GLS3′-非编码区-荧光素酶报告质粒的构建及其活性鉴定

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

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。

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-mirinhibitor and anti-mir-control were transfected at the same time. The relative luciferase activity was detected. TargetscanS.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.