A total of 47 HCC tissues and paired normal human liver specimens were obtained from liver cancer patients (stage I–IV; non-metastatic; 30 males, 17 females; age range, 37–80 years; median age, 59 years) who underwent surgery at the People's Hospital of Lishui (Lishui, China) between February 2014 and December 2014. The protocol of the present study was approved by the ethics committee of the People' Hospital of Lishui (Lishui, China). Written informed consent was obtained from all participants of this study. All experiments were performed in accordance with the Helsinki Declaration of 1975. None of these patients had received any radiotherapy or chemotherapy. The percentage of tumor cells in the liver cancer tissues was at least 70% as determined by pathological examination of histological slides. Subsequent to surgery, liver cancer and normal tissues were immediately snap-frozen in liquid nitrogen and stored at −80°C until further analysis.

The Huh7 and BEL-7404 liver cancer cell lines used in the present study were obtained from the Cell Bank of Academia Sinica (Shanghai, China) and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (both from Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA) in a humidified incubator containing 5% CO₂ in air at 37°C.

WWP2 siRNA was used to knockdown WWP2. Three siRNAs (Sangon Biotech Co., Ltd., Shanghai, China) targeting position 1,521–1,543 (5′-GGTGCTCAACCCTATGTATTT-3′, siRNA1), position 2,866–2,888 (5′-AGGAGGTTCTGCCTGTAATTT-3′, siRNA2) and position 749–771 (5′-ACGGACGTGTCTATTATGTTT-3′, siRNA3) of human WWP2 mRNA. Cells were transfected with siRNA (40 nM) using Lipofectamine 2000 (Invitrogen) following the manufacturer's protocol. Non-specific siRNA (5′-AGCCGCTTAGGAATGCTCUUU-3′; Sangon Biotech Co., Ltd.) was used as negative control, and the selective silencing of WWP2 was confirmed by reverse-transcription quantitative polymerase chain reaction (RT-qPCR) analysis. Cells were analyzed at 48 h after transfection.

Total RNA was extracted from normal tissues and HCC tissues and cells with TRIzol reagent (Invitrogen) as previously descried (19) and stored at −80°C. Complementary DNA was synthesized using a cDNA synthesis kit (Thermo Fisher Scientific). The DyNAmo Flash SYBR Green qPCR kit (Finnzymes Oy, Espoo, Finland) was used according to the manufacturer's instructions. Real-time qPCR was performed to detect the mRNA levels of the indicated genes. The primer sequences (sense/antisense) used were as follows: WWP2, 5′-GAGATGGACAACGAGAAG-3′ and 5′-CTCCTCAATGGCATACAG-3′; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-CACCCACTCCTCCACCTTTG-3′ and 5′-CCACCACCCTGTTGCTGTAG-3′ (Sangon Biotech Co., Ltd.). The PCR cycling conditions were as follows: 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 45 sec, and a final extension step of 95°C for 15 sec, 60°C for 1 min, 95°C for 15 sec and 60°C for 15 sec. Data collection was performed using an ABI 7500 (Thermo Fisher Scientific) and relative quantification of gene expression was performed using the 2^−ΔΔCq method (20) with normalization to GAPDH.

Total protein was extracted from liver cancer cell lines or from homogenized tumor samples and corresponding normal tissues using radioimmuno-precipitation buffer (JRDUN Biotechnology Co., Ltd. Shanghai, China). The protein concentration was assessed using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific; cat. no. PICPI23223). 50 µg protein lysates was separated by 10–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Sigma-Aldrich, St. Louis, MO, USA), followed by blocking in fat-free milk overnight at 4°C. The membrane was first incubated with antibody against WWP2, Bcl-2, caspase-7, caspase-8 or Bax overnight at 4°C, followed by anti-GAPDH antibody as a loading control. Subsequently, the membrane was incubated with secondary antibody labeled with horseradish peroxidase (Beyotime Institute of Biotechnology, Haimen, China; cat nos. A0208 and A0216; 1:1,000) for 1 h at 37°C, prior to being washed three times with Tris-buffered saline containing 20% Tween 20 (Amresco, Solon, OH, USA). The blots were visualized using enhanced chemiluminescence (Millipore, Billerica, MA, USA) and signal intensity was determined using Image J software version 1.46 (National Institutes of Health, Bethesda, MD, USA).

Rabbit anti-Bcl-2 polyclonal antibody (pAb) (cat. no. sc-492; 1:100 dilution) and rabbit anti-Bax pAb (cat. no. sc-493; 1:100 dilution) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and rabbit anti-GAPDH monoclonal antibody (mAb) (cat. no. 5174; 1:1,500 dilution) was from Cell Signaling Technology, Inc. (Danvers, MA, USA). Rabbit anti-WWP2 pAb (cat. no. ab103527; 1:3,000 dilution), rabbit anti-caspase-7 mAb (cat. no. ab181579; 1:5,000 dilution) and rabbit anti-caspase-8 pAb (cat. no. ab25901; 1:2,000 dilution) were purchased from Abcam (Cambridge, MA, USA).

A Cell Counting Kit-8 assay (CCK-8; Dojindo, Kumamoto, Japan) (21,22) was used to assess cell viability. In brief, cells were seeded onto 96-well plates at a density of 5×10³ cells/well and were subsequently transfected with control or WWP2 siRNA. After incubation for 0, 24, 48 and 72 h, 10 ml CCK-8 solution was added to each well, followed by incubation for 1 h. Cell viability was determined by scanning with a microplate reader (Model 680; Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 450 nm.

Briefly, the cells were seeded into six-well plates, transfected with control or WWP2 siRNA and cultured for 48 h. Approximately 1×10⁶ cells were harvested, washed twice with phosphate-buffered saline (JRDUN Biotechnology Co., Ltd.) and fixed in cold ethanol (JRDUN Biotechnology Co., Ltd.) for 30 min followed by incubation with 20 µg/ml propidium iodide (PI; Sigma-Aldrich) and 200 µg/ml RNase A (Sigma-Aldrich) for 30 min. Subsequently, cells were analyzed using a flow cytometer (BD Accuri C6 equipped with software version 1.0.264.21; BD Biosciences, San Diego, CA, USA).

Apoptosis was determined annexin V-fluorescein isothiocyanate (FITC)/PI double staining (Beyotime Institute of Biotechnology; cat. no. C1062) in accordance with the manufacturer's protocol, followed by flow-cytometric analysis. Briefly, floating as well as trypsinized adherent cells (5×10⁵) from the various treatment groups at 48 h following transfection were collected and re-suspended in 500 µl binding buffer containing 5 µl annexin V-FITC and 5 µl PI, followed by incubation for 5 min at room temperature in the dark. Analysis was immediately performed using a flow cytometer.

Values are expressed as the mean value ± standard deviation. The paired two-tailed Student's t-test was used to analyze the significance of differences between groups. Statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). P<0.01 was considered to indicate a statistically significant difference.

RT-qPCR and western blot analysis were used to assess the mRNA and protein levels of WWP2 in 47 paired HCC and adjacent tissues. As shown in Fig. 1A, the mRNA expression of WWP2 was significantly increased in HCC tissues when compared with that in paired adjacent normal tissues (P<0.0001). Similarly, WWP2 protein levels in four HCC tissues were decreased compared with those in their paired adjacent normal tissues (P<0.01) (Fig. 1B). These results indicated that WWP2 may have an important role in liver carcinogenesis.

To identify an siRNA sequence to effectively target WWP2, siRNA1, -2 and -3 were transfected into Huh7 and BEL-7404 cells. mRNA and protein expression of WWP2 following transfection with the respective siRNAs were assessed. WWP2 mRNA expression was reduced by 63±3 and 64±4% in Huh7 and BEL-7404 cells transfected with siRNA2 (P<0.01) (Fig. 2A and B). In addition, the reduction of WWP2 protein expression was consistent with that of WWP2 mRNA expression in Huh7 and BEL-7404 cells. No apparent changes in WWP2 expression were observed following transfection with negative control siRNA.

The role of WWP2 in liver carcinogenesis was then assessed in Huh7 and BEL-7404 cells. Following incubation for 72 h, the proliferation of WWP2-knockdown Huh7 and BEL-7404 cells was inhibited by 37% compared with that of the negative control cells (P<0.01) (Fig. 3A and B). Furthermore, the effect of WWP-2 knockdown on the cell cycle distribution of Huh7 and BEL-7404 cells was assessed. Treatment with WWP2 siRNA increased the G1-phase population in Huh7 cells by 25±4% and in BEL-7404 cells by 28±7% (P<0.01), indicating G1-phase arrest (Fig. 3C and D). In addition, the apoptotic rate of cells transfected with WWP2 siRNA was examined by flow cytometry at 48 h post-transfection. The results showed that the apoptotic rate was significantly increased in the WWP2-knockdown groups (>8.0-fold for Huh7 and >9.0-fold for BEL-7404 cells) compared with that in the negative control group (P<0.01) (Fig. 3E and F). These results suggested that WWP2 may function as a tumor promoter in liver cancer through enhancing cell-cycle progression and inhibiting apoptosis, and that siRNA-mediated knockdown of WWP2 may represent a novel therapeutic strategy for liver cancer.

To investigate the underlying mechanism of the effects of WWP2 siRNA on liver cancer-cell apoptosis, the protein expression of Bcl-2, caspase-7, caspase-8 and Bax was examined by western blot analysis. The protein levels of pro-apoptotic proteins caspase-7, caspase-8 and Bax were significantly increased after treatment of Huh7 and BEL-7404 cells with WWP2 siRNA (P<0.01) (Fig. 4A and B). However, the expression of anti-apoptotic protein, Bcl-2 was significantly decreased (P<0.01). These results suggested that apoptosis of Huh7 and BEL-7404 liver cancer cells induced by WWP2 knockdown may be mediated via the Bax and Bcl-2 pathways as well as caspase-7 and -8.