Let-7d靶向LOX-1逆转ox-LDL诱导血管内皮细胞凋亡观察
Effects of Let-7d on apoptosis of vascular endothelial cells induced by ox-LDL through regulating LOX-1 expression

目的:研究 Let-7d对氧化低密度脂蛋白(ox-LDL)诱导人脐静脉内皮细胞(HUVECs)凋亡以及凝集素样氧化低密度脂蛋白清除受体1(LOX-1)表达的影响。方法:用50 mg/L 的ox-LDL处理HUVECs 24 h和48 h,分别采用Western blot和RT-qPCR法检测LOX-1和Let-7d表达的变化。TargetScan和双荧光素酶报告基因实验预测LOX-1与Let-7d之间的靶向调控关系。将miRNA 模拟物对照(NC)和Let-7d单独或联合50 mg/L的ox-LDL处理HUVECs 48 h,分别记为NC组、Let-7d组、NC+ox-LDL组和Let-7d+ox-LDL组,采用Western blot和RT-qPCR法检测各组中LOX-1、Caspase 3、Bax和Bcl-2蛋白表达及LOX-1 mRNA表达,流式细胞仪检测各组细胞凋亡率。结果:用50 mg/L 的ox-LDL处理HUVECs 0 h、24 h和48 h后,各组HUVECs中LOX-1蛋白表达增加,Let-7d表达降低(P<0.05)。TargetScan和双荧光素酶报告基因实验表明LOX-1可能是Let-7d的靶基因。NC组、Let-7d组、NC+ox-LDL组和Let-7d+ox-LDL组HUVECs中LOX-1的表达、细胞凋亡率及Caspase 3、Bax、Bcl-2蛋白表达比较,差异均有统计学意义(P<0.001)。ox-LDL可诱导HUVECs中LOX-1、细胞凋亡率和促凋亡蛋白Caspase 3、Bax表达增加,抗凋亡蛋白Bcl-2表达降低; Let-7d能够显著抑制ox-LDL对HUVECs中LOX-1表达、细胞凋亡率及促凋亡蛋白Caspase 3、Bax表达的诱导作用和抗凋亡蛋白Bcl-2表达的抑制作用(P<0.05)。结论:Let-7d能够逆转ox-LDL诱导的 HUVECs凋亡,其机制可能与Let-7d靶向抑制LOX-1表达有关。
Aim:To investigate the effects of Let-7d on the apoptosis of human umbilical vein endothelial cells(HUVECs)induced by ox-LDL and the expression of LOX-1.Methods:The expressions of LOX-1 and Let-7d were detected by Western blot and RT-qPCR,respectively,after HUVECs were treated with 50 mg/L ox-LDL for 24 h and 48 h. TargetScan and dual luciferase reporter gene experiment predicted the regulating relationship between LOX-1 and Let-7d.HUVECs were treated with NC and Let-7d alone or combined with 50 mg/L ox-LDL for 48 h,which were recorded as NC group, NC+ox-LDL group,Let-7d group and Let-7d+ox-LDL group.Western blot and RT-qPCR were used to detect the protein levels of LOX-1,Caspase 3,Bax and Bcl-2,and the mRNA level of LOX-1,respectively.Flow cytometry was used to detect the apoptosis rate of the cells in each group.Results:After being treated with 50 mg/L ox-LDL for 0 h,24 h and 48 h,the protein level of LOX-1 in HUVECs was increased significantly,while that of Let-7d decreased significantly(P<0.05). TargetScan and dual luciferase reporter gene experiment showed that LOX-1 was a potential target of Let-7d.There were significant differences in the expressions of LOX-1, apoptosis rate and the protein levels of Caspase 3, Bax,Bcl-2 in HUVECs from NC,Let-7d,NC+ox-LDL and Let-7d+ox-LDL groups(P<0.001).Ox-LDL could induce the increases of expressions of LOX-1,Caspase 3,and Bax,and the apoptosis rate increased significantly, while the expression of Bcl-2 decreased remarkably; Let-7d could significantly inhibit the above changes induced by ox-LDL(P<0.05).Conclusion:Let-7d protects HUVECs from ox-LDL induced apoptosis by inhibiting the expression of LOX-1