Weighted Gene Co-Expression Network Analysis and Treatment Strategies of Tumor Recurrence-Associated Hub Genes in Lung Adenocarcinoma

Abstract

Despite the recent progress of lung adenocarcinoma (LUAD) therapy, tumor recurrence remained to be a challenging factor that impedes the effectiveness of treatment. The objective of the present study was to predict the hub genes affecting LUAD recurrence via weighted gene co-expression network analysis (WGCNA). Microarray samples from LUAD dataset of GSE32863 were analyzed, and the modules with the highest correlation to tumor recurrence were selected. Functional enrichment analysis was conducted, followed by establishment of a protein-protein interaction (PPI) network. Subsequently, hub genes were identified by overall survival analyses and further validated by evaluation of expression in both myeloid populations and tissue samples of LUAD. Gene set enrichment analysis (GSEA) was then carried out, and construction of transcription factors (TF)-hub gene and drug-hub gene interaction network was also achieved. A total of eight hub genes (ACTR3, ARPC5, RAB13, HNRNPK, PA2G4, WDR12, SRSF1, and NOP58) were finally identified to be closely correlated with LUAD recurrence. In addition, TFs that regulate hub genes have been predicted, including MYC, PML, and YY1. Finally, drugs including arsenic trioxide, cisplatin, Jinfukang, and sunitinib were mined for the treatment of the eight hub genes. In conclusion, our study may facilitate the invention of targeted therapeutic drugs and shed light on the understanding of the mechanism for LUAD recurrence.

Keywords: hub genes; lung adenocarcinoma; transcription factor; tumor recurrence; weighted gene co-expression network analysis.

FIGURE 1

Experimental design and workflow of this study.

FIGURE 2

Identification of modules associated with the clinical features of lung adenocarcinoma (LUAD). (A) Heatmap to show the correlation between modules and clinical traits with LUAD. p-Values are shown in brackets. (B–D) Scatter plot analysis to show the association between Module membership and gene significance for LUAD recurrence in green (B), purple (C), and brown modules (D).

FIGURE 3

Gene Ontology (GO) enrichment analyses of gene members in three chosen modules. The colored geometry represents GO term enrichment, dots represent biological process (BP), triangles represent molecular function (CC), rectangles represent cellular component (MF), red indicates low enrichment, and green indicates high enrichment. The sizes of the geometries represent the number of genes in each GO category.

FIGURE 4

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Genes in three modules (marked in red) could only be enriched in Tight Junction signaling pathway.

FIGURE 5

Identification of the top 15 genes from the protein–protein interaction (PPI) network of the three modules.

FIGURE 6

Survival analyses of the eight hub gene candidates identified. (A–H) Overall survival of the eight hub gene candidates in lung adenocarcinoma (LUAD) based on Kaplan–Meier plotter. (I–P) Overall survival of the eight hub gene candidates in LUAD based on the Gene Expression Profiling Interactive Analysis (GEPIA) database. p < 0.05 was considered to indicate a statistically significant difference. ACTR3, actin-related protein 3; ARPC5, actin-related protein 2/3 complex subunit 5; HNRNPK, heterogeneous nuclear ribonucleoprotein K; NOP58, NOP58 ribonucleoprotein; PA2G4, proliferation-associated 2G4; RAB13, RAB13 member RAS oncogene family; SRSF1, serine- and arginine-rich splicing factor 1; WDR12, WD repeat domain 12.

FIGURE 7

Expression of eight hub gene candidates in various clusters of human immune cells from non-small cell lung tumor (t) and peripheral blood (b). t, tumor; b, blood; DCs, dendritic cells; pDCs, plasmacytoid DCs; RBC, red blood cell. The size of dots represents the percentage of expression; red and blue represent the level of scaled mean expression.

FIGURE 8

Immunohistochemical images of eight hub genes in lung adenocarcinoma (LUAD) recurrence. The protein levels of (A–E) in LUAD tissues were compared with normal lung tissues from the Human Protein Atlas database.

FIGURE 9

Genetic alteration information and co-expression analysis of the eight hub genes. (A) A visual summary across a set of lung adenocarcinoma (LUAD) (data from Lung Adenocarcinoma, The Cancer Genome Atlas (TCGA), Nature 2014) showed the genetic alterations connected with the eight hub genes, which were altered in 75 (31.8%) of 230 sequenced patients (230 in total). (B) An overview of changes in the eight hub genes from the genomics datasets of LUAD in TCGA database. Summary for lung adenocarcinoma: Gene altered in 20% of 230 cases, Mutation 1.74% (4 in 230 cases), Amplification 17.39% (40 in 230 cases), and Multiple Alterations 0.87% (2 in 230 cases). (C) The co-expression analysis of the eight hub genes using the 230 samples above based on cBioPortal database.

FIGURE 10

Gene set enrichment analysis (GSEA) using GSE116959 showed a positive enrichment of identified hub genes associated with four Kyoto Encyclopedia of Genes and Genomes(KEGG) pathways.

FIGURE 11

The transcriptional regulatory network of eight hub genes and TFs. TFs, transcription factors. A green hexagon node represents the TFs, a pink circular node represents hub genes, a light blue diamond node represents the lung adenocarcinoma (LUAD), and the interaction is represented by an arrow. The numbers of arrows in the networks demonstrate the contribution of 1 TF to the hub genes; and the higher the degree, the more central the nodes were within the network.

FIGURE 12

Interactions between drugs and the identified hub genes based on CTD database and literature validation. Light blue circular node represents drugs, red diamond node represents hub genes, their interaction is represented by line, and the degree value represents the number of targets acted on by the drug.