Abstract：
The aim of this study was to investigate the mechanism underlying the role of a recently identified hsa_circ_0004805/hsa_miR-149-5p/transforming growth factor beta 2 (TGFB2) axis in the progression of diabetic retinopathy (DR). Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis suggested that hsa_circ_0004805 was highly expressed in aqueous humor samples of patients with DR, whereas hsa_miR-149-5p showed the opposite trend. Meanwhile, the results of a dual-luciferase reporter assay indicated that hsa_miR-149-5p directly interacted with both hsa_circ_0004805 and TGFB2. Using a variety of assays (Cell Counting Kit-8, EdU-labeling, Transwell, flow cytometric, wound healing, tube formation assays), we found that the overexpression of hsa_circ_0004805 significantly downregulated the level of hsa_miR-149-5p and promoted DNA synthesis, proliferation, migration, and tube formation in human retinal microvascular epithelial cells (hRECs) cultivated in a high-glucose environment. In contrast, hsa_miR-149-5p mimics inhibited DNA synthesis, proliferation, migration, and tube formation in hRECs by reducing the expression of its downstream target TGFB2 as well as the levels of phosphorylated SMAD2; however, these effects were reversed by the overexpression of hsa_circ_0004805. In a streptozotocin-induced Sprague-Dawley rat model of DR, retinal vascular leakage, capillary decellularization, loss of pericytes, fibrosis, and gliosis were evident, which could be reversed by vitreous microinjection of rat miR-149-5p mimics (rno-miR-149-5p agomir). Combined, our findings indicated that, under hyperglycemia, the hsa_circ_0004805/hsa_miR-149-5p/TGFB2 axis plays a critical role in the retinal pathophysiology associated with the development of DR, and has potential as a therapeutic target in the treatment of this condition.
摘要：
这项研究的目的是研究最近鉴定的hsa_circ_0004805/hsa_miR-149-5p/转化生长因子β2（TGFB2）轴在糖尿病视网膜病变（DR）进展中的作用机制。定量逆转录-聚合酶链反应（qRT-PCR）分析表明，hsa_circ_0004805在DR患者的房水样本中高表达，而hsa_miR-149-5p表现出相反的趋势。同时,双荧光素酶报告基因分析的结果表明hsa_miR-149-5p直接与hsa_circ_0004805和TGFB2相互作用。使用多种测定法(细胞计数试剂盒-8，EdU-标记，Transwell,流式细胞仪,伤口愈合，管形成测定)，我们发现，hsa_circ_0004805的过表达显著下调hsa_miR-149-5p的水平，促进DNA合成，扩散,迁移,和在高糖环境中培养的人视网膜微血管上皮细胞（hRECs）中的管形成。相比之下,hsa_miR-149-5p模拟物抑制DNA合成，扩散,迁移,通过降低其下游靶标TGFB2的表达以及磷酸化SMAD2的水平，在hRECs中形成管；然而，这些效应被hsa_circ_0004805的过表达逆转。在链脲佐菌素诱导的SD大鼠DR模型中，视网膜血管渗漏,毛细管去细胞化,周细胞的损失,纤维化，胶质增生很明显，通过玻璃体显微注射大鼠miR-149-5p模拟物（rno-miR-149-5pagomir）可以逆转。合并,我们的研究结果表明,在高血糖下，hsa_circ_0004805/hsa_miR-149-5p/TGFB2轴在与DR的发展相关的视网膜病理生理学中起关键作用，并有可能作为治疗这种疾病的治疗靶点。
