Abstract
C-terminally truncated hepatitis B virus (HBV) X (ctHBx) infection and exposure to microcystins-LR (MC-LR) can lead to human hepatitis and liver cancer, but the mechanism associated with their synergistically effects not been fully elucidated. The ctHBx (HBxΔ4 and HBxΔ32) lentivirus were constructed and transfected into the HepG2 cells. Then we investigated the function of MC-LR and ctHBx using the molecular biology approaches, including enzyme-linked immunosorbent assay, clone formation assay, scratch wound testing, transwell assays, carried out flow cytometry respectively to examine cell cycle and apoptosis in each group, and detected the related proteins of HBx, MEK/ERK/JNK/p38 in mitogen-activated protein kinase (MAPK) pathway and the downstream proteins such as cdc2, cdc25C, and p53 by western blotting. We found that the protein phosphorylase 2A (PP2A) enzyme activity in MC-LR and HBxΔ32/HBxΔ4 groups decreased more than in MC-LR and HBx group at the same time point and MC-LR concentration (P < 0.05). Meanwhile the proliferation, migration, invasion and colony formation capability of HepG2 cells were significantly enhanced in MC-LR and ctHBx groups (P < 0.05). In addition the proportion of S stage cells in the MC-LR-treated HBxΔ32/HBxΔ4 groups was significantly greater than that in the untreated groups (P < 0.05). Furthermore, the protein expression of MAPK signaling pathway including phospho-MEK1/2, ERKl/2, p38, and JNK were up-regulated by MC-LR and HBxΔ32, and the expression of cyclin-related proteins, including p53, cdc25C, and cdc2 were also activated (P < 0.05). Taken together, our findings revealed the essential significance of the MC-LR and ctHBx on the PP2A/MAPK/p53, cdc25C and cdc2 axis in the formation and development of HCC and identified MC-LR and ctHBx as potential causal cofactors of hepatocarcinogenesis.
Highlights
Hepatitis B virus (HBV) infection, aflatoxin intake and microcystin-polluted drinking water are three major risk factors for hepatocellular carcinoma (HCC) (Yu, 1995; Mittal and ElSerag, 2013)
The results of this study indicated the importance of the MC-LR and c-terminally truncated HBV X (ctHBx) phosphorylase 2A (PP2A)/mitogen-activated protein kinase (MAPK)/cdc25C and p53 axis in the formation and development of HCC and identified MC-LR and ctHBx as potential causal factors for hepatocarcinogenesis
These results confirmed that intracellular HBx 32, HBx 4, and HBx were successfully expressed and that the transfected HepG2 cell lines were available for subsequent cell function experiments
Summary
Hepatitis B virus (HBV) infection, aflatoxin intake and microcystin-polluted drinking water are three major risk factors for hepatocellular carcinoma (HCC) (Yu, 1995; Mittal and ElSerag, 2013). A hepatitis B seroepidemiological survey conducted in 2006 showed that the prevalence rates of HBsAg in individuals aged 1 to 59 years and in children aged 1 to 4 years were 7.18 and 0.96%, respectively, and that approximately 93 million individuals in China are HBV carriers (Liang et al, 2013). The results of our previous study revealed that the C-terminal deletions of four amino acids (HBx 4) and 32 amino acids (HBx 32) were the most common integration fragments of HBx, and these two ctHBx proteins have been shown to play key roles in HCC development (Zheng et al, 2015; Fang et al, 2017; He et al, 2017)
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