The Role Of Notch1and Wnt/β-catenin Signaling Pathways In Hepatitis B Virus X Protein-associated Hepatocellular Carcinoma

Objective Hepatocellular carcinoma (HCC) is a highly lethal cancer, that is mainly associated with chronic hepatitis B virus (HBV) infections. There are few effective treatments partly because the cell-and molecular-based mechanisms that contribute to the pathogenesis of this tumour type are poorly understood. Thus, it is crucial to further investigate pathogenic mechanisms and discover novel therapeutic targets for this disease. The hepatitis B virus X protein (HBx), which is encoded by the hepatitis B virus X gene (HBX), is essential for HBV-associated HCC. The Notch signaling pathway is evolutionarily conserved. Notch signaling may generate opposing effect in different steps of carcinogenesis, depending on the tumor cell type and the status of other signaling pathways, such as Wnt signaling pathway. Our previous studies have shown that activated Notch1signaling is required for hepatocarcinogenesis of the human non-tumor hepatic cell line L02induced by HBx. Here, the role and the exact mechanism of Notch1and Wnt/β-catenin signaling pathways in HBx-associated HCC was explored from the aspects of the cell cycle and apoptosis regulation, and the cross-talk between the two pathways. We hope to provide new ideas for targeting therapy of HCC.Methods Notchl short hairpin RNA (shRNA) was utilized to inhibit Notchl signaling pathway in the present study, and cell proliferation and tumor formation of L02/HBx cells in a BALB/c nude mouse model were measured. Cell cycle and apoptosis were detected by flow cytometry, and the expression of regulators about cell cycle and apoptosis were tested by qRT-PCR and Western blot. In addition, shRNA and over-expression plasmid were used respectively to down-regulate and up-regulate the activity of Wnt/β-catenin signaling pathways, cell proliferation and key factors of two pathways were measured.Results Notch1shRNA inhibited cell proliferation together with decreased activity of the Notchl pathway in vitro, and also markedly suppressed tumor formation of L02/HBx cells in a BALB/c nude mouse model in vivo. Furthermore, the blockade of Notchl was capable of arresting the cell cycle in the G0/G1phase through the downregulation of CyclinD1, CDK4, E2F1and the upregulation of p21and Rb, while all of these factors were involved in the CyclinDl/CDK4pathway. Inhibition of Notchl by shRNA markedly promoted the apoptosis of L02/HBx cells via the caspase-9-caspase-3pathway. Activated Notchl and Wnt/β-catenin signaling pathways and L02cell malignant transformation were induced by HBx. Inhibition of the Notchl pathway decreased the activation of Wnt/β-catenin pathway and cell proliferation, while inhibition of the Wnt/β-catenin pathway impaired cell proliferation, but did not significantly affect Notchl signaling pathway in L02/HBx cells. Furthermore, inhibition of the Wnt/p-catenin pathway overcame the inhibition effect of knockdown Notchl on proliferation and survival in L02/HBx cells. Additionally, the activity of Wnt/β-catenin signaling appears to be consistent with Fzd10expression.Conclusions These data suggest that Notchl signaling pathway plays an essential role in HBx-associated HCC, in which Notchl signaling pathway induces the cell cycle and apoptosis disorders via cyclingDl/CDK4and caspase-9-caspase-3pathways, and activate Wnt/β-catenin signaling pathway via Fzd10. Therefore, Notchl may be a putative therapeutic target for HBx-associated HCC, which requires its collaboration with Wnt/β-catenin signaling pathway.