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miR-223靶向RhoB抑制Aβ1-42诱导的海马神经元凋亡
作者:张峪涵1  付晖2  刘翠娟2  廖成钜1  罗昊栋1 
单位:1. 东莞市松山湖中心医院 神经内科, 广东 东莞 523326;
2. 广东医科大学 药学院, 广东 东莞 524023
关键词:miR-223 β-淀粉样蛋白1-42 RhoB 海马神经元 凋亡 
分类号:Q954.671;R338.1
出版年·卷·期(页码):2021·40·第五期(586-593)
摘要:

目的:探讨miR-223抑制β-淀粉样蛋白1-42(Aβ1-42)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Aβ1-42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Aβ1-42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:Aβ1-42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30 μmol·L-11-42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Aβ1-4230.0 μmol·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Aβ1-42诱导的海马神经元细胞凋亡。

Objective: To investigate the mechanism of miR-223 on the apoptosis of Aβ1-42-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 Aβ1-42-induced hippocampal neurons was detected by qRT-PCR and Western blotting, the survival rate of Aβ1-42-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: Aβ1-42 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. 30 μmol·L-11-42 oligopeptide fragment was selected for further grouping. After incubated for 48 hours with 30 μmol·L-11-42, 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 Aβ1-42-induced hippocampal neurons by targeting RhoB.

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