Cell lines and materials
Normal human astrocytes (NHAs) and glioma cell lines U251, A172 were purchased from Cell Resource Center of Shanghai and cultured in Dulbecco's Modified Eagle Medium (DMSO; Gibco, USA) supplemented with 10% fetal calf serum (FBS; Gibco, USA), 1% penicillin, 1% streptomycin at 37°C in 5% CO2. Curcumol and temozolomide (HY-N0104 and HY-17364, MCE, USA) were dissolved in DMSO and diluted to appropriate concentrations in culture medium immediately before use.
RNA isolation and qRT-PCR analysis
Total RNA of cells and tissues was isolated by using TRIzol reagent (Invitrogen, USA) and converted to cDNA by HiFi-MMLV cDNA Kit (Beijing ComWin BiotechCo., Ltd. Beijing, China). QRT-PCR was conducted by using UltraSYBR Mixture (Beijing ComWin Biotech Co., Ltd. Beijing, China). The primer sequences were illustrated as follows: FOXD2-AS1, F: 5¢-CTCACATCCGGCGGCT-3¢, R: 5¢-GGCTGTTCATGATATGTGCCA-3¢; CDKN1B, F: 5¢-GCCGCAACCAATGGATCTCCTC-3¢, R: 5¢-AGTCGCAGAGCCGTGAGCAA-3¢; EPHB3, F: 5¢-TGGGTAACATCTGAGTTGGCG-3¢, R: 5¢-TGGTATGTGCGGATGGGATTC-3¢; p21, F: 5¢- TGTGATGAAAGACGGCACAC-3¢, R: 5¢-CTTCCTTTGGGTATTGTTTGG-3¢; EZH2, F: 5¢- GGACCACAGTGTTACCAGCAT-3¢, R: 5¢-GTGGGGTCTTTATCCGCTCAG-3¢; β-actin, F: 5¢-GGCACCACACCTTCTACAAT-3¢, R: 5¢-GTGGTGGTGAAGCTGTAGCC-3¢. All the primers were synthesized by Nanjing GenScript Co. Ltd., China. The fold-change of each gene was calculated using the 2−ΔΔCt method.
Genome DNA extraction and Methylation-specific (MS) PCR
For DNA extraction from cells, Takara MiniBEST Universal Genomic DNA Kit (Takara, Shiga, Japan) was used, following the manufacturer’s directions. After bisulfite treatment by a DNA Methylation Kit (CoWin Biosciences, Beijing, China), 4 μL of the purified DNA was used to perform MS-PCR. Primers for the methylated MGMT were F: 5¢-TTTCGACGTTCGTAGGTTTTCGC-3¢, R: 5¢-GCACTCTTCCGAAAACGAAACG-3¢; for the un-methylated MGMT were F: 5¢-TTTGTGTTTTGATGTTTGTAGGTTTTTGT-3¢, R: 5¢-AACTCCACACTCTTCCAAAAACAAAACA-3¢. The PCR products were directly loaded onto 2.5% agarose gels, stained with ethidium bromide, and visualized with the aid of ultraviolet light. The density of each band was quantified using imaging analysis and the relative band density values were calculated as the ratio of methylated MGMT to that of methylated plus un-methylated MGMT [31]. The MS-PCR was performed in a Mastercycler Gradient (Eppendorf) under the following conditions: 95°C for 10 min; 35 cycles of 95°C for 30 sec, the annealing temperature 60°C for 30 sec, and 72°C for 30 sec; and a final extension of 10 min at 72°C.
Cell viability
Glioma cells were placed on 96-well plates at a density of 5 × 103/well and after overnight incubation treatment with curcumol for 48 hours. MTT solution (5 mg/mL) was added to each well and the plate was incubated for 4 h at 37℃. After removal of the medium, formazan crystals were dissolved in 150 μL of DMSO. The absorbance value was measured at 550 nm using a SpectraMaxM3 microplate reader (MolecularDevices, Sunnyvale, CA, USA).
Migration and invasion assays
Glioma cells were suspended in DMEM without FBS and seeded into the upper chambers of the transwell inserts (Corning, Corning, NY, USA). Afterwards, the DMEM added with 20% FBS was added to the lower chambers. The invasion assay was performed in the same way as the migration assay except that the membrane was pre-treated with matrigel (BD Biosciences, Bedford, MA, USA).The migrated or invaded cells through the member were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The stained cells were counted under microscope (ECLIPSE Ti-S, Nikon, Japan).
Colony formation assay
For colony formation assay, treated and untreated glioma cells were placed at a density of 500 cells per well into 6‐well plates and then cultured at 37°C for 2 weeks, stained with 5% crystal violet. Finally, colonies were photographed, and the number of colonies was counted manually.
Annexin V-FITC/PI apoptosis assay
Annexin V-FITC/PI staining Kit (NanJing KeyGen Biotech Co., Ltd., Nanjing, China) was utilized to detect the cell apoptotic rates. The treated and untreated cells were collected and resuspended in the binding buffer. After incubating in the dark for 15 min, the cells were analyzed using a Guava easyCyte 5 Flow Cytometer (EMD Millipore, Bellerica, MA, USA).
Neurosphere formation
Glioma cells (3× 104/well) were plated into ultra-low attached 6-well plates (Corning Costar Inc, Corning, NY, USA) and cultured in serum-free DMEM/F12 media (Gibco, USA) with 2% B27 supplement (Thermo Fisher Scientific, USA), 20 ng/mL EGF, and 10 ng/mL bFGF (PeproTech, Rocky Hill, NJ, USA). After 4–5 days, equal fresh media was added. Cells were incubated for 2 weeks, and spheres with diameter ³ 50 μm were counted.
Flow cytometric analysis (FCM)
Glioma cells (1 × 106/well) were washed twice with PBS supplemented with 0.5% BSA and 2 mM EDTA and incubated in diluted CD133-PE antibody solution (Invitrogen, USA) for 15 min. After washing the cells twice, the percentage of CD133+ cells was analyzed by Guava easyCyte 5 Flow Cytometer (EMD Millipore, Bellerica, MA, USA).
Immunofluorescence staining
For immunofluorescence staining neurospheres, the cells were fixed with 4% paraformaldehyde and incubated with antibodies against CD133 (18470-1-AP, Proteintech gruop, USA) and Nanog (ab62734, Abcam, USA) overnight at 4°C. Then, SABC (rabbit IgG)-FITC/ SABC (mouse IgG)-Cy3 Kit (Boster Bioengineering, Wuhan, China) was used. Nuclei were stained with 4′6-diamidino-2-phenylindole (DAPI). Fluorescence images were captured with fluorescent microscope (ECLIPSE Ti-S, Nikon, Japan).
Western blotting
Tissues and cells were lysed with RIPA (Boster Bioengineering, Wuhan, China) containing 1 mM phenylmethanesulfonyl fluoride (PMSF; Roche Molecular Biochemicals, Indianapolis, IN, USA). Protein samples were separated by 10%–12% SDS PAGE. Western blotting was performed based on a previous study [31]. The primary antibody to β-actin was purchased from Hua Bio (HuaAn Biotechnology Co., Ltd., Hangzhou, China), antibody to EphB3 was purchased from Invitrogen (Invitrogen, USA), the antibodies to MGMT, Nestin, SOX2, p21, CDKN1B were purchased from (Proteintech Group, Chicago, IL, USA), and the antibodies to EZH2 and Histone H3 (H3K27me3) were purchased from Abcam (Abcam, Cambridge, USA).
Generation of TMZ-resistant glioma cells
To develop a TMZ-resistant glioma cell line, U251 cells were cultured in media containing increasing concentrations of TMZ. Briefly, U251 cells were seeded in culture flasks at a density of 4~5 × 105 cells/mL and allowed to grow. After 24 h incubation, low dose TMZ was added, and the cells were incubated for another 24 h. Then, the cells were washed 3 times with PBS and the medium was changed to drug-free medium. The cells were incubated and allowed to grow until confluent. Then, the cells were subcultured and re-exposed to higher dose of TMZ. After successful development, TMZ-resistant U251 cells were maintained in 2 μg/mL TMZ.
Isobologram analysis
For combination assay, IC50 was calculated from two sets of concentration response graphs, the combination index (CI) was calculated according to the following equation [32]: CI = (d1/Dx1) + (d2/Dx2). Dx1 is the concentration of drug 1 (TMZ) required to produce x percentage effect alone, d1 is the concentration of drug 1 required to produce the same x percentage effect in combination with d2. Dx2 is the concentration of drug 2 (curcumol) required to produce x percentage effect alone, and d2 is the concentration of drug 2 required to produce the same x percentage effect in combination with d1. The CI values were defined as follows: <1, synergism; =1, additive; and >1, antagonism.
Plasmids’ construction and stable transfection
The FOXD2-AS1 sequence was synthesized from GenScript Co. Ltd., (GenScript, Nanjing, China) and subcloned into the pcDNA3.0 vector. For establishing the FoxD2-As1 stable overexpression glioma cell lines, pcDNA-FoxD2-As1 or empty vector was transfected into cells using Lipofectamin 2000. After 72h incubation, G418 was then added to the transfected cells for generating cells stably overexpressing FoxD2-As1. The transfection effect was tested by qRT-PCR.
DNA pull down assay
Biotinylated DNA probes corresponding to the EphB3, p21, and CDKN1B promoter was prepared with PCR using glioma cell genome DNA as the template. The oligonucleotides used, corresponding to the EphB3 promoter was F: 5¢-ACCCGCAGGTACTACAGTCT-3¢, R: 5¢-CACCGCGATGTATCCTGTGA-3¢; to the p21 promoter was 5¢-GAGGTCAGCTGCGTTAGAGG-3¢, R: 5¢-TGCAGAGGATGGATTGTTCA-3¢; to the CDKN1B promoter was 5¢-AAGTGCCGCGTCTACTCCTG-3¢, R: 5¢-TGGAGGCAGGGCAATGGT-3¢. The biotinylated DNA probe (1 μg) 30 μL streptavidin agarose beads (Sigma-Aldrich) were mixed and incubated in buffer (10 mM Tris-HCl (pH 8.0), 0.5 mM EDTA, 1 M NaCl) at 4 ℃ overnight. Nuclear extracts were prepared from glioma cells using the Nuclear Extraction Kit (Takara, Japan). 400 μg of cell nuclear protein extract was incubated with DNA-coupled magnetic beads at 4 ℃ for 1 h, and then the beads were washed three times with cell lysis buffer and once with PBS. After boiled for 5 min in 2×SDS-loading buffer, the samples were analyzed by western blotting using EZH2 and H3K27me3 antibodies.
Chromatin immunoprecipitation (Chip) assay
The ChiP-IT Express Enzymatic Kit (Active Motif, Carlsbad, USA) was used for Chip assay. Glioma cells were fixed with formaldehyde, harvested and chromatin was enzymatically sheared. Chromatin immune precipitates were isolated on protein G magnetic beads using 2 μg of IgG, EZH2, or H3K27me3 antibodies. After washing, the Chips were eluted, reverse cross-linked, and subjected to qRT-PCR. Chips were normalized to input DNA and the IgG control.
Mouse tumor xenografts
BALB/c nude mice (male, 5-week-old, 16-20 g) were purchased from Shanghai Slack Laboratory Animal Co. Ltd. The experiment was approved by the Institutional Animal Care and Use Committee of Zhejiang Chinese medical university laboratory animal research center, and animal studies were performed in compliance with the ARRIVE guidelines (http://www.nc3rs.org.uk/page.asp?id=1357) and all relevant guidelines and regulations. 251/TMZ cells (1× 107) were injected into each mouse. After 7 days, the mice were randomly assigned to three groups (n=5), and either treated with or without DMSO, curcumol (20 mg/kg) by intraperitoneal injections every 3 days, respectively. Tumor volume was calculated using the formula V = W2 × L × 0.5. The mice were sacrificed and tumors were harvested for the follow-up experiments.
Immunohistochemistry (IHC)
IHC was performed as described in our previous studies [33]. Briefly, the dissected tumors sections (5 mm) mounted on the glass slides were deparaffinized and rehydrated. Subsequently, the specimens were washed in 0.01 M PBS, treated with 3% hydrogen peroxide, and washed in 0.01 M PBS again. Blocking was performed with 3% BSA in 0.01 M PBS. Then the sections were incubated with antibodies for proliferation marker Ki-67, CD133, MMP2 and MGMT, followed by incubation with secondary antibodies. After the cell nuclei were labeled with hematoxylin, images were captured using a microscope (XSP-C204, Chongqing Optical Instrument Co., Ltd.).
Statistical analysis
Data were presented as mean±standard deviation (SD). The statistical analysis was performed using Student’s t-test (for comparison in two groups) or one-way analysis of variance (ANOVA; for comparison in multiple groups). Turkey’s post hoc test was performed after ANOVA. Statistical significance was determined at p< 0.05.