TRIM52-AS1通过调控miR-28-5p减轻缺氧/复氧诱导的大鼠脑皮层神经元损伤  

TRIM52 antisense RNA 1 alleviates injury of brain cortex neurons induced by hypoxia and re-oxygenation in rats through regulating miR-28-5p expression

作　　者：张广林 李天晓[1] 常晓赞 行君[1] Zhang Guanglin;Li Tianxiao;Chang Xiaozhan;Xing Jun(Cerebrovascular Department,Henan Provincial People’s Hospital Affiliated to Zhengzhou University,Zhengzhou 450000,China)

机构地区：[1]郑州大学附属河南省人民医院脑血管科,郑州450000

出　　处：《中华神经医学杂志》2021年第5期448-457,共10页Chinese Journal of Neuromedicine

摘　　要：目的探讨TRIM52反义RNA1(TRIM52-AS1)对缺氧/复氧(H/R)诱导的大鼠脑皮层神经元损伤的影响及其可能机制。方法(1)将体外培养的大鼠皮层神经元分为对照组和模型组,模型组制备H/R诱导的细胞损伤模型,采用实时定量PCR(RT-qPCR)检测2组神经元TRIM52-AS1、微小RNA(miR)-28-5p的表达。(2)将皮层神经元分为对照组、模型组、TRIM52-AS1小干扰RNA(si-TRIM52-AS1)转染组和无义序列转染组,后2组细胞分别转染si-TRIM52-AS1及其无义对照序列,转染6h后,制备H/R诱导的细胞损伤模型。采用RT-qPCR、细胞计数试剂盒-8(CCK-8)、流式细胞仪分别检测4组神经元TRIMS2-AS1的表达及细胞增殖、凋亡情况;采用酶联免疫吸附实验(ELISA),Westerm blotting分别检测培养上清中乳酸脱氢酶(LDH)、细胞中丙二醛(MDA)和超氧化物歧化酶(SOD)含量和Cyclin D1、Bcl-2、Bax蛋白的表达。(3)将皮层神经元分为miR-28-Sp转染组和无义序列转染组,分别转染miR-28-5p模拟物及其无义对照序列、转染6h后,均制备H/R诱导的细胞损伤模型。采用RT-qPCR、CCK-8、流式细胞仪分别检测2组神经元miR-28-5p的表达及细胞增殖、调亡情况;采用ELISA及Western boting分别检测培养上清中LDH、细胞中MDA、SOD含量和Cyclin D1、Bcl-2、Bax蛋白的表达。(4)将皮层神经元分别分为野生型TRIM52-ASI+miR-28-5p模拟物转染组野生型TRIM52-AS1+miR-28-5p无义对照序列转染组和突变型TRIM52-AS1+miR-28-5p模拟物转染组.突变型TRIM52-AS1+miR-28-5p无义对照序列转染组。转染6h后换新鲜培养液继续培养12h后,采用双荧光素酶报告基因实验检测各组细胞的荧光素酶活性。(5)将皮层神经元分为无义序列转染组、miR-28-5p抑制物转染组、无义序列+si-TRIM52-AS1转染组、miR-28-5p抑制物+si-TRIM52-AS1转染组,分别转染miR-28-Sp抑制物无义对照序列、miR-28-5p抑制物、miR-28-5p抑制物无义对照序列+si-TRIM52-ASI、miR-28-5p抑制物+si-TRIM52-AS1.转�Objective To investigate the effect of TRIM52 antisense RNA 1(TRIM52-AS1)on injury of brain cortex neurons induced by hypoxia/re-oxygenation(H/R)and its possible mechanism in rats.Methods(1)The cortical neurons were cultured in vitro and divided into control group and model group.In the model group,H/R-induced cell injury models were prepared.Real-time quantitative PCR(RT-qPCR)was used to detect the expressions of TR1M52-AS1 and micro RNA(miR)-28-5p in neurons of the two groups.(2)The cortical neurons were divided into control group,model group,small interfering(si)-TRIM52-AS1 transfection group,and nonsense sequence transfection group.Cells in the model group were prepared for H/R-induccd cell damage models.After cells in the latter two groups were transfected with si-TRIM52-ASl or its nonsense control sequence for 6 h.they were prepared for H/R-induced cell damage models.RT-qPCR,CCK-8,and flow cytometry were used to detect the TRIM52-ASI expression,and proliferation and apoptosis of neurons in the 4 groups;enzyme-linked immunosorbent assay(ELISA)and Western blotting were used to detect the lactate dehydrogenase(LDH),malondialdchyde(MDA)and superoxide dismutase(SOD)levels and protein expressions of Cyclin D1,Bcl-2 and Bax.(3)The cortical neurons were divided into tniR-28-5p transfection group and nonsense sequence transfection group.After the cells were respectively transfected with miR-28-5p or its nonsense control sequence for 6 h.they were prepared for H/R-induced cell damage models.RT-qPCR,CCK-8,and flow cytometry were used to detect the TRIM52-AS1 expression,and proliferation and apoptosis of neurons in the two groups;ELISA and Western blotting were used to detect the LDH,MDA and SOD levels and protein expressions of Cyclin D1,Bcl-2 and Bax.(4)The cortical neurons were divided into wild-type TRIM52-AS1+miR-28-5p mimic transfection group,wild-type TRIM52-AS1+miR-28-5p nonsense control sequence transfection group,mutant TRIM52-AS1+miR-28-5p mimic transfection group,and mutant TRIM52-ASI+miR-28-5p nonsense con

关 键 词：神经元 缺氧/复氧 TRIM52-AS1 miR-28-5p 氧化应激 细胞凋亡 

分 类 号：R743.3[医药卫生—神经病学与精神病学]