Cell lines and drugs
The THLE-3 (normal human liver cells), HepG2 (human hepatoma cells) and HepG2.2.15 (HBV transfected HepG2 cells) cell lines were obtained from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). All cell lines were authenticated by STR profiling. Entecavir was purchased from Sino American Shanghai Squib Pharmaceutical Limited (Shanghai, China) and the HepG2.2.15 cells were treated with different concentration of Entecavir (5 μg/mL, 15 μg/mL and 50 μg/mL).
EphA2-shRNA vector transfection
According to the target sequence “GAAGTTCACTACCGA GATC”, a recombinant lentiviral expression vector of EphA2-shRNA was designed and constructed by Jikai Gene Chemical Technology Company Limited (Shanghai, China). The liver cancer cells in the logarithmic stage of growth were digested with trypsin and seeded at a density of 5×105 cell per well in 6-well plates. After the fusion degree of cells reached 30%, the EphA2-shRNA lentiviral vector or the blank vector was added into the HepG2 and HepG2.2.15 cells. Three days after transfection, the EphA2 knockdown efficiency was found to be greater than 80%.
Cell migration and invasion analysis
When the cells were grown to 70% confluence, the initial scratch width (730 μm) was generated using a 96-pin Wound Maker tool. Following washing with PBS solution, the cells were cultured with DMEM/F12 medium. For invasion analysis, the matrigel was applied to create a 3D matrix on top of the cells. Time-lapse images of the cells were obtained at 36, 48, 60, 72, 84, 96, 120 and 144 h with six samples per group, and the migration length and the invasion length (the difference between the initial scratch width and the final wound width) were determined using a real-time quantitative live-cell analysis system (IncuCyte Zoom, Essen Bioscience, MI, USA). Each test process was repeated three times.
Immunofluorescence assay
The cells were fixed in 95% alcohol at 4°C for 4 h and were incubated with 0.3% H2O2 at 37°C for 10 min. Following washing with PBS, the cells were immersed into 1% bovine serum albumin at 37°C for 30 min. The cells were incubated with the primary antibody including EphA2 (CST, MA, USA), E-cadherin, Vimentin, β-catenin and p-GSK-3βSer9 (Abcam, Cambridge, UK) at 4°C overnight, which was followed by an incubation with a rhodamine-conjugated secondary antibody at 37°C for 30 min. Finally, the cells were counterstained with DAPI (4', 6-Diamidino-2-Phenylindole) and observed by a fluorescence microscopy. The density (mean) of each protein was then quantified using the image pro plus 6.0 (Media Cybernetics, DE, USA). Each test process was repeated three times with three samples per group.
Immunocytochemical staining
The cells were grown on coverslips and fixed in 4% polyformaldehyde at 4°C. After washed with PBS, the cells were incubated with 0.3% H2O2 for 10 min at room temperature. Following blocking with 5% goat serum, the cells were incubated with a rabbit anti-EphA2 monoclonal antibody (1:100; CST, MA, USA) at 4°C overnight and were immersed in a solution of horseradish peroxidase-labelled secondary antibody for at 37°C 30 min. After stained with DAB, the cells were dehydrated, cleared and mounted. The density (mean) of EphA2 was quantified using the image pro plus 6.0 (Media Cybernetics, DE, USA). Each test process was repeated three times with three samples per group.
Assessment of HBV-DNA level
After the HepG2.2.15 cells were treated with Entecavir (5 μg/mL, 15 μg/mL or 50 μg/mL) separately for 48 h with six samples per group, the load of HBV-DNA in the culture supernatant was determined using real-time fluorescence quantitative PCR. The HBV-DNA level was calculated according to the standard curve and a logarithmic transformation was applied in data analysis. Each test process was repeated three times.
Western blotting analysis
After the HepG2.2.15 cells were treated with Entecavir for 48 h, the total protein was separated in SDS-PAGE gels and electrotransferred onto PVDF membranes. The transferred membranes were blocked with 5% skim milk for 2 h and incubated with the primary monoclonal antibody including EphA2, β-catenin, p-GSK-3βSer9, E-cadherin and c-myc (1:1000; CST, MA, USA) at 4°C overnight. After washed with Tris-HCl, the membranes were incubated with the secondary antibody (1:3000) at 37°C for 1.5 h. The protein bands were quantified using a gel image analysis system (Image J). Briefly, the initial images were first transferred to gray images and then the background was subtracted. Following setting the measurement scales, all of the bands were measured. GAPDH was used as an endogenous control. Each test process was repeated three times with three samples per group.
Statistical analysis
All the data were expressed as the mean ± standard deviation and were analysed using the SPSS 11.5 software (Chicago, IL, USA). One-way analysis of variance with post hoc contrasts by Student–Newman–Keuls test and Pearson correlation analysis were performed, and P<0.05 was considered significant.