OncoTargets and Therapy Dovepress open access to scientific and medical research Open Access Full Text Article ORIGINAL RESEARCH Identification of novel biomarkers and candidate small molecule drugs in non-small-cell lung cancer by integrated microarray analysis This article was published in the following Dove Press journal: OncoTargets and Therapy Qiong Wu1,2,* Background: Non-small-cell lung cancer (NSCLC) remains the leading cause of cancer Bo Zhang1,2,* morbidity and mortality worldwide. In the present study, we identified novel biomarkers Yidan Sun3 associated with the pathogenesis of NSCLC aiming to provide new diagnostic and therapeu- Ran Xu1 tic approaches for NSCLC. Xinyi Hu4 Methods: The microarray datasets of GSE18842, GSE30219, GSE31210, GSE32863 and Shiqi Ren4 GSE40791 from Gene Expression Omnibus database were downloaded. The differential Qianqian Ma5 expressed genes (DEGs) between NSCLC and normal samples were identified by limma Chen Chen6 package. The construction of protein–protein interaction (PPI) network, module analysis and Jian Shu7 enrichment analysis were performed using bioinformatics tools. The expression and prog- Fuwei Qi7 nostic values of hub genes were validated by GEPIA database and real-time quantitative fi Ting He7 PCR. Based on these DEGs, the candidate small molecules for NSCLC were identi ed by the CMap database. Wei Wang2 Results: A total of 408 overlapping DEGs including 109 up-regulated and 296 down- Ziheng Wang2 regulated genes were identified; 300 nodes and 1283 interactions were obtained from the 1 Medical School of Nantong University, PPI network. The most significant biological process and pathway enrichment of DEGs were Nantong 226001, People’s Republic of China; 2The Hand Surgery Research Center, response to wounding and cell adhesion molecules, respectively. Six DEGs (PTTG1, TYMS, Department of Hand Surgery, Affiliated ECT2, COL1A1, SPP1 and CDCA5) which significantly up-regulated in NSCLC tissues, Hospital of Nantong University, Nantong 226001, People’s Republic of China; were selected as hub genes according to the results of module analysis. The GEPIA database 3Department of Oncology, First Teaching further confirmed that patients with higher expression levels of these hub genes experienced Hospital of Tianjin University of Traditional fi Chinese Medicine, Tianjin 300193, People’s a shorter overall survival. Additionally, CMap predicted the 20 most signi cant small Republic of China; 4Department of molecules as potential therapeutic drugs for NSCLC. DL-thiorphan was the most promising Biochemistry & Molecular Biology, Nantong small molecule to reverse the NSCLC gene expression. University, Nantong, Jiangsu 226001, People’s Republic of China; 5Emergency Office, Wuxi Conclusions: Based on the gene expression profiles of 696 NSCLC samples and 237 Center for Disease Control and Prevention, normal samples, we first revealed that PTTG1, TYMS, ECT2, COL1A1, SPP1 and Wuxi 214023, People’s Republic of China; 6Department of Oncology, The Affiliated CDCA5 could act as the promising novel diagnostic and therapeutic targets for NSCLC. Cancer Hospital of Nanjing Medical University, Our work will contribute to clarifying the molecular mechanisms of NSCLC initiation and Nanjing, People’s Republic of China; 7The First People‘s Hospital of Taicang City, Taicang progression. Affiliated Hospital of Soochow University, Keywords: non-small-cell lung cancer, novel biomarkers, candidate small molecules, Suzhou 215400, People’sRepublicofChina prognosis, bioinformatics analysis *These authors contributed equally to this work Correspondence: Ziheng Wang; Wei Wang The Hand Surgery Research Center, Introduction Department of Hand Surgery, Affiliated Lung cancer remains the leading cause of cancer morbidity and mortality worldwide. In Hospital of Nantong University, Nantong 226001, People’s Republic of China 2018, there are 234,030 newly diagnosed lung cancer patients, accounting for 13.5% of Tel +86 159 6297 0534; +86 136 1521 3504 all types of malignant tumors. In addition, lung cancer results in approximately 154,050 Email [email protected]; [email protected] death cases each year, accounting for 25.3% of all cancer-related deaths; 80–85% of all submit your manuscript | www.dovepress.com OncoTargets and Therapy 2019:12 3545–3563 3545 DovePress © 2019 Wu et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work http://doi.org/10.2147/OTT.S198621 you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Wu et al Dovepress lung cancer patients are diagnosed with non-small-cell lung basedonGPL9948(AgilentHuman0.6KmiRNA cancer (NSCLC) subtype and 80% lung cancer-associated Microarray G4471A; Agilent Technologies, Santa Clara, deaths are caused by NSCLC. The subtypes of NSCLC CA, USA) (GSE32863) and GPL570 (Affymetrix Human mainly consist of lung adenocarcinoma, lung squamous cell Genome U113 Plus 2.0 Array) (GSE18842, GSE30219, carcinoma and large cell lung cancer based on histological GSE31210 and GSE40791). A total of 696 NSCLC samples sub-classification.1–4 Although great advance has been made and 237 normal samples were included in our study, of which in the therapeutic methods for NSCLC such as surgical 46 tumor samples and 45 normal samples were in GSE18842 resection, chemotherapy, radiotherapy and targeted therapy, profile, 272 tumor samples and 14 normal samples in the patients’ prognosis is still far from ideal, with 5-year GSE30219 profile, 226 tumor samples and 20 normal samples survival rate less than 20%. For advanced patients who are in GSE31210 profile, 58 tumor samples and 58 normal sam- inoperable, chemotherapy such as platinum still remains the ples in GSE32863 profile, and 94 tumor samples and 100 most ideal and important treatment strategy for NSCLC.5–7 normal samples in GSE40791 profile. The poor long-term survival rate of NSCLC patients is mainly attributed to the lack of specific symptoms and effec- Screeningfor DEGs tive diagnostic methods at an early stage. In additionally, The matrix data of each dataset was performed log2 con- high metastasis rate and drug resistance are also vital factors version and normalization using limma package of R/ 8–10 that can not be ignored. In recent years, with our Bioconductor software.14 The limma package was also increased understanding of molecular characterization of utilized to screen and identify the DEGs between NSCLC, molecular targeting therapies especially individua- NSCLC samples and normal tissue sample. Adjust P- lized precision treatment have undergone remarkable value <0.05 and |log2FC| >1 were considered the statistical 11,12 developments. Despite the prominent progress in the significance of differential expression. molecular diagnosis and treatment for NSCLC, substantive 13 breakthroughs have not yet been made in patients’ survival. Functional enrichment analysis Therefore, there is still an urgent demand to identify the GO and Kyoto Encyclopedia of Genes and Genomes novel biomarkers correlated with NSCLC diagnosis and (KEGG) pathway enrichment analysis were performed to prognosis to elucidate the precise molecular mechanism of determine the biological functions of the overlapping NSCLC occurrence and progression. In this study, the micro- DEGs. GO enrichment analysis is an extensively used array data of GSE18842, GSE30219, GSE31210, GSE32863 method to investigate the molecular function (MF), cell and GSE40791 from Gene Expression Omnibus (GEO) data- component (CC) and biological process (BP) of genes or base was used to identify the differential expressed genes gene products. KEGG is a widely used database for systema- (DEGs) between NSCLC and adjacent normal tissues. Gene tic analysis of high-level gene functions. In this study, we Ontology (GO) and pathway enrichment analysis were per- carried out GO function and KEGG pathway enrichment formed to better understand the biological functions of these analysis based on the platform of Database for Annotation DEGs. We also established a protein–protein interaction Visualization and Integrated Discovery (DAVID, http:// (PPI) network associated the DEGs. Furthermore, we also david.ncifcrf.gov), an online database rich in comprehensive identified potential candidate small molecules for a better annotation information of gene and protein functions. P- treatment of NSCLC. Six novel biomarkers were found to – value <0.05 was considered statistically significant.15 19 be related to the pathogenesis and prognosis of NSCLC. In summary, this study aimed to exploit promising novel bio- – markers for NSCLC diagnosis, prognosis and molecular Protein protein interaction (PPI) targeting therapies from new insights. Figure 1 shows the network construction and module workflow of our study. analysis We used the online database STRING (Search Tool for the Materials and methods Retrieval of Interacting Genes, https://string-db.org/) to better Data resources illustrate the potential interactive relationships among the Series matrix files of GSE18842, GSE30219, GSE31210, overlapping DEGs.20 Then the Cytoscape