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- 2017
microRNA-181b调控精神分裂症易感基因EGR3的分子机制
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Abstract:
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-181b mimics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 verify whether early growth response-3(EGR3) gene is targeted by microRNA-181b using molecular biology methods so as to provide guidance for the subsequent study on microRNA-181b’s role in the molecular mechanisms of schizophrenia. Methods Bioinformatic methods predicted that EGR3 gene is targeted by microRNA-181b. PCR methods amplified the fragment in EGR3 gene 3’UTR including the putative microRNA-181b binding site. Then the sequence was cloned into the pmirGLO luciferase vector. 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-EGR3 vector. Finally, the pmirGLO vector, the pmirGLO-EGR3 vector, microRNA-181b mimics and negative control (NC) were divided into 5 groups and transfected into HEK393T cells; the luciferase activity was tested by dual luciferase reporter gene assay. Results The results of restriction enzyme digestion and sequencing demonstrated that the PCR fragment was successfully cloned into pmirGLO vector. The transfection results showed that the recombinant plasmid was successful transfected into HEK293T under the fluorescence microscope, with transfection efficiency being about 90%. The results of dual luciferase activity assay demonstrated that microRNA-181b significantly decreased the reporter gene’s activity compared with the NC. Conclusion At the cellular level, the schizophrenia susceptibility gene EGR3 was verified to be targeted by micorRNA-181b, which provides a new clue for the subsequent study on microRNA-181b’s role in the molecular mechanisms of schizophrenia
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