Seven surgical specimens of hepatoblastoma were collected and analyzed in the present study. The peri-tumor tissues were used as controls. Patient details are listed in Table I. Written informed consent was obtained from the patients' guardians. The present study was approved by the ethics committee of Xinhua Hospital, Affiliated to the School of Medicine, Shanghai Jiaotong University (Shanghai, China).

The human embryonic kidney (HEK)293T cell line and the Huh6 and HepG2 hepatoblastoma cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.). All cultures were maintained in a humidified atmosphere containing 5% CO₂ at 37°C.

The shRNA targeting of human EZH2 was performed as described previously (8). Briefly, the synthesized sequence, 5′-GAC TCT GAA TGC AGT TGC TTC AGT ACCC-3′, against EZH2 (GeneChem Co., Ltd., Shanghai, China) were inserted into the pGCSIL-enhanced green fluorescent protein (EGFP) vector (GeneChem Co., Ltd.) via its AgeI and EcoRI sites (New England BioLabs, Inc., Ipswich, MA, USA). A negative sequence used as a control was inserted into the identical plasmid at the identical site. The resulting lentiviral vector expressing the EZH2 siRNA or negative control siRNA was co-transfected into HEK293T cells with Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The viral titer was determined by counting EGFP-positive cells under a MicroPublisher 3.3 RTV fluorescence microscope (Olympus Corporation, Tokyo, Japan). The supernatant of the transfected HEK293T cells, which contained the lentiviral vector, was used to transduce 1×10⁵/cm² Huh6 and HepG2 cells in the presence of polybrene (10 µg/ml; GeneChem Co., Ltd.). Cells and supernatants were harvested at different time-points (24, 48, 72 and 96 h) after transduction.

The stably transfected cells were plated into 96-well plates at a density of 1×10⁴ per well. The medium in each well was replaced daily. Numbers of cells were detected at 0, 48 or 96 h post-transfection using a Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Kumamoto, Japan) according to the manufacturer's instructions. At 2 h following addition of the CCK-8 stain, the absorbance values of the wells were measured at 450 nm using a microplate reader (Synergy™ H1 Multi-Mode Reader; BioTek Instruments, Inc., Winooski, VT, USA). Each experiment was performed in triplicate.

Western blotting was used to determine EZH2 and p27 expression levels in extracts from Huh6 and HepG2 cells grown in a six-well plate. The cells were washed twice with cold phosphate-buffered saline (PBS) prior to addition of radioimmunoprecipitation assay lysis buffer containing protease inhibitors (Thermo Fisher Scientific, Inc.). Extracts were centrifuged at 12,000 × g for 15 min at 4°C and the supernatant was collected. The protein concentration was determined using a bicinchoninic acid protein assay (Thermo Fisher Scientific, Inc.). Forty micrograms of extracted protein were purified by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Invitrogen; Thermo Fisher Scientific, Inc.) and electrophoretically transferred onto nitrocellulose membranes. The membranes were blocked overnight in 5% bovine serum albumin for 2 h, washed 3 times for 5 min in PBS containing 0.1% Tween 20 (Shanghai Yeasen Biotechnology Co., Ltd., Shanghai, China). The membranes were then incubated with primary antibodies at 4°C overnight. The primary antibodies used were all obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA) and were as follows: Rabbit monoclonal anti-EZH2 (1:1,000; cat. no. 5246), rabbit monoclonal anti-p27 (1:1,000; cat. no. 3686) and rabbit polyclonal anti-α-tubulin (1:1,000; cat. no. 2148) antibodies. Subsequently, membranes were incubated with a goat anti-rabbit IgG coupled with horseradish peroxidase (1:2,000; Cell Signaling Technology, Inc.; cat. no. 7074) for 2 h at room temperature. Immunoreactivity was visualized using enhanced chemiluminescence reagents (SuperSignal West Pico; Thermo Fisher Scientific, Inc.) and Kodak X-OMAT film (Kodak, Rochester, NY, USA) with a ChemiDoc XRS molecular imager (Bio-Rad Laboratories, Inc., Hercules, CA, USA) used to capture the images.

Equal numbers of Huh6 and HepG2 cells were seeded in a 12-well plate. Seventy-two hours after transfection, cells were trypsinized and fixed with 70% ethanol overnight and stained with 100 µg/ml propidium iodide (PI; R&S Biotech, Shanghai, China) containing 100 µg/ml RNase (DNase free; Shanghai Yeasen Biotechnology Co., Ltd.). After incubation at 37°C for 30 min, the samples were analyzed on BD FACSCanto II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The average percentages of cells in G1, S or G2 phases of the cell cycle were quantified and the standard error was calculated for three experiments.

All data were reported as the mean ± standard error of the mean. Statistical analysis was performed using SPSS version 19.0 (International Business Machines, Armonk, NY, USA). Student's t-tests (two-tailed, unpaired) was performed for comparison between groups. P<0.05 was considered to indicate a statistically significant difference.

Seven patients with hepatoblastoma were enrolled in the present study (Table I). Their age ranged from five months to eight years and five of the patients were male. Their presenting symptoms included a palpable abdominal mass, abdominal pain and jaundice. Five of the patients had received four courses of pre-operative chemotherapy to enable their tumors to be resected. The remaining patients received primary surgery with post-operative chemotherapy.

The hepatoblastoma tissues and adjacent normal tissues were assessed for the expression of EZH2 using western blot analysis. As shown in Fig. 1, EZH2 was found to be overexpressed in hepatoblastoma tissues compared with that in normal peri-tumor tissues. This upregulation of EZH2 in hepatoblastoma tissues indicated its potential oncogenic roles in hepatoblastoma. Therefore, the function of EZH2 was further assessed in hepatoblastoma cell lines in vitro by using shRNA-mediated depletion of EZH2.

To further study the biological roles of EZH2 in hepatoblastoma, a lentiviral shRNA system was applied (Fig. 2A and B). Huh6 and HepG2 cells were transduced with lentivirus expressing shRNA targeting EZH2 or empty vector (Fig. 2C). A marked knockdown of EZH2 protein expression was detected at 48 h after transfection (Fig. 3).

To assess the potential effects of shRNA-mediated EZH2 silencing on the proliferation of hepatoblastoma cells, a CCK-8 assay was performed. As shown in Fig. 4, transfection with shRNA targeting EZH2 caused a significant reduction in the proliferation of the Huh6 and HepG2 cell lines compared with that in the NC groups (P<0.05). Huh6, NC, 0.36±0.05 vs. shEZH2, 0.50±0.01 and HepG2, NC, 1.17±0.02 vs. 1.49±0.01. These results suggested that EZH2 participates in the regulation of signaling cascades that positively influence hepatoblastoma cancer cell proliferation.

To further explore the effects of EZH2 repression on the cell cycle, flow cytometric cell cycle analysis following PI staining was performed. The results clearly showed that EZH2 repression significantly increased the G1 populations, accompanied by a decrease in the S-phase populations, in Huh6 and HepG2 cells (Fig. 5). These results indicated that EZH2 depletion inhibited the proliferation of hepatoblastoma cells by blocking G1-to-S-phase transition.

Western blot analysis of p27 in hepatoblastoma specimens revealed a reduced expression compared to that in normal tissues (Fig. 1). To assess the effects of EZH2 on p27 expression, hepatoblastoma cells subjected to shRNA-mediated EZH2 silencing were analyzed for p27 expression by western blot analysis. It was revealed that the protein expression of p27 was increased in cells with EZH2 repression compared to that in the NC group (Fig. 3).