Construction and primary selection of microRNA expression vector directed against Nrf2

ObjectiveTo construct microRNA(miRNA)eukaryotic expression vector via pcDNA^TM 6.2-GW/EmGFP miR eukaryotic expression vector aimed at human Nrf2 gene and to primarily select effective miRNA expression vector for in vitro experiment.MethodsComputer-designed oligonucleotide sequences expressing the pre-miRNA were cloned into plasmid pcDNA6.2-GW/Em-GFP-miR with T4 DNA ligase after annealing.Then enzyme cutting method and sequencing were performed to evaluate the four recombinants named pcDNA-Nrf2-A,pcDNA-Nrf2-B,pcDNA-Nrf2-C,and pcDNANrf2-D.Fluorescence microscope was applied to observe the transfectional efficiency after the recombinants entered MCF-7 cells via lipofectamine.The Nrf2 mRNA and Nrf2 ptotein were detected by real time quantitative reverse transcriptase polymerase chain reaction(Q-RT-PCR)and immunocytochemical detection,respectively.ResultsThe construction of the four recombinant expression vectors were successfully confirmed by the results of enzyme digestion,electrophoresis and sequencing.The transfection efficiency was 20%-40%.The ability of those vectors inhibiting Nrf2 in a transient expression experiment in MCF-7 cells was compared.Importantly,pcDNA-Nrf2-B,pcDNA-Nrf2-C,and pcDNA-Nrf2-D were able to significantly knockdown Nrf2 expression.pcDNA-Nrf2-C had the most effective activity,whereas pcDNA-Nrf2-A was inactive in the assay.ConclusionThe miRNA eukaryotic expression vector of Nrf2(pcDNA-Nrf2-C)is successfully constructed and it will be a useful tool for investigation of the function of Nrf2.