Glioma samples were collected from the 50 patients, enrolled between September 2011 and September 2012, who underwent surgery in the Department of Neurosurgery at the National Institute of Mental Health and Neurosciences (NIMHANS; Bangalore, India). Informed consent was obtained from all patients included in the study. Tissues were bisected and one half was placed in RNAlater (Sigma-Aldrich, St. Louis, Missouri, USA) and snap frozen in liquid nitrogen, then kept at −80° until the isolation of the DNA. The other half was sent for histopathological analysis. Histopathological diagnosis was made using the World Health Organization (2007) grading system (14) in the Department of Neuropathology. Control brain tissues were collected from 5 patients undergoing anterior temporal lobectomy for mesial temporal sclerosis. The study was approved by the NIMHANS Human Ethics Committee.

Tissues were analyzed histologically and those containing >95% tumor cells were used for further analysis. DNA and RNA isolation was performed using an All Prep DNA/RNA Mini isolation kit (Qiagen GmbH, Hilden, Germany) and quantified by Nanodrop ND 2000c (Thermo Fisher Scientific, Waltham, MA, USA). DNA Samples with a purity of 1.75–1.85 and RNA samples with a purity of 1.95–2.05 (A_260/280) were used in this study. RNA stability was checked by formaldehyde agarose gel electrophoresis.

Quantitative PCR amplification was performed on the Applied Biosystems 7500 (Applied Biosystems, Foster City, CA, USA), using SYBR® Select master mix (Invitrogen, Life Technologies, Carlsbad, CA, USA) with a total reaction volume of 20 µl. The PCR mixtures were then subjected to 50°C for UDG activation for 2 min and activation at 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 15 sec, annealing at 53°C for 15 sec and extension at 72°C for 60 sec. Each template-primer pair was tested in three replicates to estimate the mean Ct. The comparative Ct method was used to check the copy number variation in the BMI-1 gene. The primer sequence was as follows: β-actin forward, 5′-GCATTTAGGTAAGGGGTGGA-3′ and reverse, 5′-GGGTACACAGACGAAGCAGA-3′; and BMI-1 forward, 5′-TGTGTGCTTTGTGGAGGGTA-3′ and reverse, 5′-CATTTCCACAGATTGCAGGA-3′.

Total RNA (1 µg) was reverse transcribed into cDNA using MMLV-reverse transcriptase, oligo-dT, dNTPs and buffer following the manufacturer's instructions (Life Technologies). The sequences of the BMI-1 and GAPDH detection probes and primers were as follows: BMI-1 sense, 5′-CTGGTTGCCCATTGACAGC-3′; BMI-1 antisense, 5′-CAGAAAATGAATGCGAGCCA-3′; probe for BMI-1, 5′-CAGCTCGCTTCAAGATGGCCGC-3′, labeled with FAM and TAMRA as the reporter dye; GAPDH sense, 5′-GAAGGTGAAGGTCGGAGTCAAC-3′; GAPDH antisense, 5′-CAGAGTTAAAAGCAGCCCTGG T-3′; GAPDH probe, 5′-TTTGGTCGTATTGGGCGCCT-3′, labeled with VIC dye. The quantitative assay amplified 1 µl of cDNA in three replicates using the aforementioned primers and probes, and the Taqman Universal master mix (Applied Biosystems). Comparative analysis by the 2^−ΔΔCt method was used to compare the expression in each gene.

Whole-cell protein extracts were obtained from all 50 tissues. Cell lysates were prepared in cold lysis buffer [50 mM Tris, 150 mM NaCl, 1 mol/l EDTA, 0.1% sodium dodecyl sulfate, 1% Triton X-100, 1 mol/l phenylmethylsulfonyl fluoride (pH 8.0); SRL Ltd., Bangalore, India]. The lysate was collected and stored at −80°C. The protein content in the lysates was measured by Bradford assay. For western blot analysis, 50 µg protein was resolved on 12% SDS-PAGE gels, transferred onto polyvinyldifluoride membranes (Bio-Rad Laboratories Inc., Hercules, CA, USA) and subsequently incubated in blocking buffer (5% bovine serum albumin, 1% Tween 20 in 20 mmol/l Tris-buffered saline (pH 7.6); SRL Ltd.] for 1 h. The blots were incubated with mouse anti-human monoclonal IgG₁ antibody against BMI-1 (#sc-390443; 1:1,000 dilution), followed by rabbit anti-mouse IgG horseradish peroxidase-conjugated antibody (#sc-358914; 1:5,000 dilution) (both Santa Cruz Biotechnology Inc., Dallas, TX, USA), and detected by enhanced chemiluminescence kit (Bio-Rad Laboratories Inc.) according to the manufacturer's instructions. Equal loading of protein was confirmed by stripping the blots and re-probing with monoclonal mouse anti-human β-tubulin antibody (#sc-58882; 1:2,000 dilution; Santa Cruz Biotechnology Inc.). Bands were detected by chemiluminescence and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

R-2.3.1 (R Foundation for Statistical Computing, Vienna, Austria) was used for the statistical analysis. Due to the non-normal distribution of the samples, the statistical evaluation was performed using non-parametric tests. Comparison between mRNA and protein expression levels in the different grades of glioma was performed using the Kruskal-Wallis test. Correlation between mRNA and protein was analyzed using Spearman's correlation test. P<0.05 was used to indicate a statistically significant difference.

A total of 50 patients with varying grades of glioma, enrolled between 2011 and 2012, were included in this study. All tumors were pathologically confirmed as glioma, and the patients underwent radiological and histopathological examinations to establish the clinical profile. The age of the patients ranged from 8–60 years (mean, 35.6±14.86).

BMI-1 gene amplification analysis was performed using SYBR Green quantitative PCR in all tumor samples and non-glioma brain tissues. The copy number variation was analyzed in comparison with the β-actin gene. The comparative Ct value analysis did not show any difference between the BMI-1 and β-actin genes in the glioma samples or the non-glioma brain tissues.

The BMI-1 mRNA levels were analyzed in all the glioma samples. BMI-1 mRNA expression was found to be overexpressed in the glioma tissues compared with the non-glioma samples. In total, 36 out of 50 samples demonstrated overexpression (72%.0), which was statistically significant when compared with the non-glioma control tissues (P=0.01). The median fold-change of expression was 2.055. The mRNA expression ranged from 1.41-fold to 60.97-fold. Grade-wise gene expression analysis was performed and BMI-1 was found to be expressed more in high-grade glioma than in low-grade glioma (Kruskal-Wallis rank sum test; P=0.025), as shown in Fig. 1. The clinical details are provided in Table I.

BMI-1 protein levels were analyzed in all glioma tissue samples. BMI-1 protein expression was found to be overexpressed in the glioma tissues compared with the non-glioma samples upon western blot analysis (Fig. 2). In total, 37 out of 50 samples demonstrated overexpression (74.0%), which was statistically significant compared with the non-glioma control tissues (P=0.037). The median fold-change of expression was 2.0408. The protein expression ranged from 1.47-fold to 16.36-fold. Grade-wise gene expression analysis was performed and BMI-1 was found to be expressed more in high-grade glioma than in low-grade glioma (Kruskal-Wallis rank sum test; P=0.024), as shown in Fig. 1. There were no other clinical correlations with protein expression (Table I).

The correlation between BMI-1 mRNA and protein levels was assessed by Spearman's correlation test. A positive correlation was found with a coefficient ρ-value of 0.474 (Fig. 3); this was statistically significant (P=0.0005). This indicates that the protein expression was concordant with the mRNA expression, and suggests that there may not be much post-translational regulation in this protein in glioma.