Construction of dual luciferase reporter plasmid of HIPK3 3′UTR and verification its targeting miR-146
10.3969/j.issn.1001-6325.2018.04.004
- VernacularTitle:HIPK3基因3′UTR双荧光素酶报告质粒的构建及其与靶向miR-146的验证
- Author:
Kai-Wen YANG
1
;
Zheng QIANG
;
Wen-Yu JIN
;
Fang LIU
Author Information
1. 桂林医学院 基础医学院 人体解剖学教研室
- Keywords:
miR-146;
HIPK3;
dual-luciferase reporter assay system
- From:
Basic & Clinical Medicine
2018;38(4):439-444
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a dual luciferase reporter vector containing the 3′untranslated region(3′UTR) of HIPK3 gene and verify the relationship between HIPK3 and miR-146.Methods The binding sites of miR-146 and HIPK3 genes were predicted by miRDB database and DIANA TOOLS database. The 3′UTR region sequences of HIPK3 genes and its mutants were respectively inserted into the luciferase report plasmid psiCHECK-2 to construct a wild-type and a mutant recombinant dual luciferase report plasmid. The 293T cells were divided into 6 groups and transfected with 1) HIPK3-WT+NC negative control;2)HIPK3-WT+miR-146a mimics;3)HIPK3-WT+miR-146b mimics;4)HIPK3-MU+NC negative control; 5)HIPK3-MU+miR-146a mimics and 6)HIPK3-MU+ miR-146b mimics respectively. After 48 hours, the luciferase activity was detected.Results HIPK3-WT and HIPK3-MU re-combinant plasmid were successfully constructed. When HIPK3-WT recombinant plasmids and miR-146b mimics were transfected into 293T cells, the luciferase activity was decreased (P<0.05). Conclusions miR-146a does not have a target relationship with HIPK3 gene,whereas miR-146b can regulate the 3′UTR of HIPK3 gene.