Construction of GLS 3'-UTR-luciferase reporter vector and evaluation of its activity

We aimed to construct a luciferase reporter vector containing the 3'-untranslated region(3'-UTR) of Granulysin(GLS) and measure the correlation between GLS and the regulation sites of microRNA(miRNA).Firstly,the synthetic 3'-UTR fragment of GLS was cloned into pGL3-control reporter vector.Then the miRNA targeting GLS 3'-UTR was predicted by TargetScan 5.1 and other recognized software.The luciferase reporter vector and miRNA eukaryotic expression vector were transferred into 293T cells.To prevent the off-target effect,anti-mir-inhibitor and anti-mir-control were transfected at the same time.The relative luciferase activity was detected.Targetscan5.1,PicTar and miRBase database shared the results that miRNA(mir)-218,mir-514,mir-185,mir-611 have the complementary binding sites with 3'-UTR of GLS.Results of DNA sequencing showed that sequences of luciferase reporter vector and miRNA eukaryotic expression vector were correct.The luciferase activity of reporter vector treating miRNA-218 was decreased observably about 75%(P 0.01),and luciferase expression gradually returned to normal levels after transfected with anti-mir-218.All the result indicated that miRNA-218 is correlated with GLS expression,which lays a foundation for exploring the mechanism of miRNA regulating GLS expression.