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目的构建颗粒溶素基因3’-非编码区(3’-untranslated region,3’-UTR)-荧光素酶报告质粒,检测颗粒溶素和微小RNA(miRNA)调控位点的关联性。方法将人工合成的GLS基因3’-UTR区序列,克隆至荧光素酶报告质粒pGL3-control;通过Targetscan5.1等软件预测可能与GLS基因3’-UTR作用的miRNA;将荧光素酶报告质粒和miRNA真核表达质粒共转染293T细胞,为防止脱靶效应,同时转染anti-mir-inhibitor和anti-mir-control,用双荧光素酶检测试剂盒测定荧光素酶活性。结果用Targetscan5.1软件、PicTar软件和miRBase数据库预测交叉结果显示,miRNA(mir)-218、mir-514、mir-185、mir-611均与GLS基因3’-UTR存在互补结合位点;构建的miRNA真核表达质粒和荧光素酶报告质粒经酶切及测序鉴定正确;2种质粒共转染293T细胞后,miRNA-218可使荧光素酶报告质粒表达的荧光素酶活性降低75%左右(P<0.01);转染anti-mir-inhibitor后,荧光素酶的表达恢复到正常水平。结论成功构建了GLS基因3’-UTR荧光素酶报告质粒,通过检测荧光素酶活性,筛选出和GLS表达相关的miRNA片段即miRNA-218,为探讨miRNA调控GLS表达机制打下实验基础。
Objective To construct the 3’-untranslated region (3’-UTR) -luciferase reporter plasmid and detect the association between granulysin and miRNA regulatory sites. Methods The 3’-UTR region of synthetic GLS gene was cloned into the luciferase reporter plasmid pGL3-control. The target miRNAs that might interact with the 3’-UTR of GLS gene were predicted by software such as Targetscan5.1. The luciferase reporter plasmid And miRNA eukaryotic expression plasmid co-transfected 293T cells, in order to prevent off-target effects, while transfection of anti-mir-inhibitor and anti-mir-control, dual luciferase assay kit luciferase activity. Results The results of cross-over prediction with Targetscan5.1 software, PicTar software and miRBase database showed that miRNA-218, mir-514, mir-185 and mir-611 all had complementary binding sites with GLS gene 3’-UTR. MiRNA eukaryotic expression plasmid and luciferase reporter plasmid were identified by restriction enzyme digestion and sequencing. MiRNA-218 could reduce the luciferase activity of luciferase reporter plasmid expression by about 75% after co-transfecting 293T cells with two plasmids (P <0.01). After transfection with anti-mir-inhibitor, luciferase expression returned to normal levels. Conclusion The 3’-UTR luciferase reporter plasmid of GLS gene was successfully constructed. By detecting the luciferase activity, miRNA-218, a miRNA fragment related to GLS expression, was screened, which laid the experimental foundation for exploring the mechanism of miRNA regulation of GLS expression.