靶向Nrf2基因microRNA表达载体构建及初筛  

Construction and primary selection of microRNA expression vector directed against Nrf2

作　　者：李煌元[1] 汤章彬[1] 吴思英[2] 林炜[1] 王章敬[1] 

机构地区：[1]福建医科大学公共卫生学院环境与健康研究所职业与环境卫生学系,福建福州350004 [2]福建医科大学公共卫生学院流行病与卫生统计学系

出　　处：《中国公共卫生》2011年第9期1116-1118,共3页Chinese Journal of Public Health

基　　金：国家自然科学基金(30800936);福建省环境与健康重点学科基金(GW-10);福建省教育厅(JA07084)

摘　　要：目的利用真核表达载体pcDNATM6.2-GW/EmGFP miR载体构建针对人Nrf2基因的微小RNA（microR-NA）真核表达载体,为研究Nrf2基因在化学物毒作用提供参考依据。方法设计特异性针对Nrf2基因的寡核苷酸序列,构建重组载体并命名为pcDNA-Nrf2-A、pcDNA-Nrf2-B、pcDNA-Nrf2-C、pcDNA-Nrf2-D,转染人乳腺癌MCF-7细胞;通过荧光显微镜观察绿色荧光监测转染效率,转染48 h后用荧光定量逆转录-聚合酶链反应（RT-PCR）和免疫细胞化学检测瞬时转染细胞Nrf2基因mRNA和蛋白的表达变化观察沉默作用来初筛有效的重组载体。结果成功构建miRNA真核表达载体pcDNA-Nrf2;转染效率约为20%~40%;瞬时转染重组质粒48 h后,与空白对照和阴性对照质粒比较,pcDNA-Nrf2-A Nrf2 mRNA表达差异无统计学意义（P〉0.05）;pcDNA-Nrf2-B、pcDNA-Nrf2-C、pcD-NA-Nrf2-D mRNA表达降低,差异有统计学意义（P〈0.001）,pcDNA-Nrf2-C抑制Nrf2基因mRNA表达强于pcDNA-Nrf2-D（P〈0.05）。结论成功构建了Nrf2的microRNA表达载体pcDNA-Nrf2-C,可作为进一步研究Nrf2基因功能的工具。Objective To construct microRNA（miRNA） eukaryotic expression vector via pcDNATM 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. Methods Computer-designed oligo nucleotide 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-NrI＇2-A, pcDNA-Nrf2-B, pcDNA-Nrf2-C, and pcDNA- Nrf2-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 transcdptase poly- merase chain reaction（Q-RT-PCR） and immunocytochemical detection, respectively. Results The construction of the four recombinant expression vectors were successfully confirmed by the results of enzyme digestion, electrophoresis and sequen- cing. The transfection efficiency was 20% - 40%. The ability of those vectors inhibiting Nrf2 in a transient expression experi- ment in MCF-7 cells was compared. Importantly, pcDNA-Nrf2-B, pcDNA-Nrf2-C, and pcDNA-Nrf2-D were able to signifi- cantly knockdown Nrf2 expression, pcDNA-Nrf2-C had the most effective activity, whereas pcDNA-Nrf2-A was inactive in the assay. Conclusion The 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.

关 键 词：NRF2 微小RNA 真核表达载体 人乳腺癌细胞株MCF-7 

分 类 号：R114[医药卫生—卫生毒理学]