# An Eight-mRNA Prognostic Model to Predict Survival in Hepatic Cellular Cancer

Authors: Dong Xia; Xuebin Liao; Huamao Zhang
Journal: Computational and Mathematical Methods in Medicine
 (2023)

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## Abstract

Background. Transcriptional dysregulation plays a critical role in the onset and development of malignant tumors. Employing gene dysregulation to forecast the change of tumors is valuable for cancer diagnosis. However, the prognostic prediction for HCC using combined gene models remains insufficient. Methods. The expression profiles of GSE103512 and TCGA-LIHC were downloaded. Gene Ontology (Go) was used to evaluate the overlapping differential genes (DEG) in TCGA and GSE103512. The core genes in the critical module most significantly related to HCC were obtained by WGCNA. Eight genes most significantly related to HCC and OS were identified by reweighted coexpression network analysis and Cox regression. Results. We selected eight genes, FZEB1, CDK1, RAD54L, COL1A2, ATP1B3, CASP8, USP39, and HOXB7. Moreover, we constructed an eight-gene model and forecasted the prognosis of HCC. ROC curve of the eight-mRNA prognostic model was screened out (AUC=0.635), suggesting that this model exhibited a good prediction performance. Survival analysis showed that the survival rate of patients in the high-risk group was significantly lower than that in the low-risk group. Conclusion. The eight-mRNAs model might forecast the OS of HCC patients and advance remedial decision-making.


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## Body

## 1. Introduction

Hepatocellular carcinoma (HCC) is the world’s fifth most common malignant tumor, with more than 700000 new cancer cases worldwide every year [1, 2]. Although the primary prevention strategy, early screening and diagnosis, and more advanced treatment technologies have been applied, the overall incidence rate and mortality of HCC continue to rise [3]. The occurrence and development of HCC are regulated by multiple mechanisms and participated by multiple molecules [4, 5]. Despite the advancement of diagnostic and medicinal approaches, the forecast of HCC is relatively poor due to the elevated recurrence ratio [6]. Therefore, it is urgent to identify prognostic biomarkers, observe high-risk patients with poor prognoses, and prevent a recurrence.With the rapid development of genomics, such as genes, proteins, metabolism, and in-depth research on tumor biology, hundreds of biomarkers with prognostic value have been identified [7]. There is an urgent need to analyze prognostic markers of hepatocellular carcinoma to guide clinical treatment strategies. Gene interaction networks provide the possibility of systematic analysis [8]. Previous studies have shown that gene interaction networks might be used to explore the potential internal relationship between functional gene clusters (functional modules) and prognostic factors [9]. The gene coexpression network could be constructed by weighted gene coexpression network analysis (WGCNA). The key modules might be used to explore tumor mechanisms, predict the survival rate of patients, and establish new diagnostic or therapeutic diagnostic strategies [10, 11]. WGCNA provides a functional interpretation tool for systems biology and has been applied in breast cancer, endometrial cancer, and other tumors [12].To improve the accuracy of prognostic markers, a coexpression network was constructed with the integration of the protein interaction (PPI) data. In addition, it is well established that crucial genes have essential functions in disease development and tend to show consistent expression variations in patients. Therefore, selecting crucial genes as predictors to build a prognosis model could enhance the stability of the model and improve the biological correlation between the identified genes and disease.


## 2. Methods

### 2.1. Data Collection

The research data of patients are downloaded from the Cancer Genome Atlas (TCGA) liver hepatocellular carcinoma (LIHC) cohort and gene expression omnibus (GEO) GSE103512 dataset, which contains experimental and clinical data. The transcriptome sequencing data and clinical information of HCC patients were downloaded from this database, and the encoded gene expression was normalized.


### 2.2. Screening of Differentially Expressed Genes

R Studio was used to screen the differentially expressed genes (DEGs) with the “edger” package. The heat map was drawn for the DEGs. The false discovery rate (FDR) value of each gene was calculated by the BH method, and the fold change (FC) of gene expression level in HCC and adjacent tissues was calculated. The screening criteria are as follows:FDR<0.05and∣log2FC∣>1. log2FC>1 indicated that the gene expression was upregulated, and log2FC<−1 indicated that the expression was downregulated.


### 2.3. Screening of Prognosis-Related Genes

Cox regression analysis screened genes related to overall survival (OS). For each gene, according to its expression value, the patients were divided into a high-expression group and a low-expression group for univariate regression analysis. The regression analysis was carried out by R software, andP<0.05 was considered statistically significant.


### 2.4. Identification of the HCC Modules

The “WGCNA” package in R Studio was used to identify the modules. The “goodsamplegenes()” function was used to check whether there were abnormal genes in the data and selected an appropriate threshold to eliminate outliers. Draw a gene feature heat clustering diagram, and complete the construction of the coexpression network and the selection of modules. Choose theβ value to calculate the adjacency matrix. Construct a clustering tree to select modules, set the minimum capacity and cutting height of modules, and merge modules with high similarity. The prognosis module is identified by associating the module data with the characteristic clinical data. Scatter plots of gene significance (GS) and module membership (mm) were drawn in the prognosis module to clarify the importance of genes in the module.


### 2.5. Constructing PPI Network and Screening Hub Gene

The String database (http://string-db.org/) was used to build the PPI network. Input the filtered DEGs into the String database, and construct the PPI network matched with the totalscore>0.3. The connectivity of protein nodes was calculated using the Cytohubba plug-in. Genes with high connectivity were regarded as hub genes.


### 2.6. Construction of Coexpression Network of Prognostic Genes

The weighted gene coexpression network analysis method is used to construct the HCC prognosis network. According to the topological overlap matrix, hierarchical clustering divides the network and identifies the modules in the network. The Benjamin-Hochberg method is used to correct the correlation coefficientP value of gene pairs in the module, and the gene pairs with P<0.05 are selected to construct the module network.


### 2.7. Construction and Evaluation of the Eight-mRNA Prognostic Model

The “glmnet()” package in R Studio was used to construct and evaluate the eight-mRNA prognostic model. Draw the ROC curve of the model, and calculate the AUC to evaluate the prediction efficiency of the model. The high- and low-risk groups of HCC prognosis were divided by the median of the model risk score. The survival curve of the model was drawn by the Kaplan Meier method, and a log-rank test tested the survival difference to evaluate the ability of the model to distinguish the high and low risk of gastric cancer prognosis. IfP<0.05, the difference is statistically significant.


## 2.1. Data Collection

The research data of patients are downloaded from the Cancer Genome Atlas (TCGA) liver hepatocellular carcinoma (LIHC) cohort and gene expression omnibus (GEO) GSE103512 dataset, which contains experimental and clinical data. The transcriptome sequencing data and clinical information of HCC patients were downloaded from this database, and the encoded gene expression was normalized.


## 2.2. Screening of Differentially Expressed Genes

R Studio was used to screen the differentially expressed genes (DEGs) with the “edger” package. The heat map was drawn for the DEGs. The false discovery rate (FDR) value of each gene was calculated by the BH method, and the fold change (FC) of gene expression level in HCC and adjacent tissues was calculated. The screening criteria are as follows:FDR<0.05and∣log2FC∣>1. log2FC>1 indicated that the gene expression was upregulated, and log2FC<−1 indicated that the expression was downregulated.


## 2.3. Screening of Prognosis-Related Genes

Cox regression analysis screened genes related to overall survival (OS). For each gene, according to its expression value, the patients were divided into a high-expression group and a low-expression group for univariate regression analysis. The regression analysis was carried out by R software, andP<0.05 was considered statistically significant.


## 2.4. Identification of the HCC Modules

The “WGCNA” package in R Studio was used to identify the modules. The “goodsamplegenes()” function was used to check whether there were abnormal genes in the data and selected an appropriate threshold to eliminate outliers. Draw a gene feature heat clustering diagram, and complete the construction of the coexpression network and the selection of modules. Choose theβ value to calculate the adjacency matrix. Construct a clustering tree to select modules, set the minimum capacity and cutting height of modules, and merge modules with high similarity. The prognosis module is identified by associating the module data with the characteristic clinical data. Scatter plots of gene significance (GS) and module membership (mm) were drawn in the prognosis module to clarify the importance of genes in the module.


## 2.5. Constructing PPI Network and Screening Hub Gene

The String database (http://string-db.org/) was used to build the PPI network. Input the filtered DEGs into the String database, and construct the PPI network matched with the totalscore>0.3. The connectivity of protein nodes was calculated using the Cytohubba plug-in. Genes with high connectivity were regarded as hub genes.


## 2.6. Construction of Coexpression Network of Prognostic Genes

The weighted gene coexpression network analysis method is used to construct the HCC prognosis network. According to the topological overlap matrix, hierarchical clustering divides the network and identifies the modules in the network. The Benjamin-Hochberg method is used to correct the correlation coefficientP value of gene pairs in the module, and the gene pairs with P<0.05 are selected to construct the module network.


## 2.7. Construction and Evaluation of the Eight-mRNA Prognostic Model

The “glmnet()” package in R Studio was used to construct and evaluate the eight-mRNA prognostic model. Draw the ROC curve of the model, and calculate the AUC to evaluate the prediction efficiency of the model. The high- and low-risk groups of HCC prognosis were divided by the median of the model risk score. The survival curve of the model was drawn by the Kaplan Meier method, and a log-rank test tested the survival difference to evaluate the ability of the model to distinguish the high and low risk of gastric cancer prognosis. IfP<0.05, the difference is statistically significant.


## 3. Results

### 3.1. Identification of DEGs

A total of 89 normal hepatic samples and 101 HCC samples were identified from the GSE103512 dataset. We obtained 197 downregulated DEGs and 200 upregulated DEGs in the GSE103512 dataset (Figure1(a)). Furthermore, 1856 downregulated DEGs and 1642 upregulated DEGs were obtained from the TCGA dataset (Figure 1(b)). A total of 177 upregulated and 196 downregulated common DEGs were identified by the intersection of the two datasets (Figure 1(c)).Figure 1
(a) A heat map showed the DEGs from the GSE103512 dataset. (b) A heat map showed the DEGs from the TCGA. (c) A Venn diagram showed the common DEGs by the intersection of TCGA and GSE103512. A total of 83 upregulated and 76 downregulated were identified.
(a)(b)(c)


### 3.2. Identification of the Critical Module by WGCNA

Employing the WGCNA, the common DEGs were assessed for coexpression network analysis. The power ofβ=6 and scale-free R2=0.89 were chosen to construct a clustering tree, and a total of 13 modules were identified ultimately (Figure 2(a)). As shown in Figure 2(b), the ME of the green yellow module was the most significant gene module among the 13 modules. Furthermore, the ME in the green yellow module exhibited an advanced connection to overall survival than other modules (Figure 2(c)). The scatter diagram of GS and MM in the green yellow module suggested that the genes in the green yellow module were highly correlated with the overall survival (Figure 2(d), P<0.05). Consequently, we selected this green yellow module as the key module for further investigation.Figure 2
(a) The power value for the contiguity matrix in WGCNA. (b) The cluster dendrogram of mRNAs in GSE103512. (c) The association between modules and the clinical characters. (d) The scatter diagram of GS and MM in the green yellow module showed the correlation between OS genes.
(a)(b)(c)(d)


### 3.3. Construction of PPI Network

For the green yellow module, the protein interaction data of module genes are used for reweighting. The PPI network containing 754 module genes was constructed using the String database. We obtained the reweighted coexpression network for the prognosis module, which contained 78 nodes and 503 edges. We next analyzed the topological characteristics of the reweighted module network and observed that the reweighted coexpression network could be defined as the small world network. Its small world index is 2.033. The genes with a weight greater than 0.3 in the reweighted coexpression network were selected, and the weight network diagram was depicted using Cytoscape software (Figure3).Figure 3
The PPI network was constructed for the green yellow module.


### 3.4. Go Enrichment Analysis

We performed the Go enrichment to obtain the enriched Go terms involved in the re-weighted coexpression network. A total of 843 enriched Go terms were identified withFDR<0.05. The FDR value less than 1×10−10 in the biological process is shown in Table 1. As shown in Table 1, the DEGs in the reweighted coexpression network were enriched in the ER-associated ubiquitin-dependent protein catabolic process, regulation of cell proliferation, endoplasmic reticulum lumen, and regulation of apoptotic process pathways.Table 1
Go enrichment analysis for reweighted coexpression network.
GO:0000152~nuclear ubiquitin ligase complexNumber of allelesFDRGO:0043231~intracellular membrane-bounded organelle352.93E−13GO:0010008~endosome membrane462.95E−12GO:0019898~extrinsic component of membrane371.45E−16GO:0042405~nuclear inclusion body593.49E−12GO:0005740~mitochondrial envelope273.97E−12GO:0016020~membrane446.21E−12GO:0005788~endoplasmic reticulum lumen616.35E−14GO:0000800~lateral element533.03E−24GO:0051092~positive regulation of NF-kappaB transcription factor activity232.77E−11GO:0043123~positive regulation of I-kappaB kinase/NF-kappaB signaling354.42E−12GO:0045922~negative regulation of the fatty acid metabolic process411.45E−12GO:0042981~regulation of apoptotic process555.22E−14GO:0042127~regulation of cell proliferation766.58E−11GO:0030433~ER-associated ubiquitin-dependent protein catabolic process804.48E−13GO:0008152~metabolic process255.33E−14GO:0010803~regulation of tumor necrosis factor-mediated signaling pathway544.32E−14GO:0008625~extrinsic apoptotic signaling pathway via death domain receptors372.47E−13GO:0097190~apoptotic signaling pathway553.88E−13


### 3.5. Eight Genes Correlated with the OS of HCC Patients

Next, we calculate the score of each gene in the reweighted coexpression network and rank the genes according to the score. The top eight genes with the highest scores were selected to test their prediction performance. Among the top eight genes, four (USP39, ATP1B3, CASP8, and RAD54L) have been validated to play crucial roles in the progression of HCC and are closely related to the prognosis of HCC. According to the score ranking, a prognostic model was established for the top eight candidate prognostic markers. The prognostic model was constructed by a linear combination of the expression values of these eight prognostic biomarkers and the regression coefficients obtained from Cox regression analysis (Table2).
(1)Riskscore=−0.145894×ECSTF2+−0.43876×ECDK1+−0.14688+ERAD54L+−0.125743+ECOL1A2+−0.325682×EATP1B3+−0.35801×ECASP8+−0.6859×EUSP39+−0.259056×EHOXB7.Table 2
The eight prognostic genes and their prognostic model.
HR95% CIlogrankPCoxPAUCZEB10.860.74~0.950.0220.0430.69CDK10.670.47~0.810.0130.0020.65RAD54L0.770.62~0.940.0040.0130.66COL1A20.880.70~0.920.0350.0080.68ATP1B30.790.53~0.680.0010.0160.65CASP80.650.79~0.970.0470.0090.64USP390.720.86~0.880.0070.0370.68HOXB70.810.84~0.940.0350.0200.69


### 3.6. Evaluation of the Eight-mRNA Prognostic Model

The ROC curve of the eight-mRNA prognostic model was screened out (AUC=0.635), suggesting that this model exhibited an excellent prediction performance (Figure 4). Survival analysis showed that the survival rate of patients in the high-risk group was significantly lower than that in the low-risk group (log-rank=0.0055, Figure 5).Figure 4
The ROC curve of the eight-mRNA prognostic model was depicted.Figure 5
Survival analysis showed the survival rate of patients.


## 3.1. Identification of DEGs

A total of 89 normal hepatic samples and 101 HCC samples were identified from the GSE103512 dataset. We obtained 197 downregulated DEGs and 200 upregulated DEGs in the GSE103512 dataset (Figure1(a)). Furthermore, 1856 downregulated DEGs and 1642 upregulated DEGs were obtained from the TCGA dataset (Figure 1(b)). A total of 177 upregulated and 196 downregulated common DEGs were identified by the intersection of the two datasets (Figure 1(c)).Figure 1
(a) A heat map showed the DEGs from the GSE103512 dataset. (b) A heat map showed the DEGs from the TCGA. (c) A Venn diagram showed the common DEGs by the intersection of TCGA and GSE103512. A total of 83 upregulated and 76 downregulated were identified.
(a)(b)(c)


## 3.2. Identification of the Critical Module by WGCNA

Employing the WGCNA, the common DEGs were assessed for coexpression network analysis. The power ofβ=6 and scale-free R2=0.89 were chosen to construct a clustering tree, and a total of 13 modules were identified ultimately (Figure 2(a)). As shown in Figure 2(b), the ME of the green yellow module was the most significant gene module among the 13 modules. Furthermore, the ME in the green yellow module exhibited an advanced connection to overall survival than other modules (Figure 2(c)). The scatter diagram of GS and MM in the green yellow module suggested that the genes in the green yellow module were highly correlated with the overall survival (Figure 2(d), P<0.05). Consequently, we selected this green yellow module as the key module for further investigation.Figure 2
(a) The power value for the contiguity matrix in WGCNA. (b) The cluster dendrogram of mRNAs in GSE103512. (c) The association between modules and the clinical characters. (d) The scatter diagram of GS and MM in the green yellow module showed the correlation between OS genes.
(a)(b)(c)(d)


## 3.3. Construction of PPI Network

For the green yellow module, the protein interaction data of module genes are used for reweighting. The PPI network containing 754 module genes was constructed using the String database. We obtained the reweighted coexpression network for the prognosis module, which contained 78 nodes and 503 edges. We next analyzed the topological characteristics of the reweighted module network and observed that the reweighted coexpression network could be defined as the small world network. Its small world index is 2.033. The genes with a weight greater than 0.3 in the reweighted coexpression network were selected, and the weight network diagram was depicted using Cytoscape software (Figure3).Figure 3
The PPI network was constructed for the green yellow module.


## 3.4. Go Enrichment Analysis

We performed the Go enrichment to obtain the enriched Go terms involved in the re-weighted coexpression network. A total of 843 enriched Go terms were identified withFDR<0.05. The FDR value less than 1×10−10 in the biological process is shown in Table 1. As shown in Table 1, the DEGs in the reweighted coexpression network were enriched in the ER-associated ubiquitin-dependent protein catabolic process, regulation of cell proliferation, endoplasmic reticulum lumen, and regulation of apoptotic process pathways.Table 1
Go enrichment analysis for reweighted coexpression network.
GO:0000152~nuclear ubiquitin ligase complexNumber of allelesFDRGO:0043231~intracellular membrane-bounded organelle352.93E−13GO:0010008~endosome membrane462.95E−12GO:0019898~extrinsic component of membrane371.45E−16GO:0042405~nuclear inclusion body593.49E−12GO:0005740~mitochondrial envelope273.97E−12GO:0016020~membrane446.21E−12GO:0005788~endoplasmic reticulum lumen616.35E−14GO:0000800~lateral element533.03E−24GO:0051092~positive regulation of NF-kappaB transcription factor activity232.77E−11GO:0043123~positive regulation of I-kappaB kinase/NF-kappaB signaling354.42E−12GO:0045922~negative regulation of the fatty acid metabolic process411.45E−12GO:0042981~regulation of apoptotic process555.22E−14GO:0042127~regulation of cell proliferation766.58E−11GO:0030433~ER-associated ubiquitin-dependent protein catabolic process804.48E−13GO:0008152~metabolic process255.33E−14GO:0010803~regulation of tumor necrosis factor-mediated signaling pathway544.32E−14GO:0008625~extrinsic apoptotic signaling pathway via death domain receptors372.47E−13GO:0097190~apoptotic signaling pathway553.88E−13


## 3.5. Eight Genes Correlated with the OS of HCC Patients

Next, we calculate the score of each gene in the reweighted coexpression network and rank the genes according to the score. The top eight genes with the highest scores were selected to test their prediction performance. Among the top eight genes, four (USP39, ATP1B3, CASP8, and RAD54L) have been validated to play crucial roles in the progression of HCC and are closely related to the prognosis of HCC. According to the score ranking, a prognostic model was established for the top eight candidate prognostic markers. The prognostic model was constructed by a linear combination of the expression values of these eight prognostic biomarkers and the regression coefficients obtained from Cox regression analysis (Table2).
(1)Riskscore=−0.145894×ECSTF2+−0.43876×ECDK1+−0.14688+ERAD54L+−0.125743+ECOL1A2+−0.325682×EATP1B3+−0.35801×ECASP8+−0.6859×EUSP39+−0.259056×EHOXB7.Table 2
The eight prognostic genes and their prognostic model.
HR95% CIlogrankPCoxPAUCZEB10.860.74~0.950.0220.0430.69CDK10.670.47~0.810.0130.0020.65RAD54L0.770.62~0.940.0040.0130.66COL1A20.880.70~0.920.0350.0080.68ATP1B30.790.53~0.680.0010.0160.65CASP80.650.79~0.970.0470.0090.64USP390.720.86~0.880.0070.0370.68HOXB70.810.84~0.940.0350.0200.69


## 3.6. Evaluation of the Eight-mRNA Prognostic Model

The ROC curve of the eight-mRNA prognostic model was screened out (AUC=0.635), suggesting that this model exhibited an excellent prediction performance (Figure 4). Survival analysis showed that the survival rate of patients in the high-risk group was significantly lower than that in the low-risk group (log-rank=0.0055, Figure 5).Figure 4
The ROC curve of the eight-mRNA prognostic model was depicted.Figure 5
Survival analysis showed the survival rate of patients.


## 4. Discussion

HCC is one of the malignant tumors with a high mortality rate in China [13]. Although significant progress has been made in immunotherapy and targeted therapy, the prognosis of patients with advanced HCC is still poor [14]. Bioinformatics analysis could identify the abnormally expressed genes under pathological conditions and assist in selecting novel and more accurate diagnostic and prognostic biomarkers [15]. Based on the application and development of computer technology and artificial intelligence in medical biology, bioinformatics analysis has become necessary for studying molecular markers related to disease phenotype [16]. In this study, we analyzed biological information to identify eight mRNAs correlated with the OS of HCC patients and establish a survival prognosis risk scoring model. The ROC curve of the eight-mRNA prognostic model exhibited an excellent prediction performance of HCC outcome. Kaplan Meier analysis showed that the survival time of patients in the high-risk group was lower than that of patients in the low-risk group.As an advanced algorithm in bioinformatics analysis, weighted gene coexpression network analysis makes the calculation results more accurate by establishing a scale-free network and has been widely used in the study of tumor subtypes and prognostic markers [17]. This study constructed a coexpression network of HCC prognostic genes and identified the network modules. The prognostic module was reweighted using the PPI network data of module genes. The small world index of our reweighted module network obtained is 2.033, which is in line with the characteristics of a small world network. These findings suggested that the nodes in our reweighted module network were highly clustered.Next, we analyzed the highly clustered nodes in the reweighted module network and identified the topological importance of nodes by scoring. We selected eight genes correlated with the OS of HCC patients with the highest scores. Among the top eight genes with the highest scores, USP39, ATP1B3, AHSA1, and RAD54L have essential functions in HCC cell proliferation and tumor growth, indicating that topologically essential genes play critical roles in HCC forecasting. Zinc-finger E-box-binding homeobox 1 (ZEB1) is a critical inducer of epithelial-to-mesenchymal transition (EMT) to stimulate cancer progression. However, the effect of ZEB1 on HCC development remains insufficient [18, 19]. Caspase 8 (CASP8) is an essential protease in the apoptosis pathway of the death receptor [20]. After being recruited and activated by death signals, CASP8 might activate its downstream effector protease to induce apoptosis. The role of CASP 8 is, however, unclear.In brief, this study integrated microarray data from GEO and TCGA to identify critical genes. WGCNA combined Cox regression analysis was used to screen out genes of HCC prognosis. Eight critical genes related to the pathogenesis and progression of HCC were identified. These genes might play crucial roles in the cell cycle and abnormal behavior of HCC, indicating that these genes have great potential in the treatment and prognosis of HCC. In addition, we performed survival analysis and established a Cox proportional hazards model to identify prognostic biomarkers. A genetic marker of 8 genes was constructed to predict the overall survival rate. These results will provide a reference for a further study of the pathogenesis and drug treatment of HCC.



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*Source: 7278231-2023-02-04.xml*