用于鉴定microRNA靶基因的新型双萤光素酶报告基因系统的构建及应用

摘要

Objective: To construct reporter vector system used for identification of microRNA target gene. Methods: The CMV -promoter and region of multiple clone sites were respectively inserted into the upstream and the downstream of luc gene on pGL3-Basic vector backbone, successfully resulting in the reporter vector named pMIR-luciferase. The reporter vector containing 3'UTR of complement factor H(CFH) was subsequently constructed by inserting the target gene into the frame of multiple cloning sites of pMIR-luciferase vector. Meanwhile the control vector was subsequently constructed by replacing the luc gene of pMIR-luciferase vector with Rluc gene and was named pMIR-control. Eukaryotic expression vector of microRNA 146a was constructed basing on pIRES2-EGFP. The reporter vector containing CFH 3'UTR and the control vector were cotransfected with eukaryotic expression vector of microRNA 146a into HepC2 cell line. The relative activity of reporter gene was subsequently determined. Results: The constructed vectors, including reporter vector, control vector and eukaryotic expression vector of microRNA 146a, were confirmed by restriction enzyme digestion and plasmid sequencing. Seventy-two hours post-transfec-tion, observation under fluorescence microscope proofed that the plasmid microRNA 146a were transfected into the cells and expressed successfully. The expression level of microRNA 146a in Hep G2 cell was up-regulated significantly in the detection of real-time PCR (P<0.01). The results of detection on relative activity of reporter gene il-lustrated that miRINA significantly inhibited the activity of reporter gene containing 31JTR of target mRNA. Conclusion: A novel reporter vector system successfully constructed in our study shows great potential in identification of miRNA target gene.%目的:构建用于鉴定microRNA靶基因的报告基因系统.方法:在pGL3-Basic载体的luc基因上游插入CMV启动子,下游插入用于克隆靶基因3'UTR的多克隆位点,构建报告基因载体pMIR-luciferase；将pMIR-lu-ciferase载体的luc基因替换成Rluc基因,构建内参载体pMIR-control；将补体因子H(CFH)的3′UTR插入pMIR-luciferase载体的多克隆位点处,构建含有CFH 3′UTR的报告基因载体；用pIRES2-EGFP载体构建microRNA146a 真核表达载体；将含有CFH 3′UTR的报告基因载体、microRNA146a真核表达载体及内参载体共转染HepG2细胞,进行报告基因的活性检测.结果:构建了报告基因载体、内参载体和microRNA146a真核表达载体,经酶切和测序鉴定正确；microRNA146a真核表达载体转染细胞72 h后,经荧光显微镜观察确认载体转染及表达；用实时定量PCR检测,microRNA 146a的表达水平显著上调(P＜0.01)；用构建的报告基因系统检测,结果表明microRNA146a显著地抑制了含CFH 3'UTR的报告基因的活性(P＜0.05).结论:构建了一种新型的报告基因载体系统,该系统可用于miRNA靶基因的鉴定.