Tissue samples
This study was approved by the Ethics Committee of Second Affiliated Hospital of Shenzhen University (SSZU20180307). All samples and data were collected after obtaining the statement on informed consent from the glioma patients or the legally authorized representatives of healthy controls with brain trauma. Fifteen surgically excised glioma tissue samples were collected at our hospital from August 2018 to August 2019, and glioma was confirmed by clinical and pathological examination. Eight of the 15 samples were obtained from male patients and 7 from female patients, with an average age of 52.3 ± 6.9 years. The histological diagnosis of glioma was based on the Central Nervous System Tumor Grading Criteria[31] established by WHO in 2016. Three cases were grade I tumors, 5 grade II, 3 grade III and 4 grade IV. Furthermore, brain tissues were removed from 15 age-matched healthy controls with brain trauma (9 males and 6 females, average age 52.8 ± 7.4 years) during intracranial decompression. The inclusion criteria for the patients were: 1) primary tumor occurrence, and 2) lack of radiotherapy, chemotherapy or any treatment before surgery. The tissue specimens were flash frozen in liquid nitrogen, and stored at -80°C.
Cell lines and main reagents
The human normal glial cell line HEB and glioma cell lines U251, U87, T98-G and A172 were purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DMEM/HG, fetal bovine serum (FBS), Opti-MEM and 0.25% trypsin containing 0.02% EDTA were purchased from Gibco, MTT kit, 5-aza-2'-deoxycytosine (AZA) and histone deacetylase inhibitor (TSA) from Sigma, Trizol, reverse transcription kit from Thermo, SYBR Green Real-time PCR kit from Shanghai Solarbio Bioscience & Technology, QIAamp DNA Mini kit and EpiTect Bisulfite kit from Qiagen, Lipofectamine 3000 from Invitrogen, and the cell cycle assay kit from BD. The pcDNA3.1-miR-133a mimic, scrambled miRNA (miR-NC) and the pcDNA3.1-PPARγ overexpression plasmid were obtained from Guangzhou Ruibo Bio. Antibodies against PPARγ, cyclin D1, cyclin D2, CDK4 and β-actin were from Abcam, and the horseradish peroxidase (HRP)-labeled IgG secondary antibody from Guangzhou Jingcai. PPARγ wild type (WT) and mutant (MUT) luciferase reporter plasmids were purchased from Shanghai Jima and the luciferase assay kit from Promega. The PCR primers were synthesized by Shanghai Shenggong. Other reagents were from our laboratory and of analytical grade.
Cell culture and transfection
The cell lines were thawed, and cultured at 37°C under 5% CO2 in DMEM/HG containing 10% FBS. The cells were harvested by trypsin digestion once they were 70%-80% confluent, centrifuged at 800 rpm for 5 min at room temperature, and resuspended in DMEM/HG for further passaging. For the transfection of A172 and U251 cells, they were harvested at 80% confluency, re-suspended in Opti-MEM, and seeded in a 6-well plate at the density of 2 × 105 per well. Following overnight incubation, the cells were transfected with 100 ng pcDNA3.1-PPARγ or 50nM pcDNA3.1-miR-133a mimic and pcDNA3.1-miR-NC (using Lipofectamine 3000 according to the manufacturer’s instructions. Six hours later, the medium was replaced with DMEM/HG containing 10% FBS, and the cells were cultured for another 48h. The medium was replaced with complete DMEM/HG containing 2µg/ml puromycin, and the cells were cultured for 3 days. The transfected cells were re-plated and after 1-2 weeks, the resulting clones were expanded to establish stable miR-133a mimic and miR-NC cell lines.
miRNA microarray analysis
Three tissue samples each from glioma patients and healthy controls were sent to Shanghai Kangcheng Biotechnology Co. Ltd. for miRNA microarray analysis. Briefly, total RNA was extracted from the tissues using Trizol reagent, and purified with a miRNA Mini kit according to the instructions. The purity and concentration of the RNA samples were analyzed using a spectrophotometer, and 1µg RNA per sample was labeled using a Hy3/Hy5 Power Labeling kit according to the instructions. The labeled RNA was then hybridized with a miRCURYtm LNA Array, and the original signal intensity of the chip was tested using a GenePix 4000B chip scanner. Using intensity (int) > 50 as the normalization factor, the differences in miRNA expression levels between the glioma and normal tissue samples were analyzed by inter-chip standardization, intra-chip standardization, expression difference comparison, statistical significance test, and cluster analysis.
Cell proliferation assay
The miR-133a mimic and miR-NC-transfected cells were seeded into 96-well plates at the density of 2×104 per well in 200µl complete DMEM/HG medium. After culturing for 12, 24, 36 and 48h, 20µl MTT reagent (5mg/ml) was added to each well, and the cells were incubated further for 4h. The culture medium was removed, and 150µl dimethyl sulfoxide (DMSO) was added per well to solubilize the formazan crystals. After shaking for 10 min at room temperature, the absorbance value (OD490) of each well was measured at 490 nm. Each time point per group was tested in five replicate wells, and the mean values were calculated.
Colony formation assay
The miR-133a and miR-NC-transfected cells were seeded into 6-well plates at the density of 1×103 cells/well. The cells were cultured for 8 days, and the medium was changed every 3 days. The resulting colonies were fixed with 3.7% paraformaldehyde for 5 min, stained with 0.05% crystal violet for 20 min at room temperature, and gently washed with double distilled water five times. The number of colonies were counted under a white light microscope.
Western blotting
The suitably transfected cells were washed thrice with cold PBS at 4°C, lysed with RIPA cell lysis buffer supplemented with a protease inhibitor, and centrifuged at 4°C and 12,000 rpm for 20 min. The supernatants were aspirated and the protein concentration was determined by the BCA method. Equal amounts of protein per sample (30µg) were mixed with 5× loading buffer at the ratio of 4:1, and denatured by boiling for 10 min. The protein samples were resolved by SDS-PAGE, and the bands were transferred to PVDF membranes by the wet transfer method. The membranes were blocked with 5% skim milk at room temperature for 2h, and incubated overnight with primary antibodies against PPARγ (1:500), cyclin D1 (1:500), cyclin D2 (1:500) or CDK4 (1:500), and β-actin (1:1000) at 4°C on a shaker. After washing thrice with TBST buffer, the membranes were incubated with a horseradish-labeled secondary antibody (1:2,000) for 1h at room temperature, followed by three more washes with TBST. The blots were then developed using an ECL solution and photographed on a gel imager. The Image J software was used to measure the gray value of each band, and the ratio of the intensities of the target proteins to that of the internal control β-actin was calculated. The experiment was repeated thrice.
Dual luciferase gene reporter assay
The online database TargetScan was screened for the putative target genes of miR-133a. To validate PPARγ as a target, the dual luciferase reporter assay was performed. Briefly, A172 and U251 cells were harvested in the logarithmic growth phase and seeded in 96-well plates at the density of 2×104 cells/well. Following overnight culture, the cells were co-transfected with luciferase reporter plasmids harboring wild type (WT) or mutated PPARγ promoter sequences, and miR-133a mimic or miR-NC using Lipofectamine 3000. Each group was tested in five replicates. After 48 hours, the fluorescence intensity of firefly luciferin and Renilla fluorescein was detected according to the instructions of dual luciferase assay kit.
Propidium iodide staining
The suitably transfected A172 and U251 cells were gently washed with cold PBS, harvested, and centrifuged at 300 rpm for 5 min at room temperature. The supernatant was removed, and the cells were re-suspended in 500µl PBS. Ice-cold 70% alcohol (3.5 ml) was added immediately, and the cells were thoroughly pipetted and fixed overnight at 4°C. After washing thrice with PBS, the cells were stained with 500μl PI/RNase staining solution provided in the cell cycle flow detection kit for 30 min at 4°C in the dark. The cell cycle distribution was analyzed by flow cytometry. The experiment was repeated thrice.
Drug treatment
HEB, A172 and U251 cells were harvested and seeded in a 6-well plate at the density of 1×105/well, and cultured till 70%-80% confluency. The medium was replaced with DMEM/HG containing 1μM AZA or 300nM TSA, and the cells were cultured for 72 h. The control cells were cultured in DMEM/HG containing 1μM DMSO.
Methylation-specific quantitative PCR (MSP)
The CpG islands in the miR-133a gene were predicted using the website http://cpgislands.usc.edu, and one CpG island was detected in its promoter region. Genomic DNA was extracted from the A172 and U251 cells using a DNA extraction kit as per the manufacturer’s instructions, and the purity and concentration were determined using an ultraviolet spectrophotometer. The DNA was modified with bisulfite using the EpiTect Bisulfite kit according to the manufacturer’s instructions, and the methylated and unmethylated miR-133a were amplified using the following primers: methylated - forward 5'-GGTTGTTTGTTTTTTGGTTCG-3' and reverse 5'-ATCCTAAAACTACCCAAAATCGTA-3'; unmethylated - forward 5'-GGGATGAGGATTAGGATTTT-3' and reverse 5'-CAAACAAAACACAATAAAAACAAACA-3'. The PCR cycling conditions were: pre-denaturation at 94°C (3 min), followed by 35 cycles of denaturation at 94°C (30s), demethylation at 53°C (30s) and extension at 72°C (90s), and final extension at 94°C for 5 min. Generation of an amplified product with either methylated or unmethylated primers respectively indicated presence and absence of methylated sequences in the genome. Generation of amplified products with both primer pairs implied partial methylation. The methylation level of miR-133a gene was calculated by the ΔΔCt method. The experiment was repeated thrice.
Statistical analysis
Statistical analysis was performed using SPSS 19.0 and GraphPad Prism 5.0. The data were expressed as (X±S). One-way ANOVA was used for inter-group comparison, and independent-sample t test for comparing two groups. P values< 0.05 were considered statistically significant.