Cao Mingmei, Ren Hao, Zhao Ping, Pan Wei, Zhao Lanjuan, Qi Zhongtian
Department of Microbiology, Second Military Medical University, Shanghai 200433, China.
Sci China C Life Sci. 2005 Feb;48(1):61-9. doi: 10.1360/03yc0233.
The RNA interference (RNAi) phenomenon is a recently observed process in which the introduction of a double-stranded, small interfering RNA (siRNA) into a cell causes the specific degradation of a homologous single-stranded RNA. It represents an exciting new technology that could have therapeutic applications for the treatment of viral infections. Since hepatitis G virus (HGV) genome is a positive-sense single-stranded RNA, the replication of HGV does not lead to an integrated DNA genome, suggesting a particularly attractive target for RNAi study that could eliminate viral RNA from infected cells. The eukaryotic expression vector pVAX.EH containing the cDNA sequences of the entire HGV structural genes and hygromycin resistance gene downstream from the encephalomyocarditis virus (ECMV) internal ribosome entry site (IRES) was constructed and transfected into human hepatoma cell Huh-7. The modified cleavage products of the structural proteins of HGV expressed in hygromycin-resistant cell line Huh-7-EH were confirmed by RT-PCR and Western blot methods. Two specific HGV E2 siRNAs (1-E2 siRNA, 2-E2 siRNA) synthesized with T7 RNA polymerase by transcription in vitro were transfected into the Huh-7-EH cells. With the analyses of Western blot and the formation of hygromycin-resistant colonies, the inhibitions of expression of HGV structural protein by two HGV E2 siRNAs were detected and found lasting at least one week. The inhibition of 2-E2 siRNA was stronger and only 1% of the cells treated with 2-E2 siRNA formed hygromycin-resistant colonies. These results support that specific HGV 2-E2 siRNAs mediate the degradation of mRNA spanning from HGV structural gene cDNA to hygromycin resistance gene in a majority of cells. In conclusion, the Huh-7-EH cells expressing HGV structural proteins stably can be used as a cell model for studying the replication of HGV and RNAi and the enlargement of RNAi may exist, in mammalian cells.
RNA干扰（RNAi）现象是最近观察到的一种过程，即向细胞中引入双链小干扰RNA（siRNA）会导致同源单链RNA的特异性降解。它代表了一项令人兴奋的新技术，可能在治疗病毒感染方面有治疗应用。由于庚型肝炎病毒（HGV）基因组是正链单链RNA，HGV的复制不会导致整合的DNA基因组，这表明它是RNAi研究特别有吸引力的靶点，可从感染细胞中消除病毒RNA。构建了真核表达载体pVAX.EH，其含有整个HGV结构基因的cDNA序列和位于脑心肌炎病毒（ECMV）内部核糖体进入位点（IRES）下游的潮霉素抗性基因，并将其转染到人肝癌细胞Huh-7中。通过RT-PCR和蛋白质印迹法证实了在潮霉素抗性细胞系Huh-7-EH中表达的HGV结构蛋白的修饰裂解产物。通过体外转录用T7 RNA聚合酶合成的两种特异性HGV E2 siRNA（1-E2 siRNA，2-E2 siRNA）被转染到Huh-7-EH细胞中。通过蛋白质印迹分析和潮霉素抗性菌落的形成，检测到两种HGV E2 siRNA对HGV结构蛋白表达的抑制作用，并发现其持续至少一周。2-E2 siRNA的抑制作用更强，用2-E2 siRNA处理的细胞中只有1%形成潮霉素抗性菌落。这些结果支持特异性HGV 2-E2 siRNA在大多数细胞中介导从HGV结构基因cDNA到潮霉素抗性基因的mRNA降解。总之，稳定表达HGV结构蛋白的Huh-7-EH细胞可作为研究HGV复制和RNAi的细胞模型，并且在哺乳动物细胞中可能存在RNAi的放大作用。
