Abstract：
BACKGROUND: Previous data have shown that circ_0033596 is involved in the pathogenesis of atherosclerosis (AS). The study aims to reveal the detailed mechanism of circ_0033596 in AS.
METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with oxidized low-density lipoprotein (ox-LDL) to establish an AS cell model. Quantitative real-time polymerase chain reaction and western blot were implemented to detect the expression of circ_0033596, miR-637, growth factor receptor bound protein2 (GRB2), BCL2-associated x protein (Bax) and B-cell lymphoma-2 (Bcl-2). Cell viability, proliferation, apoptosis and tube formation were investigated by cell counting kit-8, EdU assay, flow cytometry and tube formation assay, respectively. The production of interleukin (IL-6) and tumor necrosis factor-α (TNF-α) was evaluated by enzyme-linked immunosorbent assay. Oxidative stress was evaluated by lipid peroxidation malondialdehyde assay kit and superoxide dismutase activity assay kit. Dual-luciferase reporter assay, RNA pull-down assay and RIP assay were performed to identify the associations among circ_0033596, miR-637 and GRB2.
RESULTS: The expression of circ_0033596 and GRB2 was significantly increased, while miR-637 was decreased in the blood of AS patients and ox-LDL-induced HUVECs compared with controls. Ox-LDL treatment inhibited HUVEC viability, proliferation and angiogenic ability and induced cell apoptosis, inflammation and oxidative stress, while these effects were attenuated after circ_0033596 knockdown. Circ_0033596 interacted with miR-637 and regulated ox-LDL-induced HUVEC damage by targeting miR-637. In addition, GRB2, a target gene of miR-637, participated in ox-LDL-induced HUVEC injury by combining with miR-637. Importantly, circ_0033596 activated GRB2 by interacting with miR-637.
CONCLUSIONS: Circ_0033596 depletion protected against ox-LDL-induced HUVEC injury by miR-637/GRB2 pathway, providing a therapeutic target for AS.
METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with oxidized low-density lipoprotein (ox-LDL) to establish an AS cell model. Quantitative real-time polymerase chain reaction and western blot were implemented to detect the expression of circ_0033596, miR-637, growth factor receptor bound protein2 (GRB2), BCL2-associated x protein (Bax) and B-cell lymphoma-2 (Bcl-2). Cell viability, proliferation, apoptosis and tube formation were investigated by cell counting kit-8, EdU assay, flow cytometry and tube formation assay, respectively. The production of interleukin (IL-6) and tumor necrosis factor-α (TNF-α) was evaluated by enzyme-linked immunosorbent assay. Oxidative stress was evaluated by lipid peroxidation malondialdehyde assay kit and superoxide dismutase activity assay kit. Dual-luciferase reporter assay, RNA pull-down assay and RIP assay were performed to identify the associations among circ_0033596, miR-637 and GRB2.
RESULTS: The expression of circ_0033596 and GRB2 was significantly increased, while miR-637 was decreased in the blood of AS patients and ox-LDL-induced HUVECs compared with controls. Ox-LDL treatment inhibited HUVEC viability, proliferation and angiogenic ability and induced cell apoptosis, inflammation and oxidative stress, while these effects were attenuated after circ_0033596 knockdown. Circ_0033596 interacted with miR-637 and regulated ox-LDL-induced HUVEC damage by targeting miR-637. In addition, GRB2, a target gene of miR-637, participated in ox-LDL-induced HUVEC injury by combining with miR-637. Importantly, circ_0033596 activated GRB2 by interacting with miR-637.
CONCLUSIONS: Circ_0033596 depletion protected against ox-LDL-induced HUVEC injury by miR-637/GRB2 pathway, providing a therapeutic target for AS.
摘要：
背景：先前的数据表明circ_0033596参与了动脉粥样硬化（AS）的发病机理。本研究旨在揭示circ_0033596在AS中的详细机制。
方法：用氧化低密度脂蛋白(ox-LDL)处理人脐静脉内皮细胞(HUVECs),建立AS细胞模型。实时定量聚合酶链反应和免疫印迹检测circ_0033596、miR-637、生长因子受体结合蛋白2(GRB2)的表达,BCL2相关x蛋白（Bax）和B细胞淋巴瘤2（Bcl-2）。细胞活力，扩散,细胞计数试剂盒-8，EdU测定，研究细胞凋亡和试管形成，流式细胞术和试管形成测定，分别。通过酶联免疫吸附试验评估白介素（IL-6）和肿瘤坏死因子-α（TNF-α）的产生。通过脂质过氧化丙二醛测定试剂盒和超氧化物歧化酶活性测定试剂盒评估氧化应激。双荧光素酶报告基因测定,进行RNA下拉测定和RIP测定以鉴定circ_0033596、miR-637和GRB2之间的关联。
结果：circ_0033596和GRB2的表达明显增加，而与对照组相比，AS患者和ox-LDL诱导的HUVECs血液中的miR-637降低。Ox-LDL治疗抑制HUVEC活力，增殖和血管生成能力以及诱导的细胞凋亡，炎症和氧化应激，而这些作用在circ_0033596敲低后减弱。Circ_0033596与miR-637相互作用并通过靶向miR-637调节ox-LDL诱导的HUVEC损伤。此外,miR-637的靶基因GRB2通过与miR-637结合参与ox-LDL诱导的HUVEC损伤。重要的是,circ_0033596通过与miR-637相互作用激活GRB2。
结论：Circ_0033596耗竭可通过miR-637/GRB2通路保护ox-LDL诱导的HUVEC损伤，为AS提供治疗靶点。
方法：用氧化低密度脂蛋白(ox-LDL)处理人脐静脉内皮细胞(HUVECs),建立AS细胞模型。实时定量聚合酶链反应和免疫印迹检测circ_0033596、miR-637、生长因子受体结合蛋白2(GRB2)的表达,BCL2相关x蛋白（Bax）和B细胞淋巴瘤2（Bcl-2）。细胞活力，扩散,细胞计数试剂盒-8，EdU测定，研究细胞凋亡和试管形成，流式细胞术和试管形成测定，分别。通过酶联免疫吸附试验评估白介素（IL-6）和肿瘤坏死因子-α（TNF-α）的产生。通过脂质过氧化丙二醛测定试剂盒和超氧化物歧化酶活性测定试剂盒评估氧化应激。双荧光素酶报告基因测定,进行RNA下拉测定和RIP测定以鉴定circ_0033596、miR-637和GRB2之间的关联。
结果：circ_0033596和GRB2的表达明显增加，而与对照组相比，AS患者和ox-LDL诱导的HUVECs血液中的miR-637降低。Ox-LDL治疗抑制HUVEC活力，增殖和血管生成能力以及诱导的细胞凋亡，炎症和氧化应激，而这些作用在circ_0033596敲低后减弱。Circ_0033596与miR-637相互作用并通过靶向miR-637调节ox-LDL诱导的HUVEC损伤。此外,miR-637的靶基因GRB2通过与miR-637结合参与ox-LDL诱导的HUVEC损伤。重要的是,circ_0033596通过与miR-637相互作用激活GRB2。
结论：Circ_0033596耗竭可通过miR-637/GRB2通路保护ox-LDL诱导的HUVEC损伤，为AS提供治疗靶点。
