Human glioma U251 cell line was obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (both Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) at 37°C in an atmosphere containing 5% CO₂. Methylthiazolyl-tetrazolium bromide (MTT) reagent was obtained from Sigma-Aldrich (St. Louis, MO, USA). Three pairs of β-catenin siRNA were designed by Jima Corporation (Shanghai, China) using Invitrogen Lipofectamine® transfection agent obtained from Thermo Fisher Scientific, Inc. (Waltham, MA, USA) to transfect U251 cells. Anti-β-catenin antibody was obtained from Abcam (Cambridge, UK). β-catenin shRNA(h) lentiviral particles (sc-29209) and control shRNA lentiviral particles-A (sc-108080) were obtained from Santa Cruz Biotechnology Inc for the animal experiments.

MTT assay was performed as previously described (23,24). Briefly, U251 human glioma cells (2×10³ cells/well) were seeded in 48-well plates. Following 24 h, cells were transfected with β-catenin siRNA and negative control siRNA, and cultured for 48, 72 or 96 h. Subsequently, the plate was supplemented with 20 µl MTT agent (5 mg/ml) and cultured for an additional 4 h. Finally, 200 µl dimethyl sulfoxide (Sigma-Aldrich) was added and the cells were incubated for a further 15 min by gently siphoning off the medium. Data were tested and analyzed.

Human glioma U251 cells (2×10⁵ cells/well) were cultured in a 24-well plate and, after 24 h, were transfected with three pairs of siRNA specific for β-catenin, as designed by Jima Corporation. β-catenin siRNA was transfected using Lipofectamine® according to the manufacturer's protocol. All laboratory supplies were lacking ribonucleases. siRNAs were transfected for 48 h and cell lysates were subsequently prepared for western blot analysis. Endogenous levels of β-catenin were determined by western blot analysis and the most effective siRNA was chosen for interference.

Total protein was extracted using cell protein extraction reagent (AR0103; Boster Biological Technology, Ltd., Wuhan, China). Samples (15 ng in each well) were loaded and the proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 80 V for 15 min, followed by 120 V at 1 h, and were subsequently transferred onto a nitrocellulose membrane (Beijing Biodee Biotechnology Co., Ltd., Beijing, China) for protein transfer at 400 mA for 1 h. Following this, the membrane was blocked with Tris buffered saline with Tween 20 (TBST) supplemented with 5% bovine serum albumin for 40 min prior to incubation with rabbit anti-β-catenin (1:5,000; ab32572) and mouse immunoglobulin (Ig)G₁ anti-β-actin (1:5,000; sc-47778; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) monoclonal antibodies in TBST containing 5% bovine serum albumin at 4°C overnight. The membrane was subsequently washed three times with TBST and incubated with the horseradish peroxidase-conjugated goat anti-mouse IgG (1:2,000; ab6789) and goat anti-rabbit IgG (1:2,000; ab6721; both Abcam) secondary antibodies, respectively, for 1 h at room temperature. Following this, the membranes were washed three times with TBST and the bands were detected in a dark room using chemiluminescence techniques. Images were captured using a ChemiDoc MP gel imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Scrambled siRNA was used as the negative control.

In order to further investigate the effects of the three pairs of siRNAs specific for β-catenin, RT-qPCR was performed. Scrambled siRNA was used as the negative control. Total RNA was extracted from β-catenin siRNA-transfected U251 glioma cancer cells and the negative control siRNA-transfected U251 cells using TRIzol® (Takara Biotechnology Co., Ltd., Dalian, China). RNA samples were reverse transcribed using MLV-reverse transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.) with random primers in a 20 µl final reaction volume containing 500 ng RNA, 0.5 µl PrimeScript® RT Enzyme mix, 4 µl 5X PrimeScript® buffer and 1 µl random primer. qPCR was performed to a final reaction volume of 20 µl containing 1µl template, 10 µl SYBR® Green PCR master mix (2X; Invitrogen; Thermo Fisher Scientific, Inc.), 1 µl forward and reverse specific primers (10 µm) and 7 µl water. Primer sequences were as follows: β-actin, forward 5′-CCTGTACGCCAACACAGTGC-3′ and reverse 5′-ATACTCCTGCTTGCTGATCC-3′; and β-catenin, forward 5′-AAAATGGCAGTGCGTTTAG-3′ and reverse 5′-TTTGAAGGCAGTCTGTCGTA-3′. Thermal cycling was performed on an ABI Prism 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) as follows: 40 cycles of 95°C for 30 sec and 60°C for 1 min.

A total of 30 male BALB/c (nu/nu) mice, aged 6–8 weeks old and weighing 18–20 g, were purchased from Biomed Science and Technology Co., Ltd. (Wuhan, China) and maintained in specific pathogen-free conditions. Mice were randomly divided into three equal groups: Control, β-catenin short hairpin (sh)RNA and control shRNA groups. As previously described, U251 cells were transfected with lentiviral β-catenin shRNA and control shRNA for 48 h, and the expression levels of β-catenin were subsequently detected via western blotting and RT-qPCR. The stable cell lines were screened using a blind screening method. Briefly, the transfected cells were plated into a 96-well plate containing diluted single cell solution (3–4 cells were cultured in every well). Cells were cultured for two weeks at 37°C in an atmosphere containing 5% CO₂. Once the clone had formed, western blotting was used to detect the levels of β-catenin. Cell lines with effective interfering effects were collected and used for mice injection. Control group mice were injected with untreated cells. Each group was subcutaneously injected with 6×10⁵ cells per mouse into the flank area. Survival duration was determined as follows: Survival rate: 100% × (number of survivors)/(number of challenged mice) and the data were analyzed using GraphPad 5.0. software (GraphPad software, Inc., La Jolla, CA, USA). All protocols were approved by the Institutional Animal Care and Use Committee of Renmin Hospital (Wuhan, China) in accordance with the Declaration of Helsinki outlined by the World Medical Association.

Apoptosis rates of β-catenin siRNA-transfected U251 glioma cancer cells and negative control-siRNA transfected U251 cells were analyzed using Annexin V-propidium iodide (PI) staining, according to the manufacturer's protocol (Santa Cruz Biotechnology, Inc.). Briefly, the cells in each group were washed with phosphate-buffered saline and resuspended in binding buffer containing 10 mM HEPES-NaOH (pH 7.4), 25 mM CaCl₂ and 144 mM NaCl. Subsequently, the cells were incubated with 0.1 µg/µl Annexin V and 0.05 µg/µl PI stain in the dark for 30 min on ice. In every sample, >10,000 cells were detected. The experiment was performed ≥3 times.

U251 cells were treated with negative control siRNA or β-catenin siRNA2 and were subsequently plated into a multi-well assay plate and allowed to attach, spread and form a confluent monolayer. Subsequently, a pipette tip was used to scratch the confluent monolayer in order to remove cells from a discrete area to form a cell-free zone into which cells at the edges of the wound could migrate. Cells in each group were cultured and images documenting cell migration were captured at 0 and 24 h following scratching using an Olympus CKX31/41 microscope (Olympus Corporation, Tokyo, Japan). Images were subsequently analyzed using ImageJ software (National Institutes of Health, Bethesda, MA, USA).

Experiments were performed in triplicate and the results were analyzed via one-way analysis of variance using SPSS 20.0 statistical software (IBM SPSS, Armonk, NY, USA). Data from the assay of U251 cell proliferation and mice survival were analyzed using GraphPad 5.0 software. Data were presented as the mean ± standard error of the mean. P<0.01 was considered to indicate a statistically significant difference.

In order to knockdown the expression of β-catenin in glioma cancer cells, three pairs of siRNA specific for β-catenin were designed and their effects investigated using western blot analysis and RT-qPCR. siRNA2 was demonstrated to be the most effective siRNA at silencing the β-catenin gene, as detected by western blot analysis (Fig. 1A). This was consistent with the results of RT-qPCR analysis (Fig. 1B). Scrambled siRNA was used as the negative control siRNA for RT-qPCR.

An MTT assay was performed in order to investigate the proliferation rate of U251 cells transfected with β-catenin siRNA2. The proliferation of glioma cancer U251 cells was significantly inhibited in the β-catenin siRNA2-transfected group, as compared with the negative control group (P<0.01; Fig. 2). Furthermore, the results demonstrated that the proliferation rate of U251 cells was markedly reduced in the β-catenin siRNA2-transfected group, as compared with the control group, in a time-dependent manner. Untreated cells and cells transfected with negative control siRNA were used as negative controls.

Apoptosis rates of U251 glioma cancer cells in the β-catenin siRNA transfected group were analyzed using flow cytometry with Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) dual labeling. The apoptosis rates of U251 cells were significantly elevated in the β-catenin transfected group, as compared with the untreated cells and the negative control siRNA group (P<0.01; Fig. 3). These results demonstrated that transfection with β-catenin-siRNA2 may promote the apoptosis of human glioma cancer cells.

As glioma is type of malignant tumor capable of invasion and metastasis, a scratch assay was performed to determine the effects of β-catenin siRNA on cell invasion. The relative migratory distance of glioma cells was significantly reduced in the β-catenin silencing group, as compared with the negative control group (P<0.01; Fig. 4). These results demonstrated that β-catenin siRNA may suppress the invasive activity of U251 human glioma cells.

In order to further define the potential efficacy of β-catenin, a lentiviral vector of β-catenin shRNA was used to evaluate its activity against the proliferation and metastasis of glioma cancer cells in a nude mice model. U251 cells were transfected with β-catenin shRNA using a lentiviral vector, and β-catenin expression levels were significantly inhibited, as determined by western blot analysis and RT-qPCR (Fig. 5). Notably, the survival rates of mice in the β-catenin knockdown group were significantly increased, as compared with the control shRNA group and control groups (P<0.01). Therefore, these results demonstrated that knockdown of β-catenin expression significantly inhibited the proliferation of glioma cancer cells in vivo.