Ophthalmology Department, Second People's Hospital of Quzhou, Quzhou, China.
Eur Rev Med Pharmacol Sci. 2020 Jun;24(12):6494-6504. doi: 10.26355/eurrev_202006_21632.
MiRNA has been found to have therapeutic effect on corneal damage. This paper aimed to study the effect of miR-205-3p on corneal damage induced by ultraviolet (UV) radiation.
HCE cells were exposed to UV light and transfected. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot were used to determine miRNA/mRNA and protein expression. CCK-8 assay, Edu incorporation experiment, and flow cytometry were used to separately measure cell activity, proliferation and apoptosis. LC3 puncta were researched by immunofluorescence experiment. TNF-α, IL-6 and IL-1β levels in cells were detected by enzyme-linked immunosorbent assay (ELISA) kit. MDA, SOD, and GSH-PX levels were measured using detection kits. Reactive oxygen species (ROS) level was reflected by detecting DCFH-DA density. Luciferase activity assay was performed to verify the regulating relationship between miR-205-3p and TLR4.
UV radiation decreased HCE cell viability, proliferation, and increased HCE cell apoptosis and autophagy (all p < 0.01). When exposed UV radiation, the overexpression of miR-205-3p group elevated HCE cells viability, proliferation and weakened HCE cells apoptosis and autophagy (all p < 0.01). MiR-205-3p inhibited inflammation and oxidative stress in HCE cells induced by UV radiation (p < 0.01). MiR-205-3p directly inhibited TLR4 expression. The upregulation of TLR4 significantly reversed the effects of miR-205-3p on HCE cells phenotypes induced by UV radiation (p < 0.01).
MiR-205-3p protected HCE cells from UV damage by inhibiting autophagy via targeting TLR4.
miRNA 已被发现对角膜损伤具有治疗作用。本研究旨在探讨 miR-205-3p 对紫外线（UV）辐射诱导的角膜损伤的影响。
将 HCE 细胞暴露于紫外线下并进行转染。采用实时定量聚合酶链反应（qRT-PCR）和 Western blot 检测 miRNA/mRNA 和蛋白表达。CCK-8 测定法、Edu 掺入实验和流式细胞术分别用于测量细胞活力、增殖和凋亡。免疫荧光实验用于研究 LC3 斑点。酶联免疫吸附测定（ELISA）试剂盒检测细胞中 TNF-α、IL-6 和 IL-1β 水平。采用检测试剂盒测定 MDA、SOD 和 GSH-PX 水平。通过检测 DCFH-DA 密度反映活性氧（ROS）水平。进行荧光素酶活性测定以验证 miR-205-3p 与 TLR4 之间的调节关系。
UV 辐射降低了 HCE 细胞活力、增殖，并增加了 HCE 细胞凋亡和自噬（均 p<0.01）。当暴露于 UV 辐射时，miR-205-3p 过表达组提高了 HCE 细胞活力、增殖，并减弱了 HCE 细胞凋亡和自噬（均 p<0.01）。miR-205-3p 抑制了 UV 辐射诱导的 HCE 细胞炎症和氧化应激（p<0.01）。miR-205-3p 直接抑制 TLR4 表达。TLR4 的上调显著逆转了 miR-205-3p 对 UV 辐射诱导的 HCE 细胞表型的影响（p<0.01）。
miR-205-3p 通过靶向 TLR4 抑制自噬来保护 HCE 细胞免受 UV 损伤。
