Tissues samples
Glioma patients (n = 45, 34 male and 11 females, median age = 54 years, range, 40–64 years) were included into this study, who underwent surgery at the Liaoning Cancer Hospital & Institute (Liaoning, China) after histopathological confirmation (WHO criteria). These patients were treated with radiotherapy for over 6 months and classified into radiosensitive (n = 25) and radioresistant (n = 20) groups based on radiosensitivity index (RSI) values (RSI index > 0.5 means radioresistant) as previously reported [23]. The tumor tissues were sampled from contrast enhancing regions identified by intraoperative neuronavigation (Cranial Map Neuronavigation Cart 2, Stryker, Freiburg, Germany) during resection, which were then immediately frozen in liquid nitrogen and stored at − 80 °C until analysis. The present study was approved by the ethics committee of the Liaoning Cancer Hospital & Institute (Protocol 102563, Liaoning, China) and obtained the written informed consent from all patients.
Cell culture and irradiation
Human glioma cell lines, U251 and U87 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, MA, USA) supplemented with 10% FBS (Gibco, NY, USA) in a humidified incubator containing 5% CO2 at 37 °C. For IR, U251 and U87 cells were seeded in 100-mm culture dishes at a density of 1 × 106 cells and cultured for 24 h, followed by exposed to laboratory X-ray generated by an irradiation apparatus (2100 C/D, VARIAN, CA, USA) with 0. 2. 4, 6 and 8 Gy at room temperature.
Cell transfection
MiR-153-3p mimics (miR-153-3p) and its scrambled negative control (miR-NC) were synthesized by Genepharma Company (Shanghai, China). The full length sequences of BCL2 were amplified by PCR and sub-cloned into pcDNA3.1 vector (Invitrogen) to generate pcDNA-BCL2 (BCL2) plasmids. Before transfection, U251 and U87 cells were trypsinised and seeded onto 6-well plates to reach 70% cell confluence. Then, the transfection of miR-153-3p, miR-NC, BCL2 vector and empty vector was carried out at a final concentration of 100 nM using Lipofectamine 2000 (Invitrogen, California, USA) following the manufacturer’s procedure. The transfected cells were incubated for a further 48 h prior to IR.
RNA extraction and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from tissues and cells using TRIzol (Invitrogen, Carlsbad, CA, USA). For the detection of miR-153-3p expression, cDNA synthesis was performed using TaqMan MicroRNA Array kit (Applied Biosystems, CA, USA). The relative expression level of miR-153-3p was determined by TaqMan Universal Master Mix II (Applied Biosystems) with U6 as the internal reference. For the detection of BCL2 mRNA expression, cDNA synthesis was performed by PrimeScript RT reagent kit (TaKaRa BIO, Shiga, Japan) with GAPDH as the normalization. The expression of BCL2 was measured using SYBR Premix Ex Taq™ II (TaKaRa BIO) reagent with GAPDH as the normalization. All qRT-PCR was performed in triplicate on ABI Prism 7900HT Real-Time PCR System (Thermo Fisher Scientific, Inc.). All primers were purchased from Guangzhou RiboBio Co., Ltd and listed as follows: miR-153-3p (NM_138303), 5′-ACACTCCAGCTGGGTTGCATAGTCACAAA-3′ (forward) and 5′-CAGTGCGTGTCGTGGAGT-3′ (reverse); U6 (NR_138085), 5′-CCCTTCGGGGACATCCGATA-3′ (forward) and 5′-TTTGTGCGTGTCATCCTTGC-3′ (reverse); BCL2 (NM_000657), 5′-GAACTGGGGGAGGATTGTGG-3′ (forward) and 5′-GCCGGTTCAGGTACTCAGTC-3′ (reverse); GAPDH (NM_002046), 5′-GGTGAAGGTCGGAGTCAACG-3′ (forward) and 5′-GCATCGCCCCACTTGATTTT-3′ (reverse). Data analysis was performed using the 2−ΔΔCt method.
Cell viability assay
After IR treatment, transfected cells were seeded in 96-well plates at a density of 4000 cells per well. To assess cell viability, the MTT assay was performed according to the manufacture’s instruction. In brief, each well was added 10 μl of 5 mg/ml MTT reagent in PBS, and the cells were incubated for 4 h at 37 °C. After removing the supernatant containing MTT solution, 100 μl of dimethyl sulfoxide (DMSO, Sigma-Aldrich) was added to dissolve the formazan. The optical density (OD) values at 595 nm were determined using a microplate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA).
Colony formation assay
Colony formation assay was performed to evaluate the survival fraction of cells with different treatments. Briefly, transfected cells were seeded in 6-well plates and irradiated at 4 Gy, followed by incubation for 10 days. The naturally formed colonies with more than 50 cells were fixed with 75% ethanol and stained with 0.5% (w/v) crystal violet solution (Sigma-Aldrich). The number of colonies was counted under a microscope (Leica Microsystems, Wetzlar, Germany).
Flow cytometry analysis of cell apoptosis
Cell apoptosis was detected by the Annexin V/PI Apoptosis Detection Kit (KeyGEN Biotech, Nanjing, China) according to the manufacture’s instruction. In brief, approximately 5 × 105 transfected cells irradiated with a dose of 4 Gy was harvested and re-suspended in 1 × Binding Buffer. Subsequently, cells were incubated with 5 μl Annexin V-FITC at room temperature for 15 min, followed by incubation with 10 μl propidium iodide (PI, 10 mg/ml) in the dark at room temperature for 5 min. Finally, the samples were analyzed using a FACScan flow cytometer (Becton–Dickinson; San Jose, CA, USA). Each sample was performed in triplicate.
Caspase-3 activity assay
The activity of caspase-3 activity was detected using Caspase-3 assay kit (Beyotime Institute of Biotechnology) according to the protocol of manufacturer. Briefly, transfected cells after 4 Gy IR were collected and added into 10 μl of reaction buffer with the kit, followed by incubation for 2 h at 37 °C. Then the optical density value was measured at the wavelength of 400 nm. The relative caspase-3 activity of each sample was calculated as the absorbance of the well by that of the control group.
Dual luciferase activity assay
Potential targets of miR-153-3p were searched with TargetScan (http://www.targetscan.org/), revealing that BCL2 is a potential target of miR-153-3p. Subsequently, dual luciferase activity assay was performed to verify whether BCL2 as a target of miR-153-3p. In brief, the human BCL2 3′UTR containing a putative miR-153-3p-binding site was cloned into the luciferase reporter vector psiCHECK-2 vector (Promega, Biotech Co., Ltd) to construct the wild-type-psiCHECK-2-BCL2-WT plasmid. The mutant-type luciferase vector psiCHECK-2-BCL2-MUT, harboring the mutant miR-153-3p binding site was also constructed. For cell transfection, HEK-293T cells were seeded in triplicate in 24-well plates and transfected with 100 ng of empty vector, psiCHECK-2-BCL2-WT, or psiCHECK-2-BCL2-MUT together with miR-153-3p or miR-NC using Lipofectamine 2000 (Invitrogen), followed by the detection of luciferase activity with a Dual Luciferase Reporter Assay kit (Promega). Each sample was performed in triplicate.
Western blotting
Total proteins were extracted using RIPA buffer (Beyotime, Shanghai, China) and quantified using a BCA protein assay kit (Pierce). Equal amounts of proteins (50 g) were separated on a 10% sodium dodecyl sulfate–polyacrylamide gel and transferred to polyvinylidene fluoride membranes (PVDF, Millipore, Bedford, MA, USA). Then, the membranes were blocked with 5% nonfat milk in PBS-Tween-20 for 1 h and incubated with primary antibodies against BCL2 (1: 500, Abcam) or GAPDH (1: 5000, Cell Signaling Technology) at 4 °C overnight. Next, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1: 5000, Cell Signaling Technology) at room temperature for 2 h. The protein signals were visualized using an enhanced chemiluminescence (ECL) detection reagent (Pierce).
Tumor formation in nude mice
The procedures of the animal experiments were approved by the Committee on the Use and Care of Animals of The First Hospital of Jilin University (Changchun, China). A total of 2 × 106 U251 cells transfected with equivalent amount of miR-153-3p or miR-NC were subcutaneously injected into the left flank of 4- to 6-week-old BALB/c nude mice (n = 4 per group). The mice were exposed to a dose of 8 Gy when the tumor volume was about 100 mm3. Every 5 days after injection, the tumor volumes were measured with calipers and calculated according to the formula: tumor volume (mm3) = 0.5 × length × width2. On day 35 after injection, nude mice were sacrificed and the average tumor weight was measured. Meanwhile, xenograft tumors were rapidly removed and the expression of miR-153-3p or BCL2 was determined as the above described method. All animal experiments were conducted in strict accordance with the principles and procedures approved by the Committee on the Ethics of Animal Experiments of Liaoning Cancer Hospital & Institute (Liaoning, China).
TUNEL staining
TUNEL assay was performed for detecting the apoptotic cells in xenograft tissues. In brief, paraffin cross-sectios (5-μm thick) of xenograft tissues were deparaffinized and rehydrated, followed by TUNEL staining using TUNEL Bright-Red Apoptosis Detection Kit (Vazyme) according to the manufacturer’s instructions. TUNEL-positive cells were observed and compared under fluorescence microscopy (DMI4000B, Leica).
Statistical analysis
Statistical analysis was carried out using SPSS 13.0 statistical software and the data were expressed as mean ± standard error (SD) from at least three separate experiments. Student’s t-test and one-way analysis of variance (ANOVA) were used to analyses the significant differences between the two groups and among the groups, respectively. Pearson’s correlation test was used to analyze the linear correlation between miR-153-3p and BCL2 expression. The values of p < 0.05 were considered to be statistically significant.