室旁核微小RNA 7a-5p在自发性高血压大鼠交感神经兴奋亢进中的作用及机制  

Role and mechanism of microRNA-7a-5p in paraventricular nucleus on sympathetic overactivity in the spontaneously hypertensive rats

作　　者：安美玲 徐佳琪 郝翊帆 袁月 王秀文 张敏 未晓巍[1,2] 李华[1] 王仁俊[1] AN Mei-ling;XU Jia-qi;HAO Yi-fan;YUAN Yue;WANG Xiu-wen;ZHANG Min;WEI Xiao-wei;LI Hua;WANG Ren-jun(College of Life Science,Jilin Normal University,Jilin Normal University,Siping,Jilin 136000,China;Jilin Provincial Key Laboratory of Plant Resource Science and Green Production,Jilin Normal University,Siping,Jilin 136000,China)

机构地区：[1]吉林师范大学生命科学学院,吉林四平136000 [2]吉林师范大学植物资源科学与绿色生产吉林省重点实验室,吉林四平136000

出　　处：《中华高血压杂志》2021年第12期1201-1214,共14页Chinese Journal of Hypertension

基　　金：国家自然科学基金项目(31871150)。

摘　　要：目的探讨下丘脑室旁核内微小RNA 7a-5p(miR-7a-5p)在自发性高血压大鼠(SHR)交感神经兴奋亢进中的作用及机制。方法取12周龄正常血压大鼠(WKY)和SHR各24只,分别随机分为4组,每组6只:室旁核注射miR-7a-5p-空载病毒(Null)组、过表达miR-7a-5p基因重组腺相关病毒(rAAV)+敲减miR-7a-5p基因rAAV阴性对照(miR-7a-5p+NC antimiR-7a-5p)组、过表达miR-7a-5p基因rAAV+敲减miR-7a-5p基因rAAV(miR-7a-5p+antimiR-7a-5p)组、敲减miR-7a-5p基因rAAV+过表达miR-7a-5p基因rAAV阴性对照(antimiR-7a-5p+NC miR-7a-5p)组;另取12周龄WKY大鼠和SHR各24只,分别随机分为4组,每组6只:室旁核注射γ-氨基丁酸A型受体功能性α_(1)亚基(GABRA1)-Null组、过表达GABRA1基因rAAV+敲减GABRA1基因rAAV阴性对照(GABRA1+NC siGABRA1)组、过表达GABRA1基因rAAV+敲减GABRA1基因rAAV(GABRA1+siGABRA1)组和敲减GABRA1基因rAAV+过表达GABRA1基因rAAV阴性对照(siGABRA1+NC GABRA1)组,4周后观察血流动力学指标、肾交感神经活动(RSNA)和血浆去甲肾上腺素(NE)水平变化;采用免疫荧光检测室旁核内原癌基因AP-1转录因子亚基FosB(FosB)及GABRA1阳性神经元数量;实时荧光定量聚合酶链反应(qRT-PCR)法检测室旁核内miR-7a-5p和GABRA1mRNA表达水平;免疫印迹法检测室旁核内GABRA1蛋白表达量;分离培养新生Wistar大鼠原代下丘脑神经细胞和传代培养NG108神经细胞系,并转染miR-7a-5p激动剂(agomiR-7a-5p)、抑制剂(antagomiR-7a-5p)或miR-7a-5p激动剂阴性对照(NC agomiR-7a-5p),48h后检测miR-7a-5p基因表达水平和GABRA1蛋白表达量,TargetScan软件预测和双荧光素酶报告系统检测miR-7a-5p和GABRA1的靶向关系。结果室旁核注射rAAV 4周后,过表达miR-7a-5p导致WKY和SHR的RSNA增强(均P<0.05),FosB阳性神经元数量上调,GABRA1阳性神经元数量下调(均P<0.01)。室旁核过表达GABRA1导致WKY和SHR的RSNA减弱(均P<0.05)。与WKY大鼠相比,过表达或敲减miR-7a-5p/GABRA1基因对SHR的RSNA的影响更显�Objective To investigate the role and mechanism of microRNA-7a-5p(miR-7a-5p)in paraventricular nucleus on sympathetic overactivity in the spontaneously hypertensive rats.Methods Twenty-four Wistar-Kyoto(WKY)rats and 24SHR aged 12weeks were randomly divided into 4groups with 6rats in each group,including miR-7a-5p-the empty viral vector(Null)group,recombinant adeno-associated virus(rAAV)overexpressing miR-7a-5p gene+negative control of rAAV knocking down miR-7a-5p gene(miR-7a-5p+NC antimiR-7a-5p)group,rAAV overexpressing miR-7a-5p gene+rAAV knocking down miR-7a-5p gene(miR-7a-5p+antimiR-7a-5p)group,rAAV knocking down miR-7a-5p gene+negative control of rAAV overexpressing miR-7a-5p gene(antimiR-7a-5p+NC miR-7a-5p)group.In addition,24WKY rats and 24SHR aged 12weeks were randomly divided into 4groups with 6rats in each group,including α_(1) subunit ofγ-aminobutyric acid A receptor(GABRA1)-Null group,rAAV overexpressing GABRA1 gene+negative control of rAAV knocking down GABRA1 gene(GABRA1+NC siGABRA1)group,rAAV overexpressing GABRA1gene+rAAV knocking down GABRA1gene(GABRA1+si-GABRA1)group and rAAV knocking down GABRA1gene+negative control of rAAV overexpressing GABRA1 gene(siGABRA1+NC GABRA1)group.Four weeks later,hemodynamic parameters,renal sympathetic nerve activity(RSNA)and plasma norepinephrine(NE)levels were observed.The number of FosB and GABRA1positive neurons in paraventricular nucleus was measured by immunofluorescence.Quantitative real time polymerase chain reaction was used to detect the expression levels of miR-7a-5p and GABRA1mRNA in paraventricular nucleus.The expression level of GABRA1protein in paraventricular nucleus was detected by Western blot.The primary neurons of neonatal Wistar rats were isolated and cultured.The NG108neural cell line were subcultured.Cells were treated as following,transfected with agomiR-7a-5p,antagomiR-7a-5p or agomiR-7a-5p negative control.After 48htransfection,the expression level of miR-7a-5p gene and GABRA1protein expression were detected.The target relationship betwee

关 键 词：自发性高血压大鼠 交感神经兴奋 室旁核 微小RNA 7a-5p γ-氨基丁酸A型受体α_(1)亚基 

分 类 号：R544.1[医药卫生—心血管疾病]