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 established by WHO in 2016. Three cases were grade I tumors, 5 grade II, 3 grade III and 4 grade IV（Supplementary Table-1）. 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 differences of the two groups' gender, age have no statistical significance(P>0.05), and the materials have good comparability. 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-5p mimic, scrambled miRNA (miR-NC) and the pcDNA3.1 PGC-1α overexpression plasmid were obtained from Guangzhou Ruibo Bio. Antibodies against PGC-1α, cyclin D1, cyclin D2, Cdk4 and β-actin were from Abcam, and the horseradish peroxidase (HRP)-labeled IgG secondary antibody from Guangzhou Jingcai. QuickChange Site-Directed Mutagenesis kit was purchased from Stratagene. Dual-Luciferase Reporter Assay kit and pmirGLO vector were 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. Cell transfection was performed as previously described[7, 9]. Briefly, 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- PGC-1α or 50nM pcDNA3.1-miR-133a-5p 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-5p 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. In this experiment, the seventh generation of miRCURYTMLNA hybrid chip(v.18.0) (Exiqon) was used to test the samples, containing 3100 species of probe. For the specific probe ID, please refer to http://www.kangchen.com.cn/support/supportmain.asp? Id = 21.
TCGA dataset with patient information
R2.15.3 Epicalc fuction package was used to download and preprocess the expression of miR-133a-5p miRNA SeqV2 data and the corresponding pathological data of the glioma data set from TCGA database (https://tcga-data.nci.nih.gov/tcga/). Expression of miR-133a-5p and survival analysis of glioma patients were as previously described[7, 9]. Briefly, the level 3 data of qualified miR-133a-5p expression with clinical information of glioma patients were obtained from TCGA data portal. We obtained 725 samples, which included 518 glioma samples and 207 non-tumor brain samples. And there were qualified clinical information of 479 glioma patients corresponding to miR-133a-5p expression in samples. To avoid the impact of unrelated causes of death, the cases with less than 1-month overall survival and death from other diseases or accidents were excluded in this study. As a result, 463 patients fitted this criterion for overall survival analysis. The 50% of the sorted miR-133a-5p values was set as cut-off for low/high expression of miR-133a-5p.
Cell proliferation assay
The miR-133a-5p 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
A colony formation assay was performed as previously described. Briefly the miR-133a-5p 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.
Protein levels were determined by Western blots as previously described [7-9]. Briefly, 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 PGC-1α (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 reporter assay
To obtain the luciferase construct, the full-length 3’-UTR of PGC-1α (containing the putative biding sites for miR-133a-5p) was amplified by PCR and subcloned into the downstream of luciferase gene in the pmirGLO-3 basic vector and named pmirGLO-3/PGC-1α-3’UTR-WT(PGC-1α-WT). Site-directed mutagenesis of the miR-133a-5p target-site in the pmirGLO-3/PGC-1α-3’UTR-WT , which was generated by QuikChange Site-Directed kit was also subcloned into the downstream of luciferase gene in the pGL-3 basic vector and named pmirGLO-3/PGC-1α-3’UTR-MUT (PGC-1α-MUT). For luciferase reporter assays, the 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 and then co-transfected with 100ng of PGC-1α-WT or PGC-1α-MUT and 50 nM of miR-133a-5p mimic or miR-NC using Lipofectamine 3000. Forty-eight hours after transfection, cells were harvested and assayed with Dual-Luciferase Reporter Assay kit according to the manufacturer’s instructions.
Propidium iodide staining
Propidium iodide staining was performed as previously . Briefly, 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.
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-5p 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-5p 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-5p gene was calculated by the ΔΔCt method. The experiment was repeated thrice.
Statistical analysis was performed using SPSS 19.0, R-2.15.3 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. Kaplan-Meier method was used to draw survival curve and perform Log-rank tesk. P values< 0.05 were considered statistically significant.