Cell culture and RNA interference
Human glioma cell lines U251, T98G, SHG44, U87 and U373 were obtained from the Cell Bank of Chinese Academy of Sciences. They were cultured in a 5% CO2/95% humidified air atmosphere at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Hyclone). The siRNA targeting human Rab21 (siRab21, 5’-GGCCAGGATTTCAAATCCA-3′) and nonspecific siRNA (siNC) were purchased from GenePharma Company. siRNAs or siNC were inserted into U87 and T98G cells using Lipofactamine 2000 (Invitrogen) according to the manufacturer’s instructions. Assays were performed 48 h after transfection.
RNA extraction and quantitative real-timePCR
Total RNA was extracted from samples usingTrizol reagent (TaKaRa) and the concentration wasdetected with an ultraviolet spectrophotometer. PrimeScriptRT Reagent Kit with gDNA Eraser (TaKaRa) was used to obtain cDNA according tothe manufacturer’s protocols.
The total volumesfor PCR were 20 μl,containing 10 μl of 2 × SYBR Premix Ex TaqTM (TaKaRa), 0.50 μmol/l forward primer,0.50 μmol/l reverse primer and 0.2 ± 0.02 μg cDNA template. The reaction conditions were pre-denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s, annealing at 60 °C for 20 s and elongation at 72 °C for 20 s.
The mRNA levels of the aim genes were calculated using the ΔΔCt method. β-actin was used as the housekeeping gene. Three samples were detected and three technical replicates were measured for all groups.
Western blotting
Protein expression levels were analyzed via western blot. Cells were washed twice with PBS and lysed with RIPA buffer. The protein concentration was detected using a BCA Kit (Beyotime). Equal amounts of protein (30 μg) were separated using 10% SDS-PAGE gel and transferred onto PVDF membranes (Millipore). After blocking with 5% skim milk for 2 h at room temperature, the membranes were incubated with primary antibodies overnight at 4 °C.
The specific primary antibodies were rabbit anti-Rab21 (Abcam; 1:1000 dilution), rabbit anti-Caspase7 (Abcam; 1:500 dilution), rabbit anti-Bim (Abcam; 1:500 dilution), rabbit anti-Bax (Santa Cruz Biotechnology, 1:200 dilution) and mouse anti-actin (Cell Signaling Technology; 1:1500 dilution).
The membranes were then washed with Tris-buffered and incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (Beyotime; 1:1000 dilution) for 1 h at room temperature. Immunoreactivity was visualized using a Millipore Enhanced Chemiluminescence system. Membranes were scanned with a Bio-rad Gel Doz EZ imager.
Cell proliferation assay
Cell proliferation was evaluated using the MTT colorimetric method (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; Sigma-Aldrich).The cells were placed in 96-well plates at a concentration of 1 × 104/well and transfected with siRab21 or siNC using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction. MTT (20 μl) was added into each well and incubated for 2–4 h at 37 °C (at 0, 24, 48 and 72 h post-transfection). Once the purple precipitate was visible, 150 μl isopropanol and 0.04 mol/l HCl were added and the cells incubated at room temperature in the dark for 2 h. The absorbance was recorded at 570 nm. The full procedure was repeated 3 times.
Cell cycle assay
At 48 hpost-transfection, the cells were trypsinized, washed three times with PBS, collected, and fixed with 70% ice-cold ethanol overnight at 4 °C. Then propidium iodide (PI) was added for staining, and the cells were incubated for 30 min at 4 °C in the dark. Finally, flow cytometry (BD FACScaliber) was used to evaluate the percentages of cells in each phase, including G0/G1 phase, S phase and G2/M phase.
Cell apoptosis assay
Apoptosis was analyzed with a BD AnnexinV-FITC/PI flow cytometry kit according to the manufacturer’s instructions. At 48 h post-transfection, the cells were collected and washed with ice-cold PBS three times and resuspended in 200 μl of binding buffer at a concentration of 1 × 106 cells/ml. 10 μl Annexin V-FITC and 10 μl PI were added and the cells were incubated for 30 min at 4 °C in the dark. Finally, 300 μl binding buffer was added and analyzed by flow cytometry (Beckman Coulter, Cytomics FC 500) within 1 h.
Statistical analysis
The SPSS 18.0 software package was used for the statistical analyses. Each experiment was repeated at least three times and all data are expressed asmeans ± SD. Dunnettʼs one-way analysis of variance (ANOVA) was used to evaluate the difference in the results. p < 0.05 was considered statistically significant.
