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Indian J Med Microbiol. 2018 Oct-Dec;36(4):494-503. doi: 10.4103/ijmm.IJMM_17_146.

Characterization of In vitro inhibitory effects of consensus short interference RNAs against non-structural 5B gene of hepatitis C virus 1a genotype.

Author information

1
Department of Pharmacology and Toxicology, College of Pharmacy, Umm Al Qura University, Makkah, Saudi Arabia.
2
Department of Pharmaceutical Chemistry, College of Pharmacy, Umm Al Qura University, Makkah, Saudi Arabia.
3
Infection Control Department, King Fahd Hospital, Ministry of Health, Jeddah, Saudi Arabia.
4
Department of Biochemistry, College of Medicine, Umm Al-Qura Univeristy, Makkah, Saudi Arabia.
5
Department of Laboratory Medicine, Faculty of Applied Medical Sciences, Al Baha University, Al Baha, Saudi Arabia.
6
Department of Clinical Pharmacy, College of Pharmacy, Najran University, Najran, Saudi Arabia.
7
Pharmaceutical Care Department, King Fahad Hospital, Ministry of Health, Madinah, Saudi Arabia.

Abstract

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.

KEYWORDS:

Hepatitis C virus; RNA interference; non-structural 5B protein; real-time polymerase chain reaction; stable human hepatoma-7 cells

PMID:
30880695
DOI:
10.4103/ijmm.IJMM_17_146

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