Tissue samples collection
Glioma tumor tissues and adjacent non-neoplastic tissues were collected from patients who were diagnosed with glioma by a pathologist, and none of them received any anticancer therapies, such as radiotherapy, chemotherapy or molecular targeted therapy, before underwent surgery at the Huaihe Hospital of Henan University (Kaifeng, China). All samples were stored in a freezer at −80 °C. All participants have received informed consent.
The human normal glial cell lines of HEB and the human glioma U87 cell lines were purchased from American Type Culture Collection. The cells were maintained in Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS, Hyclone) and antibiotics (100 μg/ml of streptomycin and 100 U/ml of penicillin) in a humidified incubator at 37 °C under 5% CO2.
A pcDNA3.1 overexpression vector constructed using a full-length cDNA sequence of VEGF or DNMT1 subjected to competent cell transformation. Then, the pcDNA3.1 overexpression vector was extracted using a commercial kit and identified via sequencing. MiR-148a-3p mimic and the short interfering RNA against DNMT1 (DNMT1 siRNA) or VEGF (VEGF siRNA) were designed, synthesized, and validated. The U87 cells were plated onto 24-well plates at a density of 3 × 104 cells/well. When U87 cells reached 60%-80% confluence, the vectors or siRNAs were transfected into U87 cells by using the Lipofectamine 2000 (Invitrogen) based on the manufacturer’s instruction.
Dual-luciferase reporter gene assay
MicroRNA.org (http://www.targetscan.org/) predicted the targeting relationship between miR-148a-3p and DNMT1 and was verified by the dual-luciferase reporter gene. The cells at the logarithmic growth phase were seeded into 24-well plates in triplicate. When the cell density reached 70%, the cells were transfected with Lipofectamine 2000, and the DNMT1-3'UTR-wild type, DNMT1-3' UTR-mutant and miRNA-148a-3p plasmids were mixed and co-transfected into cells. After 6 h of culture, culture in new medium containing 10% fetal bovine serum (FBS) for 48 h. After 48 h transfected, the medium was aspirated, and the DNA samples were washed twice with PBS and mixed with 100 μL of passive lysis buffer (PLB) at room temperature. Fifteen minutes after, cell lysates were collected. After pre-reading for 2 seconds, 20 μL of Luciferase Assay Reagent II Stop & Glo® was added to 100 μL of each sample, and the bioluminescence value was read for 10 seconds.
Western blot analysis
Total protein was extracted from cells using ice-cold RIPA buffer (Sigma, St. Louis, MO, USA) according to the manufacturer’s protocol. Proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene difluoride membranes (PVDF) (Sigma, St. Louis, MO, USA). The PVDF membranes were blocked with 5% skim milk in Tris-buffered saline with Tween (TBST) at room temperature for 2 h. Then, the membranes were placed into the primary antibody solution for incubation at 4 °C with gentle shaking overnight. The membranes were washed 3 times/5 min with TBST and placed into the secondary antibody solution for incubation at room temperature for 1 h and rewashed 3 times/5 min with TBST. The band development of protein was visualized using the ECL chemiluminescence reagent at a chemiluminescent detection system, and the grey values of target proteins were performed image analysis using Image J software. Antibodies used in this study are as follows: DNMT1 (1:1000), VEGF (1:1000), PI3K (1:1000), Akt (1:1000), Enos (1:1000), GAPDH (1:1000) and β-actin (1:1000). (all primary antibodies were purchased from Abcam, Cambridge, MA, USA).
Quantitative real-time polymerase chain reaction (RT-qPCR)
Total RNA isolation from cells and tissues was performed using the Trizol reagent (Invitrogen, Carlsbad, CA, U.S.A.) according to the manufacturer’s instructions. The RNA concentration was detected by NanoDrop2000 (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA) and stored at -80°C for further use. According to gene sequences published in GenBank's database, primers were designed using Primer 5.0 design software and synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). Reverse transcription was performed with approximately 0.5 μg of total RNA into cDNA with the Superscript™ reverse transcription system (Takara, Dalian, Japan). Aliquots of cDNA were used for PCR amplification with primer-specific for miR-148a-3p, DNMT1, VEGF and β-actin. RT-qPCR reaction was performed as follows: 95°C for 3 min, 35 cycles of denaturation at 95°C for 35 sec, 58°C for 45 sec, and 72°C for 30 sec and 10 min at 72°C using SYBR Green PCR master mix reagents (Takara, Dalian, Japan) in the ABI StepOne Plus Real-time PCR system. The relative quantification of mRNA expression levels was calculated using the 2−ΔΔCT method.
Bisulfite-sequencing PCR (BSP)
A total of 500 ng genomic DNA was extracted from the tissues and cells and subjected to bisulfite conversion using the EZ DNA Methylation-Gold kit (Zymo Research, CA, USA). The VEGF promoter was amplified by PCR with Phusion U Hot Start DNA Polymerase (Thermo Fisher Scientific Inc.). Sanger sequencing was performed on the PCR products. Five single molecules were sequenced for each sample.
Methylation-specific polymerase chain reaction (MSP)
MSP was performed to detect methylated (M) or unmethylated (U) alleles of the VEGF promoter CpG island. The PCR procedure was performed using Phusion U Hot Start DNA polymerase (Thermo Fisher Scientific Inc.): 98 °C for 2 min, followed by 35 cycles: 98 °C for 15 s, 58 °C for 30 s and 72 °C for 40 s.
Cell proliferation assay
After transfection, the cells were cultured in 96-well plates for 24 h, and the cell proliferation was detected by cell counting kit-8 (CCK-8) (Dojindo Laboratories, Kumamoto, Japan). According to the manufacturer's instructions, 10 μL of CCK-8 reagent was added to each well of cell plates. Then, the cells were incubated at 37 °C for 3 h. The absorbance was detected at 450 nm in each well using a multifunctional microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). The above actions were repeated 3 times.
Flow cytometry assays
The cell apoptosis was detected using a FACS Aria Sorter (Becton Dickinson, San Jose, CA, USA). The transfected cells were collected, washed three times with PBS and resuspended in 500 μL buffer solution (PBS with calcium), mixed with 5 μL of Annexin-V-fluorescein isothiocyanate (FITC) and 2.5 μL of propidium iodide (PI), followed by incubation at 4°C for 30 min. After 400 μL staining buffer was added, the cells were immediately calculated and analyzed with flow cytometry (Becton, Dickinson and Company, Franklin Lakes, New Jersey, USA). Annexin V-positive cells were apoptotic cells, and the right upper quadrant and the right lower quadrant presented the apoptotic cells. Each experiment was conducted 3 times.
Transwell invasion assay
The invasive ability of pcDNA 3.1 transfected the cells was assessed using BD FalconTM cell culture inserts coated with BD MatrigelTM Basement Membrane Matrix (BD Biosciences, Bedford, MA, USA). Transfected cells were resuspended at a density of 5 × 105 cells in serum-free medium and inoculated into the upper chamber of the assay system, while 0.5 mL of DMEM containing 10% FBS was added to the lower chamber. Following incubation for 48 h, the invaded cells were washed 3 times with PBS, fixed with 4 % paraformaldehyde solution (Affymetrix, Cleveland, OH, USA) for 15 min and stained with methyl violet (0.01% v/v; Guidechem, Shanghai, China) for 30 min. The numbers of invaded cells were then counted under a light microscope (IX73-F22FL/PH; Olympus America, Inc., Melville, NY, USA). Each experiment was conducted 3 times.
Determination of NO concentration
The cells were cultured for 48 h in colorless DMEM. Nitric oxide (NO) quantified by the accumulation of nitrite (a stable breakdown product of nitric oxide) in the medium. 500 μL of the cell culture supernatant was taken as a sample and incubated with an equal volume (500 μL) of Griess reagent for 10 min at room temperature, and the absorbance was measured by spectrophotometry at 560 nm. The sodium nitrite (NaNO2) dissolved in colorless DMEM was used as a standard. According to the manufacturer's instructions, the amount of nitrite (a stable metabolite of NO) was assessed in the cell culture supernatant using a Griess reagent kit (Promega, Madison, WI).
The data are shown as the mean ± standard error of the mean (SEM) of three or more independent experiments were performed. Statistical differences were evaluated using a one-way ANOVA, two-way ANOVA and the t-test. P-values <0.05 were considered to indicate statistically significant differences. Statistics were performed with SPSS Statistics version 20.0 software.