室旁核miR-9-5p通过靶向KCNN3介导T2D大鼠交感神经过度兴奋  

MicroRNA-9-5p mediates sympathetic overactivity by targeting KCNN3 in paraventricular nucleus of rats with T2D

作　　者：徐佳琪 安美玲 袁月 郝翊帆 王秀文 张敏 未晓巍[1,2] 李华[1] 王仁俊[1] XU Jia-qi;AN Mei-ling;YUAN Yue;HAO Yi-fan;WANG Xiu-wen;ZHANG Min;WEI Xiao-wei;LI Hua;WANG Ren-jun(College of Life Science,Jilin Normal University,Siping 136000,China;Jilin Provincial Key Laboratory of Plant Resource Science and Green Production,Jilin Normal University,Siping 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：《中国病理生理杂志》2020年第12期2148-2158,共11页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.31871150)。

摘　　要：目的:探究下丘脑室旁核(paraventricular nucleus,PVN)内微小RNA-9-5p(microRNA-9-5p,miR-9-5p)通过靶向小电导钙激活钾通道3(small-conductance calcium-activated potassium channel 3,SK3)蛋白的编码基因KCNN3(potassium intermediate/small conductance calcium-activated channel,subfamily N,member 3)介导2型糖尿病(type 2 diabetes mellitus,T2D)大鼠交感神经过度兴奋的作用机制。方法:高脂饮食配合腹腔注射链脲佐菌素制备T2D大鼠模型;real-time PCR检测miR-9-5p和KCNN3 mRNA的水平;TargetScan软件预测miR-9-5p与KCNN3的靶向关系;PVN内过表达或敲减miR-9-5p和KCNN3基因,观察血糖及交感驱动指标的变化;免疫荧光和Western blot检测FosB和SK3的蛋白表达量;细胞转染和双萤光素酶报告基因实验验证miR-9-5p与KCNN3的靶向关系。结果:与糖尿病对照(diabetes control,DC)组相比,糖尿病(diabetes mellitus,DM)大鼠的血糖和甘油三酯水平升高,交感神经活动增强,PVN内miR-9-5p上调,而SK3蛋白下调。PVN内微量注射miR-9-5p致大鼠交感驱动指标、血糖水平及FosB阳性神经元数量升高,SK3蛋白下调;而PVN内过表达KCNN3则产生相反的作用。与DC组相比,PVN内过表达或敲减miR-9-5p和KCNN3基因对DM大鼠的影响更显著。细胞转染和双萤光素酶报告基因实验证实miR-9-5p靶向调控KCNN3基因。结论:T2D大鼠PVN内上调的miR-9-5p可能通过靶向抑制SK3蛋白表达而介导交感神经兴奋亢进。AIM:To investigate whether microRNA-9-5p(miR-9-5p)mediates sympathetic overactivity by targeting KCNN3(potassium intermediate/small conductance calcium-activated channel,subfamily N,member 3)gene,which encoded small-conductance calcium-activated potassium channel 3(SK3)protein,in paraventricular nucleus(PVN)of rats with type 2 diabetes mellitus(T2D).METHODS:A rat model of T2D was established by high-fat diet combined with intraperitoneal injection of 30 mg/kg streptozotocin.The levels of miR-9-5p and KCNN3 mRNA in PVN were detected by real-time PCR.The relationship between KCNN3 and miR-9-5p was predicted by TargetScan.Recombinant adeno-associated virus(rAAV)-miR-9-5p or KCNN3 were bilaterally microinjected into the PVN to observe the changes in plasma glucose levels and sympathetic drive indicators.The number of FosB and SK3 positive cells was measured by immunofluorescence staining.The protein expression of SK3 was determined by Western blot.The relationship between KCNN3 and miR-9-5p were confirmed by cell transfection and dual-luciferase reporter assay.RESULTS:Compared with the rats in diabetes control(DC)group,the blood glucose,sympathetic drive indexes and the level of miR-9-5p in PVN were significantly increased,while the SK3 expression in PVN was obviously reduced in the diabetes mellitus(DM)rats.After microinjecion of rAAV-miR-9-5p in PVN,the sympathetic drive indexes,blood glucose,and the number of FosB-positive cells were increased significantly,but the SK3 protein expression was significantly reduced(P<0.05).However,up-regulation of KCNN3 in PVN had the opposite effect.These responses were obviously enhanced in DM rats compared with DC rats.The results of cell transfection and dual-luciferase reporter assay demonstrated that miR-9-5p bound to the 3’-UTR of KCNN3 and inhibit its expression.CONCLUSION:miR-9-5p was up-regulated in PVN of the rats with T2D,and it may mediate sympathoexcitation by targeting KCNN3.

关 键 词：2型糖尿病 交感神经兴奋 室旁核 微小RNA-9-5p 小电导钙激活钾通道 

分 类 号：R587.1[医药卫生—内分泌] R363.2[医药卫生—内科学]