Human HepG2, Bel-7402 and Huh-7 HCC cell lines were purchased from the American Type Culture Collection (Rockville, MD, USA); in addition, SMMC-7402 HCC cells were obtained from the Type Culture Collection Cell Bank, Chinese Academy of Science (Shanghai, China). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (ThermoFisher Scientific, Shanghai, China), 100 U/ml penicillin and 100 µg/ml streptomycin (Beyotime Institute of Biotechnology, Shanghai, China) at 37°C in 95% air and 5% CO₂ or when hypoxic conditions were required, cells were stored at 37°C in a sealed MIC-101 hypoxia chamber (Billups-Rothenberg, Inc., Del Mar, CA, USA) equilibrated with certified gas containing 1% O₂, 5% CO₂ and 94% N₂.

Abs against HIF-2α, β-catenin, C-Myc and proliferating cell nuclear antigen (PCNA), were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA); Ab against GAPDH was purchased from Beyotime Institute of Biotechnology (Jinan, China); and Abs against Ki-67, HIF-2α small interfering (si)RNA and control siRNA were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Lipofectamine 2000 was purchased from Invitrogen Life Technologies (Beijing, China) and sorafenib tosylate was purchased from Bayer Pharmaceuticals Co. (West Haven, CT, USA).

Silencing of HIF-2α/C-Myc was achieved via lentivival transduction of the following specific shRNA vectors (Santa Cruz Biotechnology, Inc.): HIF-2α-specific shRNA, sc-35316-v; C-Myc-specific shRNA, sc-29226-v; and scramble shRNA control, sc-108080. The process of transduction was executed according to the product protocols. Briefly, HepG2 cells (5×10⁵ cells/well) were seeded in 24-well plates. When the cell were ~50% confluent, the complete medium in the wells was replaced by complete medium with Polybrene (Santa Cruz Biotechnology, Inc., Dallas, TX, USA; sc-134220) at a final concentration of 3.5 µg/ml. In the control shRNA group, 10 µl scramble shRNA (sc-108080) was added to each well and in the HIF-2α shRNA group, 10 µl HIF-2α shRNA (sc-35316-v) was added to each well. The cells were cultured for 8 h, then the mixture of complete medium with Polybrene was replaced with complete medium, 12 h later, the cells were harvested for the following assays.

Cell viability was detected using the Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Beijing, China). In brief, cells (5×10³/well) in the control (scramble shRNA-treated group) and the group treated with HIF-2 shRNA were seeded onto 96-well plates and cultured for 12, 24, 48 and 72 h. The culture medium was then replaced with fresh medium containing 10 µl CCK-8 solution. Cells were incubated for a further 2 h at 37°C and the optical density was measured using a PowerWave HT Microplate Spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA) at 450 nm.

Cells or tumor tissues were homogenized in protein lysate buffer and debris was removed by centrifugation. Protein concentrations in cell/tissue extracts were determined using the BCA Bio-Rad protein assay dye reagent (Bio-Rad Laboratories, Inc., Richmond, CA, USA). In brief, equal amounts of protein fractions of lysates were resolved over SDS-PAGE gels (Beyotime Institute of Biotechnology) transferred to polyvinylidene fluoride membranes (Merck Millipore, Darmstadt, Germany) and immunoblotted as previously described (16,17). The primary antibodies used for western blotting included, monoclonal rabbit anti-human HIF-2α, (1:1,000 dilution, #7096), polyclonal rabbit anti-human-β-catenin (1:1,000, #9562), polyclonal rabbit anti-human c-Myc (1:1,000 dilution, #9402), monoclonal mouse anti-human PCNA (1:2,000 dilution, #2586) and monoclonal mouse anti-human GAPDH (1:2,000 dilution, AG019). The secondary antibodies included horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:1,000 dilution, #A0208; Beyotime Institute of Biotechnology) and (HRP)-conjugated goat anti-mouse IgG (1:1,000 dilution, A0216; Beyotime Institute of Biotechnology). Protein bands were developed using ECL reagent (Tiangen Biotech Co., Ltd., Beijing, China) and visualized using the AlphaImager HP System (Naturegene Corporation, Medford, NJ, USA). The protein band intensities were quantified by densitometric analysis using Image J software, version 2.0 (National Institutes of Health, Bethesda, MD, USA).

For ICC, the cells (2×10⁴) were plated on coverslips, fixed with 4% paraformaldehyde for 30 min at room temperature, and permeabilized with 0.2% Triton X-100 for 20 min at room temperature. Subsequently, the sections were incubated with primary anti-β-catenin (1:200 dilution, #9562) overnight at 4°C, followed by incubation with HRP-conjugated goat anti-rabbit IgG secondary antibody (1:1,000 dilution, #A0208). Subsequently, immunoreactivity was developed with 3,3′-diaminobenzidine (DAB; ZSGB-BIO Biotechnology Co., Ltd., Beijing, China), followed by counterstaining with hematoxylin.

All surgical procedures and care administered to the animals were in accordance with institutional ethics guidelines. The study was approved by the ethics committee of Qianfoshan Hospital, Shandong University (Jinan, China). Male BALB/c mice were purchased from the Model Animal Research Center (Nanjing, China). The mice (n=56) were housed in polycarbonate cages (5/cage), provided with food and water ad libitum and maintained on a 12–12 h light-dark cycle at 22±2°C and 55±20% relative humidity. Male BALB/c mice were randomly assigned into four groups (12/group). Eight mice were excluded for different reasons (mortality or no tumor formed). Male BALB/c (5–6 weeks old) mice underwent subcutaneous inoculation in the flank with 1.5×10⁶ HepG2 cells/mice suspended in phosphate-buffered saline (PBS). Sorafenib tosylate was used in vivo experiments. When tumors of ~100 mm³ were detected (~7 days post inoculation), mice were divided at random into the following treatment groups (n=12/group): Control, sorafenib-treated group, HIF-2α siRNA-treated and HIF-2α siRNA + sorafenib-treated. Mice in each group were administered treatment five times/week either intratumorally or orally (p.o). In the sorafenib-treated group, mice were administrated once daily with sorafenib (p.o.; 10 mg/kg; 5 times/week). Equal volumes of 20 nM siRNA and Lipofectamine 2000 were mixed and this solution was further mixed with an equal volume of serum-free medium; 50 µl siRNA transfection solution, containing 250 pmol siRNA, was injected into tumors (5 times/week). Mice in the control group were treated with equal volume PBS (p.o.) and control siRNA (intratumoral injection). The tumor volumes were estimated twice a week according to the formula: π/6xa2xb, where a is the short axis, and b the long axis. All mice were sacrificed by cervical dislocation on day 35.

As previously described (18), tumor sections were immunostained with an anti-Ki-67 Ab. Briefly, polyclonal rabbit anti-human Ki-67 (1:50 dilution, SC-15402) was applied overnight at 4°C. After washing, the sections were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:75 dilution, #A0208; Beyotime Institute of Biotechnology) for 1 h at 37°C. Immunoreactivity was developed using DAB and counterstained with hematoxylin. Ki-67 positive cells were counted under microscopy with a DM5500B equipped with HCX PL FLVOTAR 5/0.15 and HCX PL FLVOTAR 10/0.15 dry objective lenses (Leica, Solms, Germany) in ten randomly selected high-power fields (magnification, x400). The Ki-67 proliferation index was calculated according to the following formula: Proliferation rate = (number of Ki-67 positive cells/total cell count) × 100%.

Values are presented as the mean ± standard deviation and statistical analysis was performed using SPSS software version 16.0 (SPSS Inc., Chicago, IL, USA). Differences between groups were analyzed using Chi-Square test or Student's t-test according to the data type. P<0.05 was considered to indicate a statistically significant difference between values.

Western blot analysis was used to determine HIF-2α protein expression levels in various HCC cell lines, SNNC-7402, Bel-7402, HepG2 and Huh7, exposed to normoxia or hypoxia for 24 h. All of the cell lines demonstrated low expression levels of HIF-2α under normoxic conditions, while HIF-2α expression was markedly increased under hypoxic conditions in all the selected HCC cell lines (Fig. 1A). Based on this data, these HCC cell lines were further used to detect the inhibitory effects of sorafenib on the proliferation viability of cells under hypoxic conditions. Cells were transfected with HIF-2α shRNA or control shRNA and then treated with sorafenib (10 µM) for 12, 24, 48 and 72 h, following which cell viability was assessed. Compared with the control shRNA, HIF-2α shRNA further enhanced the antiproliferation activity of sorafenib in all of the four cell lines under hypoxic conditions (Fig. 1B).

β-catenin is considered to be a key signaling protein in the regulation of cell proliferation (19–21). HepG2 cells were incubated in a sealed hypoxia chamber for 24 h, which resulted in significantly higher levels of β-catenin compared with cells in normoxic conditions (Fig. 2A). Transfection of HIF-2α shRNA markedly reduced the expression of HIF-2α, β-catenin, C-Myc and PCNA in HepG2 cells compared with those transfected with control shRNA (Fig. 2B). As C-Myc is a key downstream protein in β-catenin-mediated cell proliferation (19–21), the effects of C-Myc shRNA transfection were investigated in HepG2 cells. C-Myc shRNA decreased C-Myc and PCNA expression with no effect on the expression of HIF-2α and β-catenin (Fig. 2B). These results indicated that HIF-2α regulated HCC cell proliferation in a β-catenin/C-Myc-dependent pathway under hypoxic conditions.

A gradual increase in xenograft HepG2 tumor growth was observed in the control group compared with the experimental groups. In addition, mice that underwent combination therapy had tumors that were significantly smaller than those treated with sorafenib or HIF-2α siRNA alone (Fig. 3A and B). Furthermore, a reduced number of Ki-67-positive cells were present in tumors treated with either sorafenib or HIF-2α siRNA compared with the control group; while in the combination therapy group, the number of Ki-67-positive cells was significantly reduced compared with that of the independent treatment groups (P<0.01) (Fig. 3C and D). As shown in Fig. 3E, the reduced PCNA expression levels in HIF-2α-transfected cells, as determined by western blot analysis of tumor homogenates, were comparable to the results observed in vitro (Fig. 3E).