CCNB2, NUSAP1 and TK1 are associated with the prognosis and progression of hepatocellular carcinoma, as revealed by co-expression analysis

Abstract

The mortality rate associated with hepatocellular carcinoma (HCC) is the third highest among all digestive system tumors. However, the causes of HCC development and the underlying mechanisms have remained to be fully elucidated. In the present bioinformatics study, genetic markers were identified and their association with HCC was determined. The mRNA expression datasets GSE87630, GSE74656 and GSE76427 were downloaded from the Gene Expression Omnibus (GEO) database. A total of 96 differentially expressed genes (DEGs) were screened from the 3 GEO datasets, including 25 upregulated and 71 downregulated genes. DEGs were uploaded to the database for Annotation, Visualization and Integrated Discovery to screen for enriched Gene Ontology terms in various categories and the Search Tool for the Retrieval of Interacting Genes/Proteins was used to identify the interactions and functions of the DEGs. A total of 3 genetic markers were identified in a stepwise pathway and functional analysis in a previous study. The association of the genetic markers with prognosis was analysed using the UALCAN online analysis tool. Regression analysis was also performed to identify the relationship between HCC grade and disease recurrence and the expression of genetic markers using The Cancer Genome Atlas HCC dataset. In addition, the expression of the 3 genetic markers in HCC tissues was determined using reverse transcription-quantitative PCR, the Oncomine database and the Human Protein Atlas database. The expression levels of the 3 genetic markers cyclin B2 (CCNB2), nucleolar and spindle-associated protein 1 (NUSAP1) and thymidine kinase 1 (TK1) were significantly correlated with each other and high mRNA expression of CCNB2 was significantly associated with poor overall survival of patients with HCC. Receiver operating characteristic curve analysis indicated that NUSAP1 and TK1 were capable of distinguishing between recurrent and non-recurrent HCC. Furthermore, CCNB2, NUSAP1 and TK1 were highly correlated with the HCC grade. It was also indicated that the mRNA expression of CCNB2, NUSAPA and TK1 was increased in primary HCC tissues when compared with that in adjacent tissues. The present study identified that the CCNB2, NUSAP1 and TK1 genes may serve as prognostic markers for HCC, and may be of value from the perspectives of basic research and clinical treatment of HCC.

Keywords: biological marker; gene expression profiling; hepatocellular carcinoma; hub genes; prognosis.

Figure 1

Hierarchical clustering heatmaps of differentially expressed genes screened on the basis of |FC|>2.0 and a corrected P-value <0.05. Heatmaps generated using the datasets (A) GSE87630, (B) GSE74656 and (C) GSE76427 are provided. The intensity of the color scheme is scaled to expression values (log₂FC), which are Z-score standardized per gene. The color bar above the heatmap represents the sample groups, with orange indicating the tumor sample and blue representing normal samples. FC, fold change.

Figure 2

Common differential genes in each expression microarray (GSE87630, GSE74656 and GSE76427). Red represents upregulated genes, green represents downregulated genes.

Figure 3

Enrichment analysis of DEGs in HCC. (A) GO enrichment significance items of DEGs in different functional groups. (B) Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis for DEGs in HCC. FC, fold change; HCC, hepatocellular carcinoma; DEG, differentially expressed gene; GO, gene ontology.

Figure 4

(A) PPI network. Circles represent genes, lines represent the interaction of proteins between genes and the results within the circle represent the structure of proteins. Line colors indicate the interaction between the proteins. (B) Visualization of the 15 hub genes selected from the PPI network using the maximal clique centrality algorithm and the cytoHubba plugin. Edges represent the protein-protein associations. Red octagons represent DEGs with the PPI scores. Yellow octagons represent DEGs with low PPI scores. DEG, differentially expressed gene; PPI, protein-protein interaction.

Figure 5

(A-C) CCNB2, NUSAP1 and TK1 levels were highly correlated to one another (data from TCGA). (D-F) Survival analysis of genetic markers in the TCGA datasets. (D) CCNB2, (E) NUSAP1 and (F) TK1. Red lines represent high expression of the genes and blue lines represent low/medium expression. (G-I) Correlation between the expression levels of genetic markers in the TCGA dataset and the disease progression of HCC. (G) CCNB2, (H) NUSAP1, (I) TK1. Grade means histological grade. (J-L) ROC analysis of genetic markers in TCGA database. (J) CCNB2, (K) NUSAP1 and (L) TK1. ROC curves and AUC statistics to evaluate the capacity of the distinguishing recurrent and non-recurrent hepatocellular carcinoma are provided. CCNB2, cyclin B2; NUSAP1, nucleolar and spindle-associated protein 1; TK1, thymidine kinase 1; TCGA, The Cancer Genome Atlas; ROC, receiver operating characteristic; AUC, area under curve.

Figure 6

Validation of the expression of three genes at the transcriptional and translational level by Oncomine database and The Human Protein Atlas database (immunohistochemistry). (A) CCNB2, (B) NUSAP1 and (C) TK1. Magnification, x100. CCNB2, cyclin B2; NUSAP1, nucleolar and spindle-associated protein 1; TK1, thymidine kinase 1; HCC, hepatocellular carcinoma.

Figure 7

Overexpression of markers in HCC. (A) CCNB2, (B) NUSAPA1 and (C) TK1 (n=16). ^***P<0.001 compared with the adjacent tissues. (D-F) CCNB2, NUSAP1 and TK1 levels were highly correlated with one another (n=16). CCNB2, cyclin B2; NUSAP1, nucleolar and spindle-associated protein 1; TK1, thymidine kinase 1; HCC, hepatocellular carcinoma.