MicroRNA-204-5p-Mediated Regulation of SIRT1 Contributes to the Delay of Epithelial Cell Cycle Traversal in Diabetic Corneas

PURPOSE. We investigated how the microRNA (miRNA) modifies the expression of silent mating type information regulation 2 homolog 1 (SIRT1) in diabetic corneas. METHODS. The bioinformatic assay was used to predict which miRNAs might regulate the expression of SIRT1. A lipid transfection protocol was used to upregulate or knockdown the miRNA expression in TKE2 cells. Adenovirus-expressing short interfering RNA was used to knockdown the expression of SIRT1 in TKE2 cells and Ins2(Akita/+) mice were used to evaluate how miRNA promotes diabetic corneal epithelial wound healing. Cell cycle status was determined by flow cytometry assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to analyze the cell viability. RESULTS. Nine miRNAs were selected for quantitative PCR (qPCR) detection after bioinformatics analysis. The miR-204-5p merited further investigation, because it was increased almost 5-fold in diabetic corneal epithelia compared to nondiabetic control corneal epithelia. Using luciferase activity assay, we identified SIRT1 was a direct target of miR-204-5p. The results of flow cytometry and MTT assay demonstrated that downregulation of miR-204-5p increased TKE2 cell growth and restored cell cycle progression in high glucose (HG) conditions by the regulation of Cyclin D1 and p16. Furthermore, we showed downregulation of miR-204-5p promoted HG attenuation of corneal epithelial wound healing via upregulation of SIRT1 in Ins2(Akita/+) mice. CONCLUSIONS. Our data provide firm evidence of a role for miR-204-5p in the direct regulation of SIRT1 in diabetic corneas and identified the miR-204-5p-mediated regulation of SIRT1 contributes to the delay of epithelial cell cycle traversal in diabetic keratopathy.