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.

Mesoderm/mesenchyme homeobox l may promote tumor progression in human hepatocellular carcinoma

“…The Huh7 and Hep3B cell lines were stored at the Hebei Key Lab of Laboratory Animal Science of Hebei Medical University (Shijiazhuang, China) and cultured as previously described. 21,22…”

The role of hepatitis B virus genome variations in HBV-related HCC: effects on host signaling pathways

“…The HBV X region consists of important cis- regulatory elements including enhII, CP and microRNA binding region ( Zhang et al, 2022 ). A higher ability to promote HBV replication and increase hepatocyte proliferation of some HBx mutants was confirmed in vitro compared to wild-type HBx ( Lin et al, 2019 ).…”

“…HBx mutations including AG1762/1764TA (KV130/131MI), C1653T (H94Y), C1485T (P38S), T1753C (I127T), A1383C, and G1613A, with or without substitution of amino acids, probably alter HCC survival ( Zhang et al, 2022 ). Some mutations, such as C1653T, T1753V, and A1762T/G1764A (TA), can contribute to HCC by altering the binding ability of several nuclear factors or amino acid sequence of HBx.…”

“…The C1653T mutation also had a promoting impact on the tumor growth effect of HBx in an HCC xenograft mouse model. It seems that C1653T mutation leads to HCC by promoting fibrosis and intracellular ROS production and changing the expression of monocyte chemotactic protein 1 (MCP-1) and interleukin 18 in the HepG2 cell line ( Zhang et al, 2022 ). T1753V could affect apoptosis by altering the binding capacity of HBx with bcl2 ( Geng et al, 2012 ).…”

X gene mutations of Hepatitis B virus and impact on chronic hepatitis B infection in CHB three-generations in the family

“…Speci c mutations such as C1653T, AG1762/1764TA, T1753C, C1485T, G1613A, and A1383C have been linked to HCC survival, though their functional consequences are unclear [14]. The presence of the no synonymous mutation C1653T in the EnH II region may modify binding a nity, leading to amino acid substitution in the HBx protein.…”