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

Abstract

Purpose: 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.

Keywords: C1653T mutation; HBV; HBX; HCC; VEGF; apatinib.

Graphical abstract

Figure 1

Expression of HBX in the four groups. (A) Immunostaining and Western blot analysis of HBX for Control, HBX, 1653 and 1753 groups in HepG2 cells. (B) Immunostaining and Western blotting analysis of HBX for Control, HBX, 1653 and 1753 groups in HuH7 cells.

Figure 2

The functional analysis indicated that C1653T mutation enhanced the malignancy of HepG2 cells. (A) CCK-8 assay for Control, HBX, 1653 and 1753 groups. (B) Cell invasion assay for Control, HBX, 1653 and 1753 groups. (C) Cell migration assay for Control, HBX, 1653 and 1753 groups. (D) Flow cytometry assay for Control, HBX, 1653 and 1753 groups. **P<0.01.

Figure 3

Mouse xenograft tumor model indicated that C1653T mutant enhanced growth of HepG2 cells in vivo. (A) Tumor growth was monitored by measuring tumor volume for HepG2 xenografts (n= 8 for each group). (B) Growth curves of xenograft tumours. **P<0.01, *P<0.05.

Figure 4

Measurement of fibrosis, ROS and cytokines levels. (A) Fibrosis levels of HA and Col IV in Control, HBX, 1653 and 1753 groups. (B) ROS levels in Control, HBX, 1653 and 1753 groups. (C) Cytokines levels of MCP-1 and IL-18 in Control, HBX, 1653 and 1753 groups. **P<0.01, *P<0.05.

Figure 5

Drug sensitivity test for apatinib. (A) Apatinib concentration screening showed that 2.5 and 5 μmol/L apatinib was suitable for subsequent proliferation assay. (B) 2.5 μmol/L apatinib proliferation assay for Control, HBX, 1653 and 1753 groups. (C) 5 μmol/L apatinib proliferation assay for Control, HBX, 1653 and 1753 groups. (D) Western blotting analysis of VEGF expression. **P<0.01, *P<0.05.