circSEC24A靶向miR-152-3p调控ox-LDL诱导的人脑微血管内皮细胞损伤

circSEC24A regulats ox-LDL-induced human brain microvascular endothelial cell damage by targeting miR-152-3p

目的探讨circSEC24A对氧化型低密度脂蛋白(ox-LDL)诱导的人脑微血管内皮细胞损伤的影响及其可能作用机制。方法用ox-LDL诱导人脑微血管内皮细胞HBMEC建立细胞氧化损伤模型,用脂质体转染法将si-NC、si-circSEC24A、miR-NC、miR-152-3p mimics、si-circSEC24A与anti-miR-NC、si-circSEC24A与anti-miR-152-3p分别转染至HBMEC后用ox-LDL处理24h;qRT-PCR法检测circSEC24A和miR-152-3p的表达量;利用试剂盒检测MDA的水平与SOD、CAT的活性;流式细胞术检测细胞凋亡率;双荧光素酶报告实验验证circSEC24A与miR-152-3p的靶向调控作用。Western blot法检测cleaved-caspase3、cleaved-caspase9蛋白表达量。结果ox-LDL诱导的HBMEC中circSEC24A的表达量升高(P<0.05),MDA的水平、细胞凋亡率和cleaved-caspase3、cleaved-caspase9蛋白水平升高(P<0.05),miR-152-3p的表达量降低(P<0.05),SOD、CAT的活性降低(P<0.05);转染si-circSEC24A或转染miR-152-3p mimics均可降低ox-LDL诱导的HBMEC中MDA的水平(P<0.05),并可降低细胞凋亡率和cleaved-caspase3、cleaved-caspase9蛋白水平(P<0.05),而SOD、CAT的活性升高(P<0.05);circSEC24A靶向调控miR-152-3p的表达;共转染si-circSEC24A与anti-miR-152-3p后MDA的水平升高(P<0.05),细胞凋亡率和cleaved-caspase3、cleavedcaspase9蛋白水平升高(P<0.05),而SOD、CAT的活性降低(P<0.05)。结论干扰circSEC24A表达可靶向调控miR-152-3p表达而抑制细胞氧化应激及凋亡从而减轻ox-LDL诱导的人脑微血管内皮细胞损伤。

Objective To explore the effect of circSEC24Aon human brain microvascular endothelial cell injury induced by ox-LDL and its possible mechanism.Methods Human brain microvascular endothelial cells HBMEC were induced with ox-LDL to establish a cellular oxidative damage model,and si-NC,si-circSEC24A,miR-NC,miR-152-3p mimics,si-circSEC24Aand anti-miR-NC,si-circSEC24Aand anti-miR-152-3p were transfected into HBMEC and treated with ox-LDL for 24h.qRT-PCR method was used to detect the expression levels of circSEC24Aand miR-152-3p.The level of MDA and the activity of SOD and CAT were tested using kits.Flow cytometry was used to detect the rate of apoptosis.The targeted regulation of circSEC24Aand miR-152-3p was verified by dual luciferase report assay.Western blot was used to detect the protein expression of cleaved-caspase3and cleaved-caspase9.Results The expression of circSEC24Ain ox-LDL-induced HBMEC was increased(P<0.05),the level of MDA,the apoptosis rate and the protein levels of cleaved-caspase3,cleaved-caspase9were increased(P<0.05),while the expression of miR-152-3p was decreased(P<0.05),and the activities of SOD,CAT were decreased(P<0.05)..Transfection of si-circSEC24Aor miR-152-3p mimics could reduce the level of MDA in HBMEC induced by ox-LDL(P<0.05),and could reduce the apoptosis rate and the protein levels of cleaved-caspase3,cleaved-caspase9(P<0.05),while the activities of SOD and CAT were increased(P<0.05).circSEC24Acould target and regulate the expression of miR-152-3p.After co-transfection of si-circSEC24Aand anti-miR-152-3p,the level of MDA was increased(P<0.05),the apoptosis rate and the protein levels of cleaved-caspase3,cleaved-caspase9were increased(P<0.05),while the activities of SOD and CAT were decreased(P<0.05).Conclusion Interfering with circSEC24Acould inhibit cell oxidative stress and apoptosis by targeting miR-152-3p,thereby reducing ox-LDLinduced human brain microvascular endothelial cells damage.