摘要
为了探讨CircRNA ANKRD36对脓毒症血管内皮细胞凋亡和氧化应激的影响及可能机制,该研究将CircRNA ANKRD36小干扰RNA、miR-127-5p模拟物或CircRNA ANKRD36小干扰RNA和miR-127-5p抑制剂转染至人脐静脉血管内皮细胞中,然后用1.0 μg/mL LPS干预转染后的细胞24 h,RT-qPCR法检测细胞中CircRNA ANKRD36和miR-127-5p的表达情况,流式细胞术检测细胞凋亡,蛋白印迹法检测细胞中Cleaved-Caspase-3蛋白表达水平,DCFH-DA探针法检测ROS水平,硫代巴比妥酸法检测细胞中MDA含量,黄嘌呤氧化酶法检测细胞中SOD活性。此外,用双荧光素酶报告基因实验验证了CircRNA ANKRD36和miR-127-5p的调控关系。结果显示,LPS可促进血管内皮细胞中CircRNA ANKRD36的表达(P<0.05),而抑制miR-127-5p表达(P<0.05);下调CircRNA ANKRD36或上调miR-127-5p可降低LPS诱导的血管内皮细胞凋亡率、细胞中Cleaved-Caspase-3蛋白表达水平、ROS水平及MDA含量(P<0.05),提高细胞中SOD活性(P<0.05)。CircRNA ANKRD36可靶向结合并负调控miR-127-5p。下调miR-127-5p可逆转下调CircRNA ANKRD36对LPS诱导的血管内皮细胞凋亡及氧化应激的影响。这提示,下调CircRNA ANKRD36可能通过靶向上调miR-127-5p抑制LPS诱导的血管内皮细胞凋亡及氧化应激反应,CircRNA ANKRD36/miR-127-5p轴可能为脓毒症的治疗提供了新的分子靶点。
In order to explore the effect and possible mechanism of CircRNA ANKRD36 on the apoptosis and oxidative stress of vascular endothelial cells in sepsis,CircRNA ANKRD36 small interfering RNA,miR-127-5p mimic or CircRNA ANKRD36 small interfering RNA and miR-127-5p inhibitor were transfected into human umbilical vein endothelial cells,and then 1.0 μg/mL LPS was used to intervene the transfected cells for 24 h.RT-qPCR method was used to detect the expression of CircRNA ANKRD36 and miR-127-5p in the cells.Flow cytometry was used to detect cell apoptosis.Western blot was used to detect the protein expression of Cleaved-Caspase-3 in cells.DCFH-DA probe method was used to detect the level of ROS.Thiobarbituric acid method was used to detect the content of MDA in cells.Xanthine oxidase method was used to detect the activity of SOD in cells.In addition,the dual luciferase reporter gene experiment verified the regulatory relationship between CircRNA ANKRD36 and miR-127-5p.The results showed that LPS promoted the expression of CircRNA ANKRD36 in vascular endothelial cells (P<0.05),but it inhibited the expression of miR-127-5p (P<0.05).Down-regulating CircRNA ANKRD36 or up-regulating miR-127-5p reduced LPS-induced vascular endothelial cells apoptosis,the expression of Cleaved-Caspase-3 protein expression,the level of ROS and the content of MDA in cells (P<0.05),but increased the activity of SOD in cells (P<0.05).CircRNA ANKRD36 could target to regulate the expression of miR-127-5p.Down-regulating miR-127-5p reversed the effect of down-regulating CircRNA ANKRD36 on LPS-induced vascular endothelial cell apoptosis and oxidative stress.This suggested that down-regulating CircRNA ANKRD36 might inhibit LPS-induced vascular endothelial cell apoptosis and oxidative stress by up-regulating the expression of miR-127-5p.CircRNA ANKRD36/miR-127-5p axis may provide new molecular targets for the treatment of sepsis.
作者
郭飞波
吴斌
GUO Feibo;WU Bin(Department of Clinical Laboratory,the First Peoples Hospital of Tianmen,Tianmen 431700,China;Department of Clinical Laboratory,Wuhan Hospital of Integrated Chinese And Western Medicine,Wuhan 430022,China)
出处
《中国细胞生物学学报》
CAS
CSCD
2021年第9期1756-1766,共11页
Chinese Journal of Cell Biology
