miR-199b-5p对阿霉素诱导足细胞凋亡的影响及机制  

Effect and mechanism of miR-199b-5p on adriamycin-induced podocyte apoptosis

作　　者：吴静漪 WU Jingyi(Department of Pediatrics,Jiangsu Shengze Hospital,Suzhou 215228,China)

机构地区：[1]江苏盛泽医院儿科,江苏苏州215228

出　　处：《山东医药》2023年第7期25-29,共5页Shandong Medical Journal

基　　金：苏州市科技局民生科技项目(SYSD2018043)。

摘　　要：目的 探讨miR-199b-5p对阿霉素诱导足细胞凋亡的影响及机制。方法 体外培养小鼠足细胞,用阿霉素构建足细胞凋亡模型。将足细胞随机分成对照组、阿霉素组、阿霉素+miR-199b-5p mimic组、阿霉素+NC mimic组、阿霉素+miR-199b-5p inhibitor组、阿霉素+NC inhibitor组、阿霉素+miR-199b-5p mimic+Ad-脂肪细胞质膜相关蛋白(APMAP)组、阿霉素+miR-199b-5p mimic+Ad-NC组、阿霉素+miR-199b-5p inhibitor+Ad-APMAP RNAi组和阿霉素+miR-199b-5p inhibitor+Ad-NC RNAi组,分别给予miR-199b-5p mimic/inhibitor、过表达/敲低APMAP腺病毒转染,使miR-199b-5p过表达/沉默表达以及APMAP过表达/沉默表达。用实时荧光定量PCR检测细胞内miR-199b-5p及足细胞标志蛋白(Podocin)、凋亡相关基因(Bax、Bcl-2、Cleaved caspase-3)、APMAP mRNA表达,用Western blotting法检测细胞内Podocin、Bax、Bcl-2、Cleaved caspase-3、APMAP及NF-κB信号通路相关蛋白(NIK、TRAF3)表达。用生物信息学分析miR-199b-5p的特异性下游靶基因;将细胞随机分为NC mimic+WT-APMAP组、miR-199b-5p+WT-APMAP组、NC mimic+MUT-APMAP组、miR-199b-5p+MUT-APMAP组,用双荧光素酶报告基因实验验证miR-199b-5p的直接靶基因。结果 与对照组比较,阿霉素组miR-199b-5p、Bax、Cleaved caspase-3、NIK相对表达量高(P均<0.05),Podocin、Bcl-2、APMAP、TRAF3相对表达量低(P均<0.05)。与阿霉素+NC mimic组比较,阿霉素+miR-199b-5p mimic组Podocin、Bcl-2、APMAP、TRAF3相对表达量低,Bax、Cleaved caspase-3、NIK相对表达量高(P均<0.05)。与阿霉素+NC inhibitor组比较,阿霉素+miR-199b-5p inhibitor组Podocin、Bcl-2、APMAP、TRAF3相对表达量高,Bax、Cleaved caspase-3、NIK相对表达量低(P均<0.05)。双荧光素酶报告基因实验证实,与NC mimic+MUT-APMAP组比较,miR-199b-5p+WT-APMAP组APMAP荧光素酶活性低(P<0.05)。与阿霉素+miR-199b-5p mimic+Ad-NC组比较,阿霉素+miR-199b-5p mimic+Ad-APMAP组TRAF3相对表达量高,NIK相对表达量低(P均<0.05)。与�Objective To explore the role and mechanism of miR-199b-5p in adriamycin-induced podocyte apoptosis. Methods Podocytes were cultured in vitro. Podocyte apoptosis model was induced by adriamycin(Adr) treatment.Podocytes were randomly divided into the control group, adriamycin group, adriamycin+miR-199b-5p mimic group, adriamycin+NC mimic group, adriamycin+miR-199b-5p inhibitor group, adriamycin+NC inhibitor group, adriamycin+miR-199b-5p mimic+Ad-APMAP group, adriamycin+miR-199b-5p mimic+Ad NC group, adriamycin+miR-199b-5p inhibitor+Ad-APMAP RNAi group, and adriamycin+miR-199b-5p inhibitor+Ad-NC RNAi group, respectively. They were transfected with miR-199b-5p mimic/inhibitor, overexpression/knockdown of recombinant adipocyte plasma membrane-associated protein(APMAP) adenovirus to induce the miR-199b-5p overexpression/silencing expression and APMAP overexpression/silencing expression. The expression levels of miR-199b-5p, podocyte marker protein(Podocin), apoptosis-related genes(Bax, Bcl-2, Cleared caspase-3) and APMAP mRNA in cells were detected by real-time quantitative PCR, and the expression levels of Podocin, Bax, Bcl-2, Cleared caspase-3, APMAP, and nuclear factor-κB(NF-κB)-associated protein TNF receptor-associated factor 3(TRAF3) and NF-κB inducing kinase(NIK) in cells were detected by Western blotting. The specific downstream target gene of miR-199b-5p was analyzed by bioinformatics. The cells were randomly divided into the NC mimic+WT-APMAP group, miR-199b-5p+WT-APMAP group, NC mimic+MUT-APMAP group, and miR-199b-5p+MUT-APMAP group. The direct target gene of miR-199b-5p was verified by double luciferase reporter gene experiment. Results Compared with the control group, the relative expression levels of miR-199b-5p, Bax, Cleared caspase-3 and NIK in the adriamycin group were higher(all P<0. 05), and the relative expression levels of Podocin, Bcl-2, APMAP and TRAF3 were lower(all P<0. 05). Compared with the adriamycin+NC mimic group, the relative expression levels of Podocin, Bcl-2, APMAP and TRAF3 in adr

关 键 词：慢性肾脏病 足细胞凋亡 微小RNA-199b-5p 阿霉素 脂肪细胞质膜相关蛋白 

分 类 号：R692[医药卫生—泌尿科学]