FLT3基因3′-非翻译区-荧光素酶报告载体的构建及其活性鉴定

Construction of FLT3 3′-UTR-Luciferase Reporter Vector and Evaluation of Its Activity

为分析FMS样酪氨酸激酶3(FMS-liketyrosine kinase3,FLT3)基因3′-非翻译区(3′-untranslated region,3′-UTR)可能的微小RNA(miRNA)调控位点,构建FLT3基因3′-UTR-荧光素酶报告载体,与miRNA共转染293T细胞,分析miRNA对其调控作用。采用PCR方法从HepG2细胞基因组DNA中扩增FLT3基因3′-UTR区序列,插入经PstI和EcoRI双酶切的经过改造的荧光素酶报告载体pGL3-control(pGL3-control-m);通过Target Scan5.1软件预测可能与FLT3基因3′-UTR作用的miRNA;采用FuGENEHD转染试剂包裹荧光素酶报告重组子和miRNA真核表达载体共转染293T细胞,双荧光素酶检测试剂盒测定荧光素酶活性。结果表明,成功构建了含有804bp的FLT3基因3′-UTR序列的荧光素酶报告重组子,通过酶切和基因测序的方法鉴定正确;miRNA靶位点预测显示,FLT3基因3′-UTR可能是miRNA-15a、miRNA-15b、miRNA-16、miRNA-195、miRNA-424、miRNA-497的作用靶点;与对照组相比,miRNA-15a、15b、195可使荧光素酶报告重组子的荧光素酶活性降低20%左右。结论:成功构建了FLT3基因3′-UTR-荧光素酶报告载体,而miRNA-15a、miRNA-15b、miRNA-195可抑制其荧光素酶活性。

To analyze the possible microRNA （miRNA） target sites in the 3＇ untranslated region （3＇-UTR） of FMS-like tyrosine kinase 3 （ FLT3 ） gene, a FLT3 3 ＇-UTR-luciferase reporter vector was constructed and the effect of miRNA on its activity was evaluated in 293T cell line. The 3 ＇-UTR fragment of FLT3 gene was amplified by PCR from genomic DNA of HepG2 cells. PCR products were cloned into PstL/EcoRI-modified pGL3-control reporter vector （ pGL3-control- m）. The rniRNA targeting FLT3 3＇-UTR was predicted by Target Scan 5.1 software. The luciferase reporter vector and miRNA eukaryotic expression vector were transferred into 293T cells using FuGENE~ HD transfection reagent. The Dual-Luciferase Reporter Assay System was used to quantitate the reporter activity. The results showed that a 804-bp 3＇- UTR fragment of FLT3 gene was successfully cloned into the pGL3-control-m reporter vector, which authenticated by PstI/EcoRI digestion and DNA sequencing. The predicted miRNA targeting FLT3 3＇-UTR included miRNA-15a, miRNA-15b, miRNA-16, miRNA-195, miRNA-424 and miRNA-497. The luciferase activity of reporter construct treated with miRNA-15a, miRNA-15b or miRNA-195 was decreased respectively about 20% compared with the control group. It is concluded that the FLT3 3＇-UTR-luciferase reporter vector has been successfully constructed. The luciferase activity of the reporter can be suppressed by miRNA-15a, miRNA-15b or miRNA-195.