The U87 and U251 human glioma cells were purchased from the American Type Culture Collection (ATCC; USA). Lipofectamine™ 2000 was purchased from Invitrogen (USA), the siRNAs were synthesised by GenePharma (China), the RT-PCR kit [Takara RNA PCR kit (AMV) Ver. 3.0] was purchased from Takara Biotechnology (Japan), and propidium iodide was purchased from Sigma (USA). The antibodies against Ezh2, Bax, Bcl-2, caspase 3 and 9, CDK4, CDK6, and cyclin D1 were purchased from Cell Signaling Technology, Inc., USA.

The U87 and U251 human glioma cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% foetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C with 5% CO₂. All of the experiments were performed with logarithmically growing cells.

To measure the cellular viability using the MTT assay, the cells were seeded in 96-well culture plates at a density of 1×10⁵ cells/ml, and Ezh2 siRNA was added when the cells were determined to be logarithmically growing. There were 5 groups of 6 wells of cells for each time point (24, 48, 72, 96 and 120 h) within each experiment. After 20 μl of a 5-mg/ml solution of 5-diphenyltetrazolium bromide (MTT) was added to each well, the cells were cultured for an additional 4 h. Then, the culture medium was removed, and 150 μl of dimethyl sulphoxide (DMSO) was added to each well. The cells were subsequently vortexed at room temperature for 10 min, and the OD value (570 nm) of each well was detected with a microplate reader.

For the siRNA transfections, the cells were seeded in 6-well plates at a density of 5×10⁵ cells/ml and cultured in DMEM without antibiotics. The transfection was performed when the confluence of the cells was ≥60%, and 2 individual groups of cells were either transfected with an Ezh2 siRNA or a non-targeting oligonucleotide control using Lipofectamine (Invitrogen) according to the manufacturer’s instructions. The media were changed prior to the transfection with serum-free DMEM without antibiotics and replaced 4–6 h after the transfection with serum-containing DMEM medium. The sequence of the Ezh2 siRNA was 5′-AAG ACT CTG AAT GCA GTT GCT-3′, and a non-targeting siRNA served as a negative control. The efficiency of the siRNA was determined using reverse transcription polymerase chain reaction (RT-PCR) and western blotting.

The extraction of the total-RNA was performed using an RNAiso™ Plus kit (Takara) according to the manufacturer’s instructions. After calculating the RNA concentration, the RT-PCR was performed using an RT-PCR kit (Takara) according to the product manual. The primers for the Ezh2 and β-actin RT-PCR reaction were synthesised by Invitrogen and were as follows: Ezh2, forward primer 5′-GCC AGA CTG GGA AGA AAT CTG-3′ and reverse primer 5′-TGT GCT GGA AAA TCC AAG TCA-3′; β-actin, forward primer 5′-CTG GGA CGA CAT GGA GAA AA-3′ and reverse primer 5′-AAG GAA GGC TGG AAG AGT GC-3′. The PCR reaction was then performed in a 50-μl volume with the following reaction conditions: an initial denaturing step at 94°C for 2 min and 30 cycles of denaturing at 94°C for 30 sec, annealing at 60°C for 30 sec, and extension at 72°C for 30 sec. The PCR products were separated on a 1.0% agarose gel by electrophoresis and analysed with a gel imaging scanning system.

The cells were collected by trypsinisation and washed twice in ice-cold PBS. Next, the cells were resuspended in PBS, and a single-cell suspension was prepared by pipetting the medium up and down. An Annexin-V and PI staining solution was then added according to the manufacturer’s instructions (Annexin-V-FITC kit, Biosea Biotechnology, China). The cells were stained for 15 min in the dark at room temperature, and the apoptotic cells were detected using a flow cytometer (Becton-Dickinson, USA).

JC-1 staining and flow cytometry were used to detect changes in the mitochondrial membrane potential according to a previously published protocol (17). The fluorescence signals of the JC-1 monomer and polymer were detected by the FL1 and FL2 detectors, respectively. FL1-H and FL2-H represent the green and red fluorescence intensities, respectively. CellQuest software was used for the quantification of the results.

For the cell cycle analysis, the cells were trypsinised with 0.25% trypsin, collected, washed twice in PBS, and fixed with a 70% ethanol solution at 4°C overnight. The following day, the ethanol was discarded. Subsequently, the cells were washed twice with PBS, stained with 1 ml of PI dye that contained 10 μg of RNaseA and 5 μl of Triton X-100 for 30 min at 4°C in the dark, and analysed by flow cytometry.

For the western blotting, the transfected U87 cells from each group were collected and washed twice in PBS. Then, 2 ml of lysis buffer (50 mM Tris-HCl pH 7.4, 137 mM NaCl, 10% glycerol, 100 mM sodium vanadate, 1 mM PMSF, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 1% NP-40, and 5 mM cocktail) was added to the cells. The protein concentration was determined using the BCA method, and the proteins were stained with bromophenol blue. Equal amounts of protein were loaded and separated on a 10% polyacrylamide gel by SDS-PAGE electrophoresis, and the proteins were transferred onto a PVDF membrane using the semi-dry method. The membrane was then blocked with 5% non-fat dry milk overnight. The following day, the membrane was washed with TBST, incubated for 2 h with the primary antibodies, washed with TBST, and incubated for an additional 2 h with the secondary antibody. After adding the chemiluminescence reagent, X-ray autoradiography was performed, the bands were scanned, and the gray scale images were analysed.

SPSS 16.0 statistical software was used for the statistical analysis. The values are shown as the mean ± SD. Statistical analysis was performed using the Student’s t-test, and the differences between the groups were considered to be statistically significant at p<0.05.

The Ezh2 mRNA and protein expression levels in U87 and U251 human glioma cells were examined using RT-PCR and western blotting, respectively. We found that Ezh2 mRNA was highly expressed in the U87 and U251 cells, and a higher level was present in the U87 cells. Additionally, western blot analysis demonstrated that the expression levels of the Ezh2 protein in the U87 and U251 cells correlated with the mRNA expression levels. These results suggest that Ezh2 is highly expressed in the U87 and U251 human glioma cells (Fig. 1).

An siRNA targeting Ezh2 and a non-targeting oligonucleotide were individually transfected into U87 cells, and the changes in the Ezh2 mRNA and protein expression levels after transfection were analysed by RT-PCR and western blotting, respectively. The results demonstrated that at 24 h after the transfection of the Ezh2 siRNA (55 nM), the Ezh2 mRNA expression level was significantly reduced. The silencing of Ezh2 lasted for at least 72 h after the siRNA transfection (Fig. 2). These results suggest that after the transfection of the siRNA targeting Ezh2 at 55 nM for 24 h, the Ezh2 mRNA and protein expression levels are effectively downregulated.

MTT assay was used to determine the effects of the downregulation of Ezh2 expression on the proliferation of U87 cells. We found that compared to the mock-transfected cells or the cells transfected with the non-targeting siRNA, the proliferation of the U87 cells transfected with the Ezh2 siRNA was significantly reduced at 24, 48, 72, 96, or 120 h after the transfection. These results suggest that the silencing of the Ezh2 gene inhibits the proliferation of U87 cells (Fig. 3).

To investigate whether the level of Ezh2 expression was related to the level of apoptosis found in the U87 cells, the expression of the Ezh2 gene was downregulated by RNA interference, and the indicators of apoptosis were analysed. We found that after the silencing of the Ezh2 gene, a significantly higher rate of apoptosis was present in the U87 cells as detected by flow cytometry (Fig. 4A and B). In addition, western blot analysis indicated that the protein expression levels of procaspase 9 and 3 were significantly reduced after the downregulation of Ezh2 expression (Fig. 4C and D). These results suggest that the silencing of the Ezh2 gene leads to apoptosis in U87 cells.

To verify whether the apoptosis induced by the silencing of the Ezh2 gene in the U87 cells was related to the mitochondrial pathway, the changes in the mitochondrial membrane potential were detected using JC-1 staining. The results demonstrated that the downregulation of Ezh2 expression significantly reduced the mitochondrial membrane potential (Fig. 5A). In addition, the levels of Bax, Bcl-2, and cytochrome c were analysed by western blotting. The silencing of Ezh2 expression decreased the level of Bax and increased the levels of Bcl-2 and cytochrome c in the cytoplasm. However, the changes in the levels of Bax, Bcl-2, and cytochrome c in the mitochondria were the opposite of the changes found in the cytoplasm (Fig. 5B–E). Therefore, our data indicate that the downregulation of Ezh2 expression promotes the translocation of Bax and Bcl-2, causes the release of mitochondrial cytochrome c, reduces the mitochondrial membrane potential, and results in the apoptosis of human glioma cells.

To further investigate the inhibition of proliferation caused by Ezh2 siRNA treatment, U87 cells were individually transfected with 55 nM Ezh2 siRNA or a non-targeting siRNA as a negative control, and the cell cycle was analysed by flow cytometry. We found that upon Ezh2 siRNA treatment, the percentage of cells in the G0/G1 phase was significantly higher than the percentage found in the mock-transfected cells or the cells treated with the non-targeting siRNA (Fig. 6A). Additionally, we found that the downregulation of Ezh2 expression altered the levels of several cell cycle proteins, including cyclin D1, CDK4, and CDK6, all of which have roles in the G0/G1 phase. Western blot analysis revealed that compared with the mock-transfected cells or the cells transfected with the non-targeting siRNA, the protein expression levels of cyclin D1, CDK4, and CDK6 were significantly reduced in the cells transfected with the Ezh2 siRNA (Fig. 6B and C). These results suggest that the inhibition of proliferation caused by the downregulation of Ezh2 expression in the U87 cells may be mediated by the reduced expression levels of cyclin D1, CDK4, and CDK6, which result in an arrest of the cell cycle in the G0/G1 phase.