Identification of lung adenocarcinoma biomarkers based on bioinformatic analysis and human samples
- Authors:
- Published online on: February 28, 2020 https://doi.org/10.3892/or.2020.7526
- Pages: 1437-1450
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Copyright: © Dong et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Non-small cell lung cancer (NSCLC) is the most common malignant tumor globally and is associated with an extremely high mortality rate (1). The incidence of NSCLC continues to surge globally. As with other tumors, lung adenocarcinoma which accounts for about 35–45% of all lung malignant tumors is a heterogeneous disease characterized by high rates of genetic mutation (2). Despite the emergence of diverse new approaches for the treatment of lung adenocarcinoma, such as targeted and immune therapy, long term survival is still poor (3,4). One of the main reasons for this is that most patients are diagnosed at an advanced stage. Thus, it is necessary to understand the molecular mechanisms behind lung adenocarcinoma genesis, growth and progression, and identify biomarkers that can be detected during the early stages of the disease.
Recently, high-throughput bioinformatic technologies such as microarrays have been widely used to screen for differentially expressed genes (DEGs) and identify the functional pathways involved in the genesis and development of lung adenocarcinoma. However, the reliable results are not easy to obtain due to the false-positive rates that may exist in every independent microarray analysis. Thus, we downloaded three original mRNA data sets (GSE32863, GSE63459 and GSE75037) from the Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) and aimed to identify the DEGs between normal lung and lung adenocarcinoma tissues. Next, the Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/) and Gene Ontology (GO; http://www.geneontology.org) databases were used to identify biological processes enriched in DEGs, and integrated protein-protein interaction (PPI) network analysis was used to help us understand the molecular mechanisms underlying lung adenocarcinoma genesis and development. Sixteen hub genes and 376 DEGs were identified, which could be potential target genes and candidate biomarkers for lung adenocarcinoma. To minimize the false-positive rate of the microarray analysis, the results were then confirmed in cell lines and human sample tissues.
Materials and methods
Microarray data
GEO is a public functional genomics data repository supporting MIAME-compliant data submissions. It accepts sequence based and array data. Tools are provided to help users query and download experiments and curated gene expression profiles (5). The GSE32863, GSE63459 and GSE75037 datasets produced by the Illumina HumanWG-6/Ref-8 v3.0 expression beadchip platform (Illumina Inc; http://www.illumina.com) were downloaded for further analysis. The GSE63459 dataset contains data from 33 lung adenocarcinoma tissue samples and 32 adjacent normal tissue samples (6). The GSE32863 dataset contains data from 58 lung adenocarcinoma tissue samples and 58 fresh frozen adjacent non-cancerous samples (7). Moreover, the GSE75037 dataset contains data from 84 lung adenocarcinoma and 84 adjacent non-cancerous lung tissue samples (8).
Identification of DEGs
GEO2R is an online web tool (https://www.ncbi.nlm.nih.gov/geo/geo2r/) provided by the GEO for comparing GEO series to identify DEGs across experimental conditions. The cutoff criteria were set to P-value <0.05 and logFC (fold change)>1. We excluded probe sets without exact gene symbols, and genes with two or more probe sets were averaged.
KEGG and GO enrichment analyses of DEGs
The functional annotation tools version 6.7 of the Database for Annotation, Integrated Discovery and Visualization (9) (DAVID; http://david.ncifcrf.gov) were used to extract biological information about our DEGs. KEGG is a public database used for understanding the functions and abilities of biological systems, such as cells, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets acquired by genome sequencing (10). GO was also used to annotate genes and further analyze their biological functions. The DAVID online database was used to analyze the function and biological process of the screened DEGs. P<0.05 was considered to indicate statistical significance.
PPI network construction and analysis
The Search Tool for the Retrieval of Interacting Genes (STRING; http://string-db.org; version 10.0) was used to construct the PPI network from the DEGs (11). The sources for interactions are text mining, databases, experiments, neighborhood, co-expression, gene co-occurrence and fusion. We set the minimum required interaction score to 0.4. Cytoscape version 3.4.0 software (12) was used to visualize the molecular interaction networks of the DEGs. The APP plug-in, Molecular Complex Detection (MCODE) (13), was used to arrange the network topology to cluster densely connected genes. After the PPI networks was constructed, its key modules were searched by using the MCODE application. The parameter for inclusions are MCODE score >5, degree cutoff=2, node score cutoff=0.2, node density cutoff=0.1, k-score=2 and Max depth=100. Then, DAVID was used to perform the GO and KEGG analyses for these most significant modules.
Hub gene screen and analysis
The criterion for being a hub gene selection was degree ≥10. Further analysis was performed using the cBioPortal online platform (http://www.cbioportal.org) to build the network of the DEGs and co-expressing genes (14). The mutation rates of the hub genes were also measured with the cBioPortal platform (15). Cytoscape's Biological Networks Gene Oncology tool (BiNGO) (version 3.0.3) was used for the biological process analysis and visualization (16). The University of California Santa Cruz (USCS) platform was used to analyze the hierarchical clustering of hub genes (17). Kaplan-Meier curves for overall survival and disease-free survival with these hub genes were obtained from cBioPortal. The expression profiles of DNA topoisomerase IIα (TOP2A) and aurora kinase A (AURKA) in 20 types of malignant tumors were analyzed and displayed using the Oncomine database (http://www.oncomine.com) (18).
Analysis of TOP2A and AURKA expression in cell lines
To confirm our bioinformatics results, reverse transcription and quantitative real-time PCR (RT-qPCR) were conducted on lung adenocarcinoma (HCC827, A549 and H1975) cell lines and a human bronchial epithelial (HBE) cell line. A549, HCC827 and H1975 cells were purchased from the Shanghai Cell Bank (Shanghai, China) and were cultured using Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco; Thermo Fisher Scientific, Inc.). The medium was supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.), and 10% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.) under a 5% CO2-containing humidified atmosphere at 37°C. Total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The isolated RNA was reverse-transcribed into cDNA using a reverse transcription kit (Takara, Dalian, China). RT-qPCR was performed as described in our previous research, 2 min at 50°C, 10 min at 95°C, 40 cycles at 95°C for 15 sec, and 60°C for 30 sec (19) and the results were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels. Primers were as follows: TOP2A (forward, 5-AGGATTCCGCAGTTACGTGG-3 and reverse, 5-CATGTCTGCCGCCCTTAGAA-3) (20) and AURKA (forward, 5-TTGGGTGGTCAGTACATGCTC-3 and reverse, 5-GTGAATTCAACCCGTGAT-3) (21) and GAPDH sense, 5′-CAATGACCCCTTCATTGACC-3′ and reverse, 5′-TGGAAGATGGTGATGGGATT-3′. The statistical analyses were conducted using SPSS version 21 (IBM Corp.). Results are displayed as mean ± SEM and differences between the HBE and cancerous cell lines were analyzed by one-way ANOVA. We further used the Tukey test to determine the significance between each cancer cell line and HBE. P-value <0.05 was considered to indicate statistical significance. Each experiment was repeated three times.
Analysis of TOP2A and AURKA expression in human samples
The Ethics Committee of the First Hospital of the China Medical University (Shenyang, Liaoning, China) approved our research. Written informed consent was received from all participants. Seventy-two lung adenocarcinoma and paired non-cancerous tissues were obtained between February 2013 and June 2014 from 35 women and 37 men, ranging in age from 38 to 75, with a median age of 60. Patients who had received chemotherapy, target therapy and radiotherapy or had a history of malignant tumor were excluded. All of the diagnoses were confirmed by two experienced pathologists. The resected samples were preserved at −80°C until the mRNA of TOP2A and AURKA extraction were needed. Differences between cancerous and non-cancerous tissues were compared using the paired Student's t-test.
Results
Identification of DEGs in lung adenocarcinoma
A total of 5,874 genes were found to be differentially expressed in non-cancerous and lung adenocarcinoma tissues (432 in GSE63459, 4,037 in GSE75037 and 1,405 in GSE32863) after standardizing the microarray data. A total of 376 DEGs were found in all three datasets (Venn diagram, Fig. 1A), consisting of 293 downregulated and 83 upregulated genes.
GO and KEGG enrichment analyses of the DEGs
The DAVID online database was used to further analyze the biological classification, as well as functions and pathways enriched in DEGs. GO analysis showed that the biological processes (BP) of the DEGs were mainly involved in regulation of cell proliferation, the transforming growth factor β receptor signaling pathway, cell adhesion, biological adhesion and responses to hormone stimulus (Table I). Examination of their cell component showed that the DEGs were mainly located in the proteinaceous extracellular matrix, cell surface, cell-cell junction, and cell-substrate adherent junction. KEGG pathway analysis showed that the DEGs were mainly over represented in the TGF-β signaling pathway, cell adhesion molecules (CAMs), complement and coagulation cascades and ECM-receptor interaction (Table I).
PPI network construction and module analysis
Cytoscape was used to build the DEG PPI network (Fig. 1B) and identify the most significant genes of the PPI network (Fig. 1C). Analysis of these genes with the DAVID platform found that they were mainly involved in M phases, cell cycle phase, the mitotic cell cycle and nuclear division (Table II).
Hub gene selection and analysis
Sixteen genes were identified with a degree ≥10 and were defined as hub genes. The degree of each gene was calculated by the CytoScape software and represented the number of other genes with which it was connected. The hub gene symbol, full name, function and degree are listed in Table III. cBioPortal was then used to construct a network of the 16 hub genes and their co-expressed genes. The results of this and analysis of the BP are shown in Fig. 2A and B, respectively. oncoPrint analysis with the cBioPortal showed that TOP2A and AURKA have the highest genetic mutation rates of the hub genes in lung adenocarcinoma at 8 and 14%, respectively (Fig. 2C). Hierarchical clustering analysis revealed that these 16 hub genes could generally differentiate both primary and recurrent lung adenocarcinoma tissues from their adjacent non-cancerous lung tissues (Fig. 2D).
Clinical significance of TOP2A and AURKA
Analysis of the association between these genes and disease-free survival and overall survival analysis was conducted using Kaplan-Meier curves in the cBioPortal platform. Lung adenocarcinoma patients with AURKA mutation had worse overall and disease-free survival and patients with ASPM (abnormal spindle microtubule assembly) mutation had worse disease-free survival (Fig. 3A and B). Moreover, AURKA and TOP2A had the highest node degrees at 22 and 31, respectively, implying that they may play significant roles in the genesis and development of lung adenocarcinoma. When analyzing the data from cBioPortal platform, we discovered that lung adenocarcinoma patients who had an AURKA mutation had reductions in overall survival (P=0.00192). However, this was not true for the TOP2A gene (P=0.775, Fig. 3A and B). The expression profile of AURKA and TOP2A in 20 types of human cancer tissues was displayed using the Oncomine database. TOP2A mRNA levels in bladder, brain, breast, colorectal, esophageal, kidney, gastric and sarcoma cancer tissues were higher than those in matched adjacent normal tissues (Fig. 4A). The AURKA mRNA levels in bladder, brain, breast, cervical, lung and liver cancer tissues were higher than those in adjacent matched normal tissues (Fig. 4B). When we analyzed six different datasets from the Oncomine database, we found that TOP2A and AURKA were significantly overexpressed in lung adenocarcinoma tissues compared with non-cancerous tissues (Fig. 4C and D) (7,22–28).
Expression of TOP2A and AURKA in lung adenocarcinoma cell lines
To confirm the bioinformatics results, the expression of these two genes in lung adenocarcinoma cell lines and HBE cells were assessed by RT-qPCR. The mRNA levels of both TOP2A and AURKA were significantly higher in the lung adenocarcinoma cell lines than the HBE cells (Fig. 5A and B).
Expression of TOP2A and AURKA in human lung adenocarcinoma and adjacent normal lung tissues
Table IV shows the clinicopathological characteristics of all the lung adenocarcinoma patients. TOP2A and AURKA were found to have 2.25- and 2.73-times higher expression levels in the cancer tissues than in the normal tissues, respectively (Fig. 5C and D). The 72 samples were then classified into two groups based on the relative expression levels of these two genes: The relatively high TOP2A and AURKA group (n=36, the first 36 cases having relative high TOP2A and AURKA were defined as the high-expression group; the remaining 36 cases were defined as the low-expression group) and the relatively low TOP2A and AURKA group (n=36). χ2 analysis was used to analyze the results. Analysis revealed that increased TOP2A expression was observed in tumors with a larger diameter. Increased AURKA expression was observed in tumors with a larger size and in lymphatic metastatic tumors (Tables IV and V).
Discussion
Recent statistics on cancer globally revealed that lung cancer is the most commonly diagnosed malignant tumor and is the leading cause of cancer-related mortality accounting for 11.6% of all malignant tumors and 18.4% of cancer-related deaths. Lung adenocarcinoma is the most common subtype of malignant lung cancer, and its incidence is increasing rapidly (29). Air pollution and smoking are the two main etiological factors for lung adenocarcinoma (30,31). The microarray data of Sekine et al revealed that a human lung mucoepidermoid carcinoma cell line exposed to smoke with a charcoal filter had a total of 1,582 genetic mutations (32). Still, the molecular mechanisms underlying the genesis of lung adenocarcinoma remain unclear. Without an early diagnosis most patients are not candidates for curative therapies leading to the deeply unsatisfactory prognosis for the disease. Therefore, biological markers with satisfactory efficiency for early diagnosis and therapy are desperately needed. With the development of microarray technology, we are able to efficiently screen for changes in gene expression in lung adenocarcinoma, and this approach has been proven to be a very useful method for screening early stage biomarkers in both malignant and benign diseases (33–35).
In the present research, three mRNA microarray datasets were downloaded from the GEO database and subsequently analyzed to acquire differentially expressed genes (DEGs) between lung adenocarcinoma and adjacent non-cancerous lung tissues. In addition, a total of 376 DEGs were identified in the three datasets, consisting of 293 upregulated genes and 83 downregulated genes. The biological roles of the identified DEGs were then studied using KEGG and GO enrichment analyses. The downregulated genes were mainly overrepresented in cell division, nuclear division, DNA replication and second-messenger-mediated signaling, and the upregulated genes were mainly involved in cell cycle process, DNA metabolic process, the transforming growth factor (TGF)-β signaling pathway and angiogenesis. Previous articles have revealed that dysregulation of angiogenesis and activation of the TGF-β signaling pathway are associated with the carcinogenesis and progression of lung adenocarcinoma (36–38). In addition, DNA damage and metabolic process abnormalities often play a significant role in cell cycle regulation dysfunction and are associated with malignant tumors (39). In summary, our results are consistent with these previous reports and theories. KEGG enrichment analysis showed differences mainly in the TGF-β signaling pathway, complement and coagulation cascades, Cell adhesion molecules (CAMs), and ECM-receptor interaction, while changes identified by GO terms were mainly in regulation of cell adhesion, proliferation, the TGF-β receptor signaling pathway, biological adhesion and response to hormone stimulus.
Sixteen genes with degrees ≥10 were defined as hub genes (40). Two of these hub genes, TOP2A and AURKA, had the highest node degrees at 31 and 22, respectively. Gene mutation can promote tumorigenesis, thus the mutation rates of these 16 lung adenocarcinoma hub genes were screened with the cBioPortal platform. The four genes with the highest mutation rates were AURKA, TK1, CDC45 and TOP2A with mutation rates of 14, 13, 10 and 8% respectively, which indicates that these genes may play a significant role in tumorigenesis.
DNA topoisomerase II α (TOP2A) that encodes the DNA topoisomerase, an enzyme that alters and controls the topological state of DNA during transcription, has been shown to be correlated with an increased risk of developing brain metastases, drug resistance and an abnormal cell cycle (41–43). It is regarded as a target for several anticancer agents, such as etoposide and topotecan (44). In our research, the protein-protein interaction network (PPI) network revealed that TOP2A directly interacts with maternal embryonic leucine zipper kinase (MELK), CDC20, CCNB2, UBE2T, KIAA0101 and TK1, indicating that TOP2A plays a key role in lung adenocarcinoma. Two of these genes, CDC20 and MELK, are closely involved in tumorigenesis and the cell cycle. Cell division cycle 20 (CDC20) appears to function as a regulatory protein interacting with other proteins at several important phases in the cell cycle. CDC20 is activated in lung adenocarcinoma and overexpression is correlated with poor prognosis (45). MELK plays a key role in the proliferation and self-renewal of progenitor and tumor stem cells, and is overexpressed in lung adenocarcinoma, contributing to carcinogenesis. MELK is an effective target for kinase drugs (46). Moreover, the expression of TOP2A is upregulated in various tumors, such as colon and ovarian malignant tumors, and may be considered a sensitive biomarker for early detection and therapy of these tumors (47,48).
Aurora kinase A (AURKA) is a putative oncogene, associated with cell cycle-regulated kinase (49). GO annotations associated with this gene include protein tyrosine kinase activity, transferring phosphorus-containing groups and transferase activity. Overexpression of this gene is associated with several common features of malignant tumor cells, such as chromosomal instability, aneuploidy in mammalian cells and centrosomal duplication abnormalities (50,51). It has also been found to be overexpressed in several types of malignant tumors and has been associated with poor prognosis (52,53). The aurora kinase A inhibitor, alisertib, has been approved for therapy for solid tumors especially non-small cell lung cancer (NSCLC) and breast cancer, and has achieved satisfactory results (54). In addition, Chen et al revealed that AURKA antagonists can enhance the cytotoxicity of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) (55).
The relationships between TOP2A and AURKA expression and survival were further assessed with the cBioPortal platform. We found that changes in TOP2A expression were associated with a decrease in both disease-free and overall survival, although they were not statistically significant. While changes in AURKA expression were associated with significantly worse overall survival rates, but no significant change in disease-free survival rate. This may be explained by our study of 72 paired human samples which found that upregulation of AURKA was associated with lymphatic metastasis and that upregulation of TOP2A was only associated with tumor size. One of the underlying molecular reason may be that the overexpression of TOP2A arises from amplification and mutation, while the survival analysis using the cBioPortal platform was only based on the mutation of TOP2A. Thus, amplification but not mutation may result in overexpression of TOP2A, not related to changes in prognosis, although further study is needed to prove this hypothesis. Oncomine analysis revealed that higher AURKA mRNA levels were observed in colorectal cancer, breast cancer, lung cancer and sarcoma. Additionally, higher TOP2A mRNA levels were observed in colorectal cancer, breast cancer, lung cancer and brain cancer, indicating important roles of these two genes in the carcinogenesis and development of malignant tumors. However, these results also indicate that these two genes can only be used as broad-spectrum tumor markers as they cannot differentiate lung adenocarcinoma from other malignant tumors.
The UCSC (University of California Santa Cruz) cancer platform was used to hierarchically cluster the hub genes. Their expression levels in both the primary and recurrent tumors were upregulated compared with the normal tissues. The expression levels of these hub genes in recurrent tumors were higher than in the primary tumor. TOP2A in particular was found to have the highest expression in the recurrent tumors. We therefore infer that TOP2A and other hub genes may be regarded as early biomarkers for monitoring tumor recurrence.
To confirm our results, we detected the expression of TOP2A and AURKA in lung adenocarcinoma and human bronchial epithelial (HBE) cell lines. Both of these genes were downregulated in the lung adenocarcinoma cell lines. TOP2A and AURKA had their highest expression levels in H1975 cells with approximately 15 times higher expression than that in HBEs cells. Further experiments in human samples found that TOP2A and AURKA were both upregulated in lung adenocarcinoma tissues compared with non-cancerous tissues. The upregulation of TOP2A was found to be associated with larger tumor size, and AURKA was found to be associated with both larger tumor size and positive lymphatic metastasis. Thus, we demonstrated that TOP2A and AURKA are closely involved in lung adenocarcinoma using both bioinformatics and cell experiments. The cell lines we selected have varying levels of EMT. The H1975 cell line (56) has the highest relative level of EMT and also has higher levels of AURKA and TOP2A in our study. We also showed that higher AURKA expression is correlated with poor prognosis. Thus, the higher expression of AURKA may be correlated with higher levels of EMT, which result in metastasis and lead to poor prognosis.
In conclusion, our research aimed to identify DEGs, which may be involved in the genesis and development of lung adenocarcinoma. Two of the 16 hub genes were further studied in cell lines and human samples and may be regarded as biomarkers for the diagnosis of lung adenocarcinoma. Further research is needed to elucidate the mechanisms behind their changes in expression and their biological function in lung adenocarcinoma.
Acknowledgements
Not applicable.
Funding
This research was supported by a grant (no. 81702242) from the National Science Foundation of China and Doctoral Scientific Research Foundation of Liaoning Province (no. 20170520441).
Availability of data and materials
The data used in this study are available from the corresponding author on reasonable request.
Authors' contribution
SD and SX designed the research. WM carried out the data collection and analysis. SY conducted the experiments. SD wrote the manuscript. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Ethics approval and consent to participate
The Ethics Committee of the First Hospital of the China Medical University (Shenyang, Liaoning, China) approved our research. Written informed consent was received from all of the included patients.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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