Surgical specimens of gliomas were obtained from 24 patients who had undergone tumor resection at Affiliated Hospital of Nantong University from January 2004 to December 2014. The study was approved by the local ethics committee, written informed consent was obtained from all the patients. Recurrence of glioma was defined as the presence of glioma at >3 months after surgery for primary glioma. All patients received chemoradiotherapy after the first surgical intervention. Pathological findings were determined by more than 2 pathologists and classified according to the WHO classification standard (Table I). The paired primary and recurrent glioma specimens were from the same patient. For immunohistochemistry, glioma tissue was fixed with 4% formalin, dehydrated, and then embedded in paraffin. Portions of the tumor tissues were rapidly frozen by liquid nitrogen and stored at −80°C until RNA and DNA extraction for real-time PCR, DNA methylation and western blot analysis.

The U251MG, U87MG, and A172MG glioblastoma cell lines were purchased from the Shanghai Cell Institution of Chinese Academic Sciences, which were cultured as described previously (18). They were maintained in Dulbecco's modified Eagle's medium: nutrient mixture F-12 (MDEM/F12) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco). Cells were incubated at 37°C in a humidified 5% CO₂ air atmosphere. When cell culture reached 50% confluence, U87MG, U251MG and A172MG cells were treated with 5-Aza-2′-deoxycytidine (5-Aza-dc, A3656; Sigma-Aldrich, St. Louis, MO, USA) at the final concentration of 10 nM for 72 h, respectively.

Cells or tissues were washed with cold PBS and lysed in ice-cold RIPA buffer containing protein inhibitors. Cell lysates were incubated on ice for 30 min and then centrifuged at 4°C for 10 min. Supernatants were collected, and protein concentrations were measured. Equivalent amounts of protein in each sample were separated on a 10% SDS-PAGE gel for separation and then electrotransferred to PVDF membrane. Membranes were blocked and then probed with primary CD133 antibodies (2 µg/ml, Abcam) followed by the horseradish peroxidase (HRP)-conjugated goat anti-mouse or rabbit IgG antibodies. Mouse monoclonal anti-β-actin antibody (1:5,000, Sigma) was used as an internal control. The membranes were developed using an ECL detection system (Pierce, Rockford, IL, USA). The intensity of bands was determined using the Image-Pro Plus 6.0 software. The western blot experiments were repeated at least three times.

RNA expression levels of CD133 were determined using quantitative real-time PCR with GAPDH as positive controls. Total mRNA was isolated from glioma specimens and cell lines using mRNA isolation kit (Roche, UK) following the manufacturer's instructions. The concentration and purity of mRNA was determined by ultraviolet spectrophotometry. Isolated mRNA (100 ng) from each sample was transcribed to complementary DNA (cDNA) using a First-Strand cDNA Synthesis kit Roche (Roche), which was then used as a template for quantitative real-time PCR. Primers used for CD133 were 5′-GCACTCTATACCAAAGCGTCAA-3′ (sense) and 5′-CTCCCATACTTCTTAGTTTCCTCA-3′ (antisense); and for GAPDH primers were 5′-GGAAAGCTGTGGCGTGAT-3′ (sense) and 5′-AAGGTGGAAGAATGGGAGTT-3′ (antisense). The primers were designed using Primer 5.0 software and manufactured by TIBmolbiol. After an initial denaturation at 95°C for 5 min, the samples were subjected to 40 cycles of RT-PCR (95°C for 10 sec, annealing temperature 59°C for 15 sec, and 72°C for 20 sec). At the end of each cycle, the fluorescence emitted was measured in a single step in channel F1 (gain 1). After the 40th cycle, the specimens were heated to 95°C and rapidly cooled to 59°C for 15 sec. All heating and cooling steps were performed with a slope of 20°C/sec. The temperature was subsequently raised with a slope of 12°C/sec and fluorescence was measured continuously (channel F1, gain 1) to obtain data for the melting curve analysis. The PCR reaction was subjected to a melting curve analysis to verify the presence of a single amplicon before the PCR products were visualized on agarose gels using a gel analyser (SynGene, UK). All PCR reactions were performed in triplicate and a negative control was included that contained primers without DNA.

Serial sections measuring 5 µm thick were cut from paraffin blocks and mounted on glass slides coated with 10% polylysine. Sections were dewaxed in xylene and rehydrated in graded ethanols. Endogenous peroxidase activity was blocked by immersion in 0.3% methanolic peroxide for 30 min. Immuno-reactivity was enhanced by microwaving and incubating the tissue sections for 10 min in 0.1 mol/l citrate buffer. Tissues were then incubated with an anti-CD133 antibody (1:200, Abcam) overnight at 4°C. Immuno-staining was performed using the avidin-biotin peroxidase complex method, and antigen-antibody reactions were visualized with chromogen diaminobenzadine. Appropriate positive and negative controls were used. Ten high-power fields were randomly chosen, and ≥300 tumor cells were counted per field. Percentage of cells showing positive staining in cytoplasm/membrane was designated as the CD133 labeling index, as a percentage (%). The staining procedures were repeated at least three times.

With the proteinase K digestion and phenole-chloroform method, genomic DNA was extracted from frozen tissues (19). Sodium bisulfite treatment of the extracted DNA was performed as previously described (20). In brief, 10 µg DNA in 50 µl TE was incubated with 5.5 µl of 0.3 M NaOH at 37°C for 15 min and 95°C for 2 min, and subjected to sodium bisulfite chemical treatment (2.4 M sodium metabisulfite; 0.5 mM hydroquinone, pH 5.0, both from Sigma). Following incubation at 55°C for 4 h, the treated DNA was purified using the SK1261 kit (Shenggong, China), desulfo-nated in 0.3 M NaOH, neutralized to pH 7.0 using 3 M sodium acetate (pH 5.2). The neutralized DNA was purified using SK1261 purification kit again, dissolved in TE buffer (pH 8.0).

The primers (fwd: 5′-TYGYGGTGAGTATGTTTAAGG-3′, rev: 5′-ACCCAACTACTCACCRTACACC-3′) were designed to amplify the promoter 2 from −237 to +52 for bisulfite genomic sequencing. An initial denaturation at 98°C for 4 min was followed by five PCR cycles of 94°C for 45 sec, 68°C for 45 sec and 72°C for 1 min. The PCR was then completed with 35 cycles of 45 sec at 95°C, 45 sec at 58°C. The amplified products were gel-purified using the SK1261 kit and subjected to TA-cloning using pUC18-T vector (Shenggong, Biotechnology Co.). Ten clones for each case were selected for sequencing using BigDye version 3.1, and analyzed on automated DNA sequence analyzer (ABI PRISM 3730; Applied Biosystems, Inc., Foster City, CA, USA). The cytosine or thymine residues at the CpG sites represented methylated or unmethylated status, respectively.

Statistical analysis was performed using SPSS 13.0 for Windows. Data are expressed as median and 25–75 percentiles [median (25th percentile, 75th percentile)]. Mann-Whitney U test was used to determine the differences of CD133 expression between primary and recurrent tumor. Paired t-test was used to analyze the differences of CD133 expression between the treated glioma cell lines. The correlation between CD133 DNA methylation and mRNA expression was analyzed by Pearson's correlation test. All statistical tests were calculated two-sided and values of P<0.05 was considered to be statistically significant.

CD133 mRNA and protein expression in primary and recurrent glioma specimens was assessed by real-time PCR and western blotting, respectively. While CD133 transcript was detected in all specimens, the levels were higher in recurrent (median 0.0214; range, 0.0182–0.0249) than in primary glioma (median 0.1,000; range, 0.0080–0.0119, P<0.001) (Fig. 1A). A similar trend was observed for CD133 protein level (Fig. 1B and C).

CD133 protein expression and localization in glioma specimens were evaluated by immunohistochemistry. CD133-positive cells were detected in all specimens, and expression was mainly observed in the cytoplasm and plasma membrane (Fig. 2A). The percentage of CD133-positive cells was significantly higher in recurrent (median 12.99%; range, 3.94–58.23%) as compared to primary glioma (median 4.35%; range, 2.43–14.35%, P<0.05) (Fig. 2B).

To clarify the mechanism underlying the upregulation of CD133 level in recurrent glioma, we analyzed the methylation status of CpG islands in the CD133 promoter. A sequence analysis revealed that the CD133 P2 promoter contains a 487-bp CpG island, with a GC content >50% and a CpG ratio (Obs/Exp) >0.6 (Fig. 3A). The P2 promoter region (−237 to +52) contained 32 CpG sites, as determined by bisulfite genomic sequencing (Fig. 3B and C). The 332-bp CD133 target fragment was cloned and sequenced, revealing unmethylated C residues that were completely converted to U by bisulfite treatment; these were replaced by T following PCR amplification. In contrast, methylated C residues persisted after bisulfite treatment, indicating that CpG islands of the CD133 promoter were methylated (Fig. 4A and B). However, DNA methylation levels were lower in recurrent (median, 46.88%; range, 26.56–59.69%) as compared to primary glioma (median, 58.76%; range, 42.19–67.35%, P<0.05) (Fig. 4C). Correlation analysis showed a negative relationship between DNA methylation and CD133 expression levels (r = −0.715, P<0.05).

To assess the effects of demethylation in vitro, we treated U87, U251, and A172 glioma cells with the demethylating agent 5-aza-2′-deoxycytidine (10 µM for 72 h). CD133 mRNA expression was upregulated in these cells by 2.15, 2.56, and 3.03-fold, respectively (Fig. 5A). A similar trend was observed for CD133 protein expression (Fig. 5B). These results indicate that CD133 demethylation increases CD133 level in glioma.