Patients and cell lines
Patient tumor samples were collected, with written informed consent, from the Department of Neurosurgery/neuro-oncology, Sun Yat-sen University Cancer Center (SYSUCC) between 2001 and 2008. Primary tumor cultures were derived from the tissue samples using the method described by Brassesco et al. . Human GBM cell line U87 (maintained in SYSUCC) were obtained from American Type Culture Collection. All cells were maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Hyclone) and 1% penicillin–streptomycin (Gibco) at 37°C in a humidified incubator with 5% CO2. The Ethics Review Board of SYSUCC approved this study.
Primary cell culture
Fresh tumor samples (verified from frozen sections), aseptically collected in the operating room, were minced with scissors in a petri dish. The tumor pieces were then disaggregated for 4 h at 37°C in 0.5% type IV collagenase (Sigma-Aldrich, St Louis, MO, USA) in F10 medium (Life Technologies, Carlsbad, CA, US). Then, the cells were pelleted and resuspended in the medium with 15% FBS. Cells were used for experiments after being cultured for 1 week.
RNA extraction and real-time PCR analysis
RNAs from 90 glioma samples and 4 normal brain tissues (normal adjacent tissues) were extracted with Trizol reagent (Invitrogen, USA) accordingly. RNA concentration and purity were measured using the NanoDrop ND-1000 Spectrophotometer (Thermo Fisher). One μg of total RNA was used for cDNA synthesis by the First Strand cDNA Synthesis Kit (Thermo-scientific, PA, USA). Real-time PCR reaction was performed as follows: 94°C 4 min for hot start, and then 94°C for 30 sec, 50°C for 30 sec, 72°C for 40 sec, for 40 thermal cyclesusing SYBR Premix Ex Taq II kit (TaKaRa, Dalian, China) on an ABI 7900HT instrument (ABI, NY, USA). The primers for miR-181b and U6 endogenous controls were purchased from RiboBio (Guangzhou, China). Primers for the MDM2 and GAPDH controls were from Invitrogen. All reactions were performed in triplicates. Relative gene expression was calculated using the 2-ΔΔCT method.
Cells (primary cultured cells and U87 cell line) were seeded onto 96-well plates at 2,000 cells per well in triplicates. The next day, cells were treated with teniposide at a concentration gradient of 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.39, 0.19, and 0 μg/ml. Cell viability was measured after 72 h using the Cell Counting Kit 8(CCK8, Dojindo, Japan) according to the manufacturer’s instructions. IC50 for teniposide was calculated from the dose-dependent curve.
miR-181b lentiviral transfection
The effect of miR-181b on cell sensitivity to teniposide was evaluated by constructing stable miR-181b-expressing glioma cells. The lenti-miR-181b and the corresponding empty vector were purchased from GenePharma (Shanghai, China). U87 was selected due to its low basal level of miR-181b. After being incubated with the virus for 72 h, cells were selected by medium containing 2 μg/ml puromycin for about 1 week.
Dual luciferase reporter assay
The plasmids psiCHECK-wtMDM2 and psiCHECK-mutMDM23’UTRs (carrying a mutational miR-181b binding site) were purchased from Land (Guangzhou, China). In brief, the full length MDM2 and empty psiCHECK vector were digested by same restriction endonuclease, followed with ligation, transformation, and then confirmed by both digestion and sequencing. The mutant MDM2 sequence was generated by mutagenesis PCR reaction from psiCHECK-wtMDM2. Then similar procedures (endonuclease digestion, ligation, transformation and confirmation) were performed to get psiCHECK-mutMDM2. 293 T cells were seeded onto 24-well plates. After overnight incubation, they were co-transfected with 50 nM miR-181b mimic or non-relevant control (NC), together with 0.5 μg reporter vector containing either wtMDM2 3’UTR or mutMDM23’UTR. Cells were harvested 48 h after transfection. The renilla and firefly luciferase activity were determined byDual Luciferase Assay Kit (Promega) following manufacturer’s instructions. Data were presented as mean ratio of the renilla/firefly luciferase activity obtained from at least three independent experiments .
Western blot analysis
Cells were washed twice with ice-cold PBS and lysed in RIPA buffer containing 1 mM PMSF (Beyotime, Japan) on ice . Lysates were centrifuged at 12,000 × g for 10 min at 4°C, and supernatants were collected. Equal amounts of proteins (20 μg) were fractionated by SDS-PAGEand transferred onto nitrocellulose membranes (Millipore, MA, USA). After being blocked in 5% nonfat milk at room temperature for 1 h, membranes were probed with primary antibodies at 4°C overnight. The next day, secondary antibodies were incubated for 1 h at room temperature and visualized using enhanced chemiluminescence (Millipore, MA, USA). The following antibodies were used: mouse anti-β-actin (1:1000, Santa Cruz, Dallas, TX, USA), rabbit anti-MDM2 (1:5000, Abcam, Cambridge, MA, USA), anti-phospho-MDM2 (1:1000, Cell signaling technologies, Boston, MA, USA).
The correlation between miR-181b expression and patient survival was analyzed by the Kaplan-Meier survival curve method. The correlation between miR-181b and glioma sensitivity to teniposide was determined by Spearman’s correlation coefficient. Differences between groups were calculated using paired t-test or one-way ANOVA. p < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS, version 16.0.