Elsevier

Microbial Pathogenesis

Volume 159, October 2021, 105136
Microbial Pathogenesis

C-terminal-truncated hepatitis B virus X protein promotes hepatocarcinogenesis by activating the MAPK pathway

https://doi.org/10.1016/j.micpath.2021.105136Get rights and content

Highlights

  • We investigated the role and potential mechanism of ctHBX regulated by MAPK pathway involving in HCC development.

  • ctHBx significantly enhanced the proliferation, migration, invasion and colony-forming capability of HepG2 cells.

  • ctHBx may activates the ERK/JNK/p38 MAPK pathway via regulating cdc25C and p53 axis to regulate cell viability.

  • The conclusion about the relation of ctHBX and HCC would contribute to the prevention and treatment of HBV-induced HCC.

Abstract

Purpose

C-terminally truncated hepatitis B virus X (ctHBx) is frequently detected in hepatocellular carcinoma (HCC) patients with hepatitis B virus (HBV) integrated into their genomes, but the molecular mechanisms of ctHBx-related oncogenic signaling remain unclear. In this study, the effects of ctHBx on HepG2 cells were investigated by measuring ctHBx-induced changes in the cell cycle-related target proteins cell division cycle 25C (cdc25C) and p53 downstream of the mitogen-activated protein kinase (MAPK) pathway.

Materials and methods

ctHBx lentiviruses were constructed and transfected into HepG2 cells. Then, we investigated HepG2 cell line function by conducting the Cell Counting Kit-8 (CCK8) assay, clone formation assay, scratch wound testing, Transwell assays and flow cytometry to examine cell cycle and apoptosis. Western blotting (WB) was performed to detect proteins related to and downstream of the extracellular signal-regulated kinase(ERK)/c-Jun N-terminal kinase(JNK)/p38 MAPK pathway, including cdc25C and p53.

Results

ctHBx significantly enhanced the proliferation, migration, invasion and colony-forming capability of HepG2 cells. In addition, ctHBx activated the ERK/JNK/p38 MAPK signaling pathway to regulate cell viability by affecting the expression of cyclin-related proteins, including cdc25C and p53.

Conclusion

The present study demonstrates that ctHBx promote the formation and development of HCC via regulating MAPK/cdc25C and p53 axis. ctHBx should be the driving factor of HBV-induced hepatocarcinogenesis.

Introduction

Hepatitis B virus (HBV) infection is the major risk factor for hepatocellular carcinoma (HCC) [1]. According to the latest data for 2019, the estimated carrier rate of HBsAg in China was 5%–6%, and approximately 70 million people had chronic HBV infection [2,3]. Data for an area with a high incidence of liver cancer data showed that the rates of HBsAg carriers reached 20.21% in males aged 35–64 years and 13.18% in females aged 40–64 years [4]. The high rate of HBV infection among individuals with liver cancer implies that HBV may associate with the onset of HCC.

The HBV genome is 3200 bp in length and contains four open reading frames, namely, S, C, P and X [[5], [6], [7]]. Host genome integration occurs most frequently with the X gene, followed by the S, C and P genes [8,9]. Previous studies have shown that HBV DNA fragments can be detected in the human chromosome in tissues from 80% to 90% of hepatitis B-associated liver cancer patients, and more than half of the integration fragments are derived from the HBV X gene [[10], [11], [12]]. The HBV X gene is 465 bp in length but is often partially deleted during integration, and thus is ultimately integrated into human chromosomes with a truncated C-terminus [[13], [14], [15]]. Hoare et al. found that 10 (58.8%) of 18 patients with liver cancer had HBx deletion mutations [16]. Varying truncations in HBx have been verified by several studies; these include the truncation of 20 or 35 amino acids in the C-terminus of HBx in HCC tissues [[17], [18], [19]]. Our previous study revealed that the C-terminal deletions of four amino acids (HBxΔ4) and 32 amino acids (HBxΔ32) were the most common integration fragments of HBx, and these two ctHBx proteins have been shown to play important roles in HCC development [[20], [21], [22]]. However, the exact mechanism underlying the involvement of these proteins in hepatocarcinogenesis remains largely unknown; thus, the role of ctHBx in HCC needs to be further investigated.

The mitogen-activated protein kinase (MAPK) family mainly includes ERK1/2, JNK, p38 and other subfamilies [23,24]. Furthermore, HBx reportedly activates MAPK signaling to promote oncogenesis in the early stages of chronic hepatitis B [25]. Therefore, changes in MAPK pathway activity might be related to the development of liver cancer. HBx mutants, particularly ctHBx, play a multifunctional carcinogenic role in the development of HBV-associated liver cancer, such as promoting cell cycle progression, increasing cell migration, and regulating the cell cycle and apoptosis [26]. However, the involvement of ctHBx in carcinogenesis and progression, particularly via activation of the MAPK signaling pathway in HCC is still poorly understood.

In this study, we proposed that ctHBx might cause cancer by altering MAPK signal transduction. To verify this hypothesis, we investigated the effects of ctHBx on HepG2 proliferation, invasion, migration, apoptosis and the cell cycle. Specifically, ctHBx-induced changes in the cell cycle-related target proteins cdc25C and p53 downstream of the MAPK pathway were explored.

Section snippets

Cell culture

The human hepatoma cell line HepG2 was purchased from Zhongqiao Xinzhou Biotechnology Co., Ltd. (Shanghai, China). The cells were cultured in DMEM containing 10% FBS, streptomycin (100 units/mL), and penicillin (100 units/mL; Beijing Solarbio Science & Technology Co., Ltd, Beijing, China).

Stable cell line construction

The construction of the HBx eukaryotic expression vector and the packaging of the virus were performed and confirmed by Sangon Biotech Co., Ltd. (Shanghai, China). The packaged lentiviruses were named

Intracellular expression of HBxΔ32, HBxΔ4 and HBx

The expression of HBxΔ32, HBxΔ4 and HBx in HepG2 cells was assessed by qRT-PCR and WB. The HBxΔ32, HBxΔ4 and HBx genes were successfully amplified from cDNAs isolated from the three transfected cell populations; no amplification was observed with the cDNA from control cells (Fig. 1A). The WB analysis showed the HBx-specific band in the protein samples from the three transfected cell populations, whereas no band was observed in the protein samples from the control cells (Fig. 1B). These results

Discussion

Our previous studies have demonstrated the relationships between HCC and the distribution of HBV genotypes and subtypes and the variants of HBV pre-S, S, BCP pre-C and X genes; the existence of a quasispecies-dominant strain of HBV and its relationship with HBV-related HCC; and the integration site and target genes of HBx in the chromosomes of liver cancer hosts, and revealed that ctHBx (HBxΔ32 and HBxΔ4) is a commonly integrated fragment in liver cancer tissues [[20], [21], [22]]. However, the

Conclusion

In conclusion, we demonstrated that ctHBx can promote the proliferation, migration and invasion of HepG2 cells via the MAPK/cdc25C/p53 axis (Fig. 8) and plays an important role in hepatocarcinogenesis. However, our study has several limitations: in vivo experiments using an animal model have not performed, and the specific section of the HBx gene that affects the biological function of cells has not been further clarified. Therefore, the specific carcinogenic mechanism associated with the

Declaration of competing interest

The authors declare that they have no conflict of interest.

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