Identification of potential diagnostic and therapeutic target genes for lung squamous cell carcinoma

Abstract

The purpose of this study was to identify potential molecular markers of lung squamous cell carcinoma (LUSC). Three datasets containing LUSC mRNA sequencing data were downloaded from the Gene Expression Omnibus, The Cancer Genome Atlas and the Gene Expression Profiling Interactive Analysis databases. These datasets were used to identify significantly differentially expressed genes (DEGs) in LUSC. A protein-protein interaction network of the DEGs was constructed followed by Gene Ontology, Kyoto Encyclopedia of Genes and Genomes and overall survival analyses of the DEGs. A total of 37 DEGs between LUSC and normal tissues were identified, including 26 downregulated genes and 11 upregulated genes. Biological Process enrichment analysis revealed that the DEGs were mainly enriched in 'cell adhesion', 'cell-matrix adhesion', 'anatomical structure morphogenesis', 'ECM-receptor interaction' and 'focal adhesion'. Overall survival analysis demonstrated that transcription factor 21, α-2-macroglobulin, acyl-CoA synthetase long chain family member 5, integrin subunit β8, meiotic nuclear divisions 1 and secretoglobin family 1A member 1 were significantly associated with the occurrence and development of lung cancer, and these genes were selected as hub genes. The results obtained in the present study may aid the elucidation of the molecular mechanisms involved in the development of LUSC and may provide potential targets for LUSC treatment.

Keywords: bioinformatics analysis; differentially expressed genes; hub genes; lung squamous cell carcinoma.

Figure 1.

Data preprocessing and differential expression analysis. (A) A box-plot of standardized expression data of normal and LUSC tissues. The red symbols denote normal samples, and the blue symbols represent patients with LUSC. The red diamond in each frame represents the average level of gene expression in each sample. (B) Heatmap of the common DEGs between LUSC tissues and normal controls in the GSE31552 dataset. Blue represents downregulation and red represents upregulation. The normal groups were significantly separated from the LUSC groups, indicating that the sample source data are reliable. (C) Volcano plot used to verify the rationality of the P-values and corresponding fold changes of the expression data. The red circles denote upregulation, and the green symbols denote downregulation. (D) A hierarchical clustering diagram of the DEGs. Blue represents downregulation and red represents upregulation. LUSC, lung squamous cell carcinoma; DEGs, differentially expressed genes; down, downregulated genes; up, upregulated genes; not, not differentially expressed.

Figure 2.

Venn diagram, PPI network and hierarchical clustering of significant DEGs. (A) Venn diagram drawn in FunRich (version 3.1.3). A total of 37 common significant DEGs were obtained. (B) PPI network of DEGs constructed using Gene MANIA. Red circles present DEGs and green circles present predicted genes. The size of the circle represents the level of connectivity. Purple lines represent co-expression, green lines represent genetic interactions and pink lines represent physical interactions. (C) Hierarchical clustering of DEGs was performed using the UCSC Xena database. Upregulation of genes is indicated by red and downregulation of genes is indicated by blue. The expression of DEGs in different grades and different sample types is presented (Note: TMPRSSIIE gene is not included in the lung cancer dataset of UCSC Xena database). PPI, protein-protein interactions; GEPIA, Gene Expression Profiling Interactive Analysis; TCGA, The Cancer Genome Atlas; LUSC, lung squamous cell carcinoma; DEGs, differentially expressed genes; UCSC, University of California, Santa Cruz.

Figure 3.

Overall survival analysis. (A-C) Overall survival analyses were performed using the GEPIA online platform and (D-F) Kaplan-Meier plotter online platform. The solid line represents the survival curve and the dotted line represents the 95% confidence interval. Log-rank P<0.05 was considered to indicate a statistically significant difference. Patients with expression above the median are indicated by red lines, and patients with expression below the median are indicated by (A-C) blue lines or (D-F) black lines. (A) TCF21 was negatively associated with the overall survival of patients with LUSC; (B) A2M was negatively associated with the overall survival of patients with LUSC; (C) ACSL5 was negatively associated with the overall survival of patients with LUSC; (D) ITGB8 was positively correlated with the overall survival of patients with LUSC; (E) MND1 was negatively associated with the overall survival of patients with LUSC; (F) SCGB1A1 was positively correlated with the overall survival of patients with LUSC. TCF21, transcription factor 21; A2M, α-2-macroglobulin; ACSL5, acyl-CoA synthetase long chain family member 5; ITGB8, integrin subunit beta 8; SCGB1A1, secretoglobin family 1A member 1; MND1, meiotic nuclear divisions 1; HR, hazard ratio; TMP, transcripts per million; LUSC, lung squamous cell carcinoma.

Figure 4.

Box and violin plots were produced using the Gene Expression Profiling Interactive Analysis online platform. The boxplots present the expression of hub genes in LUSC and LUAD; red, tumor tissue; gray, normal tissue. Violin plots demonstrate the association between hub gene expression and LUSC staging. (Aa) TCF21 was downregulated in LUSC and LUAD; (Ab) TCF21 was associated with LUSC stages [Pr(>F)=0.0045]. (Ba) A2M was downregulated in both LUSC and LUAD; (Bb) A2M was not significantly different among the different stages of LUSC; (Ca) ACSL5 was downregulated in LUSC but upregulated in LUAD; (Cb) ACSL5 was not significantly different among the different stages of LUSC. (Da) ITGB8 was upregulated in LUAD and LUSC; ITGB8 was associated with stages of LUSC [Pr(>F)=0.0388]; (Ea) MND1 was upregulated in LUAD and LUSC; MND1 was associated with stages of LUSC [Pr(>F)=0.000953] (Fa) SCGB1A1 was downregulated in both LUSC and LUAD; SCGB1A1 was closely related to stages of LUSC [Pr(>F)=8.06×10-7]. *P<0.01 tumor group vs. control group. LUSC, lung squamous cell carcinoma; LUAD, lung adenocarcinoma; TCF21, transcription factor 21; A2M, α-2-macroglobulin; ACSL5, acyl-CoA synthetase long chain family member 5; ITGB8, integrin subunit beta 8; SCGB1A1, secretoglobin family 1A member 1; MND1, meiotic nuclear divisions 1; T, tumor; N, normal.

Figure 5.

ROC curves of hub genes and SCGB1A1 expression in different tumors, cell lines and tissues. (A) ROC curves of hub genes were plotted based on the Gene Expression Omnibus dataset. The closer AUC value is to one, the higher the diagnostic value of the gene. (B) SCGB1A1 gene expression profiles of 20 common lung cancer cell lines based on the CCLE database. (C) Dot plot of SCGB1A1 gene expression profile across different tumor samples and paired normal tissues. Each dot represents sample expression; red denotes tumor samples and green denotes normal samples. (D) SCGB1A1 mRNA and protein expression in normal human tissues based on The Human Protein Atlas. ROC, receiver operating characteristic; SCGB1A1, secretoglobin family 1A member 1; MND1, meiotic nuclear divisions 1; TCF21, transcription factor 21; A2M, α-2-macroglobulin; ACSL5, acyl-CoA synthetase long chain family member 5; ITGB8, integrin subunit beta 8; CCLE, Cancer Cell Line Encyclopedia; GEPIA, Gene Expression Profiling Interactive Analysis; TPM, transcripts per million; AUC, area under the curve; T, tumor; N, normal.

Figure 6.

Associations between the expression of SCGB1A1 and (A) EGFR mutation, (B) EML4-ALK gene fusion, (C) expression of TP53 and (D) smoking in the Okayama and Beer dataset of the Oncomine database. No value, patients without the phenotype. SCGB1A1, secretoglobin family 1A member 1; EGFR, epidermal growth factor receptor; EML4, microtubule associated protein-like 4; ALK, anaplastic lymphoma kinase; IHC, immunohistochemistry.