Department of Neurology, Hainan General Hospital (Hainan Affiliated Hospital of Hainan Medical University), Haikou 570311, Hainan, China.
Department of Central Laboratory, Hainan General Hospital (Hainan Affiliated Hospital of Hainan Medical University), Haikou 570311, Hainan, China.
Brain Res. 2021 Dec 1;1772:147662. doi: 10.1016/j.brainres.2021.147662. Epub 2021 Sep 13.
Stroke is a fatal disease with high disability and mortality and there is no credible treatment for stroke at present. Studies on stroke are extensively developed to explore the underlying mechanisms of ischemic and reperfusion injuries. Herein, we investigated the functions of microRNA let-7f (also termed let-7f-5p) in vascular endothelial cell dysfunction. The bEnd.3 cells were stimulated with oxygen-glucose deprivation and reoxygenation (OGD/R) to mimic cell injury in vitro. CCK-8 assays, flow cytometry and western blot analyses were conducted to examine the viability and apoptosis of bEnd.3 cells. Reverse transcription quantitative polymerase chain reaction analyses were employed to measure RNA expression. Endothelial cell permeability in vitro assay was employed to assess endothelial permeability of bEnd.3 cells, and expression levels of proteins associated with cell apoptosis or blood-brain barrier (BBB) were detected by western blot analyses. Luciferase reporter assay was conducted to explore the combination between let-7f and HMGA2. We found that OGD/R induced injuries on endothelial cells (bEnd.3) by decreasing cell viability and promoting cell apoptosis. Let-7f exhibited low expression in bEnd.3 cells under OGD/R. Let-7f overexpression increased the viability of bEnd.3 cells and inhibited cell apoptosis. In addition, the endothelial permeability of bEnd.3 cells was increased by OGD/R and reversed by let-7f overexpression. The levels of tight junction proteins (ZO-1 and occludin) were downregulated by OGD/R and then reversed by let-7f overexpression. Mechanistically, HMGA2 is a target gene of let-7f and its expression was negatively regulated by let-7f. Rescue assays revealed that HMGA2 overexpression reversed the effects of let-7f overexpression on cell viability, cell apoptosis, endothelial permeability, and BBB function. In conclusion, let-7f alleviates vascular endothelial cell dysfunction by downregulating HMGA2 expression under OGD/R.
中风是一种具有高致残率和死亡率的致命疾病，目前尚无可靠的中风治疗方法。目前广泛开展中风研究，以探索缺血再灌注损伤的潜在机制。在这里，我们研究了 microRNA let-7f（也称为 let-7f-5p）在血管内皮细胞功能障碍中的作用。用氧葡萄糖剥夺和再氧合（OGD/R）刺激 bEnd.3 细胞，在体外模拟细胞损伤。通过 CCK-8 测定、流式细胞术和 Western blot 分析检测 bEnd.3 细胞的活力和凋亡。采用逆转录定量聚合酶链反应分析检测 RNA 表达。采用体外内皮细胞通透性测定法检测 bEnd.3 细胞的内皮通透性，Western blot 分析检测与细胞凋亡或血脑屏障（BBB）相关的蛋白表达水平。采用荧光素酶报告基因检测法探讨 let-7f 与 HMGA2 的结合情况。我们发现，OGD/R 通过降低细胞活力和促进细胞凋亡对内皮细胞（bEnd.3）造成损伤。bEnd.3 细胞在 OGD/R 下低表达 let-7f。let-7f 过表达增加了 bEnd.3 细胞的活力并抑制了细胞凋亡。此外，bEnd.3 细胞的内皮通透性在 OGD/R 下增加，而过表达 let-7f 可逆转这一作用。紧密连接蛋白（ZO-1 和 occludin）的水平在 OGD/R 下调，而过表达 let-7f 则可逆转这一作用。机制上，HMGA2 是 let-7f 的靶基因，其表达受 let-7f 的负调控。挽救实验表明，HMGA2 过表达可逆转 let-7f 过表达对细胞活力、细胞凋亡、内皮通透性和 BBB 功能的影响。综上所述，let-7f 通过下调 OGD/R 下的 HMGA2 表达来缓解血管内皮细胞功能障碍。
