Introduction
Hepatocellular carcinoma (HCC) is the primary form of liver cancer and the second leading cause of cancer-related deaths globally, with approximately 140,000 cases attributed to chronic hepatitis C virus (HCV) infection. HCV is a hepatotropic positive-sense RNA virus that encodes a polyprotein, generating various structural and non-structural proteins. Chronic liver damage leads to unregulated tissue regeneration, causing progressive fibrosis, which, if not reversed, can result in HCC development. Despite the availability of direct-acting antivirals to resolve HCV infection, no approved drugs exist for treating liver fibrosis. This study aims to elucidate the molecular mechanisms of fibrosis induced by HCV NS5A and Core proteins in Huh7 cells through the activation of LX2 cells.
Materials and Methods
Cell Culture
The human hepatoma cell line Huh-7 and the human hepatic stellate cell line LX-2 were cultured under specific conditions to maintain their viability and functionality. Huh-7 cells were cultured in Advanced Dulbecco’s Modified Eagle Medium, while LX-2 cells were cultured in Dulbecco’s Modified Eagle Medium, both supplemented with necessary nutrients and antibiotics.
Plasmid Transfection
Huh7 cells were transfected with plasmids pNluc-NS5A/HCV and pCore/HCV to express the respective HCV proteins. Transfection efficiency was assessed using RT-qPCR and nanoluciferase reporter assays.
Co-Culture of Transfected Huh7 Cells with LX-2 Cells
Transfected Huh7 cells were co-cultured with LX-2 cells in a transwell system to study the intercellular communication and its impact on fibrosis-related gene expression. The co-culture was maintained for 48 and 72 hours, after which cells were harvested for analysis.
Gene Expression Analysis
Fibrosis-related gene expression was evaluated using RT-qPCR and a human fibrosis PCR array. Differentially expressed genes (DEGs) were analyzed for pathway enrichment and protein-protein interactions using bioinformatics tools.
Results
NS5A and Core Protein Expression Induces LX2 Activation
Transfection of Huh7 cells with pNluc-NS5A/HCV and pCore/HCV induced the activation of LX2 cells, as evidenced by increased expression of Collagen1, a-SMA, TGFb1, and TIMP1. The activation markers showed differential expression at 48 and 72 hours of co-culture, suggesting a time-dependent response.
Fibrosis-Related Gene Regulation
Gene expression profiling revealed that NS5A and Core proteins regulate diverse fibrosis-related genes in Huh7 cells during co-culture with LX2. NS5A induced the expression of genes involved in TGFb signaling and ECM organization, while Core protein expression led to the differential regulation of genes associated with these pathways.
Pathway Enrichment and Protein-Protein Interaction Networks
Bioinformatics analysis identified significant enrichment of TGFb receptor signaling and ECM organization pathways. Protein-protein interaction networks highlighted the involvement of various cellular functions, including signal transduction and cell adhesion.
Discussion
This study demonstrates that HCV NS5A and Core proteins play a crucial role in activating hepatic stellate cells and regulating fibrosis-related gene expression. The findings underscore the importance of intercellular communication in the molecular mechanisms of fibrosis during HCV infection. Identifying genetic signatures related to fibrotic pathways provides potential therapeutic targets for developing antifibrotic drugs.
Conclusion
HCV NS5A and Core proteins induce the activation of LX2 cells and regulate fibrosis-related gene expression in Huh7 cells. These findings highlight the significance of intercellular communication in fibrosis development and suggest potential targets for antifibrotic therapy.
References
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Authors: Tania G. Heredia-Torres, Veronica Alvarado-Martínez, Ana R. Rincón-Sánchez, Sonia A. Lozano-Sepúlveda, Kame A. Galán-Huerta, Daniel Arellanos-Soto, Ana M. Rivas-Estilla