环状非编码 RNA HIPK3 介导糖尿病视网膜血管功能障碍。
Circular Noncoding RNA HIPK3 Mediates Retinal Vascular Dysfunction in Diabetes Mellitus.
机构信息
From Eye Institute, Eye and ENT Hospital, Shanghai Medical College, Fudan University, China (K.S., C.L., X.L., S.-J.Z., J.-J.W., S.-H.Z., J.-H.W., C.Z., B.Y.); Fourth School of Clinical Medicine, Nanjing Medical University, China (C.L.); Department of Pediatric Surgery, Children's Hospital of Fudan University, Shanghai, China (B.-H.L., R.D.); Department of Ophthalmology, First Affiliated Hospital of Nanjing Medical University, China (X.C.); Department of Cardiovascular Surgery, Shanghai East Hospital, Tongji University School of Medicine, China (Y.-Y.Z.); Department of Cardiology, Shanghai Tenth People's Hospital, Tongji University School of Medicine, China (B.L.); and Shanghai Key Laboratory of Visual Impairment and Restoration, China (C.Z., B.Y.).
出版信息
Circulation. 2017 Oct 24;136(17):1629-1642. doi: 10.1161/CIRCULATIONAHA.117.029004. Epub 2017 Aug 31.
BACKGROUND
The vascular complications of diabetes mellitus are the major causes of morbidity and mortality among people with diabetes. Circular RNAs are a class of endogenous noncoding RNAs that regulate gene expression in eukaryotes. In this study, we investigated the role of circular RNA in retinal vascular dysfunction induced by diabetes mellitus.
METHODS
Quantitative polymerase chain reactions, Sanger sequencing, and Northern blots were conducted to detect circular HIPK3 (circHIPK3) expression pattern on diabetes mellitus-related stresses. MTT (3-[4,5-dimethythiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assays, EdU (5-ethynyl-2'-deoxyuridine) incorporation assays, Transwell migration assays, and Matrigel assays were conducted to detect the role of circHIPK3 in retinal endothelial cell function in vitro. Retinal trypsin digestion, vascular permeability assays, and ELISA assays were conducted to detect the role of circHIPK3 in retinal vascular dysfunction in vivo. Bioinformatics analysis, luciferase activity assays, RNA pull-down assays, and in vitro studies were conducted to reveal the mechanism of circHIPK3-mediated retinal vascular dysfunction.
RESULTS
circHIPK3 expression was significantly upregulated in diabetic retinas and retinal endothelial cells following stressors related to diabetes mellitus. circHIPK3 silencing or overexpressing circHIPK3 changed retinal endothelial cell viability, proliferation, migration, and tube formation in vitro. circHIPK3 silencing in vivo alleviated retinal vascular dysfunction, as shown by decreased retinal acellular capillaries, vascular leakage, and inflammation. circHIPK3 acted as an endogenous miR-30a-3p sponge to sequester and inhibit miR-30a-3p activity, which led to increased vascular endothelial growth factor-C, FZD4, and WNT2 expression. Ectopic expression of miR-30a-3p mimicked the effect of circHIPK3 silencing on vascular endothelial phenotypes in vivo and in vitro.
CONCLUSIONS
The circular RNA circHIPK3 plays a role in diabetic retinopathy by blocking miR-30a function, leading to increased endothelial proliferation and vascular dysfunction. These data suggest that circular RNA is a potential target to control diabetic proliferative retinopathy.
背景
糖尿病的血管并发症是糖尿病患者发病率和死亡率的主要原因。环状 RNA 是一类调节真核生物基因表达的内源性非编码 RNA。在这项研究中，我们研究了环状 RNA 在糖尿病诱导的视网膜血管功能障碍中的作用。
方法
通过定量聚合酶链反应、Sanger 测序和Northern 印迹检测与糖尿病相关应激下环状 HIPK3（circHIPK3）的表达模式。MTT（3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四唑溴盐）测定、EdU（5-乙炔基-2'-脱氧尿苷）掺入测定、Transwell 迁移测定和 Matrigel 测定用于检测 circHIPK3 在体外视网膜内皮细胞功能中的作用。视网膜胰蛋白酶消化、血管通透性测定和 ELISA 测定用于检测 circHIPK3 在体内视网膜血管功能障碍中的作用。生物信息学分析、荧光素酶活性测定、RNA 下拉测定和体外研究用于揭示 circHIPK3 介导的视网膜血管功能障碍的机制。
结果
在糖尿病视网膜和糖尿病相关应激后的视网膜内皮细胞中，circHIPK3 的表达显著上调。circHIPK3 沉默或过表达改变了体外视网膜内皮细胞的活力、增殖、迁移和管状形成。体内 circHIPK3 沉默减轻了视网膜血管功能障碍，表现为视网膜无细胞毛细血管减少、血管渗漏和炎症减少。circHIPK3 作为内源性 miR-30a-3p 海绵，通过隔离和抑制 miR-30a-3p 活性，导致血管内皮生长因子-C、FZD4 和 WNT2 表达增加。外源性表达 miR-30a-3p 模拟了 circHIPK3 沉默对体内和体外血管内皮表型的作用。
结论
环状 RNA circHIPK3 通过阻断 miR-30a 功能在糖尿病性视网膜病变中发挥作用，导致内皮细胞增殖和血管功能障碍增加。这些数据表明，环状 RNA 是控制糖尿病性增殖性视网膜病变的潜在靶点。
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