Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Hyclone (Logan, UT, USA). Lipofectamine 2000, TRIzol^® reagent was purchased from Invitrogen (Carlsbad, CA, USA). M-MLV Reverse Transcriptase was purchased from Promega (Madison, WI, USA; cat. M1705). All other chemicals were obtained from Sigma (St. Louis, MO, USA). The antibodies used were as follows: anti-DNMT1 (1:500 dilution; Santa Cruz Biotechnology, Inc.); anti-MDM2 (1:500 dilution; Boster, China); anti-p53 (1:500 dilution; Boster); anti-β-actin (1:2,000 dilution; Santa Cruz Biotechnology, Inc.). DNA extraction kit was acquired from Axygen. Streptavidin peroxidase (SP) immunohistochemical kit was acquired from Zhongshan Biotechnology Corp. (Beijing, China).

Tissue samples from normal brain tissues and human glioma tumors were collected from the Neurosurgery Department of The Second Affiliated Hospital of Anhui Medical University (Hefei, China). Samples were collected and snap-frozen in liquid nitrogen immediately and preserved in −80°C until use, and their histological type was further confirmed using the standard hematoxylin and eosin staining according to the WHO criteria. A total of 71 samples were used for this study including primary-grade pilocytic astrocytomas or grade II astrocytomas (WHO I/II, n=23), grade III anaplastic astrocytomas or grade IV glioblastoma multiforme (WHO III/IV, n=36) and normal brain tissues derived from the temporal lobes and saddle area of the patients with arachnoid cyst after surgery (n=12), both glioma patients and controls were similar with respect to sex and age. This study was approved by the Research Ethics Committee of The Second Affiliated Hospital of Anhui Medical University. Informed consent was obtained from all the patients.

We studied 62 glioma patients who had been surgically treated in Department of Neurosurgery, The Second Affiliated Hospital of Anhui Medical University, Hefei, China. For immunohistochemistry, resected glioma tissues were fixed in 10% formalin solution and embedded in paraffin. Histological slices of 3 mm were prepared, then deparaffined in xylene, and dehydrated with ethanol. Endogenous peroxidase was blocked with 0.3% H₂O₂ in methanol for 20 min at room temperature (RT). Following antigen retrieval, the sections were blocked with 5% BSA for 20 min at RT and then probed with 1:500 rabbit anti-DNMT1 at 4°C overnight. After washing, the sections were incubated with biotinylated goat anti-rabbit immunoglobulins at RT for 1 h, and visualized using the peroxidase conjugated streptavidin and diaminobenzidine, followed by counterstaining with Mayer's hematoxylin. Results of the immunohistochemical (IHC) staining were evaluated by a pathologist blinded to all clinical data. The criterion for IHC scoring was that a specimen with >10% positive staining tumor cells should be considered positive, and our IHC data for pharyngeal cancer were classed as positive or negative accordingly.

U251, U87 and A172 human glioma cell lines were obtained from the Chinese Academy of Sciences Cell Bank. Cells were maintained in DMEM supplemented with 10% heat-inactivated FBS and 100 U/ml penicillin/streptomycin at 37°C in humidified atmosphere of 5% CO₂.

U251 and U87 glioma cells were seeded overnight in culture dishes, 5-AzadC (Sigma-Aldrich) was added and was refreshed every 24 until 72 h treatment finished. The medium containing PBS only was regarded as a control.

The methylation status of the MEG3 promoter region was determined by methylation-specific PCR(MSP) using bisulfite-modified DNA. Genomic DNA was extracted using the QIAamp DNA mini kit (Axygen). Two primer sets were used to amplify the promoter region of the MEG3 gene that incorporated a number of CpG sites, one specific for the methylated sequence (MEG3-M, forward, 5′-TATGAGTTGTAAGCGGTAGAGTTC-3′; reverse, 5′-TACGAACTTAACGAAAAAAAATCAT-3′) and the other for the unmethylated sequence (MEG3-U, forward, 5′-GAATATGAGTTGTAAGTGGTAGAGTTT-3′; reverse, 5′-TACAAACTTAACAAAAAAAAATCATACT-3′). The primers used in the present study detect specifically the promoter sequence of the MEG3 gene rather than that of the MEG3 pseudogene. For MSP reaction we used the HotStarTaq Master Mix kit (Qiagen). PCR reactions were carried out in a final 25 μl volume containing: HotStarTaq Master mix (final concentration 1X PCR buffer, 2.5 U Hot Star Taq DNA polymerase, 200 μm of each dNTP and 1.5 mM MgCl₂), 0.4 μM primers and bisulphite-modified DNA (~30 ng). Amplification was performed in a thermocycler with the following conditions: 94°C for 2 min, cycled at 94°C for 30 sec, 54°C or 50°C for 30 sec, and 72°C for 45 sec (36 cycles) followed by extension at 72°C for 7 min. MSP experiments were performed at least in duplicate.

RNAi experiments in glioma cells were performed by forward transfection in cultured glioma cells (2×10⁵ cells per 200 mm² dish) using Lipofectamine RNAiMax (Invitrogen) according to the manufacturer's protocol. For DNMT1, MDM2 and p53 immunoblotting, glioma cells were cultured in serum-free DMEM for 12 h and then subjected to reverse transfection with RNAiMax in Opti-MEM. Small interfering RNA (siRNA) oligonucleotides against DNMT1 genes or scrambled sequences were synthesized by the Shanghai GenaPharma Corp. The following siRNA sequences were used: si-DNMT1 (human), 5′-GGGACUGUGUCUCUGUUAUTT-3′ (sense) and 5′-AUAACAGAGACACAGUCCCTT-3′ (antisense); si-control with scrambled sequence (negative control siRNA having no perfect matches to known human genes), 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense) and 5′-ACGUGACACGUUCGGAGAATT-3′ (antisense). Transfection was allowed to proceed for various times and cells were processed for different assays. The siRNA transfection efficiency of Lipofectamine RNAiMax in cells was determined by the BLOCK-iT Alexa FluorR Red Fluorescent Oligo protocol (Invitrogen). Significance of differences was assessed using paired one tailed t-test.

Total RNA was extracted from glioma specimen or glioma cells using TRIzol reagent (Invitrogen). Real-time quantitative PCR analysis was performed using SYBR Green Master Mix kit on Thermo Fisher connect Real-Time PCR platform. In brief, each PCR reaction mixture containing 10 μl of 2X SYBR GreenMaster Mix, 1 μl of sense and antisense primers (5 μmol/μl) and 1 μl of cDNA (10 ng), was run for 40 cycles with denaturation at 95°C for 15 sec, annealing at 60°C for 30 sec, and extension at 72°C for 30 sec, in a total volume of 20 μl. For relative quantification, 2^−ΔΔCT was calculated and used as an indication of the relative expression levels, which was calculated by subtracting CT values of the control gene from the CT values of MEG3 and DNMT1. Real-time PCR was carried out under a standard protocol using the following primers: DNMT1 (forward, 5′-CGGCTTCAGCACCTCATTTG-3′; reverse, 5′-AGGTCGAGTCGGAATTGCTC-3′), MEG3 (forward, 5′-ATCATCCGTCCACCTCCTTGTCTTC-3′; reverse, 5′-GTATGAGCATAGCAAAGGTCAGGGC-3′). GAPDH was applied as an internal control. The primer sequences of GAPDH were 5′-AGCAAGAGCACAAGAGGAAG-3′ and 5′-GGTTGAGCACAGGGTACTTT-3′.

U251 and U87 glioma cells were trypsinized, resuspended, seeded into a 96-well plate with a concentration of 2,000 cells per well, and incubated at 37°C 3 days post-siRNA transfection. The number of viable cells was measured at daily intervals (at 12, 24, 48 and 72 h). At each time-point, 10 μl of 5 mg/ml MTT (Dingguo Biotechnology) was added, and incubation was continued for 4 h. Then the medium was removed carefully and 150 μl of DMSO was added at the end of incubation. The absorbance was measured at 592 nm on the spectrophotometer.

A total of 200 U251 and U87 glioma cells were seeded in 6-well plates after 3 days of siRNA transfection. The medium was changed at regular time intervals. After 11 days of culture at 37°C, the natural colonies were washed with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. The colonies were then stained with Giemsa for 10 min, washed with water and air-dried. The total number of colonies with >50 cells was counted under fluorescence microscopy.

For cell apoptosis assay, cells were plated into 6-well plates at 1×10⁵ cells per well. Several days after siRNA transfection or 5-AzadC treatment, the cells were harvested by trypsinization and washed with PBS. Annexin V-fluorescein isothiocyanate and PI double-staining (BD Biosciences) was used to detect and quantify cellular apoptosis by FCM. All the tests were performed in triplicate.

U251 and U87 glioma cells were lysed with RIPA lysis buffer (Beyotime, China). Whole extracts were prepared, and protein concentrations were determined using the BCA protein assay kit (Boster). Whole-cell extracts (20 or 40 μg) were then fractionated by electrophoresis through an 8 or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Gels were run at a 120 V for 2 h before transfer onto a PVDF membrane (Millipore Corp., Billerica, MA, USA). After blocking against non-specific protein binding, nitrocellulose blots were incubated for 1 h with primary antibodies diluted in TBS/Tween-20 (0.075% Tween-20) containing 3% Marvel. Anti-DNMT1, anti-MDM2 and anti-p53 were diluted 1:400. Following incubation with the primary antibody, blots were washed three times in TBS/Tween-20 before incubation for 1 h with goat anti-mouse or mouse anti-rabbit horseradish peroxidase conjugated antibody at a 1:10,000 dilution in TBS/Tween-20 containing 5% milk. After extensive washing in TBS/Tween-20, the blots were rinsed with distilled water and proteins were detected using the enhanced chemiluminescence system. Proteins were visualized with ECL-chemiluminescent kit (ECL-plus, Thermo Scientific).

The data are expressed as mean ± SD of three independent experiments. Statistical analysis was performed using the Student's t-test and the one-way analysis of variance (ANOVA). Pearson's test was performed to calculate the association between DNMT1 and MEG3 expression. Significance was defined as P<0.05.

To determine the expression levels of MEG3 in glioma samples and cell lines, total RNAs were extracted from glioma tissues at low-grade (grade I and II), high-grade (grade III and IV), and normal brain tissue samples, and the expression level of MEG3 was analyzed using qRT-PCR. As shown in Fig. 1A, the levels of MEG3 expression were significantly decreased in glioma tissues compared with normal brain tissues. To explore whether there is any association between the downregulation of MEG3 and the pathogenesis of glioma, we detected the expression of MEG3 in glioma samples with different grades and found marked downregulation in high-grade glioma samples and to a lesser degree, decreased in low-grade glioma samples, as compared to normal brain tissues, which gave clues that MEG3 may be involved in the disease pathogenesis in glioma (Fig. 1B). Of note, 3 glioma cell lines displayed significantly downregulated expression of MEG3 relative to that in normal brain tissues (Fig. 1C).

To investigate the mechanism underlying the decreased MEG3 expression, we analyzed whether MEG3 promoter region hypermethylation is responsible for the downregulation of MEG3 expression. Methylation-specific PCR analysis indicated that the promoter region of MEG3 from glioma tissues in which MEG3 expression was downregulated was strongly methylated, whereas normal brain tissues and low-grade glioma tissues in which MEG3 expression was present had unmethylated MEG3 promoter region (Fig. 1D). These results suggest that methylation of MEG3 promoter likely contributes to the loss of MEG3 expression in high-grade glioma.

To further confirm that the loss of MEG3 expression was caused by promoter methylation in U87 and U251 glioma cells in vitro, the DNMT inhibitor 5-AzadC was used to inhibit methylation. As illustrated in Fig. 2A and B, treatment with 10 μM 5-AzadC markedly inhibited the expression of DNMT1, and upregulated the expression of MEG3, however, expression of DNMT1 and MEG3 did not show significant changes in 5 μM 5-AzadC treated glioma cells. In addition, aberrant hypermethylation of MEG3 was diminished in 5-AzadC treated glioma cells compared with control cells (Fig. 2C). The MTT assay showed that exposure with 5-AzadC for 48 and 72 h significantly inhibited the cell viability of U251 and U87 glioma cells (Fig. 3A and B). The clone formation assay was performed to further confirm the effect of 5-AzadC on glioma cell proliferation, and data indicated that 5-AzadC treatment significantly inhibited the number of clones in U251 and U87 glioma cells (Fig. 3C). Reduction of glioma cell growth by 5-AzadC treatment suggests that these cells may undergo programmed cell death. To test this hypothesis, we analyzed cell apoptosis induced by 5-AzadC using Annexin V and PI double staining. As shown in Fig. 3D, 5-AzadC treatment significantly induced apoptosis of U251 and U87 glioma cells compare to control cells. These results suggest that the decreased expression of MEG3 is related to reversible epigenetic mechanisms such as DNA promoter region methylation in glioma.

To gain insights into the possible involvement of DNMT1 in glioma, we examined the expression of DNMT1 in glioma tissues. As shown in Fig. 4A, the levels of DNMT1 mRNA expression were significantly upregulated in gliomas tissues compared with normal brain tissues. The levels of DNMT1 mRNA expression were positively correlated to glioma grades, with lower grades exhibiting upregulation >3-fold and higher grades demonstrating a >5-fold increase in DNMT1 expression (Fig. 4B). These data provided us with experimental evidence that DNMT1 expression was upregulated in a graded manner in gliomas, with higher grades showing highest expression. In addition, immunohistochemical analyses showed that DNMT1 expression was significantly increased in high-grade glioma, and low-grade glioma compared with normal brain tissues, suggesting this gene is associated with glioma malignancy (Fig. 4C). Next, we investigated whether levels of DNMT1 mRNA expression was inversely correlated with MEG3 expression in glioma tissues. A statistically significant inverse correlation was observed between DNMT1 mRNA and MEG3 (Fig. 4D), supporting the role of DNMT1 in expression of MEG3.

To determine the role of DNMT1 in glioma, the expression of DNMT1 was inhibited by DNMT1 RNAi. Data from the MTT assay showed that transfection with DNMT1 RNAi, but not scrambled RNAi for 48 and 72 h reduced the cell viability of U251 and U87 glioma cells (Fig. 5A and B). The results from the clone formation assay verified the MTT results, and the finding that DNMT1 RNAi inhibited clone formation in U251 and U87 glioma cells (Fig. 5C). The apoptosis of glioma cells was also observed in DNMT1 knockdown U251 and U87 glioma cells (Fig. 5D). In addition, DNMT1 knockdown in U251 and U87 glioma cells ameliorated MEG3 methylation, and restored MEG3 mRNA expression (Fig. 6A–C). These results suggest that MEG3 hypermethylation was dependent on DNMT1 in glioma.

To study the potential signaling pathways affected by MEG3 methylation, the protein levels of both MDM2 and p53 were examined in glioma cells. As illustrated in Fig. 7A, treatment with 5-AzadC reduced the expression levels of DNMT1 and MDM2 proteins in U251 and U87 glioma cells, whereas upregulated the levels of p53 protein expression. Of note, the repression of MDM2 expression mediated by MEG3 may be correlated with enhanced expression of p53 protein.

Finally, the effect of DNMT1 silencing on the activation of p53 pathway was examined in glioma cells. As illustrated in Fig. 7B, knockdown of DNMT1 significantly repressed the expression of DNMT1 and MDM2 protein compared to control or scrambled U251 and U87 glioma cells, however, the expression levels of p53 protein were induced. These results suggest that an aberrant methylation occurred in MEG3 promoter contributes to the decrease in p53 expression by the activation of MDM2 in gliomas.