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

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-497 mimics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。
ABSTRACT: 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 24h and 48h 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