circUCK2靶向miR-483-5p调控oxLDL诱导的血管内皮细胞氧化应激和细胞凋亡机制研究

Effects of circUCK2 on oxidative stress and apoptosis of vascular endothelial cells induced by oxLDL and its molecular mechanism

目的探讨circUCK2对oxLDL诱导的血管内皮细胞氧化应激和细胞凋亡的影响及分子机制。方法人脐静脉血管内皮细胞(HUVEC)分为对照(Con)组、氧化低密度脂蛋白(oxLDL)组、oxLDL+pcDNA组、oxLDL+pcDNA-circUCK2组、oxLDL+anti-miR-NC组、oxLDL+anti-miR-483-5p组、oxLDL+pcDNA-circUCK2+miR-NC组、oxLDL+pcDNA-circUCK2+miR-483-5p组。实时荧光定量PCR(RT-qPCR)检测circUCK2和miR-483-5p的表达水平;试剂盒检测细胞丙二醛(MDA)含量、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活性;流式细胞术检测细胞凋亡;蛋白质印迹(Western blot)法检测蛋白表达;双荧光素酶报告实验检测circUCK2和miR-483-5p的靶向关系。结果oxLDL诱导的血管内皮细胞中circUCK2表达水平降低,miR-483-5p表达水平升高,MDA含量升高,SOD、GSH-Px活性降低,细胞凋亡率升高,Bcl-2蛋白表达水平降低,Bax蛋白表达水平升高(P<0.05)。过表达circUCK2或抑制miR-483-5p表达,oxLDL诱导的血管内皮细胞中MDA含量降低,SOD、GSH-Px活性升高,细胞凋亡率降低,Bcl-2蛋白表达水平升高,Bax蛋白表达水平降低(P<0.05)。StarBase预测发现circUCK2的序列中含有与miR-483-5p互补的核苷酸序列;WT-circUCK2与miR-483-5p共转染后的血管内皮细胞荧光素酶活性降低,而MUT-circUCK2与miR-483-5p共转染后的血管内皮细胞荧光素酶活性无显著变化(P>0.05)。过表达circUCK2后miR-483-5p表达水平降低;抑制circUCK2表达后miR-483-5p表达水平升高(P<0.05)。与oxLDL+anti-miR-NC组比较,oxLDL+anti-miR-483-5p组血管内皮细胞中miR-483-5p表达水平降低,MDA含量降低,SOD、GSH-Px活性升高,细胞凋亡率降低,Bcl-2蛋白表达水平升高,Bax蛋白表达水平降低(P<0.05)。与oxLDL+pcDNA-circUCK2+miR-NC组比较,oxLDL+pcDNA-circUCK2+miR-483-5p组血管内皮细胞中miR-483-5p表达水平升高,MDA含量升高,SOD、GSH-Px活性降低,细胞凋亡率升高,Bcl-2蛋白表达水平降低,Bax蛋白表达水平升高(P<0.05)。结论过表达circUCK2可通过靶向下调miR-483-5p抑制oxLDL诱导的血管内皮细胞氧化应激和细胞凋亡。

Objective To investigate the effects of circUCK2 on oxidative stress and apoptosis of vascular endothelial cells induced by oxLDL,and to explore its molecular mechanism.Methods Human umbilical vein endothelial cells(HUVEC)were divided into control control group,oxidized low-density lipoprotein(oxLDL)group,oxLDL+pcDNA group,oxLDL+pcDNA-circUCK2 group,oxLDL+anti-miR-NC group,oxLDL+anti-miR-483-5p group,oxLDL+pcDNA-circUCK2+miR-NC group,oxLDL+pcDNA-circUCK2+miR-483-5p group.Real-time fluorescent quantitative PCR(RT-qPCR)was used to detect the expression levels of circUCK2 and miR-483-5p;kit to detect cell MDA content,SOD and GSH-Px activity;flow cytometry to detect cell apoptosis;Western Blot to detect protein expression;dual luciferase reporter experiment to detect the targeting correlation between circUCK2 and miR-483-5p.Results The expression levels of circUCK2 in vascular endothelial cells induced by oxLDL were decreased,and the activities of SOD and GSH-Px as well as the expression levels of Bcl-2 protein were significantly decreased,howevere,the expression levels of miR-483-5p,and the content of MDA,cell apoptosis rate and The expression levels of Bax protein were significantly increased(P<0.05).After overexpression of circUCK2 or inhibition of miR-483-5p expression,the MDA content and cell apoptosis rate as well as Bax protein expression were significantly decreased,however,the SOD levels,GSH-Px activity as well as Bcl-2 protein expression were significantly increased(P<0.05).The circUCK2 targeted and regulated miR-483-5p,and overexpression of circUCK2 reversed the effects on the oxidative stress and apoptosis in vascular endothelial cells induced by oxLDL.Conclusion The overexpression of circUCK2 can inhibit the oxidative stress and apoptosis of vascular endothelial cells induced by oxLDL by targetedly down-regulating the expression of miR-483-5p.