Zhang Qin, Long Jing, Li Nannan, Ma Xuelian, Zheng Lisheng
Department of Cardiovascular, Dongying People's Hospital, Dongying, China.
Department of Critical Care Medicine, Dongying People's Hospital, Dongying, China.
Front Pharmacol. 2021 Mar 11;12:594793. doi: 10.3389/fphar.2021.594793. eCollection 2021.
Hyperglycemia exposure results in the dysfunction of endothelial cells (ECs) and the development of diabetic complications. Circular RNAs (circRNAs) have been demonstrated to play critical roles in EC dysfunction. The current study aimed to explore the role and mechanism of circRNA CLIP-associating protein 2 (circ_CLASP2, hsa_circ_0064772) on HG-induced dysfunction in human umbilical vein endothelial cells (HUVECs). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to assess the levels of circ_CLASP2, miR-140-5p and F-box, and WD repeat domain-containing 7 (FBXW7). The stability of circ_CLASP2 was identified by the actinomycin D and ribonuclease (RNase) R assays. Cell colony formation, proliferation, and apoptosis were measured by a standard colony formation assay, colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay, and flow cytometry, respectively. Western blot analysis was performed to determine the expression of related proteins. Targeted correlations among circ_CLASP2, miR-140-5p, and FBXW7 were confirmed by dual-luciferase reporter assay. High glucose (HG) exposure downregulated the expression of circ_CLASP2 in HUVECs. Circ_CLASP2 overexpression or miR-140-5p knockdown promoted proliferation and inhibited apoptosis of HUVECs under HG conditions. Circ_CLASP2 directly interacted with miR-140-5p via pairing to miR-140-5p. The regulation of circ_CLASP2 overexpression on HG-induced HUVEC dysfunction was mediated by miR-140-5p. Moreover, FBXW7 was a direct target of miR-140-5p, and miR-140-5p regulated HG-induced HUVEC dysfunction via FBXW7. Furthermore, circ_CLASP2 mediated FBXW7 expression through sponging miR-140-5p. Our current study suggested that the overexpression of circ_CLASP2 protected HUVEC from HG-induced dysfunction at least partly through the regulation of the miR-140-5p/FBXW7 axis, highlighting a novel therapeutic approach for the treatment of diabetic-associated vascular injury.
高血糖暴露会导致内皮细胞(ECs)功能障碍以及糖尿病并发症的发生。环状RNA(circRNAs)已被证明在EC功能障碍中起关键作用。本研究旨在探讨环状RNA CLIP相关蛋白2(circ_CLASP2,hsa_circ_0064772)在高糖诱导的人脐静脉内皮细胞(HUVECs)功能障碍中的作用及机制。采用定量实时聚合酶链反应(qRT-PCR)评估circ_CLASP2、miR-140-5p和含F-box和WD重复结构域7(FBXW7)的水平。通过放线菌素D和核糖核酸酶(RNase)R实验鉴定circ_CLASP2的稳定性。分别采用标准集落形成实验、比色法3-(4,5-二甲基噻唑-2-基)-2,5-二苯基-2H-四唑溴盐(MTT)实验和流式细胞术检测细胞集落形成、增殖和凋亡情况。进行蛋白质免疫印迹分析以确定相关蛋白的表达。通过双荧光素酶报告实验证实circ_CLASP2、miR-140-5p和FBXW7之间的靶向相关性。高糖(HG)暴露下调了HUVECs中circ_CLASP2的表达。在HG条件下,circ_CLASP2过表达或miR-140-5p敲低可促进HUVECs的增殖并抑制其凋亡。circ_CLASP2通过与miR-140-5p配对直接与miR-140-5p相互作用。circ_CLASP2过表达对HG诱导的HUVEC功能障碍的调节作用是由miR-140-5p介导的。此外,FBXW7是miR-140-5p的直接靶点,miR-140-5p通过FBXW7调节HG诱导的HUVEC功能障碍。此外,circ_CLASP2通过海绵吸附miR-140-5p介导FBXW7的表达。我们目前的研究表明,circ_CLASP2的过表达至少部分通过调节miR-140-5p/FBXW7轴保护HUVEC免受HG诱导的功能障碍,为糖尿病相关血管损伤的治疗突出了一种新的治疗方法。