双转基因小鼠模型中microRNA-153对RTN4的表达调控

目的探讨mir-153在阿尔茨海默病发病机制中的作用。方法通过microRNA芯片及Real-timePCR检测APPswe/PSΔE9双转基因小鼠脑内mir-153的表达;构建高表达mir-153的稳转细胞系,通过western-blot检测稳转细胞系内RTN4的蛋白表达;构建野生型及突变型RTN4的3’UTR荧光素酶报告载体,分别将其与mir-153表达载体或microRNA阴性对照载体共转染入293T细胞内,检测海肾荧光素酶的相对活性以验证mir-153对RTN4 mRNA的作用靶点。结果 microRNA芯片及Real-time PCR检测均证实3月龄APPswe/PSΔE9双转基因小鼠脑内mir-153的表达较同龄野生对照显著降低;在高表达mir-153的稳转细胞系内RTN4的蛋白水平明显降低;共转染野生型RTN4的3’UTR/mir-153表达载体可使海肾荧光素酶相对活性较共转染野生型RTN4的3’UTR/miRNA阴性对照质粒组显著降低（P〈0.05）,而共转染突变型RTN4的3’UTR/mir-153表达载体组则较之无明显差异。结论在3月龄APPswe/PSΔE9双转基因小鼠脑内存在mir-153的异常表达;mir-153可调控RTN4的蛋白表达;mir-153对RTN4的表达调控是通过结合RTN4 3’UTR 839-845碱基处的作用位点而实现的。

The Regulation of microRNA-153 on RTN4 in Double Transgenic Mice

Objective To explore the role of mir-153 in Alzheimer’s disease.Methods Detected the expression of mir-153 in the brain tissue of 3 months old APPswe /PSΔE9 mouse by means of microRNA array and Real-time PCR.Developed the stable transfection cell line that expressed mir-153 on a high level.Explored the expression of RTN4 protein by western-blot.Constructed the wild type and mutation type RTN4 3’UTR luciferase reporter vecter,cotransfected them with mir-153 expression vecter or negative control into 293T cell,detected the relative activity of Renilla Luciferase to check the binding site of mir-153 on RTN4’s mRNA.Results The result of microRNA arry and Real-time PCR all indicated in 3 months old APP swe /PSΔE9 mouse,the expression of mir-153 significantly decreased when compared to the wild contro on the same age.In stable transfection cell line expressing high level mir-153,the expression of RTN4 protein was downregulated.When compared to negative control / wild type RTN4 3’UTR contransfection group,Cotransfectionmir-153 together with wild type RTN4 3’UTR.can strikingly shutdown the relative activity of Renilla Luciferase（P〈 0.05）,however mir-153 / mutation type RTN4 3’UTR cotransfection group couldn’t reach any significant difference.Conclusions In 3 months old APPswe /PSΔE9 mouse’s brain,the expression of mir-153 is abnormal.Mir-153 can regulate the expression of RTN4 on protein level.The regulation effect of mir-153 on RTN4 is achieved by binding the specific site lie in 839-845 bp of RTN4 3’UTR.