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[微小RNA-181减轻氧化型低密度脂蛋白诱导的血管内皮细胞损伤的作用及相关机制]

[Effect and related mechanism of microRNA-181 attenuates oxidized low density lipoprotein induced vascular endothelial cell injury].

作者信息

Wang N N, Sun X, Zhang X L, Lou L, Chen K M, Li H, Tang L, Wang W G, Zhang M

机构信息

Second Department of Cardiology, Central Hospital Affiliated to Shenyang Medical College, Shenyang 110024, China.

出版信息

Zhonghua Xin Xue Guan Bing Za Zhi. 2017 Mar 24;45(3):230-234. doi: 10.3760/cma.j.issn.0253-3758.2017.03.011.

Abstract

To observe the expression level of microRNA-181 (miR-181) and importin-α3 in oxidized low density lipoprotein (ox-LDL) induced vascular endothelial cell injury models, and explore the effect and mechanism of miR-181 on endothelial cell injury. Human vein endothelial cell line CRL-1730 were cultured and vascular endothelial cell injury model was established by intervention with ox-LDL. The cells were divided into control group (intervened by double distilled water), low-dose group (intervened by 10 μg/ml ox-LDL) and high-dose group (intervened by 20 μg/ml ox-LDL). In addition, cells of low-dose group were divided into miR-181 mimic group (miR-181 mimic was transfected) and mimic control group (miR-181 mimic control was transfected). Cell viabilities, mRNA and protein expression level of interleukin-6 (IL-6), miR-181, importin-α3, and nuclear transcription factor-κB (NF-κB) were measured by methyl thiazolyl tetrazolium (MTT), real-time PCR and Western blot, respectively. (1) The cell viabilities in low-dose group and high-dose group were lower than control group (0.207±0.012 and 0.204±0.007 vs. 0.323±0.018, all <0.01). The relative IL-6 mRNA expression in low-dose group and high-dose group were higher than control group (1.24±0.16 and 1.36±0.23 vs. 0.22±0.03, all <0.01). The relative miR-181 mRNA expression in low-dose group and high-dose group were lower than control group (0.91±0.11 and 0.88±0.07 vs. 2.20±0.13, all <0.01). The relative importin-α3 mRNA expression in low-dose group and high-dose group were higher than control group (1.23±0.22 and 0.55±0.03 vs. 0.44±0.06, all <0.01). The relative NF-κB mRNA expression in low-dose group and high-dose group were higher than control group (1.67±0.34 and 0.41±0.11 vs. 0.11±0.04, all <0.01). The relative importin-α3 protein expression in low-dose group and high-dose group were higher than control group (1.44±0.23 and 1.31±0.22 vs. 0.29±0.08, all <0.01). The relative NF-κB protein expression in low-dose group and high-dose group were higher than control group (0.43±0.05 and 0.37±0.04 vs. 0.16±0.03, all <0.01). (2)The cell viabilities in miR-181 mimic group was higher than in mimic control group (0.262±0.008 vs. 0.211±0.021, <0.01). The relative miR-181 mRNA expression level in miR-181 mimic group was higher than in mimic control group (4.23±0.34 vs. 0.88±0.16, <0.01). The relative importin-α3 mRNA expression level in miR-181 mimic group was lower than in mimic control group (0.24±0.03 vs. 1.08±0.13, <0.01). The relative NF-κB mRNA expression level was lower in miR-181 mimic group than in mimic control group (0.13±0.03 vs. 0.51±0.06, <0.01). The relative importin-α3 protein expression level was lower in miR-181 mimic group than in mimic control group (0.34±0.06 vs. 1.67±0.26, <0.01). The relative NF-κB protein expression level was lower in miR-181 mimic group than in mimic control group (0.43±0.02 vs. 1.53±0.36, <0.01). Lower miR-181 expression but higher importin-α3 and its downstream NF-κB signaling are associated with ox-LDL induced vascular endothelial cell injury and up-regulation of miR-181 could alleviate ox-LDL induced vascular endothelial cell injury possibly via importin-α3/NF-κB pathway.

摘要

观察氧化型低密度脂蛋白(ox-LDL)诱导的血管内皮细胞损伤模型中微小RNA-181(miR-181)和输入蛋白-α3的表达水平,探讨miR-181对内皮细胞损伤的作用及机制。培养人静脉内皮细胞系CRL-1730,通过ox-LDL干预建立血管内皮细胞损伤模型。将细胞分为对照组(用双蒸水干预)、低剂量组(用10μg/ml ox-LDL干预)和高剂量组(用20μg/ml ox-LDL干预)。此外,将低剂量组细胞分为miR-181模拟物组(转染miR-181模拟物)和模拟物对照组(转染miR-181模拟物对照)。分别采用甲基噻唑基四氮唑(MTT)法、实时荧光定量聚合酶链反应(real-time PCR)和蛋白质免疫印迹法(Western blot)检测细胞活力、白细胞介素-6(IL-6)、miR-181、输入蛋白-α3和核转录因子-κB(NF-κB)的mRNA及蛋白表达水平。(1)低剂量组和高剂量组细胞活力均低于对照组(0.207±0.012和0.204±0.007比0.323±0.018,均<0.01)。低剂量组和高剂量组IL-6 mRNA相对表达水平均高于对照组(1.24±0.16和1.36±0.23比0.22±0.03,均<0.01)。低剂量组和高剂量组miR-181 mRNA相对表达水平均低于对照组(0.91±0.11和0.88±0.07比2.20±0.13,均<0.01)。低剂量组和高剂量组输入蛋白-α3 mRNA相对表达水平均高于对照组(1.23±0.22和(此处原文有误,应为0.55±0.03)比0.44±0.06,均<0.01)。低剂量组和高剂量组NF-κB mRNA相对表达水平均高于对照组(1.67±0.34和0.41±0.11比0.11±0.04,均<0.01)。低剂量组和高剂量组输入蛋白-α3蛋白相对表达水平均高于对照组(1.44±0.23和1.31±0.22比0.29±0.08,均<0.01)。低剂量组和高剂量组NF-κB蛋白相对表达水平均高于对照组(0.43±0.05和0.37±0.04比0.16±0.03,均<0.01)。(2)miR-181模拟物组细胞活力高于模拟物对照组(0.262±0.008比0.211±0.021,<0.01)。miR-181模拟物组miR-181 mRNA相对表达水平高于模拟物对照组(4.23±0.34比0.88±0.16,<0.01)。miR-181模拟物组输入蛋白-α3 mRNA相对表达水平低于模拟物对照组(0.24±0.03比1.08±0.13,<0.01)。miR-181模拟物组NF-κB mRNA相对表达水平低于模拟物对照组(0.13±0.03比0.51±0.06,<0.01)。miR-181模拟物组输入蛋白-α3蛋白相对表达水平低于模拟物对照组(0.34±0.06比1.67±0.26,<0.01)。miR-181模拟物组NF-κB蛋白相对表达水平低于模拟物对照组(0.43±0.02比1.53±0.36,<0.01)。miR-181表达降低但输入蛋白-α3及其下游NF-κB信号通路升高与ox-LDL诱导的血管内皮细胞损伤相关,上调miR-181可能通过输入蛋白-α3/NF-κB途径减轻ox-LDL诱导的血管内皮细胞损伤。

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