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| Construction of the AKT2 3′-UTR luciferase reporter gene vector and verification of the targeted relationship with miRNA-625 |
| WEI Bin, DENG Xia, BIAN Xiu-juan |
| (Department of Respiratory Diseases, the Affiliated Hospital of Jiangsu University, Zhenjiang Jiangsu 212001, China) |
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Abstract Objective: To identify the targetedregulating relationship between miRNA-625 and AKT2 via constructing luciferase reporter gene vector. Methods: The AKT2 was predicted as miRNA-625 target gene by biological software. The 3′-untranslated regions(3′-UTR) of the wild type AKT2 and mutant AKT2 sequence were cloned into luciferase reporter vector pMIR-Report. The HEK-293T cells were divided into four groups randomly, wild-type plasmid + miRNA625 group, wild-type plasmid + miRNA negative control group, mutant plasmid + miRNA-625 group, mutant plasmid+miRNA negative control group.The relevant plasmids and miRNAs were transfected into groups by Lipofectamine 2000. The luciferase activity was detected by dual luciferase reporter gene system. Results: The recombinant plasmids were identified correctly. Dual-luciferase reporter assay system revealed luciferase activity of wildtype plasmid+miRNA-625 group was significantly lower than that of wild-type plasmid+ miRNA negative control group(P<0.05). Conclusion: The miRNA-625 inhibited the luciferase activity of AKT2 wild type vector, which indicates AKT2 gene could be targeted regulated by miRNA-625.
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Received: 29 February 2016
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2016, Vol. 26 
