Hepatitis C Virus Core Protein-Induced Activation Of WNT/β-CATENIN Signaling And Its Molecular Pathogenic Mechanisms

Objective:Hepatocellular carcinoma (HCC) ranks among the most common cancers worldwide, its mortality rate is second only to gastric and esophageal carcinoma in China. HCV infection is a major cause of HCC, but the molecular events that lead to hepatocarcinogenesis during HCV infection are poorly understood. At present, the complex interactions between viruses, host and environmental factors are considered to be significant risk factors of HCC. HCV core protein encoded by virus is a multifunctional protein. It has been shown to transactivate several transcription factors, to induce dysregulation of some important intracellular signaling pathways and transcription factors, which results in the imbalance of cell proliferation and differentiation, thus interfering with host immunogical defense, contributing to viral persistence, and eventually involving in HCC pathogenesis.Wnt/β-catenin signaling pathway is a highly well-conserved pathway, which plays a important role in organ development and self-homeostasis. Aberrant activation of Wnt/β-catenin signaling pathway contributes to the hepatocellular carcinogenesis, which plays an important role in more than 50% of HCCs. However, whether HCV core protein exerts any effect on Wnt/β-catenin signaling pathway and hence is involved in HCV-induced liver cancer has not been elucidated. In this study, we aim to construct recombinant adenoviral vector expressing HCV core protein, use HCV core protein overexpression model in vitro to examine whether core protein induces Wnt/β-catenin signaling activation and exerts effect on the cellular biological behavior, and further investigate the molecular mechanisms of HCV-related hepatocellular carcinogenesis.Methods: HCV core gene was amplified by PCR methods,and then cloned into shuttle plasmid pAdTrack-TO4. The recombinant plasmid was homologous recombination with backbone plasmid pAdEasy-1. Subsequently, the plasmid pAdCore was transfected into HEK293 cells, and packed into adenoviral particles. The effects of core protein on Wnt/β-catenin signaling pathway were assessed by luciferase reporter assay, immunofluorescence staining, Real-time PCR. And MTS proliferation assay, crystal violet cell viability assay, cell cycle analysis and xenografts tumor model of human HCC in nude mice were used to investigate the effects of core protein on cellular biological functions. Finally, the molecular mechanisms that HCV core protein activates Wnt/β-catenin pathway were further assessed by luciferase reporter gene assay, immunofluorescence, Western blot, RT-PCR analysis. Results:Recombined adenovirus AdCore has been successfully prepared. HCV core protein acted synergistically on Wnt3a-induecd -catenin-dependent transcriptional activity in HCC cell lines Huh7 and SMMC-7721. Co-expression of core and Wnt3a leaded to stabilization and nuclear accumulation of ?-catenin, which was correlated with the induction of downstream target genes, such as c-Myc, cyclin D1, WISP2 and CTGF. In vitro, exogenous expression of the HCV core protein resulted in increases in both cell proliferation rate and S-phase fraction of cell cycle. In vivo, core protein was shown to effectively accelerate Wnt3a-induced tumor formation and elevate expression of ?-catenin and Wnt target genes in the xenograft tumor model of human HCC. The molecular events that core protein activates Wnt/β-catenin pathway were further investigated. The results showed that core protein upregulated gene expression of canonical Wnt ligands, such as Wnt2, Wnt3, Wnt3a, Wnt8b, Wnt10a, Wnt10b, frizzled receptors Fzd1, 2, 5, 6, 7, 9 and LRP5/6 co-receptors. On the other hand, the expression level of Wnt antagonists SFRP3, 5 and Dkk1 were efficiently repressed. Furthermore, ectopic expression of core protein markedly promoted cell proliferation. Soluble Fzd molecule FrzB orβ-catenin inhibitor siBC efficiently blocked cell growth stimulation by core gene.Conclusions: Our present findings demonstrated that HCV core protein activates canonical Wnt signaling through multiple regulations of several important upstream molecules ofβ-catenin, which promotes cell proliferation and hence involves in HCV-associated pathogenesis and hepatocarcinogenesis.