Identification of candidate biomarkers and pathways associated with SCLC by bioinformatics analysis

Abstract

Small cell lung cancer (SCLC) is one of the highly malignant tumors and a serious threat to human health. The aim of the present study was to explore the underlying molecular mechanisms of SCLC. mRNA microarray datasets GSE6044 and GSE11969 were downloaded from Gene Expression Omnibus database, and the differentially expressed genes (DEGs) between normal lung and SCLC samples were screened using GEO2R tool. Functional and pathway enrichment analyses were performed for common DEGs using the DAVID database, and the protein‑protein interaction (PPI) network of common DEGs was constructed by the STRING database and visualized with Cytoscape software. In addition, the hub genes in the network and module analysis of the PPI network were performed using CentiScaPe and plugin Molecular Complex Detection. Finally, the mRNA expression levels of hub genes were validated in the Oncomine database. A total of 150 common DEGs with absolute fold‑change >0.5, including 66 significantly downregulated DEGs and 84 upregulated DEGs were obtained. The Gene Ontology term enrichment analysis suggested that common upregulated DEGs were primarily enriched in biological processes (BPs), including 'cell cycle', 'cell cycle phase', 'M phase', 'cell cycle process' and 'DNA metabolic process'. The common downregulated genes were significantly enriched in BPs, including 'response to wounding', 'positive regulation of immune system process', 'immune response', 'acute inflammatory response' and 'inflammatory response'. Kyoto Encyclopedia of Genes and Genomes pathway analysis identified that the common downregulated DEGs were primarily enriched in the 'complement and coagulation cascades' signaling pathway; the common upregulated DEGs were mainly enriched in 'cell cycle', 'DNA replication', 'oocyte meiosis' and the 'mismatch repair' signaling pathways. From the PPI network, the top 10 hub genes in SCLC were selected, including topoisomerase IIα, proliferating cell nuclear antigen, replication factor C subunit 4, checkpoint kinase 1, thymidylate synthase, minichromosome maintenance protein (MCM) 2, cell division cycle (CDC) 20, cyclin dependent kinase inhibitor 3, MCM3 and CDC6, the mRNA levels of which are upregulated in Oncomine SCLC datasets with the exception of MCM2. Furthermore, the genes in the significant module were enriched in 'cell cycle', 'DNA replication' and 'oocyte meiosis' signaling pathways. Therefore, the present study can shed new light on the understanding of molecular mechanisms of SCLC and may provide molecular targets and diagnostic biomarkers for the treatment and early diagnosis of SCLC.

Figure 1.

Volcano plot and Venn diagram of DEGs in mRNA expression profiling datasets. Volcano plots of DEGs in normal lung and small cell lung cancer samples in (A) GSE6044 and (B) GSE11969 datasets. DEGs were selected by P<0.05 and |log₂ (fold-change)| >0.5. The x-axis shows the fold-change in gene expression between normal lung and small cell lung cancer samples, and the y-axis shows the statistical significance of the differences. Colors represent different genes: Red for genes without significantly different expression and blue for significantly differentially expressed genes. Venn diagrams illustrating the number of (C) downregulated and (D) upregulated genes in the two datasets, respectively. The intersection in grey represents the DEGs common between the two datasets. DEG, differentially expressed genes.

Figure 2.

GO classification of common DEGs in the listed categories (top 5 biological processes and cellular components, and top 3 molecular functions). GO enrichment analysis results of (A) downregulated and (B) upregulated common DEGs with P<0.05 and absolute fold-changes >0.5. The top × axis represents the number of genes in the marked category; the bottom × axis indicates the minus Log₁₀ (FDR) of categories. Only functional categories with P-value <0.05 are shown. GO, Gene Ontology; DEG, differentially expressed genes; FDR, false discovery rate.

Figure 3.

PPI network constructed from the common DEGs, module analysis and hub genes. (A) Using the STRING online database, a total of 123 DEGs were filtered into the DEGs PPI network complex. The nodes represent proteins, the edges represent the interaction of proteins and green circles and red circles indicate downregulated and upregulated DEGs, respectively. (B) Expression heat map of the top 10 hub genes in GSE11969. (C) The most significant module in the PPI network with MCODE score ≥4 and node >5. PPI, protein-protein interaction; DEG, differentially expressed genes; MCODE, Molecular Complex Detection plug in; SCLC, small cell lung cancer; TYMS, thymidylate synthase; PCNA, proliferating cell nuclear antigen; RFC4, replication factor C subunit 4; MCM, minichromosome maintenance protein; CHEK1, checkpoint kinase 1; CDC, cell division cycle; CDKN3, cyclin dependent kinase inhibitor 3; TOP2A, topoisomerase IIα.

Figure 4.

Topological parameters of the protein-protein interaction network. (A) Average clustering coefficient distribution. (B) Closeness centrality. (C) Average neighborhood connectivity distribution. (D) Node-degree distribution. (E) Shortest path length distribution. (F) Topological coefficients. Avg, average; topol, topological.

Figure 5.

Analysis of expression of hub genes in the Oncomine database. Gene expression data was obtained from Garber and Bhattacharjee lung datasets and analyzed with Oncomine. mRNA expression levels of (A) TOP2A, (B) PCNA, (C) RFC4, (D) CHEK1, (E) TYMS, (F) CDC20, (G) CDKN3, (H) MCM3, (I) CDC6 and (J) MCM2 in normal lung vs. SCLC were compared. Pre-processed expression levels are Log₂ normalized and median centered. Data are presented as box plot with minimum (from bottom to top), 10th percentile, 25th percentile, median, 75th percentile, 90th percentile and maximum. Ctr, control; TOP2A, topoisomerase IIα; PCNA, proliferating cell nuclear antigen; RFC4, replication factor C subunit 4; CHEK1, checkpoint kinase 1; TYMS, thymidylate synthase; CDC, cell division cycle; CDKN3, cyclin dependent kinase inhibitor 3; MCM, minichromosome maintenance protein; SCLC, small cell lung cancer.