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

Asian Pacific Journal of Tropical Medicine 2017年第7期10卷 763-770页

作者： Imran Shahid Waleed Hassan Al Malki Mohammed Wanees Al rabia Mohammed Hasan Mukhtar shaia saleh r.almalki Saad Ahmed Alkahtani Sami S.Ashgar Hani S.Faidah Muhammad Hassan Hafeez Department of Pharmacology and Toxicology College of PharmacyUmm Al Qura University Department of Medical Microbiology College of MedicineKing Abdul Aziz University Department of Biochemistry Faculty of MedicineUmm Al Qura University Department of Laboratory Medicine Faculty of Applied Medical SciencesAlbaha University College of Pharmacy Najran University Department of Microbiology College of MedicineUmm Al Qura University Department of Gastroenterology and Hepatology Fatima Memorial College of Medicine and Dentistry

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 1 000 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%(Pral rNA reduction as compared to NS3-is33, which showed a 64%(Prease in viral copy number. sirNA synergism(NS3-is33 + NS3-is44) decreased viral load by 84%(Pred to individual inhibition by each sirNA(i.e., 64%–70%(Prum-inoculated cells. Synthetic sirNAs mixture(NS5Bis88 + NS3-is33) targeting different region of HCV genom

关键词： Hepatitis C virus NS3 protein Stable Huh-7 cell culture system rNA interference NS5B HCV therapeutics

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

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Pharmacology & Pharmacy 2015年第11期6卷 502-517页

作者： Imran Shahid Waleed Hassan almalki shaia saleh r. almalki Ismail Muhammad AlTurkestany Hassan Ali AlGhamdi saleh Ali AlMenshawi Applied and Functional Genomics Laboratory Center of Excellence in Molecular Biology University of the Punjab Lahore Pakistan Department of Pharmacology and Toxicology College of Pharmacy Umm Al Qura University Makkah Saudi Arabia Laboratory Medicine Faculty of Applied Medical Sciences Albaha University Albaha Saudi Arabia Infection Control Pilgrims City Hospital Madina Saudi Arabia Toxicology Center Jeddah Saudi Arabia Toxiology research Center Ministry of Interior Jeddah Saudi Arabia

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 rly, 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 rmined 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.

关键词： Hepatitis C Virus Non-Structural Proteins Stable Cell Line Anti-HCV Drugs Short Interference rNA

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