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Identification of key biomarkers and potential molecular mechanisms in lung cancer by bioinformatics analysis

Lung cancer is one of the most widespread neoplasms worldwide. To identify the key biomarkers in its carcinogenesis and development, the mRNA microarray datasets GSE102287, GSE89047, GSE67061 and GSE74706 were obtained from the Gene Expression Omnibus database. GEO2R was used to identify the differentially expressed genes (DEGs) in lung cancer. The Database for Annotation, Visualization and Integrated Discovery was used to analyze the functions and pathways of the DEGs, while the Search Tool for the Retrieval of Interacting Genes/Proteins and Cytoscape were used to obtain the protein-protein interaction (PPI) network. Kaplan Meier curves were used to analyze the effect of the hub genes on overall survival (OS). Module analysis was completed using Molecular Complex Detection in Cytoscape, and one co-expression network of these significant genes was obtained with cBioPortal. A total of 552 DEGs were identified among the four microarray datasets, which were mainly enriched in ‘cell proliferation’, ‘cell growth’, ‘cell division’, ‘angiogenesis’ and ‘mitotic nuclear division’. A PPI network, composed of 44 nodes and 886 edges, was constructed, and its significant module had 16 hub genes in the whole network: Opa interacting protein 5, exonuclease 1, PCNA clamp-associated factor, checkpoint kinase 1, hyaluronan-mediated motility receptor, maternal embryonic leucine zipper kinase, non-SMC condensin I complex subunit G, centromere protein F, BUB1 mitotic checkpoint serine/threonine kinase, cyclin A2, thyroid hormone receptor interactor 13, TPX2 microtubule nucleation factor, nucleolar and spindle associated protein 1, kinesin family member 20A, aurora kinase A and centrosomal protein 55. Survival analysis of these hub genes revealed that they were markedly associated with poor OS in patients with lung cancer. In summary, the hub genes and DEGs delineated in the research may aid the identification of potential targets for diagnostic and therapeutic strategies in lung cancer.


Introduction
Lung cancer, one of the most common malignant tumors, is the leading cause of cancer-associated morbidity in the population worldwide; it is the most common cancer among males and the fourth most common tumor in women (1). Lung cancer is divided into different pathological subtypes, including adenocarcinoma, squamous cell carcinoma and small cell lung cancer (SCLC) (1). The occurrence, development and metastasis of lung cancer include a number of orchestrated steps, including DNA mutations and injury (2). Despite an increased understanding of the underlying molecular mechanisms of the disease and the implementation of novel therapeutic strategies, the 5-year survival rate remains low. The study of the molecular mechanism of cancer guides the classification and treatment of lung cancer, and promotes the rapid progress of targeted therapy and immunotherapy. The large-scale research and clinical trials of these new therapies provide prospects for the individualized treatment of lung cancer.
Much progress has been made with lung cancer biomarkers over the last decade, and biomarkers have been widely applied in the diagnosis, treatment and prognosis evaluation of lung cancer, with further biomarkers now being studied. For example, anaplastic lymphoma kinase (ALK) was initially identified to be abnormally downregulated in lung cancer and a fusion of echinoderm microtubule-associated protein-like 4 (EML4) and ALK genes was found in 3.7-7% of non-SCLC (NSCLC) (3). Due to ALK fusion, 57-74% of patients with lung adenocarcinoma respond well to ALK inhibitors such as crizotinib (3). The study revealed that the median progression-free survival (PFS) and response rates of patients who received crizotinib were significantly improved compared with those of patients treated with chemotherapy (4). The epidermal growth factor receptor (EGFR), a tyrosine kinase receptor, was overexpressed in 62% of patients with NSCLC (5). Tyrosine kinase inhibitors have been the standard treatment of patients with EGFR mutations due to their high response rate (55-78%) and PFS rate (1). Therefore, the discovery of new diagnostic and therapeutic targets is of great significance for the early diagnosis, drug development and targeted therapy of lung cancer.
Bioinformatics analysis has been commonly applied in cancer research to identify genetic changes associated with cancer. Previous studies have performed bioinformatics analysis to identify differentially expressed genes (DEGs) in various types of cancer, as well as to determine their roles in biological processes, molecular functions and different pathways (6,7). Accordingly, the present study analyzed data generated by microarray technology to explore the potential pathogenesis of lung cancer. Specifically, given the high number false-positives associated with the analysis of a single microarray, four public mRNA datasets were screened in the present study to identify DEGs between lung cancer and adjacent non-cancerous tissue samples. Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed, and a protein-protein interaction (PPI) network analysis was used to assist in demonstrating the molecular pathogenesis underlying the carcinogenesis and development of lung cancer. A total of 552 DEGs and 16 hub genes were identified and they may serve as candidate biomarkers in lung cancer.

Materials and methods
Public mRNA datasets. Gene Expression Omnibus (GEO; www.ncbi.nlm.nih.gov/geo) is an open platform to store genetic data (8). Four gene expression profiles (GSE102287, GSE89047, GSE67061 and GSE74706) were acquired from the GEO The GSE102287 dataset contained 32 cancer samples and 34 normal samples (9). The GSE89047 dataset consisted of 8 cancer samples and 8 normal samples. The GSE67061 contained 56 cancer samples and 17 normal samples. The GSE74706 contained 18 cancer samples and 18 normal samples (10). The datasets consisted of a number of pathological subtypes of lung cancer, including NSCLC and lung squamous cell carcinoma. In the current study, in order to be more representative, a specific pathological type was not specified when selecting datasets.
Identification of DEGs. GEO2R (www.ncbi.nlm.nih. gov/geo/geo2r) is an interactive online tool to identify DEGs from GEO series (11). GEO2R was applied to distinguish DEGs between normal and lung cancer tissue samples. Duplicate and absent probe sets were removed. The cut-off criteria for the identification of DEGs were |log 2 fold-change|>1 and adjusted P<0.05.

Functional annotation for DEGs with KEGG and GO analysis.
The Database for Annotation, Visualization and Integrated Discovery (DAVID; www.david.abcc.ncifcrf.gov) provides typical batch annotation and GO (www.geneontology.org) analysis to highlight the most relevant GO terms associated with a given gene list (12). GO covers three aspects of biology, including biological process, molecular function and cellular component. KEGG (version 90.0; www.kegg.jp), is one of the most commonly used biological information databases in the world (13). Following KEGG and GO analysis in DAVID, functional annotation for DEGs was performed. P<0.05 was considered to indicate a statistically significant difference.
Construction of the PPI network and identification of a significant module. The Search Tool for the Retrieval of Interacting Genes (version 11.0; string.embl.de) (14), an online open tool, was applied to construct a PPI network, and Cytoscape (version 3.7.1) (15) was used to present the network. Using a confidence cutoff of >0.4, a node score cutoff of 0.2, a degree cutoff of 10, a maximum depth of 100 and a k-core of 2, the significant modules in the aforementioned PPI network were identified using the Molecular Complex Detection tool (version 1.5.1) (16). Subsequently, functional annotation for the genes in this module were performed using KEGG and GO analysis in DAVID.
Analysis and identification of hub genes. Hub genes with ≥43 degrees were selected. cBioPortal (www.cbioportal.org) integrates The Cancer Genome Atlas (TCGA; portal.gdc. cancer.gov), the International Cancer Genome Consortium (icgc.org) and other cancer genome database data to provide online visualization tools. Based on the hub genes screened, a gene co-expression network was constructed and cBioportal was used to search for genes with a similar expression pattern to the hub genes in lung cancer and to investigate the interaction between genes (17). Furthermore, hub genes were analyzed with the biological process analysis, and were visualized using the BiNGO tool in Cytoscape (version 3.7.1) (18). The Kaplan-Meier plotter (kmplot.com/analysis) and the log rank test were used to plot and compared survival curves, respectively. The Kaplan-Meier plotter is an online tool that integrates gene expression data and clinical data from TCGA, GEO and the European Genome-Phenome Archive databases (www.ebi. ac.uk/ega/home). According to the different quantile expression levels of the proposed biomarkers, patients were divided into two groups to analyze the prognostic value of specific genes (19).
Construction of the PPI network and identification of a significant module. A PPI network was constructed and a significant module with 44 nodes and 886 edges was identified ( Fig. 3; Table SII). KEGG pathway and GO analysis of the DEGs involved in this module were analyzed using DAVID. Results revealed that genes in this module were significantly enriched in 'cell division', 'cell cycle' and 'mitotic nuclear division' (Table I).

Discussion
Lung cancer is one of the most common malignancies worldwide, both in terms of incidence and mortality (21,22). Despite significant advances in diagnostic and treatment strategies, the prognosis of patients with lung cancer remains unsatisfactory. Therefore, there is a requirement for the identification of lung cancer biomarkers to serve as novel diagnostic and therapeutic targets. Bioinformatics analysis has been widely applied to investigate genetic alterations in the progression of diseases, and may enable the identification of novel therapeutic targets.
Previous studies have screened biomarkers associated with the different pathological subtypes of lung cancer (23)(24)(25)(26). Similarly, the present study screened potential biomarkers of lung cancer. However, the present study differs from the previous literature in a number of ways. In the current study, research data was derived from different datasets, which allows diversification of data results. Four datasets were selected to reduce the errors associated with a single dataset and differences of sequencing platforms, so as to improve the credibility of the results. The aim of the present study was to screen common biomarkers and drug targets in various pathological types of lung cancer using bioinformatics analysis. Finally, different results were achieved due to the different data sources and statistical methods used. However, certain biomarkers identified in the current study are consistent with previously published studies (27)(28)(29)(30)(31).
In the present study, 552 common DEGs were identified in the four microarray datasets. GO enrichment analysis revealed that changes in the most significant module were mainly enriched in 'cell division', 'mitotic nuclear division' and 'G 2 /M transition of mitotic cell cycle', while changes in KEGG analysis were mainly enriched in the 'cell cycle' and 'p53 signaling pathway'. Previous studies demonstrated that dysregulation of the cell cycle is associated with carcinogenesis and the progression of tumors (32,33). In the current study, a PPI network consisting of 44 nodes and 886 edges was constructed. The 16 genes with the highest degrees in the PPI network included OIP5, EXO1, KIAA0101, CHEK1, HMMR, MELK, NCAPG, CENPF, BUB1, CCNA2, TRIP13, TPX2, NUSAP1, KIF20A, AURKA and CEP55. Subsequently, survival analysis of these genes revealed that they were significantly associated with a worse 5-year overall survival time of patients with lung cancer.
The mechanism of lung cancer is driven by specific genetic and epigenetic changes (34). In certain types of cancer, such as gastric colorectal cancer, the expression of OIP5 is upregulated and may be associated with the occurrence of cancer (35,36). However, its function in lung cancer remains unknown. EXO1 is a nuclease that modulates DNA recombination, maintains genomic stability and mediates cell cycle arrest. Several reports have indicated that functional polymorphisms of EXO1 may be associated with the occurrence of lung cancer, and it may serve as a novel biomarker for the diagnosis and treatment of lung cancer (37,38). KIAA0101 is involved in cell cycle regulation and DNA repair and is expressed at high levels in several types of cancer, including gastric and lung cancer (27,39,40). Previous studies reported that high expression levels of KIAA0101 and CHEK1 in lung cancer are associated with a poor prognosis (27,41). Man et al (28) revealed that HMMR, the receptor for hyaluronic acid, was upregulated in lung adenocarcinoma samples compared with healthy adjacent non-cancerous tissues. MELK is expressed in several types of human cancer (42,43), including SCLC. Inoue et al (42) reported that inhibition of MELK may be a therapeutic strategy for SCLC. Zhang et al (44) reported that NCAPG may be implicated in hepatocellular carcinoma cell proliferation and migration, and may provide a promising novel therapeutic target for the treatment of advanced hepatocellular carcinoma. However, the clinical significance of NCAPG in lung cancer remains unknown.
Previous studies reported that CENPF serves a role in the tumorigenesis of hepatocellular carcinoma and prostate cancer (45,46); however, its role in lung cancer requires further investigation. A number of studies demonstrated that BUB1 serves important roles in breast and endometrial cancer (47)(48)(49). However, Haruki et al (47)(48)(49) reported that the BUB gene family members, including BUB1, are not commonly associated with mitotic checkpoint defects in lung cancer. The potential association between BUB1 and lung cancer requires further investigation. Kim et al (29) reported that a functional single nucleotide polymorphism in the promoter region of CCNA2 was associated with an increased risk of lung cancer. TRIP13 is an ATPase that serves a key role in mitotic checkpoint complex inactivation and is associated with the progression of lung adenocarcinoma (30). Li et al (30) demonstrated that increased TRIP13 expression promoted lung adenocarcinoma progression and may serve as a potential therapeutic target or biomarker for the disease.
Yang et al (50,51) revealed that TPX2 was associated with lung squamous carcinoma cell radioresistance and may serve as a therapeutic target to enhance cell radiosensitivity in lung squamous carcinoma. Furthermore, Schneider et al (50,51) demonstrated that the expression of the TPX2, mitosis-associated gene, was associated with the prognosis of patients with NSCLC. Previous studies reported that overexpression of NUSAP1 was associated with a poor prognosis in prostate cancer, hepatocellular and oral squamous cell carcinoma (52,53); however; little is known about the association of NUSAP1 with lung cancer. Zhao et al (54) demonstrated that KIF20A may confer a malignant phenotype in lung adenocarcinoma by regulating cell proliferation and apoptosis. AURKA, an oncogene, encodes a serine-threonine kinase that regulates mitotic processes in mammalian cells and serves as a potential therapeutic target of NSCLC (55,56). Lo et al (55,56) reported  that AURKA upregulation is restricted to specific subtypes and poorly differentiated tumors in NSCLC. Ma et al (31) revealed that CEP55 was upregulated in lung cancer cells and was associated with poor clinical outcomes in patients with lung cancer, and that it may serve as a prognostic biomarker for the disease. The current study is only a preliminary report, and heterogeneous results due to the limitations of the source and quantity of samples may have occurred. Furthermore, statistical differences may not translate to the expected clinical significance. In order to be more representative, a specific pathological type of lung cancer was not selected in the current study. However, this may lead to poor specificity in lung cancer subtypes. The 16 hub genes identified revealed clinical significance in the validation of survival analysis. However, further validation in the subsequent basic and clinical trial studies is required. In addition to DEGs, further studies investigating differentially expressed microRNAs and their association with genes, particularly DEGs, are required.
In summary, the current study identified DEGs that may be involved in the carcinogenesis or progression of lung cancer. A total of 552 DEGs and 16 hub genes were identified, and these may serve as potential diagnostic biomarkers or therapeutic targets for lung cancer. The results suggested that data mining   and integration may be a promising tool for the identification of biomarkers in malignant tumors. As tumor biomarkers only have meaning if they are integrated with clinical data, further experiments should be conducted to verify the results obtained in the current study.

Acknowledgements
Not applicable.

Funding
No funding was received.

Availability of data and materials
The datasets generated and/or analyzed during the current study are available in the GEO repository (www.ncbi.nlm.nih. gov/geo).

Authors' contributions
ZHL and BES designed the overall research. ZL, JL and FZ collected the data. ZHL, MXS, ZQT, ZL and BES contributed to data analysis and visualization. JL and FZ drafted and revised the manuscript. All authors approved the final version of the manuscript. PCNA clamp-associated factor PCNA-binding protein that acts as a regulator of DNA repair during DNA replication. Also acts as a regulator of centrosome number. CHEK1 Checkpoint kinase 1 Required for checkpoint-mediated cell cycle arrest in response to DNA damage or the presence of unreplicated DNA. HMMR Hyaluronan-mediated motility receptor Encodes a protein involved in cell motility.

MELK
Maternal embryonic leucine zipper Serine/threonine-protein kinase involved in various processes, such kinase as cell cycle regulation, self-renewal of stem cells, apoptosis and splicing regulation. NCAPG Non-SMC condensin I complex Encodes a subunit of the condensin complex, which is responsible subunit G for the condensation and stabilization of chromosomes during mitosis and meiosis. CENPF Centromere protein F Encodes a protein that associates with the centromere-kinetochore complex. BUB1 BUB1 mitotic checkpoint Encodes a serine/threonine-protein kinase that serves a central role in serine/threonine kinase mitosis. Mutations in this gene have been associated with aneuploidy and several forms of cancer. CCNA2 Cyclin A2 Encodes a protein that binds and activates cyclin-dependent kinase 2 and promotes transition through G1/S and G2/M. TRIP13 Thyroid hormone receptor Encodes a protein that interacts with thyroid hormone receptors, interactor 13 which may serve a role in early-stage non-small cell lung cancer. TPX2 Targeting protein for Xklp2 Spindle assembly factor required for normal assembly of mitotic spindles. NUSAP1 Nucleolar and spindle-associated Nucleolar-spindle-associated protein that serves a role in spindle protein 1 microtubule organization. KIF20A Kinesin family member 20A Mitotic kinesin required for chromosome passenger complex-mediated cytokinesis. AURKA Aurora kinase A Encodes a cell cycle-regulated kinase involved in microtubule formation and/or stabilization at the spindle pole during chromosome segregation.

CEP55
Centrosomal protein 55 Serves a role in mitotic exit and cytokinesis.

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