Shahid Imran, AlMalki Waleed Hassan, AlRabia Mohammed Wanees, Mukhtar Mohammed Hasan, Almalki Shaia Saleh R, Alkahtani Saad Ahmed, Ashgar Sami S, Faidah Hani S, Hafeez Muhammad Hassan
Department of Pharmacology and Toxicology, College of Pharmacy, Umm Al Qura University, P.O. Box 13578, Makkah, Saudi Arabia.
Department of Pharmacology and Toxicology, College of Pharmacy, Umm Al Qura University, P.O. Box 13578, Makkah, Saudi Arabia.
Asian Pac J Trop Med. 2017 Jul;10(7):701-709. doi: 10.1016/j.apjtm.2017.07.011. Epub 2017 Aug 8.
To explore inhibitory effects of genome-specific, chemically synthesized siRNAs (small interference RNA) against NS3 gene of hepatitis C virus (HCV) 1a genotype in stable Huh-7 (human hepatoma) cells as well as against viral replication in serum-inoculated Huh-7 cells.
Stable Huh-7 cells persistently expressing NS3 gene were produced under antibiotic gentamycin (G418) selection. The cell clones resistant to 1000 μg antibiotic concentration (G418) were picked as stable cell clones. The NS3 gene expression in stable cell clone was confirmed by RT-PCR and Western blotting. siRNA cell cytotoxicity was determined by MTT cell proliferation assay. Stable cell lines were transfected with sequence specific siRNAs and their inhibitory effects were determined by RT-PCR, real-time PCR and Western blotting. The viral replication inhibition by siRNAs in serum inoculated Huh-7 cells was determined by real-time PCR.
RT-PCR and Western blot analysis confirmed NS3 gene and protein expression in stable cell lines on day 10, 20 and 30 post transfection. MTT cell proliferation assay revealed that at most concentrated dose tested (50 nmol/L), siRNA had no cytotoxic effects on Huh-7 cells and cell proliferation remained unaffected. As demonstrated by the siRNA time-dependent inhibitory analysis, siRNA NS3-is44 showed maximum inhibition of NS3 gene in stable Huh-7 cell clones at 24 (80%, P = 0.013) and 48 h (75%, P = 0.002) post transfection. The impact of siRNAs on virus replication in serum inoculated Huh-7 cells also demonstrated significant decrease in viral copy number, where siRNA NS3-is44 exhibited 70% (P < 0.05) viral RNA reduction as compared to NS3-is33, which showed a 64% (P < 0.05) decrease in viral copy number. siRNA synergism (NS3-is33 + NS3-is44) decreased viral load by 84% (P < 0.05) as compared to individual inhibition by each siRNA (i.e., 64%-70% (P < 0.05)) in serum-inoculated cells. Synthetic siRNAs mixture (NS5B-is88 + NS3-is33) targeting different region of HCV genome (NS5B and NS3) also decreased HCV viral load by 85% (P < 0.05) as compared to siRNA inhibitory effects alone (70% and 64% respectively, P < 0.05).
siRNAs directed against NS3 gene significantly decreased mRNA and protein expression in stable cell clones. Viral replication was also vividly decreased in serum infected Huh-7 cells. Stable Huh-7 cells expressing NS3 gene is helpful to develop anti-hepatitis C drug screening assays. siRNA therapeutic potential along with other anti-HCV agents can be considered against hepatitis C.
探讨基因组特异性化学合成的小干扰RNA（siRNA）对稳定表达丙型肝炎病毒（HCV）1a基因型NS3基因的Huh-7（人肝癌）细胞的抑制作用，以及对血清接种的Huh-7细胞中病毒复制的抑制作用。
在抗生素庆大霉素（G418）选择下产生持续表达NS3基因的稳定Huh-7细胞。挑选对1000μg抗生素浓度（G418）具有抗性的细胞克隆作为稳定细胞克隆。通过RT-PCR和蛋白质印迹法确认稳定细胞克隆中NS3基因的表达。通过MTT细胞增殖试验测定siRNA的细胞毒性。用序列特异性siRNA转染稳定细胞系，并通过RT-PCR、实时PCR和蛋白质印迹法测定其抑制作用。通过实时PCR测定血清接种的Huh-7细胞中siRNA对病毒复制的抑制作用。
RT-PCR和蛋白质印迹分析证实转染后第10、20和30天稳定细胞系中存在NS3基因和蛋白表达。MTT细胞增殖试验显示，在测试的最高浓度剂量（50nmol/L）下，siRNA对Huh-7细胞无细胞毒性作用，细胞增殖未受影响。siRNA时间依赖性抑制分析表明，siRNA NS3-is44在转染后24小时（80%，P = 0.013）和48小时（75%，P = 0.002）对稳定的Huh-7细胞克隆中NS3基因的抑制作用最大。siRNA对血清接种的Huh-7细胞中病毒复制的影响也表明病毒拷贝数显著减少，其中与显示病毒拷贝数减少64%（P < 0.05）的NS3-is33相比，siRNA NS3-is44表现出70%（P < 0.05）的病毒RNA减少。与血清接种细胞中每种siRNA单独抑制（即64%-70%（P < 0.05））相比，siRNA协同作用（NS3-is33 + NS3-is44）使病毒载量降低了84%（P < 0.05）。靶向HCV基因组不同区域（NS5B和NS3）的合成siRNA混合物（NS5B-is88 + NS3-is33）与单独的siRNA抑制作用（分别为70%和64%，P < 0.05）相比，也使HCV病毒载量降低了85%（P < 0.05）。
针对NS3基因的siRNA显著降低了稳定细胞克隆中的mRNA和蛋白表达。血清感染的Huh-7细胞中的病毒复制也明显减少。表达NS3基因的稳定Huh-7细胞有助于开发抗丙型肝炎药物筛选试验。可以考虑将siRNA与其他抗HCV药物联合用于丙型肝炎的治疗。
