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Jing Gao, Ye Wang, Xiaowen Zhao, Peng Chen, Lixin Xie; MicroRNA-204-5p–Mediated Regulation of SIRT1 Contributes to the Delay of Epithelial Cell Cycle Traversal in Diabetic Corneas. Invest. Ophthalmol. Vis. Sci. 2015;56(3):1493-1504. doi: 10.1167/iovs.14-15913.
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© ARVO (1962-2015); The Authors (2016-present)
We investigated how the microRNA (miRNA) modifies the expression of silent mating type information regulation 2 homolog 1 (SIRT1) in diabetic corneas.
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 Ins2Akita/+ 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.
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 Ins2Akita/+ mice.
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.
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