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

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...

Full description

Saved in:

Bibliographic Details
Published in	天津医药 Vol. 44; no. 9; pp. 1065 - 1068
Main Author	王珊周杰王宝利李晓霞
Format	Journal Article
Language	Chinese
Published	天津医科大学基础医学院%天津医科大学基础医学院%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 2016 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
Subjects	3′非翻译区 miR-20a Nfic 微RNAs 荧光素酶报告基因骨髓基质细胞系 miR-20a microRNAs nuclear factor I-C 3′ untranslated regions Nfic 荧光素酶报告基因骨髓基质细胞系 luciferase reporter gene marrow stromal cell line 3′非翻译区微RNAs
Online Access	Get full text
ISSN	0253-9896
DOI	10.11958/20160318

Cover

More Information
Summary:	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＆lt;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。
Bibliography:	microRNAs; 3′ untranslated regions; miR-20a; nuclear factor I-C; marrow stromal cell line; luciferasereporter gene WANG Shan, LI Xiaoxia, ZHOU Jie, WANG Baoli 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
ISSN:	0253-9896
DOI:	10.11958/20160318