microRNA-181b调控精神分裂症易感基因EGR3的分子机制

发布时间：2018-03-31 14:14

本文选题：精神分裂症　切入点：microRNA-b　出处：《西安交通大学学报(医学版)》2017年05期

【摘要】：目的用分子生物学的方法来检测microRNA-181b对EGR3基因的靶向调控作用,为后续microRNA-181b在精神分裂症(schizophrenia)中的分子功能研究指明方向。方法运用生物信息学软件预测到精神分裂症易感基因EGR3是microRNA-181b的潜在靶基因,随后采用PCR方法扩增EGR3基因3'UTR包含与microRNA-181b种子序列相结合的片段,再将该片段连接入pmirGLO质粒载体,用双酶切和测序的方法进行鉴定,与UCSC网站上EGR3序列相同的阳性重组质粒载体记为pmirGLO-EGR3。最后将该重组体与microRNA-181b阴性对照(NC)和模拟物(mimics)分为五组转染HEK293T细胞系,进行双荧光素酶报告基因活性测定。结果双酶切和测序结果表明EGR3 3'UTR成功构建入pmirGLO载体中。荧光显微镜下观察发现细胞转染实验成功,在大约90%的细胞中可检测到荧光信号。双荧光素酶报告基因活性检测结果表明,microRNA-181bmimics与NC相比,显著性降低了报告基因的活性。结论在细胞水平证明了精神分裂症易感基因EGR3是microRNA-181b的靶基因,对后续microRNA-181b的功能研究,以及其在精神分裂症中的分子作用机制研究提供了新的线索。
[Abstract]:Objective to detect the targeted regulation of microRNA-181b on EGR3 gene by molecular biology, and to point out the direction of molecular function study of microRNA-181b in schizophrenia.Methods Bioinformatics software was used to predict that the susceptible gene EGR3 of schizophrenia was a potential target gene of microRNA-181b. Then the EGR3 gene 3'UTR was amplified by PCR method, which contained a fragment combined with the microRNA-181b seed sequence, and the fragment was inserted into the pmirGLO plasmid vector.The positive recombinant plasmid vector with the same EGR3 sequence on UCSC website was identified as pmirGLO-EGR3 by double enzyme digestion and sequencing.Finally, the recombinant was divided into five groups to transfect HEK293T cell line with microRNA-181b negative control (NCX) and mimicsto detect the activity of double luciferase reporter gene.Results double enzyme digestion and sequencing showed that EGR3 3'UTR was successfully constructed into pmirGLO vector.Fluorescence microscope showed that the cell transfection was successful, and fluorescent signals could be detected in about 90% of the cells.The results of double luciferase reporter gene activity test showed that microRNA-181bmimics significantly decreased the activity of the reporter gene compared with NC.Conclusion EGR3 is the target gene of microRNA-181b, which provides a new clue to the study of the function of microRNA-181b and its molecular mechanism in schizophrenia.
【作者单位】：西安交通大学医学部附属红会医院转化医学中心;西安交通大学医学部附属陕西省人民医院精神科;西安交通大学医学部生物化学与分子生物学系;
【基金】：国家自然科学基金项目(No.31371298;81301151) 国家级大学生创新性实验项目(No.GJ201610698087) 教育部新世纪优秀人才支持计划(No.NCET-13-0452)~~
【分类号】：R749.3

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