Identification of UBE2T as an independent prognostic biomarker for gallbladder cancer

Abstract

Gallbladder cancer is the most common biliary tract malignant tumor, with unfavorable patient outcomes. The present study aimed to identify potential diagnostic or prognostic biomarkers for gallbladder cancer. To do so, differentially expressed genes in the gallbladder walls and tumor tissues of patients with gallbladder cancer were analyzed via microarray. Furthermore, a protein-protein interaction network was constructed and genes with a degree score >10 were selected as hub genes. As ubiquitin conjugating enzyme E2T (UBE2T) was considered to be a hub gene, its expression was assessed via reverse transcription-quantitative (RT-q)PCR and immunohistochemistry (IHC). In addition, the association between UBE2T expression and the clinicopathological characteristics of patients with gallbladder cancer was analyzed using the χ² test. Furthermore, all patients were divided into high- and low groups based on UBE2T expression level and overall survival analysis was performed. Univariate and multivariate Cox regression analyses were performed to determine whether UBE2T may serve as an independent risk factor for gallbladder cancer. The results demonstrated that UBE2T expression was upregulated in the gallbladder walls and tumor tissues of patients with gallbladder cancer. Furthermore, UBE2T expression level was confirmed to be upregulated following RT-qPCR, and results from IHC demonstrated that UBE2T was predominantly expressed in the cytoplasm of gallbladder cancer cells. In addition, high UBE2T expression level was associated with clinical stage, T classification, N classification and M classification. The results from Univariate and multivariate analyses indicated that UBE2T expression level may be considered as an independent risk factor for gallbladder cancer. Taken together, the findings from this study suggested that high UBE2T expression level may contribute to the poor prognosis of patients with gallbladder cancer, and that UBE2T may act as an independent prognostic biomarker for these patients.

Keywords: gallbladder cancer; hub genes; microarray analysis; prognosis; ubiquitin conjugating enzyme E2T.

Figure 1.

Work flowchart of the present study. FC, fold-change; PPI, protein-protein interaction; UBE2T, ubiquitin conjugating enzyme E2T; qPCR, quantitative polymerase chain reaction.

Figure 2.

PPI network and the key modules of DEGs. (A) Intersection between three pairs of DEGs with FC>1.5 and P<0.05 (gallbladder tumor walls vs. gallbladder adenoma walls), and three pairs of DEGs with |FC|>2 and P<0.05 (gallbladder tumor tissues vs. gallbladder adenoma tissues). For microarray analysis, gallbladder adenoma was used as the control. Blue represents DEGs in gallbladder tumor walls and pink represents DEGs in gallbladder adenoma walls. (B) PPI network of 177 DEGs was constructed using Cytoscape software. In the PPI network, each node represents a protein and each edge represents a PPI. (C) DEGs with degrees ≥10 were selected from the PPI network as the hub genes for the key modules. Hierarchical clustering analysis of the hub genes in the datasets, (D) three pairs of gallbladder tumor walls vs. gallbladder adenoma walls and (E) three pairs of gallbladder tumor tissues vs. gallbladder adenoma tissues. A10951, A10956 and A10957; gallbladder adenoma wall samples; A10958, A10960 and A10963, gallbladder tumor wall samples; A11132, A11135 and A11138; gallbladder adenoma tissue samples; A11139, A11142 and A11143, gallbladder tumor tissue samples. PPI, protein-protein interaction; DEG, differentially expressed gene; FC, fold-change.

Figure 3.

Biological processes and GO analysis of the hub genes. (A) A network of biological processes associated with the hub genes was constructed using the Biological Network Gene Ontology tool. The node color is associated with the adjusted p-value. P<0.01 was considered to indicate significantly enriched processes. GO analysis, including (B) molecular function and (C) cell component was performed using the Database for Annotation, Visualization and Integrated Discovery. GO, Gene Ontology.

Figure 4.

Co-expression network and KEGG pathway analysis of the hub genes. (A) A co-expression network was constructed using cBioPortal. Nodes with bold black outline represent the hub genes, while nodes with thin black outline represent the co-expressed genes. (B) KEGG pathway analysis of the hub genes was performed using the Database for Annotation, Visualization and Integrated Discovery. KEGG, Kyoto Encyclopedia of Gene and Genomes.

Figure 5.

Reverse transcription-quantitative PCR analysis of UBE2T expression in 30 paired gallbladder cancer tissues and adjacent noncancerous tissues. β-actin was used as the internal control. ****P<0.0001. UBE2T, ubiquitin conjugating enzyme E2T.

Figure 6.

Western blot analysis of UBE2T expression in 12 pairs of gallbladder cancer tissues and adjacent noncancerous tissues. β-actin was used as the internal control. UBE2T, ubiquitin conjugating enzyme E2T; N, normal tissues; C, cancer tissues.

Figure 7.

UBE2T was upregulated and associated with poor prognosis in patients with gallbladder cancer. (A) Representative hematoxylin and eosin staining images and (B) immunohistochemistry analysis of UBE2T expression in gallbladder cancer tissues and adjacent noncancerous tissues (magnification, ×200). (C) Kaplan-Meier overall survival curves for 127 patients with gallbladder cancer, stratified by high and low UBE2T expression levels. UBE2T, ubiquitin conjugating enzyme E2T.