The present study protocol was approved by the Ethics Committee of The First Hospital of China Medical University (Shenyang, China). Paraffin-embedded specimens of 136 glioma cases were obtained from The First Hospital of China Medical University from January 2007 to December 2009. The cases comprised 73 men and 63 women, with a mean age of 53.3 years (range, 35–72 years). The clinicopathological features of the study population are summarized in Table I. Written informed consent was obtained from each patient enrolled in the study.

Samples were fixed in 10% formaldehyde solution (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and embedded in paraffin (Sigma-Aldrich, St. Louis, MO, USA) blocks, which were subsequently cut at a thickness of 4 µm. Sections were mounted on glass slides, deparaffinized in xylene and rehydrated in a graded series of alcohol (Sigma-Aldrich), followed by boiling in 10 mmol/l citrate buffer (Santa Cruz Biotechnology, Inc.) at pH 6.0 for 10 mins for antigen retrieval. Following inhibition of endogenous peroxidase activity by incubation in methanol containing 0.3% H₂O₂ (Santa Cruz Biotechnology, Inc.) for 30 min, sections were blocked with 2% bovine serum albumin (Sigma-Aldrich) for 30 min and incubated overnight at 4°C with rabbit anti-human c-Cbl monoclonal antibody (cat. no. ab32446; dilution, 1:200; Abcam, Cambridge, UK). Upon washing three times with phosphate-buffered saline, sections were incubated with horseradish peroxidase-conjugated mouse anti-rabbit immunoglobulin (Ig)G (cat. no. 2357; dilution; 1:200; Santa Cruz Biotechnology, Inc.) for 30 min, followed by reaction with 3,3′-diaminobenzidine (Sigma-Aldrich) and counterstaining with hematoxylin (Abcam). For the negative controls, the primary antibody was substituted with a nonspecific rabbit IgG antibody (cat. no. sc-2027; Santa Cruz Biotechnology, Inc.). A Eclipse 90i microscope was used to view the sections (Nikon Corporation, Tokyo, Japan).

Immunoreactivity was evaluated and scored semi-quantitatively by two pathologists who were blinded to the patients' clinical data. Using the double scoring system (staining intensity multiplied by staining area), the staining intensity was evaluated as follows: 0, no staining; 1, definite but weak staining; 2, moderate staining; and 3, strong staining. The staining area was scored as follows: 1, <35% of cells were stained; 2, 35–75% of cells were stained; and 3, >75% of cells were stained. High c-Cbl expression was defined as a score ≥4, whereas low c-Cbl expression was defined as a score <4.

Whole cell lysates were prepared from glioma tissue, and western blotting was performed as previously described (15). Protein concentration was determined with the Protein Quantitation kit (Bradford assay; Abcam), using bovine serum albumin as a standard. Lysates (20 mg) were solubilized in Laemmli sample buffer (Abcam) by boiling, and then resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by electrotransfer (45V for 15 h) onto a nitrocellulose membrane (Amersham; GE Healthcare Life Sciences, Chalfont, UK), which was then incubated with the rabbit anti-c-Cbl antibody at 4°C overnight, followed by incubation with the peroxidase-conjugated anti-rabbit IgG at room temperature for 1 h. Rabbit anti-mouse monoclonal α-tubulin (cat. no. ab52866; dilution, 1:2,000; Abcam) antibody served as a loading control. The immune complexes were visualized with an enhanced chemiluminescence western blotting detection system (Amersham; GE Healthcare Life Sciences). Quantity One 4.6 software (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used for desitometry analysis.

Total RNA was isolated from tissues using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol. RNA was reverse transcribed using SuperScript First-Strand Synthesis System (Invitrogen; Thermo Fisher Scientific, Inc.), and PCR amplification was performed using the following sets of sense and antisense primers: c-Cbl, sense 5′-CGCTAAAGAATAGCCCACCTTAT-3′ and antisense 5′-ATGGCCTCCAGCCCAGAACTGAT-3′; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), sense 5′-TGCACCACCAACTGCTTAGC-3′ and antisense 5′-GGCATGGACTGTGGTCATGAG-3′. Primers were designed using the Primer premier software 5.0 (Premier Biosoft International, Palo Alto, CA, USA) and synthesized by Invitrogen (Thermo Fisher Scientific, Inc.). The amplification reaction consisted of 40 cycles of 94°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec, and was conducted on an Applied Biosystems 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific, Inc.) with 1.0 µl of complementary DNA and SYBR Green Master Mix (Takara Bio, Inc., Otsu, Japan). RNA from 3 non-cancerous brain tissue samples, obtained from patients that had undergone surgery for drug-resistant temporal epilepsy at The First Hospital of China Medical University, were used as the control. Data were collected and analyzed using SDS version 2.3 software (Applied Biosystems; Thermo Fisher Scientific, Inc.). The expression levels of the target gene were normalized to those of GAPDH, and determined using the 2−ΔΔCq method (16). The experiment was performed in triplicate, and repeated three times.

Statistical analysis was performed with SPSS version 19.0 (IBM SPSS, Armonk, NY, USA). The χ² test was used to assess the association between c-Cbl expression and clinicopathological parameters. Patient survival curves were generated using the Kaplan-Meier method, and Cox regression analysis and log-rank test were used to identify independent prognostic factors. Data were expressed as the mean ± standard deviation, and statistical significance was defined as a two-tailed P<0.05.

RT-qPCR analysis of the messenger (m)RNA expression levels of c-Cbl in high-grade [anaplastic astrocytoma (AA) and GBM] and low-grade [low-grade astrocytoma (LGA)] glioma revealed that the c-Cbl transcript was upregulated in high-grade glioma samples, compared with low-grade glioma samples (P<0.05; Fig. 1A). Similarly, the protein levels of c-Cbl were increased in AA and GBM, compared with LGA (Fig. 1B).

Immunohistochemical analysis demonstrated that c-Cbl was mainly localized in the cytoplasm of malignant cells, and c-Cbl immunoreactivity was higher in high- vs. low-grade glioma (Fig. 2). The protein levels of c-Cbl were upregulated in 96 of 136 (70.6%) patients with glioma, and high c-Cbl expression was correlated with World Health Organization (WHO) grade (P<0.001) and Karnofsky performance status (KPS) score (P<0.001). There were no significant differences in c-Cbl expression with respect to gender, age or extent of resection (P>0.05, Table I).

Six patients were not available for follow-up, and therefore were excluded from the survival analyses. The remaining 130 patients were followed-up for 6–68 months. Multivariate analysis revealed that c-Cbl expression was independently associated with overall survival [hazard ratio (HR)=4.923, 95% confidence interval (CI)=3.163–7.662; P<0.001], and that c-Cbl protein expression and WHO grade were independent prognostic factors of progression-free survival (HR=6.181, 95% CI=3.854–9.915; P<0.001, and HR=10.247, 95% CI=9.009–11.655; P<0.001, respectively) (Table II). The Kaplan-Meier analysis with log-rank test indicated that high c-Cbl protein expression was associated with poor overall (P<0.001; Fig. 3A) and progression-free survival (P<0.001; Fig. 3B).