Bcl-6基因3'UTR双荧光素酶报告质粒的构建及其与miR-346靶向关系的验证

Construction of Bcl-6 gene 3'UTR dual luciferase reporter vector and targeting verification between Bcl-6 and miR-346

目的:构建Bcl-6基因3'非编码区(3'untranslated region,3'UTR)荧光素酶报告质粒,分析miR-346对Bcl-6的调控作用。方法:通过PicTar数据库和miRanda数据库预测可能与Bcl-6基因3'UTR作用的miRNA;将人工合成的Bcl-6基因3'UTR区序列克隆至荧光素酶报告质粒psiCHECK-2;将重组荧光素酶报告质粒和miRNA mimics共转染293T细胞,用双荧光素酶检测试剂盒测定荧光素酶活性。结果:PicTar数据库和miRanda数据库预测交叉结果显示,miR-346与Bcl-6基因3'UTR存在互补结合位点;构建的荧光素酶报告质粒经酶切及测序鉴定正确;重组质粒和miR-346 mimics共转染293T细胞后,荧光素酶报告质粒表达的荧光素酶活性降低66%左右。结论:成功构建了Bcl-6基因3'UTR荧光素酶报告质粒,筛选出和Bcl-6表达相关的miRNA即miR-346。

Objective:To construct a luciferase reporter vector containing the 3'untranslated region (3' UTR) of Bcl-6 gene and measure the correlation between Bcl-6 and miR-346.Methods:The miRNA targeting Bcl-6 3'UTR was predicted by miRanda and PicTar.The synthetic 3'UTR fragment of Bcl-6 was cloned into PsiCHECK-2 reporter vector.The luciferase reporter vector and miRNA mimics were transfected into 293T cells.The relative luciferase activity was detected.Results:PicTar and miRanda database shared the results that miR-346 have the complementary binding sites with 3'UTR of Bcl-6.Results of double enzyme digestion and DNA sequencing showed that sequence of luciferase reporter vector was correct.The luciferase activity of reporter vector treating miR-346 was decreased observably about 66％.Conclusion:The luciferase reporter vector containing the 3'UTR of Bcl-6 was constructed successfully.