Marzieh Jamalidoust1, Maryam Shafaati2, Mehdi Kalani3, Maryam Zare3, Mazyar Ziyeayan3. 1. Department of Virology, Prof. Alborzi Clinical Microbiology Research Center, Namazi Hospital, Shiraz University of Medical Sciences, Shiraz, Iran. maryam.shafaati@gmail.com. 2. Department of Microbiology, Faculty of Sciences, Jahrom Branch, Islamic Azad University, Jahrom, Iran. 3. Department of Virology, Prof. Alborzi Clinical Microbiology Research Center, Namazi Hospital, Shiraz University of Medical Sciences, Shiraz, Iran.
Abstract
BACKGROUND: Small non-coding RNAs have emerged as essential modulators of viral infections such as hepatitis C virus (HCV). Cellular miRNAs directly regulate the viral infectivity and indirectly by targeting virus-host factors. The current study investigates the inhibitory effect of let-7b miRNA on HCV replication in the Hepatocarcinoma cell line (Huh7.5). METHODS AND RESULTS: The algorithm-based search revealed that let-7b, a high score microRNA, has target sequences on the HCV genome. The Huh7.5 cells were stably transduced with let-7b lentiviral vectors (Huh7.5/let-7b) and mock (Huh7.5/scrambled). The expression of the let-7b level was assessed by real-time PCR assay and Red fluorescence microscope. A dual-luciferase assay was conducted to evaluate the liver-specific let-7b and HCV genome interaction. In the next step, for establishing HCVcc, Full-length HCV-RNA was transduced to naïve Huh7.5, Huh7.5/scrambled, and Huh7.5/let-7b cells. The results of in silico analysis and dual-luciferase reporter assay exhibited a specific interaction of HCV-NS5B and let-7b. Real-time PCR analysis revealed that in contrast to infected naïve Huh7.5 cells and Huh7.5/scrambled, a significant decrease in HCV-RNA load was seen in Huh7.5/let-7b cells. On the other hand, the Flow Cytometry test showed that let-7b could significantly induce the apoptosis pathway in Huh7.5/let-7b. CONCLUSIONS: The results also suggest that let-7b, as a target of the HCV genome, potentially reduces HCV replication and raises cell apoptosis rate. We suggest that let-7b directly downregulates HCV replication and may serve as a unique antiviral therapy.
BACKGROUND: Small non-coding RNAs have emerged as essential modulators of viral infections such as hepatitis C virus (HCV). Cellular miRNAs directly regulate the viral infectivity and indirectly by targeting virus-host factors. The current study investigates the inhibitory effect of let-7b miRNA on HCV replication in the Hepatocarcinoma cell line (Huh7.5). METHODS AND RESULTS: The algorithm-based search revealed that let-7b, a high score microRNA, has target sequences on the HCV genome. The Huh7.5 cells were stably transduced with let-7b lentiviral vectors (Huh7.5/let-7b) and mock (Huh7.5/scrambled). The expression of the let-7b level was assessed by real-time PCR assay and Red fluorescence microscope. A dual-luciferase assay was conducted to evaluate the liver-specific let-7b and HCV genome interaction. In the next step, for establishing HCVcc, Full-length HCV-RNA was transduced to naïve Huh7.5, Huh7.5/scrambled, and Huh7.5/let-7b cells. The results of in silico analysis and dual-luciferase reporter assay exhibited a specific interaction of HCV-NS5B and let-7b. Real-time PCR analysis revealed that in contrast to infected naïve Huh7.5 cells and Huh7.5/scrambled, a significant decrease in HCV-RNA load was seen in Huh7.5/let-7b cells. On the other hand, the Flow Cytometry test showed that let-7b could significantly induce the apoptosis pathway in Huh7.5/let-7b. CONCLUSIONS: The results also suggest that let-7b, as a target of the HCV genome, potentially reduces HCV replication and raises cell apoptosis rate. We suggest that let-7b directly downregulates HCV replication and may serve as a unique antiviral therapy.