Objective: To investigate the protective effect of miR-328-3p on oxidized low-density lipoprotein (ox-LDL)-induced coronary artery endothelial cell injury and the potentially relevant mechanisms.
Methods: Human coronary artery endothelial cells (HCAECs) were induced with ox-LDL, and the cells were divided into a control group consisting of normal cells, an ox-LDL group receiving ox-LDL treatment, an ox-LDL+miR-NC group transfected with miR-NC and treated with ox-LDL, an ox-LDL+miR-328-3p group transfected with miR-328-3p and treated with ox-LDL, and ox-LDL+miR-328-3p+pcDNA group co-transfected miR-328-3p and pcDNA and treated with ox-LDL, and an ox-LDL+miR-328-3p+insulin-like growth factor 2 (IGF2) group co-transfected miR-328-3p and IGF2 and treated with ox-LDL. The expression level of miR-328-3p was determined with RT-qPCR. Cell proliferation was determined by MTT. Cell apoptosis was measured by flow cytometry. Western blot was conducted to examine the protein expression levels of cleaved cas-3 and IGF2. ELISA was performed to determine the levels of tumor necrosis factor α (TNF-α), interleukin (IL)-6, and IL-1β. Dual luciferase reporter experiment was performed to verify the targeting relationship between miR-328-3p and IGF2.
Results: Compared with those of the control group, miR-328-3p expression level and cell activity were significantly reduced in the ox-LDL group (P<0.05), while the apoptotic rate, the protein expression levels of cleaved cas-3, IGF2, Bax, and Bcl-2, and the levels of TNF-α, IL-6, and IL-1β were significantly increased (P<0.05). Compared with those of the ox-LDL+miR-NC group, miR-328-3p expression level and cell activity significantly increased in the ox-LDL+miR-328-3p group (P<0.05), while the apoptosis rate, the protein expression levels of cleaved cas-3 and IGF2, and the levels of TNF-α, IL-6, and IL-1β were significantly reduced. IGF2 was a functional target of miR-328-3p. Compared with those of the ox-LDL+miR-328-3p+pcDNA co-transfection group, the IGF2 protein level was significantly increased (P<0.05) and cell activity was significantly decreased (P<0.05) in the ox-LDL+miR-328-3p+IGF2 co-transfection group, while the apoptosis rate, cleaved cas-3 protein level, and the levels of TNF-α, IL-6, and IL-1β were significantly elevated (P<0.05).
Conclusion: miR-328-3p inhibits ox-LDL-induced apoptosis and inflammatory in coronary artery endothelial cell injury through targeted negative regulation of IGF2.
目的: 探讨miR-328-3p对氧化型低密度脂蛋白(oxidized low-density lipoprotein, ox-LDL)诱导的冠状动脉内皮细胞损伤的保护作用及可能相关的作用机制。
方法: 用ox-LDL诱导人冠状动脉内皮细胞(human coronary artery endothelial cells, HCAECs),将细胞分为对照(control)组(正常培养细胞)、ox-LDL组(ox-LDL处理)、ox-LDL+miR-NC组(转染miR-NC,用ox-LDL处理)、ox-LDL+miR-328-3p组(转染miR-328-3p,用ox-LDL处理)、ox-LDL+miR-328-3p+pcDNA组(共转染miR-328-3p和pcDNA,用ox-LDL处理)、ox-LDL+miR-328-3p+胰岛素样生长因子2(insulin-like growth factor 2, IGF2)组(共转染miR-328-3p和IGF2,用ox-LDL处理)。RT-qPCR检测miR-328-3p表达水平;MTT以及流式细胞术检测细胞增殖和凋亡;Western blot法检测cleaved cas-3、IGF2、Bax、Bcl-2蛋白含量;ELISA法检测肿瘤坏死因子-α(tumor necrosis factor α, TNF-α)、白细胞介素(interleukin, IL)-6、IL-1β含量 ;应用双荧光素酶报告实验验证分子间靶向关系。
结果: 与control组相比,ox-LDL组中miR-328-3p表达水平、细胞活性降低(P<0.05),凋亡率,cleaved cas-3、IGF2蛋白表达,TNF-α、IL-6、IL-1β水平增加(P<0.05)。与ox-LDL+miR-NC组相比,ox-LDL+miR-328-3p组miR-328-3p表达水平、细胞活性增加(P<0.05),凋亡率,cleaved cas-3、IGF2蛋白表达,TNF-α、IL-6、IL-1β水平降低(P<0.05)。IGF2是miR-328-3p的功能靶标。与共转染ox-LDL+miR-328-3p+pcDNA组比较,共转染ox-LDL+miR-328-3p+IGF2组IGF2蛋白水平升高(P<0.05),细胞活性降低(P<0.05),而凋亡率、cleaved cas-3蛋白水平以及TNF-α、IL-6、IL-1β含量升高(P<0.05)。
结论: miR-328-3p通过靶向负调控IGF2抑制ox-LDL诱导的冠状动脉内皮细胞凋亡和炎性损伤。
Keywords: Coronary artery endothelial cells; Insulin-like growth factor 2; Oxidized low-density lipoprotein; miR-328-3p.
© 2024《四川大学学报(医学版)》编辑部 版权所有Copyright ©2024 Editorial Office of Journal of Sichuan University (Medical Sciences).