Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), Lipofectamine^® 2000 and TRIzol^® reagent were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). RevertAid First Strand cDNA Synthesis kit was purchased from Fermentas (Thermo Fisher Scientific, Inc., Pittsburgh, PA, USA). SYBR Green quantitative polymerase chain reaction (qPCR) Assay kit was purchased from TOYOBO (Shanghai) Co., Ltd. (Shanghai, China). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Biosharp (Hefei, China). Annexin V-Fluorescein Isothiocyanate (FITC) Apoptosis Detection kit was purchased from BD Pharmingen (San Diego, CA, USA). Transwell chamber was obtained from Corning Inc. (Corning, NY, USA). Rabbit anti-PREX2a (1:200; ab121462), phosphorylated (p)-PTEN (1:100; ab76431), PTEN (1:100; ab79156), p-Akt (1:50; ab81283), Akt (1:100; ab32505), p-mammalian target of rapamycin (mTOR; 1:100; ab109268), mTOR (1:100; ab2732) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:50; ab181602) monoclonal antibodies, and goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:5,000; ab6721) were purchased from Abcam (Cambridge, MA, USA). Enhanced chemiluminescence (ECL) kit was purchased from Pierce Biotechnology, Inc. (Rockford, IL, USA).

The present study was approved by the Ethics Committee of Central South University (Changsha, China). Written informed consent was obtained from all of the patients. A total of 24 primary glioma tissues and six normal brain specimens were collected from the Department of Neurosurgery, Xiangya Hospital of Central South University. The glioma patients included 10 females and 14 males who ranged in age from 28 to 71 years, with a mean age of 49.5 years. None of the patients had received radiation therapy or chemotherapy prior to surgical resection. The 24 glioma samples were classified according to the World Health Organization (WHO) grading system (14), and included five pilocytic astrocytomas (WHO I), six diffuse astrocytomas (WHO II), seven anaplasia astrocytomas (WHO III), and six primary glioblastomas (WHO IV). The tissues were collected under surgical resection and the histomorphology of all of the samples was confirmed by the Department of Pathology, Xiangya Hospital of Central South University. Tissues were immediately snap-frozen in liquid nitrogen following surgical removal.

The SWO-38 human glioma cell line was purchased from the Cell Bank of Central South University. The cells were cultured in DMEM supplemented with 10% FBS at 37°C in a humidified incubator containing 5% CO₂.

Lipofectamine^® 2000 was used to perform transfection, according to the manufacturer's protocol. Briefly, the cells were cultured to 70% confluence and resuspended in serum-free medium. Non-specific (negative control) and PREX2a-specific small interfering (si)RNA (GeneChem Co., Ltd., Shanghai, China) and Lipofectamine^® 2000 were diluted with serum-free medium separately. The diluted Lipofectamine^® 2000 was added to the diluted siRNA, and incubated for 20 min at room temperature, prior to being added to the cell suspension. Following a 6 h incubation at 37°C in an atmosphere containing 5% CO₂, the medium was replaced with normal serum-containing medium. The cells were subsequently cultured for 24 h prior to further experimentation.

The tissue samples were homogenized in liquid nitrogen by grinding with a grinding rod. Subsequently, total RNA was extracted from the tissues or cells using TRIzol^® reagent, according to the manufacturer's protocol. Total RNA was reverse transcribed into cDNA using the RevertAid First Strand cDNA Synthesis kit, according to the manufacturer's protocol. Briefly, 1 µl total RNA was mixed with 1 µl of 100 mm dNTP, 1 µl reverse transcriptase, 10 µl of 10X reverse transcription buffer, 1 µl RNase inhibitor and 1 µl primer. Nuclease-free H₂O was added to obtain a final volume of 20 µl. Reverse transcription was performed at 16°C for 30 min, followed by an incubation step at 42°C for 30 min and enzyme inactivation at 85°C for 5 min. PCR was performed on 10 ng cDNA, using the SYBR Green qPCR Assay kit and the Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. The specific primer pairs used were as follows: PREX2a, sense 5′-TGG GAG GGG TCC AAC ATCA-3′, anti-sense 5′-TCT TCA ACC GTC TGT GTT TTCTT-3′; GAPDH, sense 5′-CTC CTC CTG TTC GAC AGT CAGC-3′, and anti-sense 5′-CCC AAT ACG ACC AAA TCC GTT-3′ (Sangon Biotech Co., Ltd., Shanghai, China). GAPDH was used as an internal control. The PCR cycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 sec and annealing/elongation at 60°C for 1 min. Independent experiments were repeated three times. The relative mRNA expression levels were analyzed using the 2^−∆∆Cq method (15).

Total protein was extracted from the tissues or cells using cold radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). The protein concentrations were quantified using the Bicinchoninic Acid Protein Assay kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Proteins (50 µg) were then separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was incubated with 5% milk in Tris-buffered saline containing 0.1% Tween at room temperature for 3 h, and then incubated with rabbit anti-PREX2a, p-Akt, Akt, p-mTOR, mTOR and GAPDH monoclonal antibodies at room temperature for 3 h. Subsequently, the membrane was incubated with goat anti-rabbit secondary antibody at room temperature for 40 min. Chemiluminescent detection was performed using an ECL kit. The blots were analyzed using Image-Pro Plus software 6.0 (Media Cybernetics, Inc., Rockville, MD, USA), and the relative protein expression levels were represented as the density ratio vs. GAPDH.

An MTT assay was used to measure cell proliferation. Cells in each group were cultured in 100 µl fresh serum-free medium/well in a 96-well plate. MTT (0.5 g/l) was added to the cells and incubated at 37°C for 0, 24, 48 and 72 h. Subsequently, the medium was removed by aspiration and 50 µl dimethyl sulfoxide was added to each well and incubated at 37°C for a further 10 min. The absorbance of each sample was measured at 492 nm using a plate reader (AF2200; Eppendorf, Hamburg, Germany).

Flow cytometry (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA) was used to determine the rate of cell apoptosis using the Annexin V-FITC Apoptosis Detection kit. At 24 h post-transfection, the cells were harvested and washed twice with cold phosphate-buffered saline (PBS). Subsequently, 1×10⁶ cells were resuspended in 200 µl binding buffer with 10 µl Annexin V-FITC and 5 µl propidium iodide (PI), and incubated in the dark for 30 min. Finally, 300 µl binding buffer was added to the cells, which where analyzed by flow cytometry. The flow cytometry data was analyzed using the BD Accuri C6 software (BD Biosciences).

A total of 1×10⁶ cells from each group were collected in 1X PBS and fixed with 70% ethanol overnight at −20°C. The cells were then centrifuged at 1,000 × g for 5 min, washed in 1X PBS, and centrifuged for a further 5 min at 300 × g. The cells were resuspended in 300 µl PI staining buffer and incubated for 30 min at room temperature. DNA content analyses were performed using the BD Accuri C6 Flow Cytometer (BD Biosciences).

The invasive ability of glioma cells was determined in 24-well Transwell chambers, which were coated with a layer of Matrigel (BD Biosciences). Glioma cells (1.0×10⁵ cells/ml) suspended in serum-free DMEM were seeded in the upper chamber, and DMEM supplemented with 10% FBS was added to the lower chamber. Following a 24 h incubation at 37°C, the non-invading cells and the Matrigel on the interior of the inserts were removed using a cotton-tipped swab. Invasive cells on the lower surface of the membrane were stained with gentian violet, rinsed with water and air-dried. Five fields were randomly selected, and the number of cells was counted under a microscope (CX31; Olympus, Tokyo, Japan).

All data are presented as the mean ± standard deviation. One-way analysis of variance was used to statistically analyze the data using SPSS 17 software (SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

To explore the role of PREX2a in glioma, RT-qPCR and western blotting were performed, in order to determine the mRNA and protein expression levels of PREX2a in glioma tissue. As shown in Fig. 1A and B, the mRNA and protein expression levels of PREX2a were significantly increased in glioma tissues, as compared with in normal brain tissue. In addition, PREX2a expression was positively correlated with the WHO grade of glioma. No significant difference was detected in PREX2a mRNA expression between normal brain tissue and WHO I glioma, or between WHO III and WHO IV glioma (P>0.05). However, differences between any other two groups were statistically significant (P<0.05).

To investigate the role of PREX2a in the regulation of glioma in vitro, SWO-38 glioma cells were transfected with PREX2a-specific siRNA or non-specific siRNA, which was used as a negative control (NC). Post-transfection of the SWO-38 glioma cells with PREX2a siRNA, the mRNA expression levels of PREX2a were decreased by 78%, and the protein expression levels of PREX2a were decreased by 72%, as compared with the control group (P<0.01; Fig. 2A and B). However, transfection with NC siRNA did not affect the expression levels of PREX2a, as compared with the control group (P>0.05; Fig. 2A and B).

An MTT assay was conducted to determine cell proliferation in each group. Following knockdown of PREX2a expression, the proliferation rate of SWO-38 glioma cells was significantly reduced by 32%, as compared with the control group (P<0.01; Fig. 2C). In addition, the effects of siRNA-mediated knockdown of PREX2a on apoptosis of glioma SWO-38 cells were detected. As shown in Fig. 2D, the rate of cell apoptosis was markedly increased following knockdown of PREX2a expression, as compared with in the control group (P<0.01). The percentage of apoptotic cells in the control, NC and PREX2a siRNA groups was 2.91±0.12, 3.04±0.14 and 11.87±0.46%, respectively. These results suggest that siRNA-mediated knockdown of PREX2a may inhibit proliferation and induce apoptosis of glioma cells.

Suppression of cell cycle progression was detected in SWO-38 glioma cells following knockdown of PREX2a expression. As shown in Fig. 3, post-transfection with PREX2a siRNA, the SWO-38 cells exhibited a significant increase in the percentage of cells in G₁ phase (control, 36.87±2.31%; NC, 37.65±2.57%; PREX2a siRNA, 67.03±2.92%; P<0.01) and a corresponding reduction in the percentage of cells in S phase (control, 39.36±2.54%; NC, 38.71±2.36%; PREX2a siRNA, 24.63±2.7%; P<0.01) and G₂/M phase (control, 23.77±3.86%; NC, 23.64±3.14%; PREX2a siRNA, 8.34±3.83%; P<0.01). These results suggest that siRNA-mediated knockdown of PREX2a may induce cell cycle arrest at G₁ phase.

The invasive capacity of glioma SWO-38 cells was significantly reduced following knockdown of PREX2a expression (P<0.01; Fig. 4A and B). The percentage of invasive cells in the control, NC and PREX2a siRNA groups was 100±5.27, 99.9±1.83 and 41.9±5.29%, respectively (Fig. 4B). These results suggest that PREX2a has a promoting role in the regulation of glioma cell invasion.

The present study further investigated the activity of the PI3K/Akt/mTOR signaling pathway in SWO-38 cells with or without transfection with PREX2a siRNA. Following knockdown of PREX2a expression, the phosphorylation levels of PTEN in the SWO-38 glioma cells were reduced, suggesting that the activity of PTEN was upregulated. Furthermore, the phosphorylation levels of Akt and mTOR were decreased, indicating that activity of the PI3K/Akt/mTOR signaling pathway was downregulated following knockdown of PREX2a expression (Fig. 5). These results indicate that knockdown of PREX2a may inhibit the activity of the PI3K/Akt/mTOR signaling pathway via activation of PTEN.