CCNB2, TOP2A, and ASPM Reflect the Prognosis of Hepatocellular Carcinoma, as Determined by Weighted Gene Coexpression Network Analysis

Background Hepatocellular carcinoma (HCC) is characterized by increased mortality and poor prognosis. We aimed to identify potential prognostic markers by weighted gene coexpression network analysis (WGCNA), to assist clinical outcome prediction and improve treatment decisions for HCC patients. Methods Prognosis-related gene modules were first established by WGCNA. Venn diagrams obtained intersection genes of module genes and differentially expressed genes. The Kaplan-Meier overall survival curves and disease-free survival curves of intersection genes were further analyzed on the Gene Expression Profiling Interactive Analysis website. Chi-square tests were performed to explore the associations between prognostic gene expressions and clinicopathological features. Results CCNB2, TOP2A, and ASPM were identified as both prognosis-related genes and differentially expressed genes. TOP2A (HR: 1.7, P = 0.003) and ASPM (HR: 1.8, P < 0.001) exhibited a significant difference between the high- and low-expression groups in the overall survival analysis, while CCNB2 (HR: 1.4, P = 0.052) was not statistically significant. CCNB2 (HR: 1.5, P = 0.006), TOP2A (HR: 1.7, P < 0.001), and ASPM (HR: 1.6, P = 0.003) were all statistically significant in the disease-free survival analysis. All three genes were significantly associated with race and fetoprotein values (P < 0.05). CCNB2 expression was associated with tumor stage (P = 0.01), and ASPM expression was associated with new tumor events (P = 0.03). Conclusion Overexpression of CCNB2, TOP2A, and ASPM are associated with poor prognosis, and these genes could serve as potential prognostic markers and therapeutic targets for HCC.


Introduction
Hepatocellular carcinoma (HCC) is the third most common cause of cancer mortality worldwide [1]. The mechanisms by which hepatitis B virus, hepatitis C virus, alcohol, fatty liver disease, and other environmental factors, such as aflatoxin, cause liver cancer remain unclear. However, advances in genomics provide essential information about tumor initiation and progression [2]. The complex genetic background of HCC makes current clinical staging methods, including the Barcelona Clinic Liver Cancer algorithm or the tumor node metastasis (TNM) staging, insufficient to predict patient prognosis. Indeed, patients with the same HCC stage may have significantly different outcomes. Besides, the high recurrence and metastasis rates after ablation or surgical resection lead to a low survival rate in patients with HCC. These outcomes present an urgent requirement for improved prognostic estimates other than staging [3]. To date, thousands of HCC genomes have been sequenced globally, and most driver gene mutations, structural variants, fusion genes, copy number alterations, and viral integration events have been established [4]. This vast amount of new data provides an opportunity to understand the molecular basis of HCC better [5]. However, to utilize this scientific information, knowledge of the available database resources and bioinformatics tools are indispensable. To improve the treatment decisions and the overall survival of patients with HCC, we summarized the currently available databases supporting HCC research to aid in the identification of potential prognostic markers of clinical outcome prediction.
A coexpression analysis is an efficient method to describe free-scale gene coexpression networks. The weighted gene coexpression network analysis (WGCNA), an algorithm based on large-scale datasets and modules of highly correlated genes, was used to explore associations between gene sets and clinical features and to identify candidate biomarkers [6,7]. This approach has been successfully applied in multiple tumors, such as clear cell renal cell carcinoma [8], glioblastoma [6], pancreatic carcinoma [9], adrenocortical carcinoma [10], breast cancer [11], and HCC [12,13]. We mined prognostic markers by constructing a coexpression network and performing differential gene analysis and survival analysis to verify their prognostic values.

WGCNA Analysis.
To identify hub genes associated with phenotypes, we analyzed GSE54236 by the R package "WGCNA" as follows [19,20]. (1) Data procession: the median absolute deviation (MAD) of each gene expression between samples was calculated. We selected genes with MAD greater than the 70% quantile interval of the MAD of all genes and greater than 0.01 for the subsequent analysis. Missing values were checked by the "goodSamplesGenes" function, and outlier samples were identified by the "hclust" function to eliminate possible sample interference. (2) Network construction: soft power was determined with a threshold of 0.85 by "pickSoftThreshold" function to make the constructed network in line with a scale-free network. Then, we constructed the network by "blockwiseModules" function and showed each module by a hierarchical clustering tree.
(3) Modules related to phenotypes: To explore phenotyperelated modules, we analyzed the associations between modules and phenotypes. We calculated the correlations of modules and genes (module membership), and phenotypes and genes (gene significance) by the Pearson correlation analysis. Genes highly associated with phenotypes in modules, with correlation coefficients > 0:5 and P < 0:05 were selected for further analysis. (4) Hub genes identified: the topological overlap matrix was calculated from module genes of interest, and hub nodes with an edge-adjacency threshold of 0.2 were exported to visualize in Cytoscape (version 3.7.2) by "exportNetworkToCytoscape" function.
2.3. Pathway Enrichment Analysis. We performed Gene Ontology (GO), including biological processes, cellular components, and molecular functions, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of module genes of interest by the "clusterProfiler" package [21]. Next, the top 10 most significant enrichment pathways were graphically displayed. Moreover, protein-protein interaction (PPI) analysis was performed on the Metascape website (http://metascape.org/gp/index.html#/main/step1), which determined the PPI network, Molecular Complex Detection (MCODE) components, and the top three enrichment pathways of each component.

Survival Analysis.
We performed the Kaplan-Meier overall survival curves and disease-free survival curves of intersection genes on the Gene Expression Profiling Interactive Analysis (GEPIA) website [23], a web-based tool for analyzing Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) project data from tumors and normal samples.

Correlation Analysis of Clinicopathological Features.
To further explore the associations between prognosis gene expressions and clinicopathological features, RNA-seqcounts of the TCGA liver cancer (n = 424) and phenotypes (n = 469) were downloaded from the UCSC Xena (https:// xenabrowser.net/datapages/). Genes were divided into highand low-expression groups, according to the median of gene expressions. We compared the differences between the two groups in clinicopathological features by Chisquare tests. All analyses were performed by R (version 3.6.0). Bilateral P values less than 0.05 were considered statistically significant.

Weighted Coexpression Network Construction and Key
Module Identification. To mine coexpression module genes for HCC, the top 30% MAD of all genes in GSE54236 were extracted, which provided 5,612 genes for further analysis. No outlier samples were identified in the sample clustering tree, so no samples were removed from the analysis 2 BioMed Research International ( Figure 1(a)). Next, we constructed the gene-gene similarity network. We set the soft power as 7 to ensure a scale-free network ( Figure 1(b)). By the "blockwiseModules" function, we divided the network into 11 modules with similar gene expressions ( Figure 1(c)). Then, we analyzed the relationships between modules and phenotypes. Results showed that the red module had a significant negative correlation with doubling time and survival time ( Figure 1(d)). Figures 1(e) and 1(f) further proved the associations between the red module genes and doubling time and survival time with correlation coefficients > 0:5 and P < 0:001. Finally, we identified 153 hub genes in the red module as key genes by Cytoscape.

Gene Function and Pathway Enrichment Analyses.
To further understand the biological functions and signaling pathways in which red module genes participate, we performed enrichment analysis for 153 hub genes by GO and KEGG. Biological processes were mainly enriched in chromosome segregation, nuclear division, and organelle fission (Figure 2(a)). Associated cell components were mainly present in the chromosomal region, chromosome, centromeric region, and condensed chromosome (Figure 2(b)). Molecular functions occurred in microtubule motor activity, tubulin binding, and microtubule activity (Figure 2(c)). KEGG pathways were considerably enriched in the cell cycle and oocyte meiosis (Figure 2(d)). PPI and MCODE analyses revealed that red module genes were divided into five components (Figures 2(e) and 2(f)). The top three enrichment pathways for each component were shown in Supplementary Table 1. These genes mainly participated in the cell cycle and divisions.

Identification of Intersection Genes.
We explored the intersection genes both related to prognosis and differentially expressed in tumors and paracancerous nontumor tissues by the Venn diagram. There were 510, 75, and 415 DEGs for GSE60502, GSE64041, and GSE45114, respectively. We found three intersection genes (CCNB2, TOP2A, and ASPM) which were both red module genes and DEGs in the Venn diagram ( Figure 3(a)).

Associations between Gene Expressions and
Clinicopathological Features. To explore the associations between CCNB2, TOP2A, ASPM, and clinicopathological features, we divided three genes into the low-and highexpression groups according to the median of gene expressions in the TCGA. Through Chi-square tests, we found that all three genes were significantly associated with race and fetoprotein (AFP) values (P < 0:05). CCNB2 was significantly associated with tumor stage (P = 0:01), while there was no significant association between TOP2A or ASPM and tumor stage. Moreover, a significant association between ASPM and new tumor events was observed (P = 0:03), as shown in Table 1. We did not found significant associations between CCNB2, TOP2A, ASPM, and BMI, adjacent hepatic tissue inflammation, fibrosis Ishak score, residual tumor, vascular tumor, and Child-Pugh classification, as shown in Supplementary Table 2.

Discussion
Clinical prognostic information is limited for patients of HCC. Several staging systems that attempt to predict patient prognosis and guide treatment modality have been proposed for HCC. However, the clinical features of these systems have drawbacks concerning the efficacy of treatment guidance and prognostic accuracy. Staging systems based on clinical features lack universal applicability, having been designed using data mostly acquired from a Western population or by being too rigid in suggesting therapy for some problematic prognostic factors [24]. Therefore, it is necessary to identify prognostic markers for patients with HCC. Patients with HCC present with features of high heterogeneity, metastasis, recurrence, and mortality and provide potential targets for prognosis prediction and treatment monitoring [25]. On the other hand, there have been very few reports detailing data mining by combining coexpressed and differentially expressed genes to explore HCC prognostic markers.
In this study, we explored genes associated with HCC prognosis using WGCNA, a method more frequently used to investigate phenotype-related coexpression module genes than to analyze DEGs. We identified 153 hub genes in the red module that were significantly negatively associated with doubling time and survival time, symbolizing a poor prognosis. Besides, the strong correlations of modules and genes and phenotypes and genes further proved the robust prognostic efficacy of red module genes. Second, gene function and pathway enrichment analyses indicated that red module genes were mainly engaged in the cell division and proliferation of HCC. DEGs were mined by combining GSE60502, GSE64041, and GSE45114, making results more credible. Further combining overall and disease-free survival analysis using the GEPIA website, we identified that TOP2A, ASPM, and CCNB2 were promising prognostic markers and potential therapeutic targets for HCC. It is worth noting that the overall survival analysis was based on patient all-cause mortality, and disease-free survival analysis was based on disease progression or death. Therefore, overall survival analysis endpoints were optimal observation endpoints without subjective judgment bias. In our study, both overall and disease-free survival analyses were evaluated by public databases.

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We further investigated associations between TOP2A, ASPM, CCNB2, and clinicopathological features by Chisquare tests. We found significant associations between three prognostic genes and AFP levels; therefore, we speculated that these three genes were potential markers of the development and progression of HCC. However, whether these genes might become favorable diagnostic markers for HCC and specific mechanisms required further experimental verification. Importantly, high CCNB2 expression was strongly associated with tumor stage (P = 0:01), and high ASPM expression was associated with new tumor events (P = 0:03). These findings indicated that the overexpression of CCNB2 and ASPM accelerated the possibility of HCC deterioration and metastasis, suggesting a worse prognosis for HCC patients; hence, more aggressively integrated treatment and assessment were needed when these genes were found to be overexpressed.
TOP2A, DNA topoisomerase II alpha encoding a DNA topoisomerase, regulates the topologic states of DNA and controls tumor cell response. Wong et al. [26] showed that TOP2A overexpression in HCC tumors, relative to adjacent nontumors, correlated with histological grading, microvascular invasion, tumor deterioration, shorter survival, and chemoresistance, which was consistent with our clinicopathological features correlation analysis [27]. TOP2A targeted therapeutic drugs, such as doxorubicin, were suggested when

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TOP2A was overexpressed in HCC. Overexpression of microRNA-23a potentiated the response of HCC to the TOP2A targeted drugs etoposide and doxorubicin in vivo and in vitro, while suppressing another topoisomerase, TOP1, but not altering TOP2A expression levels [28]. Another study showed that TOP2A was targeted for proteasomal degradation by histone deacetylase inhibitors by activating casein kinase 2α and GSK3β double phosphorylation, highlighting a novel potential mechanism of HCC treatment [29]. The significance of TOP2A in tumor aggressiveness and chemoresistance indicate that the mechanisms through which it functions require further exploration. ASPM encodes an abnormal spindle-like, microcephalyassociated protein that participates in mitosis and is reported to be a recurrence, invasion, metastasis, and prognostic marker for HCC. However, the specific molecular mecha-nism and pathways through which ASPM function have not been reported [30,31]. CCNB2 encodes cyclin B2, the cell cycle mediated by transforming growth factor β, which, according to the GEPIA website, did not have a statistically significant influence on overall survival. However, we further analyzed the influence on CCNB2 on the UALCAN website [32] (http://ualcan.path.uab.edu/index.html) and found a statistical significance with P < 0:001. The variation in these results could be attributed to the different sample sizes and sequencing databases. A review of the literature revealed that CCNB2 expression is increased in HCC tissues compared to adjacent nontumor tissues [33], and CCNB2 is a target molecule following knockdown of XPOT [34], TPX2 [35], KPNA2 [36], and TMEM9 [37]. The prognostic value and target therapy of the hub genes for HCC patients remain to be confirmed by further clinical studies.  There were some limitations in our study. All of these sequencing data were from tissue samples and may not be suitable for unresectable patients whose liver tissues may be unavailable. Given that bioinformatics predictions are obtained in silico, further experimental validation is required. Additionally, we analyzed expression profiling by the array in the GEO datasets and concluded that TOP2A, ASPM, and CCNB2 were promising prognostic markers and potential therapeutic targets. However, other prognostic markers, such as methylation, microRNA, lncRNA, and circRNA, should be explored, and the data should be integrated with the findings of the present study to propose a new treatment algorithm that incorporates these markers in determining how aggressive is the treatment approach.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Ethical Approval
This article does not contain any studies with human participants performed by any of the authors.