摘要
目的探讨微小RNA-34b-3p(miR-34b-3p)和Kelch样环氧氯丙烷相关蛋白(Keap1)对脑缺血再灌注氧化应激损伤的作用及调控机制。方法制备脑缺血再灌注(I/R)动物模型,实时定量PCR(qPCR)检测miR-34b-3p的表达。I/R动物模型大脑中注射miR-34b-3p慢病毒,检测miR-34b-3p对氧化应激的影响。免疫印迹(WB)和萤光素酶报告基因检测miR-34b-3p对Keap1表达的调控。B35细胞中过表达或干扰Keap1,检测Keap1对氧化应激损伤的影响。B35细胞中共表达Keap1和miR-34b-3p,检测miR-34b-3p对Keap1导致的氧化应激的影响。结果miR-34b-3p在I/R动物模型大脑中表达降低,I/R动物模型大脑中过表达miR-34b-3p,使3-硝基酪氨酸(3-NT)、一氧化氮(NO)含量降低,总超氧化物歧化酶(SOD)和锰超氧化物歧化酶(MnSOD)含量升高,表明miR-34b-3p减弱氧化应激。动物模型和细胞中过表达miR-34b-3p减少Keap1蛋白表达,增加核因子E2相关因子2(Nrf2)和血红素氧合酶1(HO-1)蛋白表达。萤光素酶报告基因结果显示miR-34b-3p可靶向Keap13'非翻译区(3'-UTR)。细胞中过表达Keap1增强氧化应激,而干扰Keap1则结果相反。共表达实验表明,miR-34b-3p减弱了Keap1导致的氧化应激。结论miR-34b-3p通过调控Keap1表达减轻脑缺血再灌注氧化应激损伤。
Objective To investigate the effect of miR-34b-3p and Keap1 on oxidative stress damage induced by cere⁃bral ischemia-reperfusion and its regulatory mechanism.Method Incerebral ischemia reperfusion(I/R)mice model was established.The expression of miR-34b-3p was detected by qPCR.The effect of miR-34b-3p on oxidative stress was detected in I/R mice injected miR-34b-3p lentivirus.The expression of Keap1 regulated by miR-34b-3p was detected by both western blot and dual-luciferase reporter assay.The effect of Keap1 on oxidative stress was detected through overexpression or interference of Keap1 in B35 cells.The effect of miR-34b-3p on oxidative stress was detect⁃ed through induced by Keap1 was detected by the co-expression of Keap1 and miR-34b-3p in B35 cells.Results The expression of miR-34b-3p was decreased in the I/R mice model.Overexpression of miR-34b-3p decreased the contents of 3-NT and NO,while increased the contents of total SOD and MnSOD,indicating that miR-34b-3p alleviat⁃ed oxidative stress.Overexpression of miR-34b-3p decreased the expression of Keap1 and increased the expression of Nrf2 and HO-1 in mice and cells simultaneously.Dual-luciferase reporter assay results showed that miR-34b-3p tar⁃geted the 3'-UTR of Keap1.Overexpression of Keap1 enhanced oxidative stress,while interference with Keap1 had the opposite effect.Co-expression experiments showed that miR-34b-3p attenuated oxidative stress induced by Ke⁃ap1.Conclusion miR-34b-3p attenuates cerebral I/R oxidative stress injury by down-regulating the expression of Keap1.
作者
刘晓蓓
马娟
牛博
李晋
常剑
刘鹏
陈婷
刘硕
黄荣
LIU Xiaobei;MA Juan;NIU Bo;LI Jin;CHANG Jian;LIU Peng;CHEN Ting;LIU Shuo;HUANG Rong(CT/MRT Room,the Fifth Affiliated Hospital of Xinjiang Medical University,Urumqi 830054,China;Department of Neurosurgery,the First Affiliated Hospital of Xinjiang Medical University,Urumqi 830011,China;Department of Neurosurgery,the Fifth Affiliated Hospital of Xinjiang Medical University,Urumqi 830054,China)
出处
《新疆医科大学学报》
CAS
2021年第10期1114-1120,共7页
Journal of Xinjiang Medical University
基金
新疆维吾尔自治区自然科学基金(2018D01C302)。
