analysis (IPA). In addition, potential miRNA-mRNA interactions involved in atropine-treated corneal epithelial cells were predicted and validated using different miRNA target prediction databases. <i>Results:</i> Our results showed 0.003% atropine might suppress the apoptosis of corneal epithelial cells, potentially through Ras and protein kinase A signaling pathways. We also validated the possible miRNA regulations by using TargetScan and miRDB databases. Hsa-miR-651-3p-<i>EPHA7</i>, hsa-miR-3148-<i>TMEM108</i> and hsa-miR-874-5p-<i>TBX6</i> were validated as possible miRNA regulations involved in corneal epithelial cells treated with 0.003% atropine. <i>Conclusions:</i> These findings may contribute novel insights into therapeutic strategies for treating cornea with 0.003% atropine.
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