Inhibition of Hepatitis C Virus Genotype 1a Non-Structural Proteins by Small Interference RNA in Human Hepatoma Cell Lines

Imran Shahid^1,2*, Waleed Hassan AlMalki², Shaia Saleh R. Almalki³, Ismail Muhammad AlTurkestany⁴, Hassan Ali AlGhamdi⁵, Saleh Ali AlMenshawi⁶

¹Applied and Functional Genomics Laboratory, Center of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan.
²Department of Pharmacology and Toxicology, College of Pharmacy, Umm Al Qura University, Makkah, Saudi Arabia.
³Laboratory Medicine, Faculty of Applied Medical Sciences, Albaha University, Albaha, Saudi Arabia.
⁴Infection Control, Pilgrims City Hospital, Madina, Saudi Arabia.
⁵Toxicology Center, Jeddah, Saudi Arabia.
⁶Toxiology Research Center, Ministry of Interior, Jeddah, Saudi Arabia.

DOI: 10.4236/pp.2015.611053                

Abstract

Hepatitis C virus (HCV) infection and associated liver diseases are still challenging and represent a significant health care burden around the world. Although, the treatment strategies have been improved by the development of novel direct-acting antivirals, but such therapeutic options are still expensive and beyond the financial range of the most infected individuals in developing or even in resource replete countries. It demands an urgent need to search novel and improved alternate treatment strategies to treat the infection. The present study was aimed to develop an in vitro stable cell culture system, persistently expressing HCV genotype 1a non-structural genes and to characterize the inhibitory effects of synthetic siRNAs (short interference RNA) directed against the most conserved regions of nonstructural genes in an in vitro cell culture model. The continuous expression of nonstructural genes for more than 30 days post transfection was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis in stable human hepatoma cell line (Huh-7). The gene expression studies revealed significantly reduced gene expression of HCV nonstructural genes (i.e., NS2, NS4A and NS5A) both at mRNA and protein levels when treated against genome specific synthetic siRNAs in stable cell lines (51%, 47% and 54% respectively, p < 0.05). Similarly, a vivid decrease in HCV viral titer was exhibited by synthetic siRNAs in an in vitro viral replicate cell culture model (58%, 48% and 50%, respectively, p < 0.05) determined by quantitative Real-Time PCR (qPCR). Our data indicate that siRNA mediated gene silencing may be considered a promising alternate treatment strategy against HCV in combination with other effective therapeutic regimens in future.

Keywords

Hepatitis C Virus, Non-Structural Proteins, Stable Cell Line, Anti-HCV Drugs, Short Interference RNA

Conflicts of Interest

The authors declare no conflicts of interest.

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