Analysis of ARHGAP4 Expression With Colorectal Cancer Clinical Characteristics and Prognosis

Abstract

Background: This study aims to analyze the correlation between ARHGAP4 in the expression and clinical characteristics of colorectal cancer (CRC), and the influence of ARHGAP4 expression on the prognosis of CRC, and to evaluate whether ARHGAP4 is a potential prognostic oncotarget for CRC.

Methods: ARHGAP4 was identified using the Gene Expression Omnibus database through weighted gene coexpression network analysis. Using the Gene Expression Profiling Interactive Analysis to perform and analyze the expression and prognosis of ARHGAP4 in CRC. The expression of AGRGAP4 and immune cells was analyzed by the Tumor IMmune Estimation Resource online database. Finally, immunohistochemistry was used to analyze the expression difference and prognosis of ARHGAP4 in CRC and adjacent normal tissues, as well as the relationship between AGRGAP4 expression and clinical features of CRC.

Results: We identified ARHGAP4 that is related to the recurrence of CRC from GSE97781 data. ARHGAP4 has not been reported in CRC. The high expression of ARHGAP4 in select colon adenocarcinoma indicates a poor prognosis by database analysis. In our clinical data results, ARHGAP4 is highly expressed in CRC and lowly expressed in normal tissues adjacent to cancer. Compared with the low-expression group, the high-expression group has a significantly poorer prognosis. In colon cancer, the B-cell, macrophage, neutrophil, and dendritic-cell levels are downregulated after ARHGAP4 gene knockout; the levels of CD8⁺ and CD4⁺ T cells, neutrophils, and dendritic cells are upregulated after the amplification of the ARHGAP4 gene. In addition, ARHGAP4 expression is related to N,M staging and clinical staging.

Conclusion: ARHGAP4 is highly expressed in CRC, and the high expression of ARHGAP4 has a poor prognosis. The expression of ARHGAP4 in CRC is related to the immune cells such as B cells, CD8⁺ and CD4⁺ T cells, macrophages, neutrophils, and dendritic cells. ARHGAP4 is correlated with N,M staging and clinical staging in CRC. ARHGAP4 may be a potential biomarker for the prognosis of CRC.

Keywords: ARHGAP4; CRC; WGCNA; immune; prognostic.

Figure 1

Construction of weighted co-expression network (WGCNA) and WGCNA module analysis. (A, B) Soft threshold selection process: scale independence and mean connectivity. (C) Cluster module dendrogram. Each color represents one specific co-expression module, and branches above represent genes. (D) Heat map of the correlation between clinical traits including pre-treatment, post-treatment, recurrence, and module eigengenes (E) Visualizing gene networks. Select genes for the network heat map plot. (F) Heat map of the correlation between clinical traits and a module. (G) Analysis of the relevancy between blue, brown, gray, and turquoise gene modules and clinical parameters. (H) Correlation between the module membership of modules of interest and gene significance with clinical traits in the brown module. Four clinically significant genes with high connectivity were identified in the brown module.

Figure 2

Gene ontology (GO) and gene set variation analysis (GSVA). (A) Using the Metascape database for annotation and visualization, GO analysis was performed on the ARHGAP4 enrichment correlation genes from the brown module. (B) GSVA divergence bar chart. Using GSVA, the signal pathways of the ARHGAP4 enrichment correlation genes. Bar graph of enriched terms across input gene lists, colored by p-values.

Figure 3

Expression and prognosis of ARHGAP4 in CRC. (A) ARHGAP4 is expressed in colorectal adenocarcinoma (COAD) and READ (rectal adenocarcinoma). (B) ARHGAP4 is expressed in each clinical stage. (C, D) The relationship between ARHGAP4 expression and OS and DFS in COAD. (E, F) The relationship between ARHGAP4 expression and overall survival (OS) and disease-free survival (DFS) in READ. *Indicates that the P value is less than 0.05.

Figure 4

ARHGAP4 gene and immune cells. (A) ARHGAP4 expression was highly correlated with CD4⁺ T-cell infiltration in CRC and dendritic cell infiltration in READ. (B) After ARHGAP4 gene knockout, the levels of B cells, macrophages, neutrophils, and dendritic cells are downregulated, after the high-amplification ARHGAP4 gene. The levels of CD8⁺ and CD4⁺ T cells, neutrophils, and dendritic cells are upregulated in COAD. *Indicates that the P value is less than 0.05, **Indicates that the P value is less than 0.01, ***Indicates that the P value is less than 0.001.

Figure 5

ARHGAP4 expression and prognosis in clinical colorectal cancer (CRC). (A) Comparison of ARHGAP4 in CRC tissues and in normal tissues by two pathologists. (B) Comparison of ARHGAP4 in CRC tissues and in normal tissues performed quantitative analysis by ImageJ. (C) The relationship between ARHGAP4 expression and OS in CRC. (D, E) ARHGAP4 is lowly expressed in normal tissues adjacent to CRC. (F, G) ARHGAP4 is highly expressed in CRC tissues. ****Indicates that the P value is less than 0.0001.