Trejo-Avila Laura, Elizondo-González Regina, Trujillo-Murillo Karina Del C, Zapata-Benavides Pablo, Rodríguez-Padilla Cristina, Rivas-Estilla Ana María
Laboratorio de Inmunología y Virología, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, Monterrey, Nuevo León, México.
Ann Hepatol. 2007 Jul-Sep;6(3):174-80.
Hepatitis C virus (HCV) is a major public health problem with 170 million chronically infected people throughout the world. Currently, the only treatment available consists of a combination of pegylated interferon (INF-alpha) and ribavirin, but only half of the patients treated show a sufficient antiviral response. Thus there is a great need for the development of new treatments for HCV infections. RNA interference (RNAi) represents a new promising approach to develop effective antiviral drugs and has been extremely effective against HCV gene expression in short-term cell culture. Our aim was to determine the effect of RNAi directed against the NS5B-HCV region on HCV expression in a human hepatoma cell line that expresses HCV-subgenomic replicon (Huh7 HCV replicon cells).
We transfected Huh7 HCV replicon cells with different concentrations of RNAi (100-200 nM) targeting the NS5B region of the viral genome. 2-6 days post-transfection HCV-RNA was quantified by semiquantitative and real-time RT-PCR, and HCV NS5B protein levels were assayed by western blot. Cell viability was also quantified by MTT assay.
Our results indicate that the NS5B-siRNAs used in this study can specifically inhibit HCV-RNA replication and protein expression (more than 90%) compared to control cells.
Synthetic siRNA against NS5BHCV inhibited HCV replication and viral proteins levels and thereby becomes a powerful strategy to combat hepatitis C virus.
丙型肝炎病毒（HCV）是一个重大的公共卫生问题，全球有1.7亿人慢性感染该病毒。目前，唯一可用的治疗方法是聚乙二醇化干扰素（INF-α）和利巴韦林联合使用，但只有一半接受治疗的患者显示出足够的抗病毒反应。因此，迫切需要开发针对HCV感染的新治疗方法。RNA干扰（RNAi）是开发有效抗病毒药物的一种新的有前景的方法，并且在短期细胞培养中对HCV基因表达极为有效。我们的目的是确定针对NS5B-HCV区域的RNAi对表达HCV亚基因组复制子的人肝癌细胞系（Huh7 HCV复制子细胞）中HCV表达的影响。
我们用不同浓度（100-200 nM）靶向病毒基因组NS5B区域的RNAi转染Huh7 HCV复制子细胞。转染后2-6天，通过半定量和实时RT-PCR对HCV-RNA进行定量，并通过蛋白质印迹法检测HCV NS5B蛋白水平。细胞活力也通过MTT法进行定量。
我们的结果表明，与对照细胞相比，本研究中使用的NS5B-siRNAs可以特异性抑制HCV-RNA复制和蛋白质表达（超过90%）。
针对NS5B HCV的合成siRNA抑制了HCV复制和病毒蛋白水平，从而成为对抗丙型肝炎病毒的有力策略。
