In vitro inhibitory analysis of consensus siRNAs against NS3 gene of hepatitis C virus 1a genotype.

OBJECTIVE

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.

METHODS

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.

RESULTS

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).

CONCLUSIONS

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.