The U251, A172, U87-MG and SHG-44 human glioma cell lines were purchased from the Shanghai Institute of Biological Sciences, Chinese Academy of Sciences (Shanghai, China). The U373 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The U251, U373, A172 and U87-MG cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco Life Technologies, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS, GE Healthcare, Logan, UT, USA) at 37°C and 5% CO₂. The SHG-44 cells were cultured in RPMI-1640 medium (Gibco Life Technologies) containing 15% FBS (GE Healthcare) at 37°C and 5% CO₂.

The TCTP interfering sequences were designed, according to the TCTP mRNA sequence in GenBank using shRNA designing software, as shown in Table 1. The obtained interfering sequences (Wanleibio, Shenyang, China) were ligated into the pRNA-H1.1 eukaryotic expression vector (GenScript, Nanjing, China) using the restriction sites of HindIII and BamHI, and the resulting plasmid was termed pRNA-H1.1-TCTP. U251 cells in the logarithmic growth phase were seeded into 6-well plates. pRNA-H1.1-TCTP was transfected into the U251 cells using Lipofectamine 2000, according to the manufacturer's instructions (Invitrogen Life Technologies, Carlsbad, CA, USA). After 24 h, complete DMEM, containing 400 µg/ml G418 (Invitrogen Life Technologies) was added into each well for screening for 7–14 days, and the clones exhibiting positive expression of TCTP were selected and identified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. In the process of the experiment, an untransfected group (parental) and an empty vector-transfected group (pRNA-H1.1-control) were set up as the controls.

Following lysis of the cells using NP-40 lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China), the total cellular proteins were extracted, and the protein concentrations were determined using a Bicinchoninic Acid Protein Assay kit (Beyotime Institute of Biotechnology). Equal quantities of proteins (40 µg) were subjected to 10 or 12% SDS-PAGE, and the proteins were then transferred onto a polyvinylidene difluoride membrane (EMD Millipore, Bedford, MA, USA), followed by incubation overnight at 37°C with rabbit anti-human poly clonal antibodies against TCTP antibody (1:200; cat. no. sc-133131; Santa Cruz biotechnology Inc., Santa Cruz, CA, USA), cyclin D1 antibody (1:1,000; cat. no. WL0205), cyclin E antibody (1:1,000; cat. no. WL0055), cyclin B antibody (1:1,000; cat. no. WL0023), Bax polyclonal antibody (1:1,000; cat. no. WL0101), Bcl-2 antibody (1:1,000; cat. no.WL0104), cleaved-caspase-3 antibody (1:1,000; cat. no. WL0146), MMP-2 antibody (1:1,000; cat. no. WL0657), MMP-9 antibody (1:1,000; cat. no. WL0884) or β-actin antibody (1:1,000; all from Wanleibio, Shenyang, China) at 4°C. Subsequently, the membrane was incubated with the corresponding horseradish peroxidase-labeled goat anti-rabbit IgG secondary antibody at a 1:5,000 dilution (Beyotime Institute of Biotechnology) for 45 min at 37°C. Signals were detected using enhanced chemiluminescence (ECL) solution (Qihai Biotec, Shanghai, China) and the band intensities were normalized against those of β-actin using Gel-Pro-Analyzer version 4.0 software (Media. Cybernetics, Inc., Bethesda, MD, USA).

Total cellular RNA was extracted from the cells in each group, strictly according to the instructions of the total RNA extraction kit (Tiangen Biotech, Co., Ltd., Beijing, China). The cDNA (20 µl) was obtained by RT using M-MLV Reverse Transcriptase (BioTeke, Beijing, China), and quantitative fluorescence analysis was performed using SYBR Green MasterMix (10 µl; Solarbio, Beijing, China) in an Exicycler™ 96 quantitative fluorescence analyzer (Bioneer, Daejeon, Korea). The concentration of each primer was 10 µM. The reaction conditions were as follows: 95°C for 10 min; 95°C for 10 sec, 60°C for 20 sec and 72°C for 30 sec, for a total of 40 cycles. With β-actin as the internal control, the 2^−ΔΔCt method (22) was used to analyze the mRNA expression levels of TCTP. The sequences of the primers used are listed in Table II.

Cells in the logarithmic growth phase were harvested and seeded into 96-well plates at a density of 3×10³ cells/well. Following culture of the cells for 12, 24, 48, 72 and 96 h, MTT solution (final concentration, 0.2 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) was added into the each well. The cells were continuously cultured at 37°C for 4 h, and the supernatant was then discarded. The purple crystals were dissolved using 200 µl dimethyl sulfoxide (Sigma-Aldrich), and the optical density (OD₄₉₀) values were measured using a microplate reader (ELX-800; Bio-Tek Instruments Inc, Winooski, VT, USA). A cell growth curve was plotted and statistical analysis was performed.

The cells in each group were seeded (200 cells/dish) into a 35 mm petri-dish for culture. Subsequent to the formation of colonies, 4% paraformaldehyde (Guoyao, Shenyang, China) was used to fix the cells for 20 min, followed by Wright-Giemsa staining (Jiancheng Bioengineering Institute, Nanjing, China) for 5–8 min. The number of colonies, defined as a cell group with ≥50 cells, were counted and recorded under an inverted microscope (AE31; Motic Electric, Xiamen, China). The colony formation rate was then calculated according to the following formula: Colony formation rate = (number of colonies / number of seeded cells) × 100%.

The cells were cultured in DMEM medium and were harvested on reaching ~90% confluence. The cell cycle was detected using a flow cytometer (FACSCalibur; BD Biosciences, San Jose, CA, USA) using a Cell Cycle Analysis kit (Beyotime Institute of Biotechnology). In brief, the cells were harvested, washed with phosphate-buffered saline (PBS), and fixed with pre-cooled 70% ethanol at 4°C for 2 h. The fixed cells were then resus-pended in 500 µl binding buffer, containing 25 µl propidium iodide (PI) and 10 µl RNase A, at 37°C in the dark for 30 min, and detected using flow cytometry.

A Hoechst staining kit (Beyotime Institute of Biotechnology) was used to observe the apoptotic nuclear morphology of the cells. Briefly, cells in the logarithmic growth phase were seeded onto cell slides in 12-well plates, at a density of 1×10⁵ cells per well, and were cultured for 24 h. Then, the supernatant was discarded. The cells were fixed with 0.5 ml fixing solution at room temperature for 20 min and were then treated with Hoechst staining solution for 5 min. Anti-fluorescence quenching mounting solution (Beyotime Institute of Biotechnology) was added dropwise onto the slide, which was then covered with the coverslip containing the cells. The cells were visualized and images were captured under a fluorescence microscope (Olympus BX61; Olympus Corporation, Tokyo, Japan).

The cells were seeded into T25 cell culture flasks. When they had reached 90% confluence, the cells were harvested. The cells were resuspended in 500 µl Binding Buffer, according to the instructions of the apoptosis detection kit (KeyGEN Biotech, Co., Ltd., Nanjing, China). 5 µl Annexin V-FITC and 5 µl PI were immediately added with thorough mixing. Following 15 min incubation at 37°C in the dark, the cells were detected using flow cytometry (FACSCalibur; BD Biosciences).

The cells in each group were seeded into 6-well plates for culture. At a confluence of 80–90%, the culture medium was discarded. Subsequently, a scratch was created on the cell layer using a sterile 200 µl pipette tip. Images of the cells in each group were captured (AE31; Motic Electric) following being washed twice with serum-free medium. After 12 and 24 h of continuous culture, the migration distances of the cells were measured, and the cell migration rates were calculated using the following formula: Cell migration rate = (1−distance following healing / distance prior to healing) × 100%. Each experiment was repeated three times.

Following harvesting, 200 µl cell suspension containing 2×10⁴ cells (resuspended in serum-free culture medium) were seeded in upper Transwell chambers (Corning Incorporated Life Sciences, Tewksbury, MA, USA), which were coated with Matrigel (BD Biosciences), while 800 µl DMEM medium containing 30% FBS was added to the lower chamber. Following culture for 24 h, the uninvaded cells on the upper side of the microporous membrane were removed using a cotton swab. The remaining cells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet staining solution (Amresco, Solon, OH, USA). The numbers of invaded cells were recorded under an inverted microscope (AE31; Motic Electric) in five randomly-selected fields (magnification, ×200), to obtain the average.

The data are presented as the mean ± standard deviation. One-way analysis of variance was used for comparisons between groups, and Bonferroni's post hoc-test was used for the comparison of multiple variables. The image and data were processed using Graphpad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

In the present study, total proteins were extracted from the U373, U251, A172, U87-MG and SHG-44 cells to analyze the expression of TCTP in these cells. The results of the western blotting revealed that the expression levels of TCTP in the A172, U87-MG and SHG-44 cells were essentially the same as that in the U373 cells (Fig. 1A; P>0.05), while the level of expression in the U251 cells was significantly higher than that in the U373 cells (P<0.01). Thus, U251 cells with high expression levels of TCTP were selected to investigate TCTP gene function. The TCTP shRNA eukaryotic expression plasmid, pRNA-H1.1-TCTP, was subsequently transfected into the U251 cells. Western blot and RT-qPCR analyses revealed that the protein and mRNA expression levels of TCTP in the pRNA-H1.1-TCTP group were significantly decreased, and were 0.28-fold (Fig. 1B; P<0.01) and 0.18-fold (Fig. 1C; P<0.01) lower than that in the pRNA-H1.1-control group, respectively. Thus, a U251 glioma cell line stably transfected with TCTP shRNA was successfully established.

The impact of the downregulated expression of TCTP on glioma cell proliferation was investigated. The MTT results demonstrated that at 48, 72 and 96 h, cell proliferation in the pRNA-H1.1-TCTP group was significantly lower, compared with that in the pRNA-H1.1-control group (Fig. 2A; P<0.05). The effect of downregulated expression of TCTP on the clonogenic capacity of glioma cells, detected using a colony formation assay, is shown in Fig. 2B. The colony formation rate of the cells in the pRNA-H1.1-TCTP group was significantly lower than that in the pRNA-H1.1-control group (32.0±4.9, vs. 60.3±6.9%, respectively; P<0.01). The present study further detected the cell cycle distribution using flow cytometry. The percentage of G2/M phase cells in the pRNA-H1.1-TCTP group was significantly reduced, compared with that in the pRNA-H1.1-control group (0.56±0.08, vs. 8.97±0.94)%, respectively; Fig. 2C; P<0.01). The percentage of S phase cells was essentially unchanged (26.17±2.48, vs. 27.00±2.23%), while the percentage of G0/G1 phase cells was significantly increased (73.27±2.47, vs. 63.03±2.11%; P<0.01). Western blotting revealed that the expression levels of Cyclin D1, Cyclin E and Cyclin B in the pRNA-H1.1-TCTP cells were significantly reduced (Fig. 2D; P<0.01). These results suggested that downregulation of the expression of TCTP caused cell cycle arrest in the G0/G1 phase and ultimately inhibited glioma cell proliferation.

The apoptosis and expression levels of apoptosis-associated factors were analyzed using flow cytometry and Hoechst staining. The Hoechst staining revealed that the nuclei of the cells in the parental group and pRNA-H1.1-control group were homogeneous blue. Typical apoptotic morphological changes, including condensed chromatin and shrunken, crumpled and condensed nuclei, were observed in the pRNA-H1.1-TCTP group. The apoptotic rate of the cells in the pRNA-H1.1-TCTP group was significantly higher than that in the pRNA-H1.1-control group (29.6±3.03, vs. 6.36±0.79%, respectively; Fig. 3A; P<0.01). Flow cytometry consistently indicated that the apoptotic rate of the cells in the pRNA-H1.1-TCTP group was significantly higher than that in the pRNA-H1.1-control group (24.60±2.68, vs. 6.88±0.86%, respectively; Fig. 3B; P<0.01). Western blot analysis demonstrated that the expression levels of Bax and cleaved-caspase-3 in the cells in the pRNA-H1.1-TCTP group were significantly increased (Fig. 3C; P<0.01), whereas the expression of Bcl-2 was significantly decreased (P<0.01). These results suggested that downregulation of the expression of TCTP effectively induced apoptosis in the glioma cells.

The present study investigated the impact of downregulated expression of TCTP on glioma cell migration. The cell migration rate of the cells was examined using a wound healing assay, the results of which revealed that the migration rate of the cells in the pRNA-H1.1-TCTP group 12 h (Fig. 4A; P<0.01) and 24 h (P<0.05) post-wounding were significantly lower than those in the pRNA-H1.1-control group. The effect of TCTP shRNA on the invasiveness of the glioma cells was further detected using a Transwell assay, the results of which revealed that the number of invaded cells in the pRNA-H1.1-TCTP group was significantly lower than that in the pRNA-H1.1-control group (32.40±6.07, vs. 73.40±9.04), respectively; Fig. 4B; P<0.01). Western blot analysis revealed that the protein expression levels of MMP-2 and MMP-9 in the cells in the pRNA-H1.1-TCTP group were significantly lower than those in the pRNA-H1.1-control group (Fig. 4C; P<0.01). Gelatin zymography revealed bands at 72 and 92 kDa (corresponding to MMP-2 and MMP-9, respectively) in the cells of all groups. Additionally, the activities of MMP-2 and MMP-9 in the cells of the pRNA-H1.1-TCTP group were significantly decreased (Fig. 4D; P<0.01). These results demonstrated that downregulation of the expression of TCTP inhibited glioma cell migration and invasion.