摘要
目的:探讨miR-223抑制β-淀粉样蛋白1-42(Aβ_(1-42))诱导海马神经元凋亡的分子机制。方法:体外培养新生SD大鼠的海马神经元,随机分为空白组(不作任何转染处理)、阴性组(转染miR-NC)、miR-223-m组(转染miR-223 mimics)、miR-223-m+LV组(转染miR-223 mimics+慢病毒骨架)、miR-223-m+LV-RhoB组(转染miR-223 mimics+RhoB过表达载体)。qRT-PCR和Western blotting检测Aβ_(1-42)诱导的海马神经元中miR-223和RhoB的表达水平;MTT法检测不同浓度Aβ_(1-42)诱导海马神经元的存活率;流式细胞术检测各组细胞的凋亡;双荧光素酶报告基因检测细胞的荧光活性。结果:Aβ_(1-42)可显著抑制海马神经元的存活率,且具有一定的剂量依赖性;同时其可导致海马神经元miR-223相对表达量下降,同时RhoB表达水平升高。选择30μmol·L^(-1) Aβ_(1-42)寡聚肽片段作用48 h用于后续分组实验。经流式细胞术检测,经Aβ_(1-42)30.0μmol·L^(-1)孵育48 h后,与空白组及阴性组相比,miR-223-m组细胞凋亡率降低;而与miR-223-m组及miR-223-m+LV组相比,miR-223-m+LV-RhoB组细胞凋亡率升高(P<0.05)。经Western blotting法检测,与空白组及阴性组相比,miR-223-m组细胞RhoB、Bax和caspase-3蛋白表达降低,同时Bcl-2蛋白表达升高(P<0.05);而与miR-223-m组及miR-223-m+LV组相比,miR-223-m+LV-RhoB组细胞RhoB、Bax和caspase-3蛋白表达升高,同时Bcl-2蛋白表达降低(P<0.05)。双荧光素酶报告基因分析结果显示,miR-223 mimics和野生型RhoB 3′UTR共转染后可抑制荧光素酶活性(P<0.05)。结论:miR-223可通过靶向RhoB抑制Aβ_(1-42)诱导的海马神经元细胞凋亡。
Objective:To investigate the mechanism of miR-223 on the apoptosis of Aβ_(1-42)-induced hippocampal neurons.Methods:The hippocampal neurons of newborn SD rats were cultured in vitro,and randomly divided into blank group(without any transfection),negative group(transfected with miR-NC),miR-223-m group(transfected with miR-223 mimics),miR-223-m+LV group(transfected with miR-223 mimics+lentiviral skeleton),miR-223-m+LV-RhoB group(transfected with miR-223 mimics+Rhob overexpression vector).The expression of miR-223 and RhoB in Aβ_(1-42)-induced hippocampal neurons was detected by qRT-PCR and Western blotting,the survival rate of Aβ_(1-42)-induced hippocampal neurons with different concentrations was detected by MTT assay,and the apoptosis of hippocampal neurons was detected by flow cytometry assay.Dual-luciferase reporter gene was used to detect the fluorescence activity of cells.Results:Aβ_(1-42)could significantly inhibit the survival rate of hippocampal neurons in a dose-dependent manner,and it also decreased the relative expression of miR-223 and increased the expression level of RhoB.30μmol·L^(-1)Aβ_(1-42)oligopeptide fragment was selected for further grouping.After incubated for 48 hours with 30μmol·L^(-1)Aβ_(1-42),the apoptosis rate of the miR-223-m group was lower than that of blank group and negative group,and the apoptosis rate of the miR-223-m+LV-RhoB group was increased compared with the miR-223-m group and the miR-223-m+LV group(P<0.05).In addition,the expression of RhoB protein,Bax and caspase-3 protein was decreased and the expression of Bcl-2 protein was increased in the miR-223-m group compared with the blank group and the negative group(P<0.05),while the expression of Rhob,Bax and caspase-3 protein increased and the expression of Bcl-2 protein decreased in the miR-223-m+LV-RhoB group compared with the miR-223-m group and the miR-223-m+LV group(P<0.05).Dual-Luciferase reporter gene analysis showed that the miR-223 mimics and wild type RhoB 3′UTR could inhibit luciferase activity(P<0.05).Conclusion:miR-223 can inhibit apoptosis of Aβ_(1-42)-induced hippocampal neurons by targeting RhoB.
作者
张峪涵
付晖
刘翠娟
廖成钜
罗昊栋
ZHANG Yuhan;FU Hui;LIU Cuijuan;LIAO Chengju;LUO Haodong(Department of Neurology, Dongguan Songshanhu Central Hospital, Dongguan 523326, China;School of Pharmacy, Guangdong Medical University,Guangzhou 524023, China)
出处
《东南大学学报（医学版）》
CAS
2021年第5期586-593,共8页
Journal of Southeast University(Medical Science Edition)
基金
广东省医学科学技术研究基金资助项目(A2020209,B2021446)。
