Mir-106b对阿尔茨海默病昼夜节律调控的初步研究  

Mir-106b Regulation of the Circadian Rhythm of Alzheimer's Disease and Neuronal PAS Domain Protein 2 Expression

作　　者：王海林[1] 刘嘉琳[1] 宗园媛[1] 张连峰[1] 秦川[1] 

机构地区：[1]中国医学科学院医学实验动物研究所,北京协和医学院比较医学中心,北京100021

出　　处：《中国比较医学杂志》2010年第4期19-23,87,共6页Chinese Journal of Comparative Medicine

基　　金：中央级公益性科研院所基本科研业务费专项基金(DWS200814)

摘　　要：目的探讨mir-106b在阿尔茨海默病发病中的作用。方法取6月龄APPswe/PSΔE9小鼠脑组织,进行microRNA芯片的检测;利用real-time PCR对芯片检测结果进行验证;构建mir-106b表达载体,将其转染至SH-SY5Y细胞中构建mir-106b稳定转染的稳转细胞系。用Targetsan、Pictar、miRanda等靶基因预测软件,对mir-106b的靶基因进行预测,根据靶基因的功能选择可能与AD发病相关的靶基因,并用Western blot对所选择的靶基因在稳转细胞中的表达进行验证。用双荧光素酶报告检测系统检测mir-106b与其靶基因的结合位点。结果芯片结果显示,与野生型小鼠相比,6月龄APPswe/PSΔE9小鼠脑组织中mir-106b的表达下降,经real-time PCR验证,mir-106b的表达在APPswe/PSΔE9小鼠脑组织中的表达较野生型小鼠升高,差别具有统计学意义(P=0.03)。mir-106b稳转细胞系较对照mir-106b的表达升高2～5倍。神经PAS结构域蛋白2(neuronal PAS domain protein2,NPAS2)在mir-106b稳转细胞中的表达明显下降。双荧光素酶报告实验证实:与对照相比,带有NPAS2的3'UTR的荧光素酶报告载体与mir-106b表达载体共转染,海肾荧光素酶/萤火虫荧光素酶的相对活性降低;荧光素酶报告载体的NPAS2-3'UTR突变后与mir-106b表达载体共转染,海肾荧光素酶/萤火虫荧光素酶的相对活性升高。结论mir-106b可能通过调节机体生物钟基因NPAS2的表达,影响阿尔茨海默病人的生活节律,参与阿尔茨海默病的发生。Objective To explore the role of mir-106b in Alzheimer＇s disease.Methods A microRNA array was used for the analysis of microRNA expression from brain tissue of 6-month old APPswe /PSΔE9 mice.The results of microRNA array were validated by real-time PCR.We constructed the mir-106b expression vector and transfected it into SH-SY5Y cell line to construct a mir-106b stable expression cell line.Then,we predicted the target genes of mir-106b by Targetsan,Pictar,miRanda and so on,and analyzed the functions of target genes to select genes that are associated with the pathogenesis of AD.The protein from mir-106b stable expression cell line and its control cell line was extracted,and the expression level of target genes of mir-106b was detected by western blot.The luciferase activity was detected by dual-luciferase reporter assay system to identify the binding site of mir-106b and its target genes.Results The data of microRNA array showed that the expression level of mir-106b in APPswe / PSΔE9 mice was reduced compared with the age-matched controls.Validated results of mir-106b expression by real-time PCR was opposite to the results of microRNA assay,with a statistically significant difference（P=0.03）.The expression level of NPAS2 in mir-106b stable expression cells was reduced compared with that of control cells.Dual-luciferase reporter assay confirmed that co-transfection of mir106b expression vector and dual-luciferase reporter vector with the 3＇UTR of NPAS2 made the renilla luciferase activity /firefly luciferase activity decreased,compared with that of the control.Co-transfection of mir-106b expression vector and dual-luciferase reporter vector with the mutant 3＇UTR of NPAS2 made the renilla luciferase activity / firefly luciferase activity restored.Conclusion Mir-106b may play a role in pathogenesis of Alzheimer＇s disease by affecting the expression of NPAS2 which fuctions as a clock gene.

关 键 词：阿尔茨海默病 MICRORNA 转基因小鼠 microRNA芯片 神经PAS结构域蛋白2 

分 类 号：R-33[医药卫生]