The multidrug resistant human hepatic cancer cell line HepG2/DPP was obtained from The Third Affiliated Hospital of Xinxiang Medical University (Xinxiang, China) and cultured with RPMI 1640 (containing 10% calf serum; Corning Life Sciences, Manassas, VA, USA) in an incubator under 37°C, 5% CO₂ and saturated humidity conditions. Trypsin-EDTA [0.25% in phosphate-buffered saline (PBS); Corning Life Sciences] was used for digestion and passaging. Cells in the logarithmic growth phase were used in all experiments. The present study was approved by the ethics committee of Luoyang Central Hospital Affiliated to Zhengzhou University (Luoyang, China).

The cells were cultured in 24-well plates, at a density of 3×10⁴ cells/well. The culture was continued until ~70% cells reached confluence. The wee1 shRNA (h) viral vectors (Lentiviral Particles; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were prepared in accordance with the procedures described in the manufacturer's instructions. The cells were transfected with different multiplicity of infection (MOI) values (10 and 20). The culture medium was switched to virus-free medium (PT3414-1; Corning Life Sciences) 24 h following transfection. The culture was continued for 48 h for passaging. The silencing status of WEE1 was evaluated by western blotting and quantitative polymerase chain reaction (qPCR). The HepG2/DDP cells were the parental control group, the HepG2/DDP cells transfected with blank vector were the negative control group and the HepG2/DDP cells transfected with WEE1 short hairpin (sh)RNA were the shRNA#1 (MOI, 10) and shRNA#2 (MOI, 20) silencing groups.

Cells (5×10⁴ cells/ml) were seeded in 96-well microplates, at 100 µl/well and cultured overnight to allow cell adherence. Next, different concentrations of cisplatin (0, 30, 60, 90, 120, 150, 180, 210, 240, 270 and 300 µM; Sigma-Aldrich, St. Louis, MO, USA) were added and the culture was continued for 72 h. Following aspiration of the culture medium, 50 µl MTS (Promega Corporation, Madison, WI, USA) was added in accordance with the reagent instructions and cultured for 4 h. The optical density (OD) was measured at 490 nm wavelength with a Bio-Rad 3550 microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and the inhibition rate of the drug on the cells was calculated as follows: Inhibition rate = (1 - OD of experimental group/OD of control group) × 100. Using the cisplatin concentration as abscissa and the inhibition rate as ordinate, the inhibition curve was plotted and fitted to obtain the half maximal inhibitory concentration (IC₅₀). Reversal fold (RF) was defined as RF = IC₅₀ (silence group)/IC₅₀ (parental group).

Following addition of 100 µl of 10 µM Rh-123 staining solution (Beyotime Institute of Biotechnology, Shanghai, China), the cells in logarithmic growth phase were cultured for 1 h and harvested. The fluorescence intensity of intracellular Rh-123 was detected with a flow cytometer (FACSAria™ I; BD Biosciences, Franklin Lakes, NJ, USA) to indicate intracellular Rh-123 content. P-gp expression was determined with flow cytometry using specific procedures as follows: 10⁶ cells were added to 0.1 ml culture medium for each sample and stained in accordance with the kit instructions [Anti-P-gp/fluorescein isothiocyanate (FITC) fluorescent antibody kit; Abcam, Cambridge, MA, USA]; cell fluorescence intensity was detected with a flow cytometer to indicate the P-gp expression level. Apoptosis was detected using the annexin V-FITC/propidium iodide (PI; BD Biosciences, Franklin Lakes, NJ, USA) double-staining technique; the cells were treated with 20 µM cisplatin for 24 h and harvested to determine apoptosis in accordance with the manufacturer's instructions. Following similar cisplatin treatment, intracellular caspase-3 and −8 activities were determined with phycoerythrin-labeled anti-active rabbit IgG caspase-3 (cat no. sc-7147) and −8 antibodies (cat no. sc-7890; 1:1,000 dilution; Santa Cruz Biotechnology, Inc.) using the flow cytometer in accordance with the manufacturer's instructions.

Cells in the logarithmic growth phase were lysed using a cell lysis kit (Beyotime Institute of Biotechnology) to extract the total protein content. The proteins were separated in 12% SDS-PAGE (Beyotime Institute of Biotechnology) at 220 V and transferred onto a polyvinylidene difluoride (PVDF) membrane (Beyotime Institute of Biotechnology). Antibodies for WEE1 (cat no. sc-9037; rabbit anti-human IgG; 1:2,000 dilution), MDR1 (cat no. sc-55510; mouse anti-human IgG; 1:2,000 dilution), MRP1 (cat no. sc-13960; rabbit anti-human IgG; 1:2,000 dilution), LRP (cat no. sc-390134; mouse anti-human IgG; 1:2,000 dilution), BCL-2 (cat no. sc-492; rabbit anti-human IgG; 1:2,000 dilution), survivin (cat no. sc-10811; rabbit anti-human IgG; 1:2,000 dilution), GST (cat no. sc-459; rabbit anti-human IgG; 1:2,000 dilution), p-MEK (cat no. sc-130203; rabbit anti-human IgG; 1:1,500 dilution) and p-ERK (cat no. sc-13073; rabbit anti-human IgG; 1:1,500 dilution) were used to detect the target proteins, and β-actin (cat no. sc-1616; goat anti-human IgG; 1:5,000 dilution) and ERK (cat no. sc-94; rabbit anti-human IgG; 1:2,000 dilution) were used as the internal reference genes. All antibodies were purchased from Santa Cruz Biotechnology, Inc. Cells were incubated with the antibodies at 4°C overnight. Subsequent to washing off the primary antibody with 10% PBS, horseradish peroxidase (HRP)-conjugated secondary antibody (cat no. sc-2005; goat anti-mouse IgG-HRP; 1:2,000 dilution; and cat no. sc-2004; goat anti-rabbit IgG-HRP; 1:2000 dilution) was added to the cells and incubated for 1 h. Cells were washed with 10% PBS, then an enhanced chemiluminescence (ECL) kit (Millipore, Boston, MA, USA) was used to identify the immunoreactive bands using methods according to the manufacturer's instructions.

Following the extraction of total RNA from each group of cells in the logarithmic growth phase using TRIzol (Invitrogen Life Technologies, Carslbad, CA, USA), RT was conducted with Real-Time PCR kit (Ambion, Austin, TX, USA) to obtain the cDNA. The primer sequences were as follows: WEE1, F 5′-GATGAGCAGAACGCTTTGAGAG-3′ and R 5′-CAGAGGCAGCATTTGGGATT-3′; MDR1, F 5′-AAAAAGATCAACTCGTACCACTC-3′ and R 5′-GCACAAAATACACCAACAA-3′; MRP1, F 5′-ACTTCCACATCTGCTTCGTCAGTG-3′ and R 5′-ATTCAGCCACAGGAGGTAGAGAGC-3′; LRP, F 5′-AGTCAGAAGCCGAGAAAG-3′ and R 5′-CCCAGCCACAGCAAGGT-3′; BCL-2, F 5′-ACGGGGTGAACTGGGGGAGGA-3′ and R 5′-TGTTTGGGGCAGGCATGTTGACTT-3′; survivin, F 5′-GCATGGGTGCCCCGACGTTG-3′ and R 5′-GCTCCGGCCAGAGGCCTCAA-3′; GST, F 5′-ACGTGGCAGGAGGGCTCACTC-3′ and R 5′-TACTCAGGGGAGGCCAGCAA-3′; GAPDH, F 5′-CTTAGATTTGGTCGTATTGG-3′ and R 5′-GAAGATGGTGATGGGATT-3′. Following denaturation at 94°C for 3 min, 40 cycles of amplification were conducted under the following conditions: 95°C for 5 sec, 65°C for 35 sec, 72°C for 60 sec, and extension at 72°C for 5 min.

The experimental data are presented as the mean ± standard deviation and analyzed with SPSS software, version 13.0 (SPSS, Inc., Chicago, IL, USA). One-way analysis of variance was used for comparison; P<0.05 was considered to indicate a statistically significant difference.

Transfection of HepG2/DDP cells with different MOI values resulted in varying degrees of WEE1 silencing. Subsequent to screening, cells transfected with the MOI values of 10 and 20 were selected for subsequent studies. As presented in Fig. 1, WEE1 expression levels in the shRNA#1 (MOI, 10) and shRNA#2 (MOI, 20) groups were 53 and 22% of the parental group cells, respectively (P<0.05). Blank vector shRNA cells (negative group) were observed to have no effect on WEE1 expression levels (P>0.05).

MTS assay results (Fig. 2A) demonstrated that the IC₅₀ of cisplatin-mediated inhibition on HepG2/DPP cell proliferation was 156 µM, while the IC₅₀s for the shRNA#1 and shRNA#2 groups were 70.2 and 43.5 µM, respectively, suggesting that silencing WEE1 enabled an increase in the sensitivity of HepG2/DPP cells to cisplatin. The RFs calculated based on the IC₅₀s for shRNA#1 and shRNA#2 groups were 2.2 and 3.6 fold, respectively.

The fluorescence intensity of intracellular Rh-123 in the shRNA#1 and shRNA#2 groups increased by 1.75- and 2.23-fold compared with the parental control group (Fig. 2B). By contrast, P-gp expression was downregulated to 73.2 and 34.5% of the control group, respectively (Fig. 2C), suggesting that elevation of intracellular Rh-123 was associated with the downregulation of P-gp, which may increase intracellular cisplatin. In addition, following treatment with 20 µM cisplatin for 24 h, the apoptosis rate of the HepG2/DDP parental group was 15.3%, while those of the shRNA#1 and shRNA#2 groups were 28.4 and 39.8% (Fig. 2D; P<0.05).

RT-qPCR analysis demonstrated that the gene transcription levels of MDR1, MRP1, LRP and GST decreased significantly following WEE1 silencing (Fig. 3A). Western blot results demonstrated that, compared with the control group, the protein levels were also reduced (Fig. 3B). These results indicate that the downregulation of protein expression was regulated at the transcriptional level.

Since WEE1 silencing enhanced apoptosis, the expression of 2 anti-apoptotic proteins, BCL-2 and survivin, were assessed. Western blotting indicated that WEE1 silencing was accompanied by downregulation of BCL-2 and survivin expression. RT-qPCR analysis demonstrated that the downregulation of expression was achieved at transcriptional level (P<0.05).

In order to explain these results, the MEK/ERK pathway was considered as the possible intracellular signaling pathway regulated by WEE1. Western blotting demonstrated that levels of phosphorylated MEK and ERK decreased following WEE1 silencing, indicating that the activity of this pathway was significantly suppressed.

Furthermore, the shRNA#1 and shRNA#2 groups were treated with 20 µM cisplatin for 24 h, and the activated caspase-3 content was increased by 10.3 and 18.7-fold, respectively, compared with the control group; the activated caspase-8 content increased 12.1 and 21.5-fold, compared with the control group (Fig. 3).