ONCOLOGY LETTERS 10: 543-549, 2015 Bioinformatics analysis of the target gene of fibroblast growth factor receptor 3 in bladder cancer and associated molecular mechanisms XING AI1*, ZHUO-MIN JIA1,2*, JUAN WANG3, GUI-PING DI1, XU ZHANG2, FENGLING SUN1, TONG ZANG1 and XIUMEI LIAO1 1Department of Urology, Military General Hospital of Beijing PLA, Beijing 100700; 2Department of Urology, Chinese PLA General Hospital, Beijing 100853; 3Department of Medicine, Military General Hospital of Beijing PLA, Beijing 100700, P.R. China Received August 4, 2014; Accepted April 24, 2015 DOI: 10.3892/ol.2015.3231 Abstract. The aim of the present study was to elucidate CCNB1 and H2AFZ were significantly associated with BC, the molecular mechanisms of fibroblast growth factor as determined by the protein-protein interaction network of receptor 3 (FGFR3) activation via overexpression or muta- DEGs and co-expressed genes. In conclusion, the present tion of the FGFR3 target gene in bladder cancer (BC). study revealed the involvement of FGFR3 in the regulation The transcription profile data GSE41035, which included of sterol biosynthesis and metabolism in the maintenance of 18 BC samples, containing 3 independent FGFR3 short BC; in addition, the present study provided a novel insight hairpin (sh)RNA, and 6 control samples, containing enhanced into the molecular mechanisms of FGFR3 in BC. These green fluorescent protein (EGFP) shRNA, were obtained results may therefore contribute to the theoretical guidance from the National Center of Biotechnology Information into the detection and therapy of BC. Gene Expression Omnibus database. The Limma package with multiple testing correction was used to identify differen- Introduction tially expressed genes (DEGs) between FGFR3 knockdown and control samples. Gene ontology (GO) and pathway Bladder cancer (BC) is estimated to be among the top five enrichment analysis were conducted in order to investigate most common types of cancer in western countries and ranks the DEGs at the functional level. In addition, differential number 13 in terms of cancer-associated mortality world- co-expression analysis was employed to construct a gene wide (1,2). Histologically, BC may be classified based on the co-expression network. A total of 196 DEGs were acquired, depth of invasion: pTa, papillary; pT1, lamina propria invasion; of which 101 were downregulated and 95 were upregulated. pT2, muscle invasive; pT3, invasion to peri-vesical fat; and pT4, In addition, a gene signature was identified linking FGFR3 locally advanced (3,4). BC results from long-term exposure to signaling with de novo sterol biosynthesis and metabolism contaminants or other environmental factors involving gene using GO and pathway enrichment analysis. Furthermore, mutations and progressive cellular damage. Parkin et al (5) the present study demonstrated that the genes NME2, demonstrated that the incidence of BC is almost four times higher in men than in women and inducing factors include tobacco smoke, prolonged exposure to chemical substances and race (6,7). Genetic mutations in gene expression may lead to the Correspondence to: Dr Zhuo-Min Jia, Department of Urology, malignant transformation of bladder cells. High-throughput Military General Hospital of Beijing PLA, 5 Nanmeng Cang Road, Dongcheng, Beijing 100700, P.R. China DNA microarray analyses have identified multiple DNA muta- E-mail: [email protected] tions and alterations in the genesis of BC; these include genes encoding for B cell lymphoma‑2, p53, H‑Ras and fibroblast Dr Xu Zhang, Department of Urology, Chinese PLA General growth factor receptor 3 (FGFR3) (8,9). FGFR3 is a tyrosine Hospital, 28 Fuxing Road, Haidian, Beijing 100853, P.R. China kinase receptor that regulates fundamental developmental E-mail: [email protected] pathways and triggers a range of cellular processes, including *Contributed equally proliferation, differentiation, migration and apoptosis (10). The FGFR3 gene is located on chromosome region 4p16.3 (11) Key words: bladder cancer, differentially expressed genes, and it is made up of 9 exons and 18 introns (12). The structure gene ontology and pathway enrichment analysis, protein-protein of FGFR3 is composed of an extracellular domain consisting interaction network, differentially co-expressed genes of two or three immunoglobulin-like domains, a hydrophobic transmembrane domain and an intracellular tyrosine kinase domain (13). Upon ligand binding, FGFR3 forms dimers 544 AI et al: THE ROLE OF FGFR3 IN BLADDER CANCER and activates the intracellular kinase domain, resulting in of all probes for a given gene were reduced to a single autophosphorylation of this domain. The phosphorylated value by taking the average expression value. Subsequently, residues are the binding targets of the adaptor proteins and missing data was imputed and quartile data normalization their binding results in the activation of several signal trans- was performed by robust multichip averaging using Affy duction pathways, including the Ras mitogen-activated protein package in R software (version 3.1; http://www.bioconductor. kinase (MAPK) signaling pathway and phosphoinositide org/packages/release/bioc/html/affy.html) (24). The Limma 3-kinase (PI3K) Akt mammalian target of rapamycin (mTOR) package version 3.24.2 (http://www.bioconductor.org/pack- pathway (14). ages/release/bioc/html/limma.html) (25) in R language with There are two mechanisms to explain the abnormal acti- multiple testing correction was then used according to the vation of FGFR3: Overexpression or activating mutations. Benjamini & Hochberg method (26) in order to identify DEGs FGFR3 mutations have been identified in multiple dwarf- between BC samples and normal controls. P<0.05 and |log(fold isms (15), such as hypochondroplasia, and in multiple types of change; FC)|>1 were defined as the thresholds. cancer, including prostate cancer (16), cervical cancer (17) and BC. FGFR3 mutations were reported in BC for the first time by Gene ontology (GO) enrichment analysis. In order to inves- Cappellen et al (18). There is evidence to suggest that codons tigate DEGs at the molecular and functional level, the online 248, 249 and 375 are the major mutation hot spots in BC (19); biological tool, Database for Annotation, Visualization and in addition, activating mutations of FGFR3 have been revealed Integrated Discovery (DAVID) version 6.7 (http://david. primarily in pTa (60-70%) and in pT1-4 (16-20%) (19). Over- abcc.Ncifcrf.gov/), was used for GO term enrichment and expression of FGFR3 has been frequently identified in BC; genes were clustered according to GO. GO is a collection furthermore, Jebar et al (20) demonstrated that the expression of controlled vocabularies, which include molecular func- of FGFR3 was higher in low stage BC. Du et al (21) identified tion, cellular component and biological process, to describe a gene pathway linking FGFR3 with sterol and lipid metabo- the biology of a gene product in any organism. P<0.05 was lism through transcriptional profiling of BC cells subjected selected as the cut-off criterion during the analysis. to short hairpin (sh)RNA knockdown of FGFR3 (21). FGFR3 has been demonstrated to be a promising therapeutic target for Pathway enrichment analysis. The theoretical principle for BC (22,23). However, the molecular mechanisms of FGFR3 enrichment analysis is that associated functional genes are activation, via overexpression or activating mutation, in BC more likely to be selected in the abnormal biological process remain to be elucidated. by the high-through screening technologies (27). Based on The present study aimed to analyze microarray data in the selected genes, researchers are able to correctly identify order to investigate the changes in gene expression profiles that the biological processes involved. In order to identify the occur following loss of FGFR3; in addition, the current study enriched pathways of DEGs, DAVID was used with P<0.05 aimed to explore the target genes and molecular mechanisms as the threshold. The pathways used as DAVID input for of FGFR3 The genes that were differentially expressed in cluster analysis were from Kyoto Encyclopedia of Genes FGFR3-deleted cell lines as compared with the control cell and Genomes (KEGG; http://www.genome.jp/kegg/) and lines were considered to be potential transcriptional targets BIOCARTA (http://www.biocarta.com/). of over-expressed FGFR3 in bladder cancer. Furthermore, a protein-protein interaction (PPI) network was constructed and PPI network construction. PPIs are crucial for all biological the disturbed biological pathways were identified following processes. In the present study, the PPI network was FGFR3 knockdown in order to explore the pathogenesis and constructed based on the Protein Interaction Network Anal- occurrence of BC associated with FGFR3. ysis platform (PINA2) database (http://cbg.garvan.unsw.edu. au/pina/). PINA2 (28) is a database containing known and Materials and methods predicted associations of protein interaction. The interactions include direct (physical) and indirect (functional) associations. Messenger RNA expression profile data of BC. The transcrip- Of note, the protein names in the Universal Protein Resource tion profile dataset of BC was obtained from National Center database (http://www.ebi.ac.uk/uniprot/remotingAPI/), which of Biotechnology Information Gene Expression Omnibus correspond to the DEGs, were submitted to construct the database (http://www.ncbi.nlm.nih.gov/geo/). The accession PPI network. Here