Hepatitis B Virus X Protein Induces Hepatic Oval Cell Malignant Transformation

BackgroundHepatocellular carcinoma (HCC) is the fifth most common form of cancer worldwideand the third most common cause of cancer-related deaths. China is a high incidence area ofHCC. The chronic infection of hepatitis B virus (HBV) is the main cause of HCC in China.The carcinogenic mechanism of HBV is not clear, but the hepatitis B virus x protein (HBx)is considered playing an important role in the occurrence and development of HCC. HBx iscoded by hepatitis B virus x gene and plays a regulatory role in gene transcription, cellsignal transduction, cell cycle and cell proliferation.The cancer stem cell (CSC) theory provides a new way to explain the occurrence ofHCC. It is considered that the occurrence of HCC is resulted from the disturbeddifferentiation of CSC. The hepatic oval cell (HOC) is considered as the liver stem cellbecause of the ability of self-renewal and multipotential differentiation. A lot of studysuggested that the oval cell plays a significant role in the in the occurrence anddevelopment of HCC.ObjectiveThe aim of this study was to detect the malignant transformation of HBx on hepaticoval cell, and to find out the correlation between HBx and oval cell in the occurrence ofHCC.Methods1.The WB-F344oval cell which was infected with lentivirus expressing HBx andpuromycin(PM)-resistant gene was defined as experimental group (WB-F344-HBx). TheWB-F344cell which was infected with lentivirus only expressing puromycin-resistant genewas defined as negative control group (WB-F344-PM). The WB-F344cell without infectingwith lentivirus was defined as blank control group (WB-F344-Mock). The expressions ofHBx in the three groups were confirmed by real-time RT-PCR and Western blot analysis. 2.The cell cycles of the three groups were detected with flow cytometry.3.The cell proliferations of the three groups were detected with MTT assay.4.The abilities of cell metastasis of the three groups were detected with cell migrationassay.5.The left axillary subcutaneous inoculations of WB-F344-HBx, WB-F344-PM andWB-F344-Mock oval cells were performed in nude mice, respectively. There were six nudemice in each group. The conditions of tumorigenicity were observed after six weeks.6.HE stain of the tumor was performed for histological observation. The expressionsof HBx and AFP in the tumor were confirmed by immunohistochemistry (IHC).Results1.WB-F344-HBx highly stable expressed HBx at both gene level and protein level.There were no expressions of HBx in WB-F344-PM or WB-F344-Mock.2.Compared with WB-F344-PM, WB-F344-HBx had a lower G0/G1phase cellproportion [(55.98±0.39)%vs.(63.73±2.02)%，P<0.01] and a higher G2/M phase cellproportion [(15.05±1.67)%vs.(7.41±0.38)%，P<0.01]. There was no significant differencein cell cycle between WB-F344-PM and WB-F344-Mock.3.WB-F344-HBx had a stronger ability of cell proliferation compared withWB-F344-PM (P<0.01). There was no significant difference in cell proliferation betweenWB-F344-PM and WB-F344-Mock.4.The numbers of the cells moved across the membrane were249.60±17.28pervision in WB-F344-HBx group，42.50±14.34per vision in WB-F344-PM group and35.30±10.00per vision in WB-F344-Mock group. WB-F344-HBx had a stronger ability of cellmetastasis compared with WB-F344-PM (P<0.01). There was no significant difference inability of cell metastasis between WB-F344-PM and WB-F344-Mock.5.There was only1nude mouse which belonged to WB-F344-PM group died duringthe following six weeks. Subcutaneous tumorigenic rate of WB-F344-HBx, WB-F344-PMand WB-F344-Mock were5/6,0/5,0/6, respectively. And the tumor size was7±1.9mm ofWB-F344-HBx group. Histopathology shown that the subcutaneous tumorwas characterized by invasive growth. The subcutaneous tumor had a positive expression ofHBx, a negative expression of AFP by IHC. The subcutaneous tumor was confirmed notHCC by histopathology and IHC. Conclusions1.The stable hepatic oval cell strain of transfection with HBx gene was successfullyestablished.2.HBx significantly accelerate the process of G1/S cell cycle, promote the cellproliferation, and enhance ability of metastasis.3.HBx can induce hepatic oval cell malignant transformation.