环状 RNA 0006667 通过调控 miR-7-5p/TGFA 轴介导高糖诱导的视网膜色素上皮细胞功能障碍在糖尿病视网膜病变中的作用。
Circ_0006667 contributes to high glucose-induced retinal pigment epithelial cell dysfunction by mediating miR-7-5p/TGFA axis in diabetic retinopathy.
机构信息
Department of Endocrinology, Xiantao First People's Hosptial, No. 29, Mianzhou Avenue, First People's Hospital, Xiantao City, 433000, Hubei Province, China.
Department of Ophthalmology, Xiantao First People's Hosptial, No. 29, Mianzhou Avenue, First People's Hospital, Xiantao City, 433000, Hubei Province, China.
出版信息
Int Ophthalmol. 2023 Jul;43(7):2383-2396. doi: 10.1007/s10792-023-02636-y. Epub 2023 Jan 30.
BACKGROUND
Diabetic retinopathy (DR) is a common complication of diabetes mellitus and it can lead to visual impairment and blindness. The loss of retinal pigment epithelial (RPE) cells is associated with the etiology of DR. Moreover, dysregulated circular RNAs (circRNAs) are implicated in DR progression. Therefore, this project aims to explore the role and potential mechanism of circ_0006667 in DR.
METHODS
RPE cells (ARPE-19) were stimulated with high glucose (33 mM; HG group) for 24 h to establish the DR cell model. Circ_0006667, microRNA-7-5p (miR-7-5p), and transforming growth factor alpha (TGFA) expression was determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cell viability, proliferation, and apoptosis were analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine (EdU), and flow cytometry. CyclinD1, Cleaved-caspase-3, and TGFA protein levels were detected using western blot. Using Circinteractome and starBase analysis, the binding miR-7-5p and circ_0006667 or TGFA was predicted, and then validated using dual-luciferase reporter and RNA Immunoprecipitation (RIP).
RESULTS
Circ_0006667 expression was up-regulated in DR patients and HG-induced ARPE-19 cells. HG stimulation suppressed ARPE-19 cell proliferation and promoted cell apoptosis and inflammation, which were alleviated via circ_0006667 silence. Circ_0006667 acted as a molecular sponge for miR-7-5p, and circ_0006667 absence-mediated protective effects in HG-induced ARPE-19 cells were largely overturned by the interference of miR-7-5p. miR-7-5p directly targeted TGFA, and miR-7-5p overexpression protected ARPE-19 cells from HG-induced dysfunction largely by down-regulating TGFA. Circ_0006667 can up-regulate the expression of TGFA by sponging miR-7-5p in ARPE-19 cells.
CONCLUSION
Circ_0006667 silencing protected ARPE-19 cells from HG-induced dysfunction by mediating miR-7-5p/TGFA axis.
背景
糖尿病视网膜病变（DR）是糖尿病的常见并发症，可导致视力损害和失明。视网膜色素上皮（RPE）细胞的丢失与 DR 的病因有关。此外，失调的环状 RNA（circRNA）与 DR 的进展有关。因此，本项目旨在探讨 circ_0006667 在 DR 中的作用和潜在机制。
方法
用高糖（33 mM；HG 组）刺激 RPE 细胞（ARPE-19）24 h 建立 DR 细胞模型。通过逆转录定量聚合酶链反应（RT-qPCR）测定 circ_0006667、微小 RNA-7-5p（miR-7-5p）和转化生长因子α（TGFA）的表达。通过 3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴盐（MTT）、5-乙炔基-2'-脱氧尿苷（EdU）和流式细胞术分析细胞活力、增殖和凋亡。使用 Western blot 检测细胞周期蛋白 D1、Cleaved-caspase-3 和 TGFA 蛋白水平。通过 Circinteractome 和 starBase 分析预测 miR-7-5p 与 circ_0006667 或 TGFA 的结合，并通过双荧光素酶报告和 RNA 免疫沉淀（RIP）验证。
结果
DR 患者和 HG 诱导的 ARPE-19 细胞中 circ_0006667 表达上调。HG 刺激抑制 ARPE-19 细胞增殖，促进细胞凋亡和炎症，而沉默 circ_0006667 可减轻这种作用。circ_0006667 作为 miR-7-5p 的分子海绵，而 circ_0006667 缺失介导的 HG 诱导的 ARPE-19 细胞中的保护作用在 miR-7-5p 干扰后大部分被推翻。miR-7-5p 直接靶向 TGFA，miR-7-5p 过表达通过下调 TGFA 可在很大程度上保护 ARPE-19 细胞免受 HG 诱导的功能障碍。circ_0006667 可通过海绵吸附 miR-7-5p 来上调 ARPE-19 细胞中 TGFA 的表达。
结论
沉默 circ_0006667 通过介导 miR-7-5p/TGFA 轴来保护 ARPE-19 细胞免受 HG 诱导的功能障碍。
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