ONCOLOGY LETTERS 16: 6663-6669, 2018 Identification of the key genes and pathways in prostate cancer SHUTONG FAN1*, ZUMU LIANG1*, ZHIQIN GAO1, ZHIWEI PAN2, SHAOJIE HAN3, XIAOYING LIU1, CHUNLING ZHAO1, WEIWEI YANG1, ZHIFANG PAN1 and WEIGUO FENG1 1College of Bioscience and Technology, Weifang Medical University, Weifang, Shandong 261053; 2Department of Internal Medicine, Laizhou Development Zone Hospital, Yantai, Shandong 261400; 3Animal Epidemic Prevention and Epidemic Control Center, Changle County Bureau of Animal Health and Production, Weifang, Shandong 262400, P.R. China Received March 5, 2018; Accepted September 17, 2018 DOI: 10.3892/ol.2018.9491 Abstract. Prostate cancer (PCa) is one of the most common Introduction malignancies in men globally. The aim of the present study was to identify the key genes and pathways involved in the Prostate cancer (PCa) is one of the most common malignancies occurrence of PCa. Gene expression profile (GSE55945) in men globally and the second leading cause of cancer was downloaded from Gene Expression Omnibus, and associated mortality in developed countries (1,2). Like other the differentially expressed genes (DEGs) were identified. cancers, PCa is considered to be a disease which caused by Subsequently, Gene ontology analysis, KEGG pathway age, diet and gene aberrations (3). Accumulating evidences analysis and protein-protein interaction (PPI) analysis of have demonstrated that a series of genes and pathways involved DEGs were performed. Finally, the identified key genes were in the occurrence, progression and metastasis of PCa (4). At confirmed by immunohistochemistry. The GO analysis results present, the underlying mechanism of PCa occurrence is still showed that the DEGs were mainly participated in cell cycle, unclear, which limits the diagnosis and therapy. Therefore, it is cell division, cell development and cell junction. The KEGG urgent to identify the key genes and pathways involved in the pathway analysis showed that the DEGs were mainly enriched occurrence of PCa (2,5). in proteoglycans in cancer, endocytosis, focal adhesion and Microarray is a useful tool for analysis of gene expression that hippo signaling pathway. The PPI analysis results showed can be applied to disease diagnosis and targeted therapy (6,7). that RPS21, FOXO1, BIRC5, POLR2H, RPL22L1 and NPM1 During the past decade, hundreds of differentially expressed were the key genes involved in the occurrence of PCa, and genes (DEGs) participated in biological processes, cell compo- the Module analysis indicated that the occurrence of PCa nent, molecular functions and pathways of PCa were identified was associated with cell cycle, oocyte meiosis and ribosome by microarray technology (8,9). However, previous studies of biogenesis. IHC result showed that the expression of RPS21, DEGs analysis have shown relative limitations, for example, no BIRC5, POLR2H, RPL22L1 and NPM1 were significantly reliable biomarker was identified that could distinguish tumors upregulated in PCa, while the expression of FOXO1 was from normal tissues (10). Therefore, gene expression in the significantly downregulated in PCa, matching with the occurrence of PCa needs to be further analyzed by microarray bioinformatics analysis. Taken together, several key genes combining bioinformatics technology at present. and pathways were identified involved in PCa, which might In the present study, Gene expression profile (GSE55945) provide the potential biomarker for prognosis, diagnosis and was downloaded from Gene Expression Omnibus (GEO) drug targets. (http://www.ncbi.nlm.nih.gov/geo/). The gene expression profile was analyzed, and the DEGs were identified between PCa group and normal group. Subsequently, gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and protein-protein interaction (PPI) analysis of DEGs were performed. Finally, the expression Correspondence to: Dr Weiguo Feng or Dr Zhifang Pan, College of screened key genes was verified by immunohistochemistry of Bioscience and Technology, Weifang Medical University, (IHC). The present study aimed to identify key genes and path- 7166 Baotong Street, Weifang, Shandong 261053, P.R. China ways which involved in the occurrence of PCa and explored the E-mail: [email protected] E-mail: [email protected] potential biomarker for prognosis, diagnosis and drug targets. *Contributed equally Materials and methods Key words: bioinformatics analysis, prostate cancer, differentially Expression Profile Microarray. Gene expression profile expressed gene, pathways, identification (GSE55945) was downloaded from GEO (http://www.ncbi. nlm.nih.gov/geo/). GSE55945 was based on GPL570 platform (Affymetrix Human Genome U133 Plus 2.0 Array), which 6664 FAN et al: IDENTIFICATION OF THE KEY GENES AND PATHWAYS was submitted by Arredouani et al, and contained 21 samples, positive staining. In addition, an independent sample t-test was including 18 PCa samples and 8 normal prostate samples (8). applied to the results of staining. Identification of DEGs. The gene expression profile was Results analyzed by Morpheus online tools (https://software.broadin- stitute.org/morpheus/) as previous study (11). The DEGs were Identification of DEGs. A total of 13 PCa samples and identified between PCa group and normal group. The signifi- 8 normal samples were analyzed using Morpheus online tools cance of DEGs was identified by classical t-test. The change (https://software.broadinstitute.org/morpheus/). A total of ≥twofold and P<0.05 was considered to indicate a statistically 2,000 DEGs were identified in PCa compared to normal group, significant difference. including 1000 upregulated and 1,000 downregulated genes respectively. The heat map of DEGs expression (including top GO analysis and KEGG pathway analysis of DEGs. In order 40 upregulated and downregulated genes) was shown in Fig. 1. to analyse the function and pathway of the DEGs, DAV I D database (https://david.ncifcrf.gov/) was used for GO analysis GO analysis of DEGs. All DEGs were uploaded to DAVID and KEGG pathway analysis as previous study (7). P<0.05 was database (https://david.ncifcrf.gov/), and then GO analysis was considered to indicate a statistically significant difference. conducted. The results showed that upregulated DEGs were enriched in biological processes, including cellular response PPI network and module analysis. Search Tool for the Retrieval of to growth factor stimulus, phosphate metabolic process, cell Interacting Genes (STRING; https://string-db.org/cgi/input.pl) development, cell cycle and cell division, while downregulated was a database that could assess the protein-protein interac- DEGs were enriched in biological processes, including signal tion. The DEGs were mapped to STRING, and a score >0.4 transduction, cell communication, response to stimulus, cell was considered to be significant. Then, the Cytoscape software projection organization and cell development (Table I). For cell (version 3.3.0) was used to construct PPI networks. Finally, component, upregulated DEGs were enriched in contractile the modules of PPI network were screened by the plug-in fiber, actin cytoskeleton, anchoring junction, adherens junction Molecular Complex Detection (MCODE). In addition, the and focal adhesion, while downregulated DEGs were enriched in pathway analysis was performed in the modules. P<0.05 was membrane raft, actin cytoskeleton, adherens junction, anchoring considered to indicate a statistically significant difference. junction and membrane-bounded vesicle (Table I). Furthermore, for molecular function, upregulated DEGs were enriched in Patients and tissue samples. The tissue microarray was enzyme binding, cytoskeletal protein binding, enzyme regulator purchased from Alenabio Co., Ltd (Xian, China) including activity, macromolecular complex binding and protein kinase 60 PCa samples from patients and 10 normal prostate tissue binding, while downregulated DEGs were enriched in RNA samples from healthy donors. The procedures performed binding, cytoskeletal protein binding, enzyme regulator activity, in this study involving human patients were in accordance transcription factor binding and calcium ion binding (Table I). with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration KEGG pathway analysis of DEGs. The KEGG pathway and its later amendments or comparable ethical standards. analysis results showed that upregulated DEGs were enriched The present study was approved by the Research Ethics in proteoglycans in cancer, endocytosis, focal adhesion, Committee of Weifang Medical University (Weifang, hippo signaling pathway and cGMP-PKG signaling pathway, China). whereas downregulated DEGs were enriched in proteogly- cans in cancer, endocytosis, hippo signaling pathway, thyroid IHC validation. IHC was performed as previous study (12). hormone signaling pathway and sulfur relay system (Table II). The tissue sample was blocked by 0.3% H2O2 and blocked by 10% bovine serum albumin (BSA) for 25 min. Then, the tissue Module screening from the PPI network. The top 6 hub nodes sample was incubated overnight with anti-ribosomal protein with high degrees were screened by the STRING database. S21 (RPS21), anti-forkhead box O1 (FOXO1), anti-baculoviral These hub genes included RPS21, FOXO1, BIRC5, POLR2H, IAP repeat containing 5 (BIRC5), anti-RNA polymerase II RPL22L1 and NPM1. In addition, total nodes were analyzed by subunit H (POLR2H), anti-ribosomal protein L22 like 1 plug-ins