Seventy cases of surgically resected GBMs were collected from the 2007–2010 pathology files of the Third People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine and Zhongnan Hospital of Wuhan University. Approval was obtained from the Ethics committee of the Third People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine and Zhongnan Hospital of Wuhan University. Specimens were handled and made anonymous according to ethical and legal standards. Written informed consent was obtained from all patients. There were 40 males and 30 females with a mean age of 46 years (range, 29–68). None of the patients had received chemical therapy or radiotherapy prior to surgery. Control brain tissues were obtained from 10 individuals, who had died in traffic accidents exhibiting no prior pathologically detectable condition. Based on the results of hematoxylin-eosin staining, histopathological diagnosis was performed by various neuropathologists.

Frozen sections were immersed in a solution of 30% hydrogen dioxide and methanol for 30 min following brief warming at room temperature, then incubated at 37°C with pepsin diluted by 3% citric acid. Sections were postfixed for 10 min in 1% paraformaldehyde and were incubated at 38–42°C with DIG-labeled antisense cRNA probes overnight in a humidified chamber. Following multiple washes in 4× SSC at room temperature, the slides were incubated at 37°C in a blocking reagent for 30 min, a biotinylated anti-digoxin antibody for 60 min, SABC for 20 min and the biotinylated peroxydase for 20 min, at 37°C, followed by staining with DAB (Sigma, St. Louis, MO, USA). Sections were then covered with glycerol-gelatin and coverslips for microscopic examination (11,12). The SATB1 probe (sequence: 5′-TCTTTAATTTCTAATATATTTAGAA-3′) was synthesized and labeled with biotin at the 5′ end by Sangon Bioengineering Technology and Services Co., Ltd. (Shanghai, China). The probe was replaced with the dilution solution in control samples.

Antigen retrieval was performed in boiling citrate buffer for 15 min. Peroxide blocking was performed with 0.3% peroxide in absolute methanol. The slides were then incubated with anti-Bcl-2 polyclonal antibody (diluted 1:300; Sigma) or mouse anti-PCNA monoclonal antibody (diluted 1:100; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at 4°C overnight and washed twice with phosphate-buffered saline prior to incubation with a secondary antibody (Santa Cruz Biotechnology, Inc.) at room temperature for 30 min. After washing, the sections were incubated with immunoglobulins conjugated with horseradish peroxidase. The reaction was then developed with 3,3′-diaminobenzidine substrate (2,13). Tissue sections were counterstained with hematoxylin or methyl green.

The nucleoli of SATB1-positive cells, cytoplasm of Bcl-2-positive cells (14) and nuclei of PCNA-positive cells were stained brown-yellow. Images of the sections were obtained (magnification, ×100) using the HPIAS-1000 High Resolution Color Pathological Image Analysis System (Tongji Medical College Qianping Imaging Engineering Co., Ltd., Shanghai, China) (15). Specimens with a positive cell ratio <30% were defined as negative.

Tissues collected in RPMI-1640 medium supplemented with 10% fetal bovine serum were processed routinely to generate single-cell suspensions (16,17). Suspensions were then fixed in 70% cold ethanol, treated with 10 g/l RNase and suspended and stained with 10 g/l propidium iodine (PI). After washing with PBS, the cells were stained directly with PI at a final concentration of 10 μg/ml and 2% Annexin-V Flous (Roche, Basel, Swizerland) in incubation buffer for 10 min. The cells were collected by FACSCalibur (BD Pharmingen, San Diego, CA, USA) following instrument set-up with controls (non-treated, stained cells) and two washes in PBS. In this experiment, cells with early apoptotic signals (stained with Annexin V) and cells with late death signals (stained with PI) were quantified and apoptotic cells were analyzed using the CellQuest software (18,19). Each assay was performed in triplicate.

Quantitative values were expressed as the mean ± SD. Statistical analysis was performed using the Pearson method and a Student's or Chi-squared test using SPSS 12.0 (for Windows; SPSS, Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The majority of brown-positive staining for in situ hybridization of SATB1 mRNA was homogeneously distributed within the nucleolus (Fig. 1A). SATB1 mRNA expression levels were found to be significantly higher in GBM than in the normal brain tissues (Fig. 1B; P<0.01).

Immunohistochemical staining revealed Bcl-2 protein expression in both the cytoplasm and cell membrane (Fig. 1A). PCNA protein expression was primarily detected in the nucleolus (Fig. 1A). Expression of Bcl-2 and PCNA protein was noted at significantly higher levels in GBM tissues than in normal brain tissues (Fig. 1B, P<0.01).

To quantify apoptotic cell death in tissues, ~1×10⁶ cells were double stained with Annexin V-FITC and PI at various times post-transfection. Apoptotic cell death was detected in GBM and normal brain tissues (Fig. 2A). FACS analysis identified a significantly higher number of apoptotic cells in GBM tissues than normal brain tissues (Fig. 2B; P<0.01).

SATB1 mRNA and Bcl-2 protein expression levels were associated with the survival rate of patients (P<0.01), but were not associated with patient gender, age and tumor size and site (Table I).

Statistical analysis revealed a positive correlation between SATB1 mRNA and Bcl-2 protein levels (P<0.05) and between SATB1 mRNA and PCNA protein levels (P<0.01). A negative correlation was identified between SATB1 and apoptosis (P<0.01) and between Bcl-2 and apoptosis (P<0.01). However, a positive correlation was observed between Bcl-2 and PCNA (P<0.01), whereas a negative correlation was found between PCNA and apoptosis (P<0.01; Table II).

SATB1-positive cases (+) were divided into two groups consisting of a survival rate <1 and ≥1 year. Differences between the groups were evaluated according to the Bcl-2 protein expression and identified as χ²=20.95 (P<0.001; Table III).