本文選題：精神分裂癥　切入點：microRNA-b　出處：《西安交通大學(xué)學(xué)報(醫(yī)學(xué)版)》2017年05期

【摘要】：目的用分子生物學(xué)的方法來檢測microRNA-181b對EGR3基因的靶向調(diào)控作用,為后續(xù)microRNA-181b在精神分裂癥(schizophrenia)中的分子功能研究指明方向。方法運用生物信息學(xué)軟件預(yù)測到精神分裂癥易感基因EGR3是microRNA-181b的潛在靶基因,隨后采用PCR方法擴增EGR3基因3'UTR包含與microRNA-181b種子序列相結(jié)合的片段,再將該片段連接入pmirGLO質(zhì)粒載體,用雙酶切和測序的方法進行鑒定,與UCSC網(wǎng)站上EGR3序列相同的陽性重組質(zhì)粒載體記為pmirGLO-EGR3。最后將該重組體與microRNA-181b陰性對照(NC)和模擬物(mimics)分為五組轉(zhuǎn)染HEK293T細胞系,進行雙熒光素酶報告基因活性測定。結(jié)果雙酶切和測序結(jié)果表明EGR3 3'UTR成功構(gòu)建入pmirGLO載體中。熒光顯微鏡下觀察發(fā)現(xiàn)細胞轉(zhuǎn)染實驗成功,在大約90%的細胞中可檢測到熒光信號。雙熒光素酶報告基因活性檢測結(jié)果表明,microRNA-181bmimics與NC相比,顯著性降低了報告基因的活性。結(jié)論在細胞水平證明了精神分裂癥易感基因EGR3是microRNA-181b的靶基因,對后續(xù)microRNA-181b的功能研究,以及其在精神分裂癥中的分子作用機制研究提供了新的線索。
[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.
【作者單位】： 西安交通大學(xué)醫(yī)學(xué)部附屬紅會醫(yī)院轉(zhuǎn)化醫(yī)學(xué)中心;西安交通大學(xué)醫(yī)學(xué)部附屬陜西省人民醫(yī)院精神科;西安交通大學(xué)醫(yī)學(xué)部生物化學(xué)與分子生物學(xué)系;
【基金】：國家自然科學(xué)基金項目(No.31371298;81301151) 國家級大學(xué)生創(chuàng)新性實驗項目(No.GJ201610698087) 教育部新世紀(jì)優(yōu)秀人才支持計劃(No.NCET-13-0452)~~
【分類號】：R749.3

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