14-3-3β exerts glioma-promoting effects and is associated with malignant progression and poor prognosis in patients with glioma.

Glioma is a type of tumor that affects the central nervous system. It has been demonstrated that 14-3-3β, a protein that is mainly concentrated in the brain, serves an important role in tumor regulation. However, the mechanism of action of 14-3-3β that underlies the pathogenesis of glioma remains to be elucidated. In the present study, 14-3-3β was silenced by RNA interference in the human glioma cell line U373-MG. Following knockdown of 14-3-3β, the proliferation, colony formation, cell cycle progression, migration and invasion of U373-MG cells were significantly decreased (P<0.01), whereas cell apoptosis was increased (P<0.01). Furthermore, in a tumor xenograft experiment, silencing 14-3-3β significantly inhibited the in vivo tumor growth of U373-MG cells (P<0.01). The results demonstrated that 14-3-3β levels were significantly higher in human glioma tissues compared with normal brain tissues (P<0.01) and high 14-3-3β expression was significantly associated with advanced pathological grade (P<0.03) and low Karnofsky performance scale (P<0.003). Patients with glioma who had high 14-3-3β levels had a significantly shorter survival time compared with those with low expression of 14-3-3β (P=0.031), suggesting that 14-3-3β may be an effective predictor of the prognosis of patients with glioma. The results of the present study indicate that 14-3-3β serves an oncogenic role in glioma, suggesting that 14-3-3β may have potential as a promising therapeutic target for glioma.

Introduction

Glioma is considered to be a serious health problem that affects the central nervous system (1). Based on the degree of malignancy, gliomas are graded from I to IV according to the World Health Organization classification (2). Grade IV glioblastoma (GB) are the most common and malignant type of glioma, accounting for >50% of glial neoplasms (2). Despite improvements in the therapeutic strategies available, including adjuvant chemotherapy, radiation therapy and microsurgical resection, the prognosis for patients with glioma remains poor; 75% of patients with GB succumb within 18 months of diagnosis (3). Therefore, investigating the underlying mechanisms of glioma progression is important to improve patient outcome.

14-3-3 proteins are a family of highly conserved acidic polypeptides found in eukaryotic organisms and researchers have identified seven isoforms in mammalian cells: α, β, δ, γ, σ, θ and ζ (4). As 14-3-3 proteins have multiple binding partners, they have widely diverse intracellular functions, including cell cycle regulation, proliferation, apoptosis, differentiation and senescence (4,5). In addition to these normal physiological processes, 14-3-3 proteins are associated with oncogenesis (6). For example, six isoforms of 14-3-3 have been identified as being overexpressed in human lung cancer tissues (7). Upregulation of 14-3-3ζ promotes cell growth in lung cancer cells (8) and several 14-3-3 proteins act as tumor suppressors (8,9). It has been demonstrated that downregulating 14-3-3σ leads to increased metastasis in human lung cancer (9), nasopharyngeal carcinoma (10) and ovarian cancer (11).

14-3-3β, which is an important member of the 14-3-3 protein family, is associated with tumor initiation (7,12). Increased 14-3-3β expression has been observed in human lung cancer tissues (7) and the expression of 14-3-3β is associated with increased aggressiveness in human astrocytoma (12). Additionally, a previous study reported that 14-3-3β mRNA was overexpressed in various murine tumor cell lines and that reducing the expression of 14-3-3β suppressed tumor cell growth in vitro and in vivo (13). Overexpression of 14-3-3β in NIH3T3 cells stimulates cell proliferation and tumor formation (14), and previous studies have demonstrated that upregulating 14-3-3β expression mediates cell proliferation and tumor progression in Kaposi's sarcoma and papillary thyroid carcinomas (15,16). These results suggest that 14-3-3β serves a vital role in tumor growth and progression.

Although 14-3-3 proteins have been identified in different organs, including the liver and intestines, the highest concentration of 14-3-3 proteins is located in the brain (17,18). Accordingly, the effects of 14-3-3 proteins in brain tumors has been widely studied. A previous study reported that 14-3-3 proteins were highly expressed in primary human nervous system tumors (19). Furthermore, downregulation of 14-3-3 increases the sensitivity of glioma cells to apoptosis and suppresses glioma growth (20). Park et al (21) demonstrated that 14-3-3β modulates the migration and invasion of the human malignant glioma cell line U87-MG. However, the mechanism of action of 14-3-3β in the pathogenesis of glioma remains to be elucidated.

In the present study, RNA interference (RNAi) was used to silence the expression of 14-3-3β in the human glioma cell line U373-MG. The proliferation, apoptosis, migration and invasion of pretreated cells were measured to evaluate the effects of 14-3-3β expression. A tumor xenograft experiment was performed to assess the in vivo tumor formation of pretreated cells. Human glioma tissues were collected from patients with tumors and the expression of 14-3-3β was assessed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. Subsequently, the association between 14-3-3β expression and the prognosis of patients with glioma was investigated. The results of the present study may help to improve understanding of the role and mechanism of 14-3-3β during the progression of glioma.

Materials and methods

Clinical tissue collection

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.

Table I.

Association between 14-3-3β expression and clinicopathological characteristics in glioma.

Variables	Cases (n=76)	Low 14-3-3β expression (n=37)	High 14-3-3β expression (n=39)	χ2 test P-values
Age				0.368
<55	35	15	20
≥55	41	22	19
Sex				0.241
Male	48	26	22
Female	28	11	17
WHO grade				0.030[a]
I–II	27	18	9
III–IV	49	19	30
KPS				0.003[a]
>90	26	19	7
≤90	50	18	32

P<0.05; KPS, Karnofsky performance scale; WHO, world health organization.

Cell culture

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).

Knockdown of 14-3-3β by small interfering (si)RNA

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.

RT-qPCR

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).

Western blotting

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 assay

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.

Analysis of cell cycle distribution

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).

Cell apoptosis assay

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).

Wound-healing assay

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).

Cell migration assay

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.

Tumor xenograft experiment

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.

Statistical analysis

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.

Results

Knockdown of 14-3-3β inhibits the proliferation and induces apoptosis of glioma U373-MG cells

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.

Figure 1.

U373-MG cells were transfected with NC siRNA or 14-3-3β siRNA. (A) Western blotting was used to examine the protein expression of 14-3-3β. (B) An MTT assay was performed to examine cell proliferation. (C) Colony formation ability was assessed. **P<0.01 vs. NC siRNA. NC, negative control; siRNA, small interfering RNA.

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.

Figure 2.

U373-MG cells were transfected with NC siRNA or 14-3-3β siRNA. Flow cytometry was used to assess the (A) cell cycle and (B) cell apoptosis. **P<0.01 vs. NC siRNA. NC, negative control; siRNA, small interfering RNA.

Inhibition of 14-3-3β decreases the migratory capacities of U373-MG cells

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.

Figure 3.

U373-MG cells were transfected with NC siRNA or 14-3-3β siRNA. (A) Wound-healing and (B) Transwell assays were performed to assess cell migration. Magnification, ×400. **P<0.01 vs. NC siRNA. NC, negative control; siRNA, small interfering RNA.

Downregulation of 14-3-3β represses the tumor growth of U373-MG cells in vivo

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.

Figure 4.

U373-MG cells were stably transfected with NC siRNA or 14-3-3β siRNA, and these cells were injected into nude mice to determine tumor growth in vivo. (A) Representative images of tumors from the 14-3-3β siRNA and NC siRNA groups. Tumor (B) volume and (C) weight. **P<0.01 vs. NC siRNA. NC, negative control; siRNA, small interfering RNA.

High expression of 14-3-3β is associated with malignant progression and poor prognosis for patients with glioma

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.

Figure 5.

(A) The expression of 14-3-3β mRNA was examined in 76 glioma tissues and 10 normal brain tissues using reverse transcription-quantitative polymerase chain reaction. **P<0.01 vs. Normal. (B) Comparison of the survival time of patients with glioma and high or low 14-3-3β levels. Kaplan-Meier analysis was used for the survival analysis. Normal, normal brain tissue.

Discussion

14-3-3 proteins are able to bind to a wide variety of ligands associated with the transduction of signaling pathways, the cell cycle and apoptosis (25). The oncogenic proteins that 14-3-3 binds include Raf, breakpoint cluster region (Bcr), Bcr-Abelson murine leukemia viral oncogene homolog 1 and polyomavirus middle tumor antigens (25). Furthermore, the proteins that regulate cell survival and apoptosis, including B-cell lymphoma 2-associated death receptor, B-cell lymphoma 2-associated X protein, apoptosis signal regulating kinase 1 and the Foxo family forkhead transcription factors, are also ligands of the 14-3-3 family (25). Tumor suppressors, including p53 and tuberous sclerosis complex 2 and p27, are also regulated by 14-3-3 (6,26,27). Therefore, different 14-3-3 isoforms may act as oncogenes or tumor suppressors in different types of cancer and further research is required to identify the role of different 14-3-3 family members in different tumors.

Malignant proliferation is an important characteristic of cancer cells; therefore, the 14-3-3 family's regulatory effects on tumor cell proliferation have been investigated. For example, 14-3-3ζ serves important roles in lung cancer cell proliferation and chemotherapy drug resistance (28), whereas 14-3-3ó functions as a tumor suppressor in breast cancer (29). Previously, 14-3-3β has been demonstrated to promote tumorigenesis in nude mice (14) and silencing 14-3-3β expression in liver cancer cells inhibits cell proliferation and oncogenicity in nude mice (13). It has also previously been reported that 14-3-3β promotes cell proliferation and tumor formation in NIH3T3 cells (14). In accordance with previous studies, the results of the present study demonstrated that the downregulation of 14-3-3β in a mixed glioma cell line leads to decreased tumor growth in vivo, suggesting that 14-3-3β promotes the growth and proliferation of glioma cells.

Glioma is considered to be a serious tumor of the central nervous system and the prognosis of patients remains poor, despite medical advances (1–3). These previous studies demonstrated that surgical resection of glioma in the brain is an invasive procedure. Furthermore, residual tumor cells are invasive and not amenable to further surgery, radiotherapy or chemotherapy. These residual cells typically cause tumor recurrence and patient morbidity within 15 months of initial diagnosis (30). Regulating the invasive ability of glioma is therefore an important aspect to consider. It has been reported that 14-3-3 proteins may be associated with tumor malignancy and invasion (6–11). The 14-3-3 family of proteins is mainly distributed in the brain (17,31), thus the regulatory effects of 14-3-3β on glioma migration were investigated in the present study. The results demonstrated that the migration of glioma U373-MG cells was inhibited following 14-3-3β silencing, indicating that 14-3-3β promotes the migration of glioma. This is in accordance with the results of Park et al (21), who reported that 14-3-3β modulated the migration and invasion of the human malignant glioma cell line U87MG. Therefore, the development of novel therapeutic strategies should focus on preventing further spreading or targeting these invasive cells (32). The current study indicates that 14-3-3β may be used as a novel target. It has previously been demonstrated that 14-3-3β expression increases with the severity of human astrocytoma (33). In the present study, increased expression of 14-3-3β was significantly associated with advanced grade and low KPS, and the high expression of 14-3-3β in primary tumors was an important predicting parameter of a shorter survival time in patients with glioma. This suggests that 14-3-3β serves important roles in human glioma and targeting 14-3-3β expression may be a promising method for the treatment of glioma in humans.

In conclusion, the results of the present study revealed that 14-3-3β is associated with the promotion of glioma proliferation, migration and tumor growth in vivo. In addition, the expression of 14-3-3β was associated with advanced histopathological grading, as well as poor patient prognosis. Given the high expression of 14-3-3β in glioma tissues, future studies should identify novel therapeutic strategies aimed at 14-3-3β for the effective treatment of human glioma.