The present study was approved by the Ethics Committee of the Fourth Military Medical University (Xi’an, China). Fifty-three glioma tissue samples and 26 adjacent normal tissue samples were collected from patients who underwent surgery at the Xijing and Tangdu Hospital of the Fourth Military Medical University (Xi’an, China) between November 2006 and September 2007. The patients included 31 males and 22 females. The median patient age was 42 years (range, 13–70 years). None of the patients had received radiotherapy or chemotherapy prior to surgery. Among the patients included in the present study, there were 24 cases of astrocytoma, 19 cases of anaplastic astrocytoma and 10 cases of glioblastoma multiform astrocytoma. Tumors were graded according to the pathological classification criteria of glioma established by the World Health Organization (7). There were 24, 19 and 10 cases of grade II, III and IV tumors, respectively. All patients provided informed consent according to institutional guidelines and remained under continuous medical supervision and assistance in accordance with the Declaration of Helsinki. All samples were snap-frozen in liquid nitrogen and stored at −70°C until analysis, or fixed in 10% formaldehyde and embedded in paraffin for subsequent analysis.

U251 human glioblastoma cells were obtained from American Type Culture Collection (Rockville, MD, USA) and were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen Life Technologies, Carlsbad, CA, USA) with 10% fetal bovine serum in a humidified 5% CO₂ atmosphere at 37°C. U251 cells served as a typical glioblastoma sample for the subsequent methylation analysis.

Total RNA was extracted from each tissue sample (50–100 μg) using TRIzol^® reagent (Invitrogen Life Technologies) according to the manufacturer’s instructions. Total RNA was reverse-transcribed using a ThemoScript™ RT-PCR system (Invitrogen Life Technologies). Primers were designed from the conserved region of their respective cDNA sequences and were synthesized by Beijing Aoke Biotechnology Co., Ltd. (Beijing, China). The primer sequences were as follows: Forward, 5′-GAG ATA TGC TCT TAA CCA CCC G-3′ and reverse, 5′-GCT GCC CAA TCC ATC CAA-3′ for NDRG2 (GenBank accession no. AF159092; amplicon size, 90 bp); and forward, 5′-ATC ATG TTT GAG ACC TTC AAC A-3′ and reverse, 5′-CAT CTC TTG CTC GAA GTC CA-3′ for β-actin (GenBank accession no. NM001101; amplicon size, 318 bp). The ΔΔCT method (8) was used to calculate the fold gene expression relative to the lowest tumor grade in the specific analysis. Human β-actin mRNA served as the reference transcript.

Total protein was extracted from the 100-μg tissue samples from each of the eight cases of grade II, III and IV tumors using 1 ml lysis buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, 0.25% sodium taurodeoxycholate, 50 mM NaF, 5 mM EDTA, 1 mM phenylmethanesulfonyl fluoride, 1 mM Na₃NO₄, 20 μg/ml aprotinin, 1 μg/ml leupeptin and 1 μg/ml pepstatin) as described previously (9). Equal quantities of protein were resolved using discontinuous SDS-PAGE (6% lamination gel and 12% separation gel). Proteins were transferred from the gel onto a pyroxylin membrane (Takara, Dalian, China) for 2.5 h at a constant voltage of 100 V. The membranes were blocked with Tris-buffered saline (TBS; pH 7.0) containing 5% non-fat dried milk for 1 h at room temperature. The membrane was subsequently washed in TBS containing Tween-20 (TBST) and incubated overnight at 4°C with anti-NDRG2 (Abnova Co., Ltd., Taipei, Taiwan) and -tubulin antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Following agitation for 1 h using a Vortex Genie 2 (Scientific Industries, Inc., Bohemia, NY, USA) the membranes underwent four 5 min washes with TBST. The membranes were incubated with two horseradish peroxidase-conjugated secondary antibodies (Beijing Zhongshan Jinqiao Biotechnology Co. Ltd., Beijing, China) at room temperature and were agitated for 1 h in a shaker and washed with TBST four times. Immunoreactive bands were visualized using an enhanced chemiluminescence (ECL) method and ECL reagents (Pierce Biotechnology, Rockford, IL, USA).

In order to detect the expression of NDRG2, the tissue samples were subjected to immunohistochemistry as previously described (10). In brief, 5-μm sections of paraffin-embedded, formalin-fixed tissue samples were treated with 3% H₂O₂ at room temperature for 10 min to inhibit any endogenous peroxidase activity. Sections were washed three times with phosphate-buffered saline (PBS) and pretreated for antigen retrieval by microwaving in 10 mM citrate buffer (pH 6.0) for 15 min at high power. Sections were subsequently incubated with primary antibodies against NDRG or Tubulin (Santa Cruz Biotechnology) overnight at 4°C and underwent three 5 min washes with PBS containing 0.1% Tween-20. Following washing, slides were incubated with biotin-labeled secondary antibodies (Santa Cruz Biotechnology, Inc.) for 10 min at room temperature and washed again. The staining was visualized using an avidin-biotin complex and counterstaining was performed using hematoxylin (11).

DNA was extracted from the tumor tissues and U251 cells using a DNA extraction kit (Tiangen Biotechnology Co., Ltd., Beijing, China) according to the manufacturer’s instructions and was stored at −20°C. Normal lymphocyte DNA was modified using a SssI methyltransferase kit (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions. Genomic DNA was modified using bisulfite (Sigma-Aldrich, St. Louis, MO, USA) according to the method described by Herman et al (12). Non-methylated genomic DNA from normal human peripheral blood lymphocytes served as negative control. Genomic DNA was obtained from human lymphocytes, whose CpG sites had been completely methylated, using the SssI methyltransferase and served as positive control. Deionized water was the blank control. Two overlapping fragments from the NDRG2 promoter region, spanning 32 CpG sites between nucleotides 20,564,110 and 20,562,476 (Genbank accession no. NC_000014) were PCR-amplified from sodium bisulfite-modified DNA using the following primers: Forward, 5′-AAG GAG AGT TTA TTT TAG GGT GTG-3′ and reverse 5′-TAC CCA AAA TCC TAAT ACC TCT C-3′ (9). The amplified sequence was 530 bp and included 32 CpG sites. The recovered PCR products were ligated into the pMD19-T Vector (Takara Bio Inc., Shiga, Japan) and transformed into E. coli competent cells (GM109). Extracted plasmids were digested using the PstI and EcoRI restriction endonucleases (Takara) according to the manufacturer’s instructions. Restriction digests were ethanol precipitated prior to gel analysis.

Data are presented as the mean ± standard deviation. Paired t-tests were performed to assess the differences in NDRG2 mRNA expression between tumor and adjacent normal tissue samples. Mean mRNA levels were compared between the three tumor grades using variance analysis and P<0.05 was considered to indicate a statistically significant difference. Mean protein levels were compared between the three tumor grades using variance analysis. The proportion of promoter methylation in normal and tumor tissues among the three grades was analyzed using Pearson’s χ² test. The number of methylation sites in each sample was compared between grades using the Kruskal-Wallis H test.

Immunohistochemistry was used to analyze NDRG2 expression in 26 human glioma samples (grade II, n=9; grade III, n=9; and grade IV, n=8). As shown in Fig. 1A, the expression of NDRG2 was observed to be significantly higher in adjacent normal tissues (Fig. 1A-a) compared with glioma tissues (Fig. 1A-b) and NDRG2 was observed to be primarily expressed in the cytoplasm. NDRG2 staining was also found to decrease with increasing glioma malignancy (data not shown). Western blot analysis was performed to further assess the protein expression of NDRG2 in glioma tissues. The protein expression of NDRG2 in the grade II and III glioma samples was found to be 1.75- and 1.7-fold of that in the grade IV glioma samples, respectively. This difference in NDRG2 protein expression among the glioma grades was considered to be statistically significant (P<0.05; Fig. 1B).

Furthermore, NDRG2 mRNA expression was assessed and the findings were in accordance with those that were identified via immunohistochemistry and western blot analysis. In 26 human brain normal tissue samples, the expression of NDRG2 mRNA was found to be 1.7-fold that in the glioma samples (P<0.05). Furthermore, NDRG2 mRNA expression in the grade II and III glioma samples was observed to be 2.7- and 2.5-fold of that in grade IV glioma samples. Moreover, a significant negative correlation was identified between NDRG2 mRNA expression and the glioma grade (P<0.05; Fig. 1C).

To further investigate the mechanisms underlying the decrease in NDRG2 expression observed in the glioma tissue samples, NDRG2 promoter methylation was detected in glioma tissue samples using bisulfite sequencing PCR. In bisulfite sequencing PCR, cytosine (C) residues in methylated CpG sites of the NDRG2 promoter are not deaminated into uracil (U); therefore, remain as a C or guanine in the PCR product. C residues of non-methylated CpG sites are deaminated into U residues and are transformed into thymine or adenine in the PCR product. Sequencing revealed that none of the CpG sites in the NDRG2 promoter region in the genomic DNA from normal human lymphocytes were methylated; however, following treatment with the SssI methyltransferase, all of the CpG sites were methylated, thus, validating the experimental procedures. Methylated CpG sites were detected in 19 out of 41 of the glioma tissue samples (46.3%), compared with four out of the 22 adjacent normal tissues (18.2%), which was considered to be a significant difference (P<0.05; Table I). In addition, 66.7% of the 15 grade IV glioma samples, 33.3% of the 12 grade III glioma samples and 35.7% of the 14 grade II glioma samples were observed to be methylated.

Further assays were performed in order to analyze the distribution of methylated CpG sites in the NDRG2 promoter region. As shown in Fig. 2A, methylation was predominantly observed in the frontal 16 CpG sites, which were 63 of 87 (72.4%) as compared with 24 of 87 (27.6%) in the latter 16 CpG sites (P<0.05). The average number of methylation sites in 19 methylation-positive glioma tissues was 5.53±1.21, compared with 4.85±1.18 in the four adjacent normal tissue samples. No significant difference was observed in the average number of methylation sites in glioma tissue compared with that in the normal tissue (P>0.05). The average number of methylation sites in the 10 methylated grade IV, four methylated grade III and five methylated grade II samples was 6.04±1.76, 3.44±1.12 and 5.22±1.14, respectively (Fig. 2B). The average number of methylation sites in the grade IV samples compared with that in the grade III and II samples was significantly different (P<0.05).

To further investigate the methylation of the NDRG2 promoter region, DNA sequencing of the NDRG2 promoter was performed in different tissues and cells. Sequencing revealed that the CpG sites in the NDRG2 promoter region were methylated in the U251 cells and glioma tissues (Fig. 3).