The present study was approved by the Legislation and Ethical Board of the People's Hospital of Ningxiang County (Changsha, China). A total of 76 glioma tissues and 10 normal brain tissues were collected from patients at the People's Hospital of Ningxiang County between April 2008 and September 2011. The 10 normal brain tissues were used as controls and obtained from 10 patients (male:female, 6:4; mean age, 31.4 years; age range, 24–51 years) as they were treated for severe head injuries. These tissues were collected by partial resections of normal brain tissue in order to reduce intracranial pressure. Written informed consents were obtained and the clinical information of patients is summarized in Table I. All tissues were formalin fixed at room temperature for 48 h and paraffin-embedded.

The human glioma cell line U373-MG was purchased from the Cell bank of Chinese Academy of Sciences (Shanghai, China). The U373 cell line has been demonstrated to be contaminated/misidentified and to be a U-251MG derivative (22). As such, U373-MG is considered to be a mixed astrocytoma cell line in the present study. Cells were maintained at 37°C in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; both Thermo Fisher Scientific, Inc., Waltham, MA, USA).

U373-MG cells were transfected with 14-3-3β siRNA (cat. no. sc-29186) or non-specific siRNA (cat. no. sc-36869) (both Santa Cruz Biotechnology, Inc., Dallas, TX, USA) using Lipofectamine RNAiMAX (Thermo Fisher Scientific, Inc.), following the manufacturer's protocol. Briefly, siRNA (30 pmol) was diluted in 500 µl DMEM and mixed with 5 µl Lipofectamine RNAi MAX for 15 min at room temperature. The mixture was then added to U373-MG cells. Following 48 h transfection, cells were harvested for further analysis.

Total RNA was extracted from cells using TRIzol Reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. A total of 800 ng RNA was converted to cDNA using a RevertAid RT Reverse Transcription kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. qPCR was performed using a SuperScript^® III Platinum^® SYBR^® Green One-Step qRT-PCR kit (Thermo Fisher Scientific, Inc.) on an ABI 7300 plus thermocycler (Thermo Fisher Scientific, Inc.). The primer sequences were as follows: 14-3-3β forward 5′-GGCAAAGAGTACCGTGAGAAG-3′ and reverse 5′-CTGGTTGTGTAGCATTGGGAATA-3′, GAPDH forward 5′-CTGGGCTACACTGAGCACC-3′ and reverse 5′-AAGTGGTCGTTGAGGGCAATG-3′. The thermo cycling conditions were as follows: 95°C for 10 min, 40 cycles of denaturation at 95°C for 15 sec and annealing/elongation at 60°C for 60 sec. GAPDH was used as an internal control. The relative expression was analyzed using the 2^−ΔΔCq method (23).

U373-MG cells were lysed in ice-cold buffer (0.5 mol/l Tris-HCl, pH 7.4, 1.5 mol/l NaCl, 2.5% deoxycholic acid, 10% NP-40 and 10 mmol/l EDTA; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) with a protease inhibitor cocktail (Thermo Fisher Scientific, Inc.). Cell lysates underwent centrifugation at 16,000 × g for 20 min at 4°C. Protein concentrations were determined using Protein Assay kit I (Bio-Rad Laboratories, Inc., Hercules, CA, USA), according to the manufacturer's protocol. A total of 50 µg protein was separated by 12% SDS-PAGE and immunoblotted onto a polyvinylidene difluoride membrane (Thermo Fisher Scientific, Inc.). The membrane was blocked at room temperature for 1 h in PBS with Tween 20, [0.1% Tween 20, 2.67 mmol/l KCl, 1.47 mmol/l KH2PO4, 137.93 mmol/l NaCl and 8.1 mmol/l Na2HPO4 (PBST; pH 7.4)] containing 5% nonfat dry milk. The membrane was subsequently incubated with rabbit polyclonal anti-14-3-3β antibody (1:50; cat. no. ab15260) or rabbit polyclonal anti-GAPDH antibody (1:50; cat. no. ab9485) at room temperature for 3 h and washed 3 times with PBST. The membrane was subsequently incubated with horseradish-peroxidase-conjugated goat anti-rabbit secondary antibody (1:5,000; cat. no. ab7090) at room temperature for 1 h. All antibodies were purchased from Abcam (Cambridge, MA, USA). Protein expression was determined using Pierce ECL Plus Western Blotting Substrate (Thermo Fisher Scientific, Inc.) and analyzed using Image-Pro Plus software (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA).

Cell proliferation was measured using an MTT assay. U373-MG cells (5,000 cells/well) were seeded in 96-well plates in 200 µl serum-free DMEM. Following the attachment of cells, the medium was replaced with fresh medium containing 10% FBS. At 24, 48 and 72 h, cells were treated with 20 µl MTT solution (5 mg/ml). MTT solution was replaced with 150 µl dimethylsulfoxide after 4 h to dissolve the tetrazolium crystals for 10 min. Absorbance was measured at 570 nm using a microplate reader.

U373-MG cells were collected, fixed with 70% ethanol at room temperature for 30 min and washed twice with PBS. Analysis of cell cycle distribution was conducted using Vybrant™ DyeCycle™ Violet Ready Flow™ Reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Cells were analyzed using BD Accuri™ C6 Flow Cytometer and BD Accuri™ C6 software (version 1.0) (both BD Biosciences, San Jose, CA, USA).

An Alexa Fluor 488 Annexin V and PI kit (Invitrogen; Thermo Fisher Scientific, Inc.) was used to assess apoptosis. Briefly, 1×10⁵ U373-MG cells were harvested, washed twice with cold PBS and resuspended in 100 µl binding buffer (Thermo Fisher Scientific, Inc.). A total of 5 µl Annexin V-fluorescein isothiocyanate and 1 µl PI were added to the solution. Following 15 min incubation at 4°C, 400 µl binding buffer was added to the solution and cells were analyzed using the Accuri™ C6 Flow Cytometer. The results were analyzed using CellQuest™ software 1.0 (BD Biosciences). A quadrant dot plot was used to identify whether cells were in the early or late phase of apoptosis and whether they were living or necrotic, as previously described (24).

U373-MG cells were plated at a density of 1×10⁵ cells/well with 1 ml DMEM in 24-well plates and incubated at 37°C for 24 h. Scratch wounds were generated using a 200 µl pipette tip and the medium was replaced with fresh DMEM with 10% FBS. The wound size was recorded using a camera at 0 and 24 h. Wound size was quantified using ImageJ software (version 1.48; National Institutes of Health, Bethesda, MD, USA).

To determine the migration of glioma cells, 24-well plate-matched Corning™ BioCoat™ Matrigel™ Invasion Chamber with BD Matrigel Matrix (BD Biosciences) were used. Briefly, 1.0×10⁵ cells were seeded in upper Transwell chambers with serum-free DMEM. A total of 500 µl DMEM containing 10% FBS was plated in the lower chamber. Following incubation at 37°C for 24 h, cells that did not migrate through the pores were carefully removed using a cotton-tipped swab. The filters were then fixed at room temperature for 30 min in 90% alcohol, followed by staining with 0.1% crystal violet at room temperature for 30 min. Following 3 washes with PBS, migrating cells were observed and images were captured using an inverted microscope.

The animal experiment was also approved by the Legislation and Ethical Board of the People's Hospital of Ningxiang County. Male BALB/c nu/nu nude mice (n=6; age, 8 weeks old; weight, 22–25 g) were purchased from the Laboratory Animal Research Center (Shanghai, China). The mice were housed at 22–25°C, 40–60% humidity with a 12 h light/dark cycle, and access to clean food and water ad libitum. Control and 14-3-3β silenced cells were trypsinized, washed and re-suspended in serum free DMEM. A total of 2×10⁷ cells/mouse in 0.1 ml DMEM were subcutaneously injected into the left and right flanks of nude mice. Tumor development was assessed in each mouse individually on a daily basis. Tumor volume was determined by sequential caliper measurements of length (L) and width (W) every 2 h, calculated as 1/2 LW and a growth curve was constructed. Mice were sacrificed 30 days post-injection. Tumors were harvested and weighed to record wet tumor weight.

The quantitative data are presented as the mean ± standard deviation. Statistical analysis was performed using SPSS version 17.0 software package (SPSS, Inc., Chicago, IL, USA). Kaplan-Meier analysis was used for the survival analysis. The χ² test was used to examine the association between 14-3-3β expression and clinic pathological characteristics in patients with glioma. Student's t test was used to analyze differences between two groups and P<0.05 was considered to indicate a statistically significant difference.

To analyze the function of 14-3-3β, cells were transfected with antisense siRNA to silence the expression of 14-3-3β. The expression of 14-3-3β was significantly reduced following transfection of 14-3-3β siRNA compared with the negative control (NC) siRNA group (P<0.01; Fig. 1A), indicating that the 14-3-3β-silenced U373-MG cells were successfully established.

The potential role of 14-3-3β in glioma growth in vitro was further assessed. Data from the MTT assay revealed that the growth curve of U373-MG cells at 72 h was significantly lower in the 14-3-3β siRNA group compared with the NC siRNA group (P<0.01; Fig. 1B). Furthermore, cells with reduced expression of 14-3-3β formed significantly fewer colonies than the NC group, indicating that inhibition of 14-3-3β suppresses the colony forming capacity of U373-MG cells (P<0.01; Fig. 1C). Flow cytometry analysis of the cell cycle determined that the number of cells from the 14-3-3β siRNA group in the G1 phase was significantly higher than with the NC siRNA group (P<0.01; Fig. 2A), indicating that knockdown of 14-3-3β induces a cell cycle arrest at G1. Flow cytometry was also performed to assess cellular apoptosis. Apoptosis of U373-MG cells was significantly higher in the 14-3-3β siRNA group compared with the NC siRNA group (P<0.01; Fig. 2B), indicating that knockdown of 14-3-3β induces apoptosis in U373-MG cells. These results suggest that inhibition of 14-3-3β decreases the proliferation of glioma U373-MG cells, as well as inducing cell apoptosis, at least in part by inducing cell cycle arrest at the G1 phase.

To investigate whether 14-3-3β serves a role in glioma metastasis, cell migration was observed using wound-healing and Transwell assays. Glioma cells with decreased 14-3-3β expression had a significantly diminished migration ability compared with the NC siRNA group (P<0.01; Fig. 3A). In addition, the number of glioma cells migrating through the pores in the Transwell assay was significantly decreased in the 14-3-3β siRNA group compared with the NC siRNA group (P<0.01; Fig. 3B). These results suggest that 14-3-3β may serve a role in promoting glioma cell migration.

To further investigate the potential role of 14-3-3β on glioma growth, an in vivo xenograft model was constructed to detect the growth of glioma cell tumors with or without knockdown of 14-3-3β. U373-MG cells were injected into nude mice and tumor volume and weight were examined. Tumors removed from the nude mice were markedly smaller in the 14-3-3β siRNA group compared with those in the NC siRNA group (Fig. 4A). Significantly lower tumor volumes and weights were observed in mice in the 14-3-3β siRNA group compared with the NC siRNA group (P<0.01; Fig. 4B and C). These results suggest that silencing 14-3-3β inhibits glioma growth in vivo.

The expression of 14-3-3β in the 76 glioma tissues and 10 normal brain tissues was examined using RT-qPCR and levels of 14-3-3β mRNA were demonstrated to be significantly higher in glioma tissues compared with normal brain tissues (P<0.01; Fig. 5A). Subsequently, patients with glioma were divided into two groups: A high 14-3-3β expression group and a low 14-3-3β expression group, according to their mean value of 14-3-3β expression. Further investigation demonstrated that the high expression of 14-3-3β was significantly associated with advanced grade (P=0.03) and low Karnofsky performance scale (KPS; P=0.003; Fig. 5B), suggesting that the upregulation of 14-3-3β may contribute to the malignant progression of glioma (Table I). Furthermore, patients with high 14-3-3β levels had significantly shorter survival times compared with those with low expression of 14-3-3β (P=0.031; Fig. 5B), suggesting that 14-3-3β may be used as an effective predictor of prognosis for patients with glioma.