Nonsynonymous C1653T Mutation of Hepatitis B Virus X Gene Enhances Malignancy of Hepatocellular Carcinoma Cells

Abstract

Cuifang Zhang,1,2 Ying Xie,3 Ruixue Lai,1 Jianhua Wu,4 Zhanjun Guo1 1Department of Rheumatology and Immunology, The Fourth Hospital of Hebei Medical University, Shijiazhuang, People’s Republic of China; 2Department of Oncology, The Pingshan County People’s Hospital, Shijiazhuang, People’s Republic of China; 3Hebei Key Laboratory of Laboratory Animal Science, Hebei Medical University, Shijiazhuang, People’s Republic of China; 4Animal Center, The Fourth Hospital of Hebei Medical University, Shijiazhuang, People’s Republic of ChinaCorrespondence: Zhanjun Guo, Department of Rheumatology and Immunology, The Fourth Hospital of Hebei Medical University, 12 Jiankang Road, Shijiazhuang, 050011, People’s Republic of China, Tel + 86 311 8609 5734, Fax + 86 311 8609 5237, Email [email protected]: Functional analysis was performed to elucidate the mechanism by which hepatocellular carcinoma (HCC) outcome-associated mutation in the hepatitis B virus X (HBx) gene modifies the HCC process.Methods: Proliferation, invasion, migration, and apoptosis assays were performed, and changes in fibrosis, intracellular reactive oxygen species (ROS), and cytokine levels were measured. The differences between variables were evaluated by Student’s t-test.Results: The influence of two previously identified nonsynonymous mutation, C1653T and T1753C, on HCC cells was assessed. With regard to HBX-induced promotion of proliferation (p < 0.01), invasion (p < 0.01) and migration (p < 0.01), the C1653T mutation displayed a significant additive effect in these assays (P < 0.05). The subsequent apoptosis assay indicated that HBX could inhibit apoptosis (P < 0.01), whereas the C1653T mutation markedly amplified this effect in HCC cells (P < 0.01). Furthermore, the tumor growth-promoting effect of HBX was confirmed in a mouse xenograft model of HCC (P < 0.05), and the C1653T mutation was observed to amplify this effect (P < 0.05). To further investigate the mechanism by which the C1653T mutation enhances malignancy in HCC cells, fibrosis, intracellular ROS, and cytokine levels were measured. The C1653T mutant increased fibrosis and intracellular ROS level, and altered monocyte chemotactic protein-1 and interleukin-18 expression in HepG2 cells. Drug sensitivity test revealed that the C1653T mutation is sensitive to apatinib treatment and that overexpression of vascular endothelial growth factor might be involved in this process.Conclusion: Our data indicate that the C1653T mutation of HBx promotes HCC malignancy by altering the levels of fibrosis, ROS, and some cytokines. This mutation could serve as a potential biomarker for screening HCC patients to determine apatinib treatment efficacy.Graphical Abstract: Keywords: HCC, HBV, HBX, C1653T mutation, apatinib, VEGF

Keywords

• hcc
• hbv
• hbx
• c1653t mutation
• apatinib
• vegf