All human normal brain and glioma tissues were collected from patients treated at the Department of Neurosurgery, The Second Affiliated Hospital of Soochow University. Normal brain tissues were obtained from patients with cerebral trauma. Glioma tissues were obtained and verified following diagnosis of the clinical and pathological grade. Prior consent was obtained from all patients, and the study was approved by the institutional research boards of the affiliated institutions.

The human glioma cell lines U87, U251, and U343, and the human prostate cancer cell line PC3 were purchased from the Cell Bank of the Chinese Academy of Science. PC3 cells were used as the opposite control for maspin expression and methylation status studies. All cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/ml penicillin and 100 U/ml streptomycin; Gibco, Grand Island, NY, USA). The cells were grown in a 37°C incubator with 5% CO₂.

Total RNA from tissues and cell lines was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and the isolated RNA was reverse transcribed into complementary DNA using the RevertAid™ First Strand cDNA Synthesis kit (Fermentas, Vilnius, Lithuania) following the manufacturer's instructions. The primers for the PCR were as follows: maspin forward, 5′-GCTTTTGCCGTTGATCTGTTC-3′ and reverse, 5′-GATCTGACCTTT CGTTTTCTTC-3′; and GAPDH forward, 5′-GGAAGGTGAAGGTCGGAGTC-3′ and reverse, 5′-GAGGCATTGCTGATGATCTTGA-3′. The PCR conditions were as follows: an initial denaturation at 94°C for 5 min followed by 33 cycles of 94°C for 30 sec, 52°C for 30 sec, and 72°C for 30 sec, and a final extension at 72°C for 7 min. The RT-PCR products were analyzed using Quantity One software (Bio-Rad, Hercules, CA, USA), and the images were collected and saved. The housekeeping gene GAPDH was used as the internal control.

Tissues and cells were lysed in lysis buffer, and whole proteins were extracted by incubation with the western blot assay buffer (Beyotime, Nantong, China). Protein concentration was measured using a BCA protein assay kit (Beyotime, Nantong, China). A total of 50 µg of each protein sample was subjected to 10% SDS-PAGE, and the proteins were transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk in TBST for 1 h at room temperature, and then incubated with a 1:1,200 dilution of anti-maspin monoclonal antibody (BD Pharmingen, San Diego, CA, USA) overnight at 4°C. The HRP-conjugated secondary antibody was incubated for 1 h at 37°C. Specific bands were visualized using the enhanced chemiluminescence detection kit for HRP (Biological Industries, Kibbutz Beit Haemek, Israel) and Kodak X-OMAT LS film (Eastman Kodak, Rochester, NY, USA). Quantitative data were obtained using Quantity One software.

Lentiviral vectors expressing maspin (LV-maspin) and a non-silenced control lentivirus (LV-NC) were designed and constructed by GenePharma (Shanghai, China). Cell infection was conducted according to the recommendations of GenePharma. U87-LV-maspin (U87-maspin) cells represent U87 cells infected with LV-maspin, and the negative control U87-LV-NC (U87-NC) cells represent U87 cells infected with LV-NC. GFP expression was observed under a fluorescence microscope after infection. For further validation, the expression of maspin was confirmed by RT-PCR and western blotting.

For the migration assay, U87, U87-NC and U87-maspin cells were suspended in 120 µl of serum-free medium. A total of 2×10⁴ cells/well were seeded into the upper chambers of the 24-well Transwell inserts with 8-µm pores (Corning, Corning, NY, USA). Next, 600 µl of DMEM containing 10% FBS was added to the lower chambers. After 48 h, the cells on the upper surface were removed, and the cells on the lower surface were fixed using a crystal violet cell colony staining kit (Genemed, Shanghai, China). The stained cells were counted in five randomly selected fields per filter under a microscope (×400 magnification). For the invasion assay, the Transwell inserts were coated with Matrigel (40 µl/well; BD Biosciences, San Jose, CA, USA). The following procedures were the same as those for the migration assay.

The number of viable cells was assayed using the Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) as per the manufacturer's protocol. U87, U87-NC and U87-maspin cells were seeded onto the 96-well plates at a density of 2×10³ cells/well. After 24 h, 10 µl of CCK-8 solution was added to each well. After 2 h, the optical density (OD) at 450 nm was measured using a microplate reader (Bio-Rad).

Cell proliferation was also determined using 5-ethynyl-2-deoxyuridine (EdU) using the Cell-Light™ EdU Apollo^® 567 In Vitro Imaging kit (RiboBio, Guangzhou, China) according to the manufacturer's protocol. Briefly, cells seeded in 96-well culture plates were incubated with 50 µM EdU for 3 h before fixation, permeabilization, and Apollo staining. Cell nuclei were stained with Hoechst 33342 at a concentration of 5 µg/ml for 30 min. The cells were observed using a fluorescence microscope (Olympus, Tokyo, Japan).

U87 and U251 cells were treated with various concentrations of 5-Aza-2′-deoxycytidine (5-Aza-dC; Sigma-Aldrich, St. Louis, MO, USA). U87 cells were treated with 12 µM 5-Aza-dC for 96 h. U251 cells were treated with 15 µM 5-Aza-dC for 96 h. After treatment with 5-Aza-dC, the maspin mRNA expression level was investigated in the U87 and U251 cell lines.

Genomic DNA from cells and tissues was isolated using the Genomic DNA purification kit (Thermo, Waltham, MA, USA). Bisulphite modification was performed using the EZ DNA Methylation-Gold™ kit (D5005; Zymo Research, Orange, CA, USA) following the manufacturer's instructions.

The primers for maspin promoter methylation analysis by PCR were designed by MethPrimer (http://www.urogene.org/methprimer/). The primers were as follows: methylated maspin (Mmaspin) forward, 5′-TTTTATCGAATATTTTATTTTTCGG-3′ and reverse, 5′-GATAACTCACCTAAACAACACCG-3′; and unmethylated Maspin (Umaspin) forward, 5′-TTTTATTTTATTGAATATTTTATTTTTTGG-3′ and reverse, 5′-CAATAACTCACCTAAACAACACCAC-3′. The DNA for this PCR was treated as previously described. The PCR reaction conditions were as follows: 94°C for 5 min; 40 cycles of 94°C for 30 sec, 57°C for 30 sec, 72°C for 30 sec; and a 72°C - extension for 7 min. MSP products were analyzed by Quantity One software.

DNA CpG islands of the maspin promoter were analyzed by EMBOSS Cpgplot (http://www.ebi.ac.uk/). The parameters for CpG island searching were set at Mayor >100 bp, CpG/expected CpG >0.6, GC% >50% for the EMBOSS Cpgplot. The primers for this PCR were designed from the predicted sequence of the maspin promoter CpG island using MethPrimer as previously described. The primers were 5′-GAGAAATTTGTAGTGTTATTATTATTATAT-3′ (forward) and 5′-ATAACTCACCTAAACAACACC-3′ (reverse), amplifying a 321-bp product including 16 expected CpG sites. The PCR conditions were as follows: 94°C for 5 min; 40 cycles of 94°C for 30 sec, 57°C for 30 sec, 72°C for 30 sec; and a 72°C extension for 7 min. Bisulphite-sequencing PCR products were analyzed using Quantity One software. The PCR products were purified directly using the TIANgel Midi Purification kit (Tiangen Biotech, Beijing, China). The purified products were sequenced by Suzhou Genewiz, Inc. (Suzhou, China).

Data are presented as the mean ± standard error of the mean (mean ± SEM). Statistical analyses were conducted using Statistical Package for the Social Sciences (SPSS) software version 19.0 (Chicago, IL, USA). The differences between groups were analyzed using the Student's t-test or one-way analysis of variance (ANOVA). Differences were considered statistically significant at a P-value <0.05.

We first investigated the expression of maspin in glioma cells and tissues. The mRNA and protein levels of maspin in the glioma cell lines and tissues were explored by RT-PCR and western blotting, respectively. PC3 cells were used as the positive control for maspin expression. As shown in Fig. 1A and C, maspin transcripts were present in the PC3 cells and normal brain tissues, but not in the glioma cell lines and tissues. Consistent with this result, maspin protein was not detected in the above glioma cell lines and tissues (Fig. 1B and D). These results indicated that the expression of maspin was silenced in glioma.

To explore the function of maspin, the coding region of maspin was cloned and then subcloned into an overexpression vector (with GFP tag). To perform the cell function experiments, the stable glioma cell lines with overexpression of maspin and the control cells were screened (Fig. 2A). Expression of maspin in the parental U87, U87-NC and U87-maspin cells was verified by RT-PCR and Western blotting. As shown in Fig. 2B–E, the mRNA and protein levels of maspin were both significantly upregulated in the U87-maspin cells compared with the control cells (P<0.01). These results indicated that the stable cell line with overexpression of maspin and the control cell line were successfully established.

We next investigated the effect of maspin overexpression on cell migration and invasion. Parental U87 cells, U87 cells infected with the control lentivirus (U87-NC) and U87 cells with maspin overexpression (U87-maspin) were used for the Transwell migration and invasion assays. The numbers of invasive U87, U87-NC and U87-maspin cells were 45.0±4.0, 46.0±4.6 and 44.3±2.5, respectively. As shown in Fig. 3A, no significant difference was noted between the U87-maspin cells and control cells regarding the invasion capability (P>0.05). The numbers of migrated U87, U87-NC and U87-maspin cells were 47.7±9.5, 52.3±4.5 and 53.7±3.5, respectively. The number of U87-maspin cells that transferred through the chambers was almost the same as that of the control group (P>0.05; Fig. 3B). These data indicate that maspin had no effect on the invasion and migration of the U87 cells.

We next investigated the effect of maspin on the proliferation of glioma cells. To this end, the CCK-8-based assay was used. As shown in Fig. 4A, U87-maspin cells grew markedly slower than the control group starting from the second day. Thus, maspin inhibited cell proliferation in the U87 cells in vitro. To further characterize the effect of maspin on the proliferation of glioma cells, the EdU proliferation assay was also performed (Fig. 4B). The percentage of EdU-positive cells was quantified. The percentage of EdU-positive cells was significantly downregulated in the U87-maspin cells when compared with the control cell lines (P<0.01; Fig. 4C). These results indicated that maspin contributed to the reduced proliferation of the U87 cells.

Gene sequences of maspin were analyzed using EMBOSS Cpgplot (http://www.ebi.ac.uk/). After bioinformatic analysis, a CpG island in the 5′ promoter region and 5 CpG islands in the gene body were predicted (Fig. 5A). The presence of a CpG-rich island in the 5′ promoter region suggested the possibility that the maspin gene might be regulated through DNA methylation. To demonstrate a functional association between maspin promoter methylation and its gene inactivation, a DNA-demethylating agent, 5-Aza-dC, was used to treat the U87 and U251 cells. After treatment with 5-Aza-dC, a significant increase in maspin mRNA expression was noted in both the U87 and U251 cells (Fig. 5B).

Since maspin could be induced by 5-Aza-dC in the U87 and U251 cells, it could be suggested that the aberrant promoter methylation might contribute to maspin inactivation in glioma. To test this hypothesis, MSP was used to examine the methylation status of the maspin promoter. The results from MSP showed that the maspin promoter was methylated in the U87 and U251 cells but not in the PC3 cells (Fig. 5C). Furthermore, to determine a more detailed map of the methylation in the maspin promoter, we performed bisulphite sequencing around the promoter region of the maspin gene in the glioma cell lines and tissues. The maspin gene was amplified with bisulphite-treated DNA dinucleotides in the promoter, including 16 putative CpG sites. In the U87 and U251 cells as well as three glioma tissues, 16 CpG dinucleotides of the promoter were found to be methylated, whereas in the PC3 cells and three normal brain tissues, the CpG dinucleotides in the maspin promoter were unmethylated. Representative sequencing results are shown in Fig. 5D and E. Thus, DNA methylation is involved in the suppression of maspin promoter activity in glioma.