Applied and Functional Genomics Lab, Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan.
Virol J. 2011 May 13;8:221. doi: 10.1186/1743-422X-8-221.
Hepatitis C virus (HCV) is a major causative agent of liver associated diseases leading to the development of hepatocellular carcinoma (HCC) all over the world and genotype-3a responsible for most of the cases in Pakistan. Due to the limited efficiency of current chemotherapy of interferon-α (IFN-α) and ribavirin against HCV infection alternative options are desperately needed out of which the recently discovered RNAi represent a powerful silencing approach for molecular therapeutics through a sequence-specific RNA degradation process to silence virus infection or replication. HCV translation is mediated by a highly conserved internal ribosome entry site (IRES) within the 5'UTR region making it a relevant target for new drug development.
The present study was proposed to assess and explore the possibility of HCV silencing using siRNA targeting 5'UTR. For this analysis full length HCV 5'UTR of HCV-3a (pCR3.1/5'UTR) was tagged with GFP protein for in vitro analysis in Huh-7 cells. siRNA targeting 5'UTR were designed, and tested against constructed vector in Huh-7 cell line both at RNA and Protein levels. Furthermore, the effect of these siRNAs was confirmed in HCV-3a serum infected Huh-7 cell line.
The expression of 5'UTR-GFP was dramatically reduced both at mRNA and protein levels as compared with Mock transfected and control siRNAs treated cells using siRNAs against IRES of HCV-3a genotype. The potential of siRNAs specificity to inhibit HCV-3a replication in serum-infected Huh-7 cells was also investigated; upon treatment with siRNAs a significant decrease in HCV viral copy number and protein expression was observed.
Overall, the present work of siRNAs against HCV 5'UTR inhibits HCV-3a expression and represents effective future therapeutic opportunities against HCV-3a genotype.
丙型肝炎病毒（HCV）是导致世界各地肝脏相关疾病发展为肝细胞癌（HCC）的主要病原体，在巴基斯坦，基因型 3a 是大多数病例的原因。由于目前针对 HCV 感染的聚乙二醇干扰素-α（IFN-α）和利巴韦林的化疗效率有限，因此迫切需要替代方案，其中最近发现的 RNAi 通过序列特异性 RNA 降解过程代表了一种强大的沉默方法，用于分子治疗，以沉默病毒感染或复制。HCV 的翻译是由 5'UTR 区域内高度保守的内部核糖体进入位点（IRES）介导的，使其成为新药开发的相关靶点。
本研究旨在评估和探索使用针对 5'UTR 的 siRNA 沉默 HCV 的可能性。为此，在 Huh-7 细胞中对 HCV-3a 的全长 5'UTR（pCR3.1/5'UTR）进行 GFP 蛋白标记，进行体外分析。设计针对 5'UTR 的 siRNA，并在 Huh-7 细胞系中针对构建的载体进行 RNA 和蛋白质水平的测试。此外，还在 HCV-3a 血清感染的 Huh-7 细胞系中证实了这些 siRNA 的效果。
与 Mock 转染和对照 siRNA 处理的细胞相比，使用针对 HCV-3a 基因型 IRES 的 siRNA，5'UTR-GFP 的表达在 mRNA 和蛋白质水平上均显著降低。还研究了 siRNA 抑制血清感染的 Huh-7 细胞中 HCV-3a 复制的特异性潜力；在用 siRNAs 处理后，观察到 HCV 病毒拷贝数和蛋白表达显著下降。
总的来说，针对 HCV 5'UTR 的 siRNAs 抑制 HCV-3a 的表达，并代表针对 HCV-3a 基因型的有效未来治疗机会。
