Characterization of In vitro Inhibitory Effects of Consensus Short Interference RNAs against Non-Structural 5B Gene of Hepatitis C Virus 1a Genotype

Purpose: Chronic hepatitis C has infected approximately 170 million people worldwide. The novel direct-acting antivirals have proven their clinical efficacy to treat hepatitis C infection but still very expensive and beyond the financial range of most infected patients in low income and even resourc...

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Published in:	Indian journal of medical microbiology Vol. 36; no. 4; pp. 494 - 503
Main Authors:	Shahid, Imran, Almalki, Waleed Hassan, Ibrahim, Munjed M., Alghamdi, Sultan Ahmad, Mukhtar, Mohammed H., Almalki, Shaia Saleh R., Alkahtani, Saad Ahmed, Alhaidari, Mohammad S.
Format:	Journal Article
Language:	English
Published:	United States Elsevier B.V 01-10-2018 Wolters Kluwer India Pvt. Ltd Elsevier Limited
Subjects:	Antiviral agents Antiviral Agents - metabolism Cell culture Cell Line Cloning Cytoplasm Deoxyribonucleic acid DNA Drug resistance Drug screening Enzymes Gene Expression Genomes Genotype Genotype & phenotype Genotypes Gentamicin Hepacivirus - classification Hepacivirus - enzymology Hepacivirus - genetics Hepatitis Hepatitis C Hepatitis C virus Hepatocytes - drug effects Hepatoma Humans In vitro methods and tests Infections Interference Liver cancer non-structural 5B protein NS5B protein Nucleotide sequence Pathogenesis Polymerase chain reaction Proteins real-time polymerase chain reaction Replication Reverse transcription Ribonucleic acid RNA RNA Interference RNA polymerase RNA, Small Interfering - metabolism RNA-mediated interference siRNA stable human hepatoma-7 cells Studies Viral infections Viral Nonstructural Proteins - antagonists & inhibitors Viral Nonstructural Proteins - biosynthesis Viruses United States > US stable human hepatoma-7 cells RNA interference Hepatitis C virus real-time polymerase chain reaction non-structural 5B protein
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Summary:	Purpose: Chronic hepatitis C has infected approximately 170 million people worldwide. The novel direct-acting antivirals have proven their clinical efficacy to treat hepatitis C infection but still very expensive and beyond the financial range of most infected patients in low income and even resource replete nations. This study was conducted to establish an in vitro stable human hepatoma 7 (Huh-7) cell culture system with consistent expression of the non-structural 5B (NS5B) protein of hepatitis C virus (HCV) 1a genotype and to explore inhibitory effects of sequence-specific short interference RNA (siRNA) targeting NS5B in stable cell clones, and against viral replication in serum-inoculated Huh-7 cells. Materials and Methods:In vitro stable Huh-7 cells with persistent expression of NS5B protein was produced under gentamycin (G418) selection. siRNAs inhibitory effects were determined by analysing NS5B expression at mRNA and protein level through reverse transcription-polymerase chain reaction (PCR), quantitative real-time PCR, and Western blot, respectively. Statistical significance of data (NS5B gene suppression) was performed using SPSS software (version 16.0, SPSS Inc.). Results: siRNAs directed against NS5B gene significantly decreased NS5B expression at mRNA and protein levels in stable Huh-7 cells, and a vivid decrease in viral replication was also exhibited in serum-infected Huh-7 cells. Conclusions: Stable Huh-7 cells persistently expressing NS5B protein should be helpful for molecular pathogenesis of HCV infection and development of anti-HCV drug screening assays. The siRNA was effective against NS5B and could be considered as an adjuvant therapy along with other promising anti-HCV regimens.
ISSN:	0255-0857 1998-3646
DOI:	10.4103/ijmm.IJMM_17_146