Epigenetic harnessing of HCV via modulating the lipid droplet-protein, TIP47, in HCV cell models

AbstractThis study aimed at identifying potential microRNAs that modulate hepatic lipid droplets (LD) through targeting the Tail interacting protein of 47kDa (TIP47) in HCV infection.Bioinformatics analysis revealed that miR-148a and miR-30a potentially target TIP47. Expression profiling showed that both microRNAs were downregulated, while TIP47 was upregulated in liver biopsies of HCV-infected patients. Forcing the expression of both microRNAs in JFH-I infected, oleic acid-treated Huh7 cells, significantly suppressed TIP47 expression and reduced cellular LDs with marked decrease in viral RNA. This study shows that miR-148a and miR-30a, regulate TIP47 expression and LDs in HCV infected cells

Repressed induction of interferon-related microRNAs miR-146a and miR-155 in peripheral blood mononuclear cells infected with HCV genotype 4

AbstractMicroRNAs regulate the expression of many genes and subsequently control various cellular processes, such as the immune response to viral infections mediated by type I interferon (IFN). In this study, the expression pattern of two interferon-related microRNAs, miR-146a and miR-155, was examined in healthy and HCV-genotype-4-infected peripheral blood mononuclear cells (PBMCs) using qRT-PCR. In contrast to other viral infections, the expression pattern was similar in both healthy and infected PBMCs. This could be attributed to attenuation of IFN pathway by HCV, which was assessed by investigating the expression of MxA, an interferon-stimulated gene, that showed lower expression in HCV-infected PBMCs. To determine the site of interference of HCV in the IFN pathway, expression of both microRNAs was examined following stimulation of PBMCs with IFN-α2a, an activator of the JAK/STAT pathway as well as with imiquimod, a toll-like receptor-7 (TLR-7) agonist that promotes interferon release. IFN stimulation induced the expression of miR-146a and miR-155 in HCV-infected and healthy PBMCs. Stimulation with imiquimod led to a down-regulation of both microRNAs in infected PBMCs, while it increased their expression in healthy PBMCs, indicating that HCV might interfere with miR-146a and miR-155 expression at sites upstream of interferon release, specifically in the TLR-7 pathway. The pattern of expression of both miR-146a and miR-155 was very similar with a strong positive correlation, but showed no correlation to the patients’ clinical or histopathological parameters or response to treatment. In conclusion, HCV infection might repress the induction of miR-146a and miR-155 by interfering with TLR-7 signaling

Methylation in MIRLET7A3 Gene Induces the Expression of IGF-II and Its mRNA Binding Proteins IGF2BP-2 and 3 in Hepatocellular Carcinoma

miR-let-7a is a tumor suppressor miRNA with reduced expression in most cancers. Methylation of MIRLET7A3 gene was reported to be the cause of this suppression in several cancers; however, it was not explicitly investigated in hepatocellular carcinoma (HCC). We aimed at investigating miR-let-7a expression and molecular mode in HCC, identifying drug-targetable networks, which might be affected by its abundance. Our results illustrated a significant repression of miR-let-7a, which correlated with hypermethylation of its gene of origin MIRLRT7A3. This was further supported by the induction of miR-let-7a expression upon treatment of HCC cells with a DNA-methyltransferase inhibitor. Using a computational approach, insulin-like growth factor (IGF)-II and IGF-2 mRNA binding proteins (IGF2BP)-2/-3 were identified as potential targets for miR-let-7a that was further confirmed experimentally. Indeed, miR-let-7a mimics diminished IGF-II as well as IGF2BP-2/-3 expression. Direct binding of miR-let-7a to each respective transcript was confirmed using a luciferase reporter assay. In conclusion, this study suggests that DNA hypermethylation leads to epigenetic repression of miR-let-7a in HCC cells, which induces the oncogenic IGF-signaling pathway

Disruption of Claudin-1 Expression by miRNA-182 Alters the Susceptibility to Viral Infectivity in HCV Cell Models

HCV entry involves a complex interplay between viral and host molecules. During post-binding interactions, the viral E2 complexes with CD81 receptor for delivery to the tight junction proteins CLDN1 and OCLN, which aid in viral internalization. Targeting HCV entry receptors represents an appealing approach to inhibit viral infectivity. This study aimed at investigating the impact of targeting CLDN1 by microRNAs on HCV infectivity. miR-155 was previously shown to target the 3′UTR of CLDN1 mRNA. Therefore, miR-155 was used as a control in this study. In-silico analysis and luciferase reporter assay were utilized to identify potential targeting miRNAs. The impact of the identified miRNAs on CLDN1 mRNA and protein expression was examined by qRT-PCR, indirect immunofluorescence and western blotting, respectively. The role of the selected miRNAs on HCV infectivity was assessed by measuring the viral load following the ectopic expression of the selected miRNAs. miR-182 was identified in-silico and by experimental validation to target CLDN1. Both miR-155 and miR-182 inhibited CLDN1 mRNA and protein expression in infected Huh7 cells. Ectopic expression of miR-155 increased, while miR-182 reduced the viral load. In conclusion, despite repressing CLDN1, the impact of miR-155 and miR-182 on HCV infectivity is contradictory. Ectopic miR-182 expression is suggested as an upstream regulator of the entry factor CLDN1, harnessing HCV infection

MiR-29a promotes lipid droplet and triglyceride formation in HCV infection by inducing expression of SREBP-1c and CAV1

Aims: To examine the regulation of SREBP-1c and CAV1 by microRNA-29a (miR-29a) in cells infected with hepatitis C virus (HCV) in an attempt to control HCV-induced non-alcoholic fatty liver disease. Methods: In order to examine the manipulation of SREBP-1c and CAV1 by miR-29a, oleic acid (OA)-treated JFH-I-infected Huh-7 cells were used. OA was added 24 h post-transfection and gene expression was investigated by qRT-PCR at 48 h post treatment. The functional impact of the observed alteration in SREBP-1c and CAV1 expression was analyzed by examining lipid droplet (LD) and triglyceride (TG) content at 72 h post-OA treatment using light microscopy and spectrophotometry, respectively. Viral load was quantified by qRT-PCR at 72 h post-transfection. Results: OA treatment induced the expression of miR-29a and SREBP-1c, as compared to untreated cells. Forced miR-29a expression led to a significant up-regulation of SREBP-1c as well as CAV1 compared to mock untransfected cells. Ectopic expression of miR-29a resulted in a marked increase in LDs and their respective TGs, while miR-29a antagomirs decreased both the LD and TG content compared to mock untransfected cells. Moreover, forcing the expression of miR-29a in JFH-1 HCV-infected Huh-7 cells resulted in 53% reduction in viral titers compared to mock untransfected Huh-7 cells. Conclusion: Inducing miR-29a expression significantly induces SREBP-1c and CAV1 expression, thereby increasing LDs as well as their respective TGs. Nonetheless, forcing the expression of miR-29a resulted in reduction of HCV RNA levels in Huh-7 cells

DataSheet1.DOC

<p>HCV entry involves a complex interplay between viral and host molecules. During post-binding interactions, the viral E2 complexes with CD81 receptor for delivery to the tight junction proteins CLDN1 and OCLN, which aid in viral internalization. Targeting HCV entry receptors represents an appealing approach to inhibit viral infectivity. This study aimed at investigating the impact of targeting CLDN1 by microRNAs on HCV infectivity. miR-155 was previously shown to target the 3′UTR of CLDN1 mRNA. Therefore, miR-155 was used as a control in this study. In-silico analysis and luciferase reporter assay were utilized to identify potential targeting miRNAs. The impact of the identified miRNAs on CLDN1 mRNA and protein expression was examined by qRT-PCR, indirect immunofluorescence and western blotting, respectively. The role of the selected miRNAs on HCV infectivity was assessed by measuring the viral load following the ectopic expression of the selected miRNAs. miR-182 was identified in-silico and by experimental validation to target CLDN1. Both miR-155 and miR-182 inhibited CLDN1 mRNA and protein expression in infected Huh7 cells. Ectopic expression of miR-155 increased, while miR-182 reduced the viral load. In conclusion, despite repressing CLDN1, the impact of miR-155 and miR-182 on HCV infectivity is contradictory. Ectopic miR-182 expression is suggested as an upstream regulator of the entry factor CLDN1, harnessing HCV infection.</p

Image1.PNG

<p>HCV entry involves a complex interplay between viral and host molecules. During post-binding interactions, the viral E2 complexes with CD81 receptor for delivery to the tight junction proteins CLDN1 and OCLN, which aid in viral internalization. Targeting HCV entry receptors represents an appealing approach to inhibit viral infectivity. This study aimed at investigating the impact of targeting CLDN1 by microRNAs on HCV infectivity. miR-155 was previously shown to target the 3′UTR of CLDN1 mRNA. Therefore, miR-155 was used as a control in this study. In-silico analysis and luciferase reporter assay were utilized to identify potential targeting miRNAs. The impact of the identified miRNAs on CLDN1 mRNA and protein expression was examined by qRT-PCR, indirect immunofluorescence and western blotting, respectively. The role of the selected miRNAs on HCV infectivity was assessed by measuring the viral load following the ectopic expression of the selected miRNAs. miR-182 was identified in-silico and by experimental validation to target CLDN1. Both miR-155 and miR-182 inhibited CLDN1 mRNA and protein expression in infected Huh7 cells. Ectopic expression of miR-155 increased, while miR-182 reduced the viral load. In conclusion, despite repressing CLDN1, the impact of miR-155 and miR-182 on HCV infectivity is contradictory. Ectopic miR-182 expression is suggested as an upstream regulator of the entry factor CLDN1, harnessing HCV infection.</p