Differences of core genes in liver fibrosis and hepatocellular carcinoma: Evidence from integrated bioinformatics and immunohistochemical analysis

Abstract

Background: Liver fibrosis and hepatocellular carcinoma (HCC) are common adverse consequences of chronic liver injury. The interaction of various risk factors may cause them to happen. Identification of specific biomarkers is of great significance for understanding the occurrence, development mechanisms, and determining the novel tools for diagnosis and treatment of both liver fibrosis and HCC.

Aim: To identify liver fibrosis-related core genes, we analyzed the differential expression pattern of core genes in liver fibrosis and HCC.

Methods: Gene expression profiles of three datasets, GSE14323, GSE36411, and GSE89377, obtained from the Gene Expression Omnibus (GEO) database, were analyzed, and differentially expressed genes (DEGs) between patients with liver cirrhosis and healthy controls were identified by screening via R software packages and online tool for Venn diagrams. The WebGestalt online tool was used to identify DEGs enriched in biological processes, molecular functions, cellular components, and Kyoto Encyclopedia of Genes and Genomes pathways. The protein-protein interactions of DEGs were visualized using Cytoscape with STRING. Next, the expression pattern of core genes was analyzed using Western blot and immunohistochemistry in a carbon tetrachloride (CCl₄)-induced liver cirrhosis mouse model and in patient liver samples. Finally, Kaplan-Meier curves were constructed using the Kaplan-Meier plotter online server.

Results: Forty-five DEGs (43 upregulated and 2 downregulated genes) associated with liver cirrhosis were identified from three GEO datasets. Ten hub genes were identified, which were upregulated in liver cirrhosis. Western blot and immunohistochemical analyses of the three core genes, decorin (DCN), dermatopontin (DPT), and SRY-box transcription factor 9 (SOX9), revealed that they were highly expressed in the CCl₄-induced liver cirrhosis mouse model. The expression levels of DCN and SOX 9 were positively correlated with the degree of fibrosis, and SOX 9 level in HCC patients was significantly higher than that in fibrosis patients. However, high expression of DPT was observed only in patients with liver fibrosis, and its expression in HCC was low. The gene expression profiling interactive analysis server (GEPIA) showed that SOX9 was significantly upregulated whereas DCN and DPT were significantly downregulated in patients with HCC. In addition, the Kaplan-Meier curves showed that HCC patients with higher SOX9 expression had significantly lower 5-year survival rate, while patients with higher expression of DCN or DPT had significantly higher 5-year survival rates.

Conclusion: The expression levels of DCN, DPT, and SOX9 were positively correlated with the degree of liver fibrosis but showed different correlations with the 5-year survival rates of HCC patients.

Keywords: Bioinformatical analysis; Decorin; Dermatopontin; Hepatocellular carcinoma; Liver cirrhosis; SRY-box transcription factor 9.

Figure 1

Identification of differentially expressed genes. A-C: The volcano plots of GSE14323, GSE36411 and GSE89377. The red dots and blue dots represent up-regulated and downregulated genes, respectively; D: The Venn diagram software identified 45 common differentially expressed genes (DEGs) in three datasets (GSE14323, GSE36411 and GSE89377), including 43 upregulated genes and 2 downregulated genes; E: protein-protein interaction network of DEGs was constructed by STRING online database and drew by Cytoscape software; F: Top 10 hub genes of DEGs were identified by cytoHubba plug-in of Cytoscape and their importance were represented by their color’s shade.

Figure 2

Expression of hub genes in the liver tissue of CCl₄-induced mouse mice. A: Masson’s trichrome and HE staining of control and CCl₄-induced liver cirrhosis mouse liver tissues; B: Collagen area in Masson’s trichrome staining (n = 7 or 8); C: The mRNA expression levels of 10 hub genes of control and CCl₄-induced cirrhosis mouse liver tissues (n = 6); D: The protein expression levels of α-SMA, Decorin (DCN), Dermatopontin (DPT), and SRY-box transcription factor 9 (SOX9) in liver tissues of mice in two groups. The right panel showed the result of quantitative analysis (n = 4). All data were presented as mean ± SE. Two-tailed Student’s t test were performed. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001.

Figure 3

Comparison of decorin, dermatopontin, and SRY-box transcription factor 9 expression in CCl₄-induced mouse model. A: Immunohistochemical (IHC) analyses of the expression of Decorin (DCN) and the percentage of positive area were shown, n = 9 or 11; B: IHC analyses of the expression of Dermatopontin (DPT) and the percentage of positive area were shown (n = 9 or 10); C: IHC analyses of the expression of SRY-box transcription factor 9 (SOX9) and the percentage of positive area were shown (n = 9 or 10). All data were presented as mean ± SE. Two-tailed Student’s t test were performed. ^cP < 0.001, ^dP < 0.0001.

Figure 4

Comparison of decorin, dermatopontin, and SRY-box transcription factor 9 expression in human liver tissues. The expression levels of Decorin (DCN), Dermatopontin (DPT) and SRY-box transcription factor 9 (SOX9) in normal, S0, S1-S2, S3-S4, Hepatocellular carcinoma (HCC) groups were analyzed by immunohistochemistry. The H&E staining and Masson’s trichrome staining were shown also.

Figure 5

Expression levels analysis of decorin, dermatopontin, and SRY-box transcription factor 9 in human liver tissues. A-C: The percentage of positive area of decorin (DCN) , dermatopontin (DPT) and SRY-box transcription factor 9 (SOX9) among normal (n = 5 or 7), S0 (n = 3 or 4), S1-S2 (n = 10 or 12), S3-S4 (n = 8, 9 or 11) and hepatocellular carcinoma (HCC) (n = 11, 12 or 14) groups were counted; D-F: The percentage of positive area of DCN , DPT and SOX9 in normal (n = 5 or 7), liver fibrosis (n = 22, 23 or 26) and HCC (n = 11, 12 or 14) groups were counted. All data were presented as mean ± SE.One-way ANOVA with multiple comparisons and Tukey’s post-test were performed, ^aP < 0.05, ^bP < 0.01, ^cP < 0.001.

Figure 6

The relationship between decorin, dermatopontin, SRY-box transcription factor 9 expression and survival rate of hepatocellular carcinoma patients. A-C: Decorin (DCN), dermatopontin (DPT), and SRY-box transcription factor 9 (SOX9) were analyzed by gene expression profiling interactive analysis server (GEPIA) to determine their expression level differences between hepatocellular carcinoma (HCC) and normal liver tissues. Red box represents tumor tissue and gray box represents normal tissue; D-F: Prognostic information of hub genes. Kaplan-Meier plotter online tool was used to identify the prognostic information of DCN, DPT and SOX9, which associated with the survival rate of HCC patients (^aP < 0.05).