Anti-COX-2 antibody was purchased from Abcam (Cambridge, UK; cat no. ab151571). Anti-β-catenin antibody was purchased from OriGene Technologies, Inc. (Rockville, MD, USA; cat no. sc-7963). Anti-c-myc antibody was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA; cat no. SC-40). Anti-cyclin D1 antibody was purchased from Santa Cruz Biotechnology, Inc. (cat no. sc-70899). Anti-β-actin antibody was purchased from OriGene Technologies, Inc. (cat no. TA-09). Peroxidase-conjugated goat anti-rabbit immunoglobulin (Ig)G (H+L) was purchased from OriGene Technologies, Inc. (cat no. ZB-2301). Peroxidase-conjugated goat anti-mouse IgG (H+L) was purchased from OriGene Technologies, Inc. (cat no. ZB-2305). XAV939 was purchased from Selleck Chemicals (Houston, TX, USA). Cells were treated with 20 µmol/l XAV939 (a β-catenin inhibitor) and DMSO (as a control) for 24 h in cell proliferation assay. NS-398 was purchased from Beyotime Institute of Biotechnology (Haimen, China). Cells were treated with 50 µmol/l NS-398 (a selective COX-2 inhibitor) and DMSO (as a control) for 72 h in western blot analysis and colony formation assay. Primers were synthesized by Biosune Biotechnology Co., Ltd (Shanghai, China; www.biosune.com). The enhanced chemiluminescence kit was purchased from OriGene Technologies, Inc. TRIzol reagent was purchased from Life Technologies (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Moloney murine leukemia virus (MMLV) transcriptase was purchased from New England BioLabs., Inc. (Ipswich, MA, USA). Dulbecco's Modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from HyClone Company; GE Healthcare (Chicago, IL, USA).

The human hepatocyte HL-7702 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). HL-7702-HBx cells (HL-7702 cells transfected with lentiviral vector pLOV.flag-HBx to stably express the HBX gene: HBV subtype ayw) and HL-7702-mock cells (HL-7702 cells transfected with lentiviral vector pLOV.flag) were constructed previously by the authors of the present study (5). HBx, mock and control represent HL-7702-HBx, HL-7702-mock and HL-7702 cells, respectively. Cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in a humidified atmosphere of 5% CO₂.

Total RNA was isolated using TRIzol reagent. The primer sequences of each gene are listed in Table I. First-strand cDNA was generated using MMLV transcriptase (2 µg total RNA, 1 µg primer, 5 µl dNTP, 5 µl M-MLV 5X Reaction Buffer and final volume 25 µl), incubated for 60 min at 37°C. The extension temperature is 37°C, and qPCR was performed using FastStar universal SYBR Green master mix (Roche Applied Science, Penzberg, Germany) in triplicate using an Applied Biosystems Step one plus Real time PCR system (Life Technologies; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Thermocycling conditions of the qPCR reaction are as follows: 95°C for 3 min, then 40 cycles of 95°C for 15 sec followed by 60°C for 30 sec. Specific primers were used to detect the relative mRNA expression by the 2^−ΔΔCq method. The expression level was normalized against endogenous β-actin (16).

Cells were harvested and lysed by RIPA buffer from Beyotime Institute of Biotechnology. The protein concentration of each sample was determined using a bicinchoninic protein assay kit. A total of 20 ug protein per lane was loaded on a 10% SDS/PAGE gel, and subsequently transferred to a nitrocellulose membrane. The membrane was blocked in 5% milk in TBS-Tween 20 [0.1% Tween 20, 20 mM Tris (pH 7.4) and 150 mM NaCl] for 2 h at room temperature, followed by overnight incubation with the primary antibody at 4°C. Primary antibodies were used at 1:1,000 (Anti-COX-2 antibody, Anti-β-catenin antibody, Anti-c-myc antibody and Anti-β-actin) or 1:500 (Anti-cyclin D1 antibody), and secondary antibodies conjugated with horseradish peroxidase were used at a 1:2,000 dilution in 5% nonfat dry milk at room temperature for 1 h. Following a final wash, proteins were visualized using the enhanced chemiluminescence kit. Images of the blots were captured using an Image Scanner (Epson, Nagano, Japan). Expression levels of protein were semi-quantitatively analyzed by using Image J Launcher (Broken Symmetry Software 1.4.3.67), normalized to β-actin density.

A Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Laboratories, Inc., Kumamoto, Japan) was used to analyze cell proliferation. Cells were seeded into 96-well plates at 3×10³ cells/well in 100 µl complete DMEM and incubated at 37°C and 5% CO₂. The plates were incubated for 24, 48 and 72 h, the medium was removed and 100 µl fresh complete DMEM was added, containing 10 µl CCK-8, to each well. The plates were incubated for 3 h at 37°C. The absorbance was measured at a wavelength of 450 nm using an ELISA plate reader. Proliferation inhibition rate was calculated according to the following formula: Proliferation inhibition rate (%)=(1-OD₄₅₀/OD_{450 with XAV939}) ×100%.

Cells were seeded into 6-well plates at 500 cells/well in triplicate and incubated at 37°C and 5% CO₂ for 14 days. Clones were observed in the dish. Each well was washed with PBS twice, fixed with 1 ml 100% methanol for 15 min, then washed with PBS for three times and stained with crystal violet staining solution (Beyotime Institute of Biotechnology) for 10 min at room temperature. Directly counted by naked eyes based on experience, in case of doubting whether 50 or more cells formed the suspected colony, counted the colony by stereomicroscope under lower magnification (×40). Clone formation rate and Clone formation inhibition rate were calculated according to the following formula: Clone formation rate=number of formed colonies/number of seeded cells ×100. Clone formation inhibition rate=(1-number of formed colonies/number of formed colonies with NS398) ×100%.

Cells were harvested and resuspended in PBS (1×10⁶ cells/ml). For analysis of the cell cycle, cells were fixed in 70% ethanol overnight at 4°C. Subsequently, cells were digested with 50 µl RNase (TakaRa Biotechnology Co. Ltd. Dalian, China; 10 mg/ml) at 37°C for 30 min and stained with 20 µl 50 µg/ml propidium iodide (Beyotime Institute of Biotechnology) at 37°C for 10 min in the dark. The DNA histograms were determined by flow cytometry (C6; Becton-Dickinson; BD Biosciences, San Jose, CA).

Experiments were performed in triplicate. All statistical analyses were performed using the SPSS version 16.0 statistical software package (SPSS, Inc., Chicago, IL, USA). Continuous variables are expressed as the mean ± standard deviation. One-way analysis of variance was used for the statistical analyses, followed by the Fisher's Least Significant Difference. P≤0.05 was considered to indicate a statistically significant difference.

Considerable efforts have been made to detect the role of HBx in cell proliferation (17). A cell viability assay and plate colony formation assay were performed in the present study to observe the role of HBx in HL-7702 cell proliferation. As demonstrated in Fig. 1A, HL-7702-HBx cells grew faster compared with the mock and control groups on days 1, 2 and 3 (P<0.05). As presented in Fig. 1B, following incubation for 2 weeks, HL-7702-HBx cells formed more colonies and had higher colony formation efficiency compared with the mock and control groups. In addition, HL-7702-HBx cells exhibited a shortened G1 phase of the cell cycle compared with the HL-7702-mock and HL-7702-control cells (Fig. 1C). These results suggested that HBx promoted the proliferation of HL-7702 cells.

Previous studies have demonstrated that HBx promoted the levels of mitochondrial ROS in HL-7702 cells (4,5). ROS from mitochondria lead to COX-2 induction (18). As demonstrated in Fig. 2A and B, HBx enhanced the mRNA and protein expression levels of COX-2 in HL-7702 cells. COX-2 increases proliferation in various types of tumor and its expression appears to be associated with early HCC events (19). The present study investigated whether upregulation of COX-2 promoted the proliferative ability of HL-7702 cells and treated cells with NS-398. COX-2 protein expression levels and colony formation efficiency were analyzed. As presented in Fig. 2C and D, NS-398 suppressed the colony formation efficiency in the three groups investigated, the colony formation inhibition rate of HL-7702-HBx cells was significantly higher than that of other two groups after downregulation of COX-2 at the protein level (Fig. 2E). Therefore, HBx may promote cell proliferation through upregulation of COX-2.

The Wnt/β-catenin signaling pathway is involved in cell proliferation, differentiation and oncogenesis (10,12). The present study questioned whether aberrant activation of the Wnt/β-catenin signaling pathway occurs in HBx-transfected cells. The mRNA expression level of β-catenin was increased in HL-7702-HBx cells compared with the control and mock groups (Fig. 3A). In support of this upregulation at the mRNA level, the protein expression of β-catenin was increased in HL-7702-HBx cells compared with the other two cell groups (Fig. 3B). The results demonstrated that the mRNA and protein expression levels of β-catenin responsive genes, c-myc and cyclin-D1, were activated compared with the mock and control groups (Fig. 3C-E). These data suggested that HBx may activate Wnt/β-catenin signaling in HL7702 cells.

To further examine the association between β-catenin and COX-2 in HL-7702-HBx cell proliferation, cells were treated with NS-398 (50 µmol/l) for 72 h, and as presented in Fig 3B and Fig. 4A, NS398 attenuated the effects of HBx on β-catenin expression in HL7702-HBx cells. The present study considered whether the upregulation of β-catenin resulted in increased proliferation of HL-7702-HBx cells. To support this hypothesis, cell viability assays were performed following treatment of the cells with a β-catenin inhibitor, XAV939 (20 µmol/l), for 24 h. As indicated in Fig. 4B, treatment with XAV939 suppressed cell proliferation in the three groups. HL7702-HBx cells treated with XAV939 demonstrated higher proliferative inhibition rate than the mock and control groups (Fig. 4C). Therefore, it was concluded that upregulation of COX-2 may promote the proliferation of HL-7702-HBx cells via activation of the Wnt/β-catenin signaling pathway.