Identifying a Novel Biomarker TOP2A of Clear Cell Renal Cell Carcinoma (ccRCC) Associated with Smoking by Co-Expression Network Analysis

Abstract

Although it is well known that smoking is one of pathogenesis of clear cell renal cell carcinoma (ccRCC), the underlying molecular mechanism is still unclear. In our study, the microarray dataset GSE46699 is analyzed by weighted gene co-expression network analysis (WGCNA). Then we identify 15 co-expressed gene modules in which the lightcyan module (R² = 0.30) is the most significant. Combined with the protein-protein interaction (PPI) network and WGCNA, two hub genes are identified. Meanwhile, linear regression analyses indicate that TOP2A has a higher connection with smoking in ccRCC, survival analysis proved that overexpression of TOP2A in ccRCC could lead to shorter survival time. Furthermore, bioinformatical analyses based on GSE46699 and GSE2109 as well as qRT-PCR experiment show similar results that TOP2A is significantly up-regulated in smoking ccRCC compared to non-smoking ccRCC samples. In addition, Functional analysis, pathway enrichment analysis and gene set enrichment analysis (GSEA) indicate that high expression of TOP2A is related to cell cycle and p53 signaling pathway in ccRCC samples. Moreover, in vitro experiments revealed that TOP2A induced cell cycle arrest at G2 phase and proliferation inhibition via p53 phosphorylation. Taken together, by using WGCNA, we have identified a novel biomarker named TOP2A, which could affect the development of smoking-related ccRCC by regulating cell cycle and p53 signaling pathway.

Keywords: TOP2A; biomarker; clear cell renal cell carcinoma (ccRCC); smoking; weighted gene co-expression network analysis (WGCNA).

Figure 1

Description of the study. (A) Flow chart of this study. (B) Sample dendrogram and cigarette smoking heatmap (GSE46699).

Figure 2

Determination of soft-thresholding power and analysis of hub genes as well as protein-protein interaction network (PPI). (A) Analysis of the scale-free fit index for various soft-thresholding powers (β). (B) Analysis of the mean connectivity for various soft-thresholding powers. (C) Histogram of connectivity distribution when β = 11. (D) Checking the scale-free topology when β = 11. (E) Scatter plot of module eigengenes in lightcyan module. (F) PPI network of genes in the lightcyan module.

Figure 3

Identify modules associated with the smoking of ccRCC. (A) Dendrogram of all differentially expressed genes clustered. (B) Distribution of average gene significance in the modules associated with smoking of ccRCC. (C) The correlation between module eigengenes and the smoking group.

Figure 4

Validate the relationship between TOP2A and smoking ccRCC samples. (A) TOP2A is significantly overexpressed in the ccRCC according to GEPIA database. (B) Linear regression analysis between the expressions of TOP2A and smoking group. (C) The expression of TOP2A is correlated with smoking of ccRCC in GSE46699 (N represents non-smoking patients, S represents smoking patients). (D) TOP2A expression is correlated with smoking of ccRCC in GSE2109. (E) TOP2A expression is positive correlation with smoking status in lung adenocarcinoma in GSE10072 (NNS: Normal never smoked, NFS: Normal former smoker, NCS: Normal current smoker, TNS: Tumor never smoked, TFS: Tumor former smoker, TCS: Tumor current smoker). (F) qRT-PCR indicates the expression of TOP2A is up-regulated in smoking ccRCC tissues. (G-H) Kaplan-Meier survival curve downloaded from GEPIA database demonstrate that up-regulation of TOP2A have a significantly shorter overall survival time and disease-free survival time. (I) Biological processes and (J) KEGG pathway enrichment analysis of TOP2A.

Figure 5

Gene set enrichment analysis (GSEA). “Viral myocarditis”, “Antigen processing and presentation”, “Leishmania infection”, “Cell cycle”, “Autoimmune Thyoid disease”, “P53 signaling pathway” were enriched in ccRCC samples with TOP2A highly expressed.

Figure 6

Downregulation of TOP2A inhibited cell proliferation by triggered cell cycle arrest at G2 phase in ccRCC cells. (A) The expression of TOP2A in transcriptional level (upper picture) and translation level (lower picture) by treating with siTOP2A-1, siTOP2A-2 and siTOP2A-3 in 786-O cell line. The proliferative capacity was measured by (B) MTT assay and (C) colony formation assays in 786-O cell line. (D) Flow cytometry analysis of TOP2A deficiency group and NC group in 786-O cell line. (E) Cell cycle related proteins such as Cyclin A1/2 and CDK1/2 were significantly decreased in the 786-O cell line in TOP2A knockdown group. (F) TOP2A deficiency strongly induced phosphorylated p53 in 786-O cell line.