Zhang Tao, Cheng Tong, Wei Li-Hua, Zhang Ya-Li, Wang Ying-Bing, Cai Yi-Jun, Zhang Jun, Xia Ning-Shao
National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Sciences, Xiamen University, Xiamen 361005, China.
Bing Du Xue Bao. 2010 Jan;26(1):8-15.
Effective and specific RNA interference (RNAi) elements are essential for the RNAi-based anti-HIV-1 research which has achieved extensive application. vif37 targeted to HIV-1 vi f is a highly effective and conserved RNAi target obtained from the previous study on screening. In this study, we explored the construction of artificial miRNAs to induce RNAi targeted to vif37, which had advantages on inhibition efficiency and flexibility of promoter selection. Three artificial miRNA targeted to vif37 were constructed by walking method using native miR-155 as a backbone and expressed by RNA polymerase II promoter. Then, expression vectors of artificial miRNA were co-transfected with HIV-1 infectious clone pNL4-3 to score its inhibition ability and showed that only miR-vif37 had the significant inhibition efficiency similar to shRNA-vif37. Subsequently, co-transfections with luciferase reporter plasmids into which different target sequences were inserted proved the specificity of miR-vif37 H. The replication of HIV-1 was inhibited in MT-4-miR37 H cells which could express miR-vif37 H stably and were cloned from MT-4 cells transducted with recombinant lentiviral vectors containing the miR-vif37 H expression element. Real-time RT-PCR revealed that miR-vif37 H had much lower expression level than shRNA-vif37. Results also showed that intracellular miR-181 and miR-16 expression levels and stat1 mRNA levels were not effected by the expression of miR-vif37 H in MT-4-miR37 H cells. We conclude miR-vif37 is a specific and highly effective artificial miRNA which will promote the further application of vif37 target.
有效的特异性RNA干扰（RNAi）元件对于已得到广泛应用的基于RNAi的抗HIV-1研究至关重要。靶向HIV-1病毒感染因子（vif）的vif37是先前筛选研究中获得的一种高效且保守的RNAi靶点。在本研究中，我们探索构建人工微小RNA（miRNA）以诱导针对vif37的RNAi，其在抑制效率和启动子选择灵活性方面具有优势。以天然miR-155为骨架，通过步移法构建了3种靶向vif37的人工miRNA，并由RNA聚合酶II启动子进行表达。然后，将人工miRNA的表达载体与HIV-1感染性克隆pNL4-3共转染以评估其抑制能力，结果显示只有miR-vif37具有与shRNA-vif37相似的显著抑制效率。随后，将其与插入了不同靶序列的荧光素酶报告质粒共转染，证实了miR-vif37 H的特异性。在能够稳定表达miR-vif37 H的MT-4-miR37 H细胞（从转导了含有miR-vif37 H表达元件的重组慢病毒载体的MT-4细胞中克隆得到）中，HIV-1的复制受到抑制。实时逆转录聚合酶链反应（RT-PCR）显示miR-vif37 H的表达水平远低于shRNA-vif37。结果还表明，MT-4-miR37 H细胞中miR-vif37 H的表达对细胞内miR-181和miR-16的表达水平以及stat1 mRNA水平没有影响。我们得出结论，miR-vif37是一种特异性高效的人工miRNA，将促进vif37靶点的进一步应用。
