关键词: 【关键词】 miRNA-146 , HIPK3, 双荧光素酶报告系统

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 inserted respectively 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 recombinant 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.

Key words: Keywords: miRNA-146, HIPK3, luciferase reporter assay