Zhuang Pei, Muraleedharan Chithra K, Xu Shunbin
Department of Ophthalmology/Kresge Eye Institute, Wayne State University, Detroit, Michigan, United States 2Department of Anatomy and Cell Biology, School of Medicine, Wayne State University, Detroit, Michigan, United States.
Invest Ophthalmol Vis Sci. 2017 Mar 1;58(3):1646-1655. doi: 10.1167/iovs.16-21223.
Previously, we showed that microRNA-146 (miR-146) is a pivotal negative feedback regulator of multiple nuclear factor kappa-B (NF-κB) activation pathways in retinal endothelial cells (RECs). We hypothesized that miR-146 plays an important role in diabetic retinopathy (DR) by inhibiting diabetes-induced inflammatory response in the retina. The purpose of the current study is to test this hypothesis in vivo.
Lentiviruses expressing rno-miR-146a, lenti-miR-146a, and negative control oligonucleotide with scrambled sequence, lenti-miR-neg ctl, were produced. Young male Sprague-Dawley rats were injected with a single dose of streptozotocin ([STZ] 65 mg/kg) to induce diabetes. One week after diabetes, animals were injected with lentivirus intravitreally (4 μl, ∼106 CFU/mL). Three months after diabetes, retinal microvascular leakage was tested by Evans blue assay; retinal function by electroretinogram (ERG). Total RNA and protein lysate were isolated from the retina for quantitative (q)RT-PCR and Western blot analyses.
Lenti-miR-146a robustly transduced human retinal endothelial cells (HRECs) and increased the expression of miR-146a in vitro. In vivo, intravitreal injection of lenti-miR-146a increased the expression of miR-146a in the retina, while its key downstream target genes, including CARD10, IRAK1, and TRAF6, were downregulated. Intravitreal delivery of miR-146 inhibited diabetes-induced upregulation of NF-κB downstream gene, Intercellular Adhesion Molecule 1 (ICAM1), as well as microvascular leakage and retinal functional defects.
Intravitreal delivery of miR-146 inhibited diabetes-induced NF-κB activation and retinal microvascular and neuronal functional defects in a diabetic rat model.
此前，我们发现微小RNA - 146（miR - 146）是视网膜内皮细胞（REC）中多种核因子κB（NF - κB）激活途径的关键负反馈调节因子。我们推测miR - 146通过抑制视网膜中糖尿病诱导的炎症反应在糖尿病视网膜病变（DR）中发挥重要作用。本研究的目的是在体内验证这一假设。
制备表达大鼠miR - 146a的慢病毒（lenti - miR - 146a）以及带有随机序列的阴性对照寡核苷酸慢病毒（lenti - miR - neg ctl）。对年轻雄性Sprague - Dawley大鼠单次注射链脲佐菌素（[STZ] 65 mg/kg）以诱导糖尿病。糖尿病发生一周后，通过玻璃体腔内注射慢病毒（4 μl，~106 CFU/mL）。糖尿病发生三个月后，通过伊文思蓝试验检测视网膜微血管渗漏；通过视网膜电图（ERG）检测视网膜功能。从视网膜中分离总RNA和蛋白质裂解物用于定量（q）RT - PCR和蛋白质印迹分析。
lenti - miR - 146a在体外能有效转导人视网膜内皮细胞（HREC）并增加miR - 146a的表达。在体内，玻璃体腔内注射lenti - miR - 146a可增加视网膜中miR - 146a的表达，而其关键下游靶基因，包括CARD10、IRAK1和TRAF6的表达则下调。玻璃体腔内递送miR - 146可抑制糖尿病诱导的NF - κB下游基因细胞间黏附分子1（ICAM1）的上调，以及微血管渗漏和视网膜功能缺陷。
在糖尿病大鼠模型中，玻璃体腔内递送miR - 146可抑制糖尿病诱导的NF - κB激活以及视网膜微血管和神经元功能缺陷。
