《中国康复理论与实践》

《中国康复理论与实践》 ›› 2017, Vol. 23 ›› Issue (2): 166-172.doi: 10.3969/j.issn.1006-9771.2017.02.010

• 基础研究 • 上一篇    下一篇

大鼠细胞外信号调节激酶1基因3ʹ非编码区双荧光素酶报告载体的构建及rno-miR-15b-5p对其活性的影响

罗汉将1, 徐云峰2, 李晓晓1, 杨予涛1, 徐志卿1   

  1. 1.首都医科大学基础医学院,北京市 100069;
    2. 黄岛出入境检验检疫局,山东青岛市 266555
  • 收稿日期:2016-09-05 修回日期:2016-09-18 出版日期:2017-02-05 发布日期:2017-03-06
  • 通讯作者: 杨予涛(1972-),男,博士,副教授,主要研究方向：抑郁症的发病机制;徐志卿(1963-),男,博士,教授,主要研究方向：神经递质受体和重大脑病。E-mail: yutaoy@ccmu.edu.cn(杨予涛); zhiqingx@ccmu.edu.cn(徐志卿)。
  • 作者简介:罗汉将(1990-),男,汉族,浙江余姚市人,硕士研究生,主要研究方向：miR-15b参与抑郁症发生的机制研究。
  • 基金资助:
    1.北京市自然科学基金面上项目(No.7162016); 2.国家自然科学基金面上项目(No.31271154; No.31171032)

Construction of Rat Extracellular Signal-regulated Kinase 1 Gene 3ʹ Untranslated Regions Dual-luciferase Reporter Plasmids and Effect of rno-miR-15b-5p on Its Activitiy

LUO Han-jiang1, XU Yun-feng2, LI Xiao-xiao1, YANG Yu-tao1, XU Zhi-qing1   

  1. 1. Captial Medical University School of Basic Medical Sciences, Beijing 100069, China;
    2. Huangdao Entry-Exit Inspection and Quarantine Bureau, Qingdao, Shandong 266555, China
  • Received:2016-09-05 Revised:2016-09-18 Published:2017-02-05 Online:2017-03-06
  • Contact: YANG Yu-tao, XU Zhi-qing. E-mail: yutaoy@ccmu.edu.cn (YANG Yu-tao), zhiqingx@ccmu.edu.cn (XU Zhi-qing)

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摘要： 目的 以大鼠细胞外信号调节激酶1(ERK1)基因3ʹ非编码区(UTR)为研究对象,构建含有ERK1基因3ʹ UTR区的野生型以及突变型重组双荧光素酶报告载体,通过分析双荧光素酶报告载体的活性,明确rno-miR-15b-5p对报告载体的调节作用。方法 采用miRNeasy Mini Kit提取大鼠肾上腺嗜铬细胞瘤PC12细胞的总RNA,以PC12细胞的cDNA为模板,利用聚合酶链式反应(PCR)将ERK1基因3ʹ UTR克隆到pmiR-RB-ReportTM vector 中。利用重叠延伸PCR,将ERK1基因3ʹ UTR中的rno-miR-15b-5p潜在的靶序列TGCTGCT分别突变成CGAACGT和GTACACG,并将突变的ERK1基因3ʹ UTR克隆到pmiR-RB-ReportTM vector中。结果 成功构建了包含ERK1基因3ʹ UTR区的野生型报告载体pmiR-ERK1 3ʹ UTR和突变型报告载体pmiR-ERK1-mut 3ʹ UTR。利用荧光素酶活性检测系统,发现rno-miR-15b-5p mimic可以降低野生型报告载体的活性(P<0.001),但不影响突变型报告载体的活性。结论 成功构建ERK1基因3ʹ UTR区野生型和突变型双荧光素酶报告载体,初步证明ERK1基因3ʹ UTR区是rno-miR-15b-5p的结合靶点。

关键词: rno-miR-15b-5p, 细胞外信号调节激酶1, 重叠延伸聚合酶链式反应, pmiR-ERK1 3'UTR, pmiR-ERK1-mut 3'UTR, 荧光素酶活性检测

Abstract: Objective To construct dual-luciferase reporter plasmids containing the wild type and mutant rat extracellular signal-regulated kinase 1 (ERK1) gene 3ʹ untranslated regions (UTR) which were used to detect rno-miR-15b-5p's putative target gene. Methods The rat ERK1 gene 3ʹ UTR fragment was amplified by polymerase chain reaction (PCR) from PC12 cell cDNA and cloned into pmiR-RB-ReportTM vector. The mutant rat ERK1 gene 3ʹ UTR fragment was obtained by overlap PCR and inserted into pmiR-RB-ReportTM vector. Successful wild type and mutant recombinant plasmids were confirmed by DNA sequencing. PC12 cells were co-transfected with rno-miR-15b-5p mimic and pmiR-ERK1 3ʹ UTR or pmiR-ERK1-mut 3ʹ UTR and then analyzed by dual-luciferase reporter assay system. The achieved mutation sequence of the target site TGCTGCT was mutated to CGAACGT and GTACACG, respectively. Results The wild-type reporter vector pmiR-ERK1 3ʹ UTR and the mutant reporter vector pmiR-ERK1-mut 3ʹ UTR were successfully constructed. The rno-miR-15b-5p mimic decreased the activity of pmiR-ERK1 3ʹ UTR plasmid (P<0.001) but did not decrease the activity of pmiR-ERK1-mut 3ʹ UTR plasmid. Conclusion The recombinant pmiR-ERK1 3ʹ UTR and pmiR-ERK1-mut 3ʹ UTR plasmids were constructed successfully, and luciferase activities demonstrated that the 3ʹ UTR of ERK1 gene might be a potential target of rno-miR-15b-5p.

Key words: rno-miR-15b-5p, extracellular signal-regulated kinase 1, overlap polymerase chain reaction, pmiR-ERK1 3'UTR, pmiR-ERK1-mut 3'UTR, luciferase reporter assay

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