目的 观察miR-27 a-3p在七氟烷诱发的神经认知功能障碍中的作用.方法 生物信息学预测及验证:通过生物信息学数据库预测miR-27a-3p的靶基因,用双荧光素酶报告基因法和蛋白质印迹法验证miR-27 a-3p与靶基因的相互作用.细胞实验:将细胞分为2组,miR-27a-3p干涉对照组转染miR-27a-3p干涉对照质粒,miR-27a-3p干涉处理组转染miR-27a-3p干涉质粒,转染质粒之前先用4％的七氟烷处理6 h.以蛋白质印迹法检测各组SY5Y细胞中微管相关蛋白tau(tau)和磷酸化的tau蛋白(p-tau)的表达水平.动物实验:小鼠被随机分为对照组(不做任何处理)、七氟烷处理组(用4％七氟烷处理)、miR-27a-3p干涉对照组(用4％七氟烷处理后注射miR-27a-3p干涉对照质粒)、miR-27a-3p干涉处理组(用4％七氟烷处理后注射miR-27a-3p干涉质粒).通过水迷宫实验测试小鼠的神经认知能力,用免疫荧光法检测小鼠海马组织中的p-tau水平.结果 生物信息学预测及其验证:生物信息学的预测提示早老素1(PSEN1)可能是miR-27a-3p的靶基因.双荧光素酶报告基因法和蛋白质印迹法表明miR-27a-3p与PSEN1存在相互作用.细胞实验:miR-27a-3p干涉对照组和干涉处理组的p-tau水平分别为0.69±0.08和0.21±0.05.动物实验:对照组、七氟烷处理组、miR-27a-3p干涉对照组和miR-27a-3p干涉处理组的逃逸潜伏时间分别为(27.54±3.67)、(52.38±6.12)、(55.16±5.79)和(38.46±4.78)s,新事物探索指数结果分别为0.78±0.11、0.31±0.07、0.33±0.06 和 0.57±0.08,免疫荧光检测结果显示小鼠海马组织中p-tau水平也显著下降(P＜0.05).结论 miR-27 a-3p靶向PSEN1基因调控tau蛋白的磷酸化,干涉miR-27a-3p可缓解七氟烷诱发的小鼠神经认知功能障碍.
Objective To investigate the role of miR-27a-3p in sevoflurane-induced neurocognitive dysfunction.Methods Bioinformatics prediction and validation:Predicted the target genes of miR-27a-3p using bioinformatics databases,and verified the interaction between miR-27a-3p and target genes using dual-luciferase reporter gene assay and Western blot.Cell experiments:Cells were divided into two groups,the miR-27a-3p interference control(Sevo+NC)group was transfected with miR-27a-3p interference control plasmid,and the miR-27a-3p interference treatment(Sevo+anti-miR-27a-3p)group was transfected with miR-27a-3p interference plasmid.Before transfection,the plasmids were treated with 4％sevoflurane for 6 h.Western blot was used to detect the protein expression levels of tau and phosphorylaed tau(p-tau)in SY5Y cells of each group.Animal experiments:Mice were randomly divided into control group(no treatment),sevoflurane group(treated with 4％sevoflurane only),miR-27a-3p interference control group(Sevo+NC,injected with miR-27a-3p interference control plasmid after 4％sevoflurane treatment)and miR-27a-3p interference treatment group(Sevo+anti-miR-27-3p,injected with miR-27a-3p interference plasmid after 4％sevoflurane treatment).The neurocognitive abilities of mice were tested using the water maze experiment,and the level of tau phosphorylation in the hippocampal tissue of mice was detected by immunofluorescence.Results Bioinformatics prediction and validation:Bioinformatics prediction suggested that presenilin 1(PSEN1)might be a target gene of miR-27 a-3p.Dual-luciferase reporter gene assay and Western blot showed that miR-27 a-3p interacted with PSEN1.Cell experiments:The levels of p-tau in Sevo+NC group and Sevo+anti-miR27-3p group were 0.69±0.08 and 0.21±0.05,respectively.Animal experiments:The escape latency times of the control group,sevoflurane group,Sevo+NC group and Sevo+anti-miR-27-3p group were(27.54±3.67),(52.38±6.12),(55.16±5.79)and(38.46±4.78)s,respectively;the results of the novel object exploration index were 0.78±0.11,0.31±0.07,0.33±0.06,and 0.57±0.08,respectively.Immunofluorescence detection showed a significant decrease in p-tau levels in the hippocampal tissue of mice(P＜0.05).Conclusion miR-27 a-3p regulates the p-tau protein by targeting the PSEN1 gene,and interfering with miR-27 a-3p can alleviate sevoflurane-induced neurocognitive dysfunction in mice.
韩晶;万亚会;杨晓霞;姚小娟;程焱
天津医科大学总医院神经内科,天津 300052
药学
微小核糖核酸27a-3p;早老素1;微管相关蛋白;七氟烷;神经认知功能障碍
microRNA-27a-3p;presenilin 1;microtubule-associated protein tau;sevoflurane;neurocognitive dysfunction
《中国临床药理学杂志》 2024 (011)
1583-1587 / 5
10.13699/j.cnki.1001-6821.2024.11.009
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