天津医药

天津医药 ›› 2016, Vol. 44 ›› Issue (9): 1065-1068.doi: 10.11958/20160318

• 细胞与分子生物学 • 上一篇    下一篇

Nfic 基因 3′UTR 双荧光素酶报告质粒的构建及其与 miR-20a 靶向关系的验证

王珊 1,2， 李晓霞 2， 周杰 1， 王宝利 1△   

  1. 1 天津医科大学代谢病医院内分泌研究所、卫生部激素与发育重点实验室（邮编 300070）；2天津医科大学基础医学院
  • 收稿日期:2016-04-19 修回日期:2016-05-09 出版日期:2016-09-15 发布日期:2016-09-28
  • 通讯作者: 王宝利 E-mail:18202692040@163.com
  • 基金资助:
    国家自然科学基金

Construction of Nfic gene 3′UTR dual luciferase reporter vector and targeting verification between Nfic and miR-20a

WANG Shan1,2, LI Xiaoxia2, ZHOU Jie1, WANG Baoli1△   

  1. 1 Key Laboratory of Hormones and Development (Ministry of Health), Metabolic Diseases Hospital & Institute of Endocrinology, Tianjin Medical University, Tianjin 300070, China; 2 Basic Medical College of Tianjin Medical University
  • Received:2016-04-19 Revised:2016-05-09 Published:2016-09-15 Online:2016-09-28
  • Contact: WANG Baoli E-mail:18202692040@163.com

PDF (PC)

4346

摘要： 摘要： 目的 构建核因子 C（nuclear factor I-C， Nfic）基因 3′非编码区（3′UTR）荧光素酶报告质粒， 利用双荧光素酶报告基因验证 microRNA-20a（miR-20a）与其潜在靶基因 Nfic 的靶向关系。 方法 通过 microRNA 靶基因预测软件 Targetscan 获取 miR-20a 与 Nfic 基因 3′UTR 潜在的互补结合位点； PCR 扩增出 Nfic 基因 3′UTR 序列， 将此序列克隆至荧光素酶报告载体 pMIR-Report Luciferase； 将重组荧光素酶报告质粒与 miR-20a mimics（ 实验组） 或 NC mimics（对照组）共同转染 293-AD 细胞， 收集细胞后通过双荧光素酶报告系统检测 2 组细胞的荧光素酶活性， 从而对 Nfic 与 miR-20a 的靶向调节关系进行鉴定。 将 miR-20a mimics 和 NC mimics 分别转染骨髓基质细胞系 ST2， 裂解细胞提取蛋白后采用 Western blotting 检测 NFIC 蛋白的表达水平。 结果 构建的重组荧光素酶报告质粒经酶切及测序鉴定正确。 双荧光素酶报告基因检测显示， 与对照组相比， miR-20a 可以抑制 Nfic 3′UTR 报告基因载体的荧光素酶活性（P < 0.05）；Western blotting 结果显示， 与对照组相比， ST2 细胞转染 miR-20a mimics 后 NFIC 蛋白表达水平明显下调。 结论 成功构建了 Nfic 基因 3′UTR 荧光素酶报告质粒,而 miR-20a 可以直接作用于 Nfic 基因 3′UTR，抑制其荧光素酶活性。

关键词: 微 RNAs, 3′非翻译区, miR-20a, Nfic, 骨髓基质细胞系, 荧光素酶报告基因

Abstract: Abstract: Objective To construct a luciferase reporter vector containing the 3′ untranslated region (3′ UTR) of nuclear factor I-C (nuclear factor I-C, Nfic), and apply dual luciferase reporter gene system to determine the association between microRNA-20a (miR-20a) and its potential target gene Nfic. Methods The potential complementary binding sites of miR-20a and Nfic were predicted by Targetscan. The 3′UTR of Nfic fragment amplified by PCR was cloned into luciferase reporter vector MIR- Report Luciferase. The luciferase reporters containing 3′ UTR of Nfic and miR- 20 mimics (experimental group) or NC mimics (control group) were co-transfected into 293-AD cells. Cells were collected, and then dual-luciferase reporter assay was performed to detect the luciferase activity of the two groups of cells, consequently the relationship between miR-20a and Nfic was identified. The miR-20a mimics and NC mimics were transfected into marrow stromal cell line ST2 respectively. The total cell lysates were collected, and the expression level of NFIC was detected by Western blotting assay. Results Results of double enzyme digestion and DNA sequencing showed that sequence of luciferase reporter vector was correct. miR-20a specificity bounded to Nfic 3′UTR and inhibited the luciferase activity of the reporter construct (P < 0.05). Western blotting assay showed that the NFIC protein level was obviously down-regulated in ST2 cells after the transfection of miR- 20a mimics compared with that of control. Conclusion The luciferase reporter vector containing the 3′UTR of Nfic is constructed successfully, which confirms that miR- 20a can direct effect on Nfic3′ UTR and repress its luciferase activity.

Key words: microRNAs, 3&prime, untranslated regions, miR- 20a, nuclear factor I-C, marrow stromal cell line, luciferase reporter gene