miR-497靶向调控疾病易感基因EFNB2的分子机制研究  

Molecular mechanism of miR-497 in regulating the schizophrenia susceptibility gene EFNB2

作　　者：孟凡波 张蕊[2] 李琦[2] 邓红利 马建兵[2] 王玉琛 许华阳 王一心 马捷 MENG Fan-bo;ZHANG Rui;LI Qi;DENG Hong-li;MA Jian-bing;WANG Yu-chen;XU Hua-yang;WANG Yi-xin;MA Jie(Second Department of Clinical Medicine,Shaanxi University of Chinese Medicine,Xianyang 712046;Hong Hui Hospital,Xi'an Jiaotong University,Xi'an 710054;School of Basic Medical Sciences,Xi'an Jiaotong University Health Science Center,Xi'an 710061,China)

机构地区：[1]陕西中医药大学第二临床医学系,陕西咸阳712046 [2]西安交通大学附属红会医院,陕西西安710054 [3]西安交通大学医学部基础医学院,陕西西安710061

出　　处：《西安交通大学学报（医学版）》2019年第1期17-22,共6页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：国家自然科学基金资助项目(No.31371298);国家级大学生创新性实验项目(No.GJ201710698121)~~

摘　　要：目的用分子克隆和双荧光素酶报告基因的方法研究miR-497对精神分裂症易感基因EFNB2的靶向调控作用,为miR-497在精神分裂症中的分子功能研究奠定基础。方法运用生物信息学在线软件预测,发现与精神分裂症相关的miR-497可能直接调控疾病易感基因EFNB2。随后采用分子生物学技术扩增EFNB2基因3′-UTR中包含与miR-497种子序列相结合的片段,将该片段连接入pmirGLO质粒载体构建野生型的重组体(WT),并突变EFNB2基因3′-UTR与种子序列相结合的碱基序列,构建突变型的重组体(MUT)。最后将miR-497阴性对照(NC)和模拟物(mimics)分组转染HEK293T细胞系,并在24、48h后分别进行双荧光素酶报告基因活性测定。结果测序结果表明EFNB2基因3′-UTR的野生型(WT)和突变型(MUT)分别成功构建入pmirGLO载体中,表明WT和MUT的重组体均构建成功。转染24、48h后的双荧光素酶报告基因活性检测结果表明,miR-497mimics与NC相比,显著性降低了报告基因的活性。结论本研究在细胞水平证明了miR-497可直接调控精神分裂症易感基因EFNB2,为后续miR-497在精神分裂症中的功能研究提供了新思路和新线索。Objective To verify whether EFNB2 gene is targeted by miRNA-497 using molecular biology methods.Methods Bioinformatic method predicted that EFNB2 gene is targeted by miRNA-497.PCR methods amplified the fragment in EFNB2 gene 3′-UTR including the putative miRNA-497 binding site.Then the sequences of wide type(WT)and mutant(MUT)were cloned into the pmirGLO luciferase vector,respectively.The DNA sequences of the amplified fragments were identified by restriction enzyme digestion and sequencing,and were consistent with the reference sequence from UCSC.This constructed vector was marked as pmirGLO-EFNB2 vector.Finally,the pmirGLO vector,the pmirGLO-EFNB2 vector,miRNA-497 mimics and negative control(NC)were divided into five groups and transfected into HEK393T cells,and the luciferase activity was tested after 24 h and 48 h by dual luciferase reporter gene assay.Results The results of DNA sequencing demonstrated that the PCR fragment was successfully cloned into pmirGLO vector.The transfection results showed that the recombinant plasmid of WT and MUT was successfully transfected into HEK293T.The results of dual luciferase activity assay demonstrated that miRNA-497 significantly decreased the reporter gene activity compared with the NC.Conclusion At the cellular level,the schizophrenia susceptibility gene EFNB2 was verified to be targeted by miRNA-497,which provides a new idea and new clue for subsequent studies on miRNA-497 function in the molecular mechanism of schizophrenia.

关 键 词：精神分裂症 miR-497 EFNB2 靶基因 分子机制 

分 类 号：R749.3[医药卫生—神经病学与精神病学]