The present study was approved by the Ethics Committee of Beijing Tiantan Hospital (Beijing, China). Written informed consent was obtained from all patients. Tissue samples were obtained from patients during surgery at Beijing Tiantan Hospital between January 2006 and December 2009. In total, 220 adult patients diagnosed with glioma were enrolled in the present study. The glioma tumor grade of patients was diagnosed histologically according to the 2007 WHO classification (2), as follows: 97 patients possessed grade II disease; 34 patients possessed grade III disease; and 89 patients possessed grade IV disease. Control non-cancerous brain tissue specimens were obtained from 5 patients with craniocerebral injuries.

All patients had undergone prior surgical resection, followed by adjuvant radiation and alkylating agent-based chemotherapy. Post-operative follow-up data was gathered for all patients, and the clinical characteristics and pathological findings, including the age at diagnosis, gender, extent of resection, isocitrate dehydrogenase-1 (IDH1) mutation status and temozolomide (TMZ) chemotherapy, were obtained.

Total RNA was extracted from frozen tumor tissues using the Ambion mirVana™ miRNA Isolation kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol. RNA concentration and quality were measured using the NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Inc., Wilmington, DE, USA), and its integrity was determined using the Agilent Bioanalyzer 2100 (Agilent Technologies, Inc., Santa Clara, CA, USA). Gene expression analysis was performed using Agilent Gene Expression oligo microarrays (Agilent Technologies, Inc.) containing >41,000 probe sets for ~27,958 human genes using the Agilent One-Color Microarray-Based Gene Expression Analysis (catalog no., G4140–90040; Agilent Technologies, Inc.), according to the manufacturer's protocol. Average values of the replicate spots for each gene were background subtracted, normalized, log₂-transformed and subjected to additional analysis.

Formalin-fixed, paraffin-embedded (FFPE), 5 µm-thick tissue sections were used for immunohistochemical analysis, which was performed using a streptavidin-biotin immunoperoxidase assay, as previously described (19). Immunohistochemical staining was performed using anti-human mouse monoclonal anti-UBE2C antibody (catalog no., ab201979; dilution, 1:100; Abcam, Cambridge, MA, USA). Staining for UBE2C in the nucleus or the cytoplasm was scored as absent, weak, moderate or strong. Absent or weak staining was categorized as negative, while moderate or strong staining was categorized as positive. Controls consisted of tissues without primary antibody and non-cancerous control tissues, and were included in all experiments to ensure the quality of staining. In total, 2 pathologists performed immunohistochemistry scoring independently and blindly.

Statistical analyses were performed using a χ² test or Fisher's exact test with 2×2 tables. Kaplan-Meier survival time analysis was used to estimate the survival time distributions, and the log-rank test was used to assess the statistical significance between different groups. Multivariate analysis was performed using Cox's proportional hazards model, and the risk ratio and 95% confidence intervals were calculated for each characteristic. Statistical analysis was performed using SPSS version 13.0 for Windows (SPSS, Inc., Chicago, IL, USA). Two-tailed tests were performed. P<0.05 was considered to indicate a statistically significant difference.

A microarray was performed to detect the UBE2C mRNA expression in 220 glioma tissues and 5 non-cancerous brain specimens. The present study observed significant differences in the UBE2C expression between non-cancerous brain, low-grade glioma and high-grade glioma tissues. Fig. 1 reveals that the expression levels of UBE2C mRNA in grade II tumor tissues were significantly decreased compared to grade III (P<0.0001) and IV (P<0.0001) glioma tissues. However, there is no significant difference in UBE2C expression levels between grade III and IV gliomas.

To additionally detect the expression of the UBE2C protein in glioma tissues, immunohistochemistry was performed. As illustrated in Fig. 2, UBE2C was not expressed in non-cancerous brain tissues. There was significant UBE2C protein expression in 46 of 97 (47.4%) low-grade gliomas. In total, 73.5% of patients (25 out of 34 patients) with anaplastic (grade III) gliomas demonstrated significant UBE2C protein expression, whereas 78.8% (63 of 80) of glioblastomas (GBM; grade IV) exhibited significant UBE2C staining (2).

The present study evaluated the association of UBE2C expression with clinicopathological variables (Table I). According to the strength of staining for UBE2C in cells, which was classified as absent, weak, moderate or strong staining, the glioma tissues were divided into two groups, negative and positive. UBE2C expression was not associated with the patient age, patient gender, extent of resection, IDH1 mutation status and TMZ chemotherapy.

The Kaplan-Meier method was performed to elucidate the association of UBE2C expression with the prognosis of glioma patients. The median expression of UBE2C in anaplastic glioma patients was 2.1061 and the median expression of UBE2C in GBM patients was 1.3937. When the median expression values of UBE2C (the relative expression level detected by microarray) was used as the cut-off point, patients with a higher UBE2C expression possessed a significantly decreased overall survival time in patients with anaplastic glioma (P<0.01; Fig. 3A) and those with GBM (P=0.035; Fig. 3B).

Multivariate analysis of 80 patients with GBM was performed for UBE2C expression and pathological predictors for survival time using Cox's proportional hazard regression analysis. The results demonstrated that the UBE2C expression, extent of resection, TMZ chemotherapy and IDH1 mutation status were independent prognostic factors of patient prognosis (Table II). Therefore, the expression level of UBEC1 may be a valuable prognostic marker for glioblastoma patients.