1746 MOLECULAR MEDICINE REPORTS 13: 1746-1764, 2016

Differential expression analysis and network construction of recurrent cardiovascular events

JIANGQUAN LIAO1,2*, ZHONG CHEN1,2*, QINGHONG HE1*, YONGMEI LIU1 and JIE WANG1

1Department of Cardiology, Guang'anmen Hospital, China Academy of Chinese Medical Sciences, Beijing 100053; 2Graduate School, Beijing University of Chinese Medicine, Beijing 100059, P.R. China

Received January 10, 2015; Accepted November 2, 2015

DOI: 10.3892/mmr.2015.4707

Abstract. Recurrent cardiovascular events are vital to the with and without recurrent cardiovascular events, including prevention and treatment strategies in patients who have DEGs, their biological function, signaling pathways and key experienced primary cardiovascular events. However, the in the PPI network. With the use of bioinformatics and susceptibility of recurrent cardiovascular events varies among genomics these findings can be used to investigate the patho- patients. Personalized treatment and prognosis prediction are logical mechanism, and improve the prevention and treatment urged. Microarray profiling of samples from patients with of recurrent cardiovascular events. acute myocardial infarction (AMI), with or without recur- rent cardiovascular events, were obtained from the Gene Introduction Expression Omnibus database. Bioinformatics analysis, including Gene Oncology (GO) and Kyoto Encyclopedia of With the significant advances in medication, reperfusion Genes and Genomes (KEGG), were used to identify genes therapy, cardiac rehabilitation and organ transplantation, and pathways specifically associated with recurrent cardio- cardiovascular disease remains one of the major causes of vascular events. A ‑protein interaction (PPI) network mortality worldwide (1). Evaluation of cardiovascular disease was constructed and visualized. A total of 1,329 genes were based on risk factors is important in the clinical prevention differentially expressed in the two group samples. Among and treatment of cardiovascular disease, which may alter the them, 1,023 differentially expressed genes (DEGs; 76.98%) risk stratification and guide the treatment and prognosis (2,3). were upregulated in the recurrent cardiovascular events group More and more indexes are included in the risk stratification and 306 DEGs (23.02%) were downregulated. Significantly as clinical and experimental research develops, including enriched GO terms for molecular functions were brain natriuretic , C reactive protein and blood homo- binding and nucleic acid binding, for biological processes cysteine. However, the prediction of cardiovascular disease is were and regulation of not so satisfying (4), particularly in personalized prevention (DNA‑dependent), and for cellular component were cyto- and treatment. Sensitivity of risk factors varies in different plasm and nucleus. The most significant pathway in our individuals, and clinical doctors must be aware of this and KEGG analysis was Pathways in cancer (P=0.000336681), objective to the current risk factors and stratification (5). More and regulation of was also significantly superior and systematic algorithms for stratification remain to enriched (P=0.00165229). In the PPI network, the signifi- be elucidated (6). cant hub nodes were GNG4, MAPK8, PIK3R2, EP300, The evaluation and stratification of cardiovascular CREB1 and PIK3CB. The present study demonstrated the diseases depend more on primary cardiovascular events, underlying molecular differences between patients with AMI, which elevate the stratification and enhance the treatment once they occur. However, recurrent cardiovascular events are also vital, which indicate that the current intervention is not marked enough to prevent disease progression. Although patients receive standard treatment based on the risk factors Correspondence to: Professor Jie Wang, Department of Cardiology, stratification, recurrent cardiovascular events still occur, Guang'anmen Hospital, China Academy of Chinese Medical Sciences, which indicates that certain individuals are more prone to 5 Beixiange, Xicheng, Beijing 100053, P.R. China recurrent cardiovascular events. These patients may require E‑mail: [email protected] more aggressive therapies, involving susceptibility screening *Contributed equally and personalized treatment (7). With the development and application of clinical genomics technology and bioinfor- Key words: recurrent cardiovascular events, microarray profile, matics, novel biomarkers are used in the diagnosis and differential expressed genes, protein‑protein interaction network, prognosis of cardiovascular disease (8,9). Previous research bioinformatics revealed that the expression of different genes varies in different stages of cardiovascular diseases, and these genes are involved in the pathological process, and may even predict LIAO et al: DIFFERENTIAL ANALYSIS AND NETWORK CONSTRUCTION 1747 the cardiovascular events (10). With the help of genomics and Further analysis of the key nodes in the PPI network were bioinformatics, patient susceptibility to recurrent cardiovas- processed. cular events may be screened out, and personalized treatment can be made. This may reduce the recurrence of cardiovas- Results cular events and improve the prognosis. The present study used genomics and bioinformatics technology, and associ- Clinical characteristics of the two group samples. According ated software to analyze the differentially expressed genes to Suresh et al (11), the clinical characteristics between the (DEGs) associated with recurrent cardiovascular events. recurrence and no recurrence groups, including age, sex, body The present study also aimed to identify the key genes in mass index, cardiovascular risk factor score, lipid profile and the pathological process and provide alternative guidance severity of AMI, were similar with the exception of the usage in the preventions, and personalized treatment of recurrent of beta‑blockers. The clinical characteristic details are listed in cardiovascular events. Table I.

Materials and methods Recurrent cardiovascular event‑associated DEGs. By comparing the two group samples of with or without recurrent Microarray data and clinical characteristics. The micro- cardiovascular events in the 18 month follow‑up following array dataset, GSE48060 with GPL570 [HG‑U133_Plus_2] primary AMI, 1,329 genes (2.43% of total probe set) were Affymetrix U133 Plus 2.0 Array platform, identified to be differentially expressed and annotatable. A was obtained from the Gene Expression Omnibus (GEO) total of 1,023 DEGs (76.98%) were upregulated and 306 DEGs database (11). The data samples were peripheral blood (23.02%) were downregulated in the recurrent cardiovascular samples collected from patients with acute myocardial infarc- events group. The top 10 markedly up or downregulated genes tion (AMI) 48 h within the primary AMI. All 27 samples with a fold change >1.5 are listed in Tables II and III, respec- were divided into two groups, according to the recurrence tively. of cardiovascular events in the 18 month follow‑up. A total of five patients exhibited recurrent cardiovascular events Significant GO enrichment. To gain insights into the biological and 22 did not. The definition of recurrent cardiovascular roles of the DEGs in recurrent cardiovascular events, a GO cate- events is recurrent myocardial infarction, re‑vascularization, gories enrichment analysis was performed using GeneCoDis. evidence of restenosis, hospitalization for unstable angina GO categories are predominantly in three groups: Biological or heart failure, cardiovascular mortality, stroke or transient process, cellular component and molecular function. The ischemic attack, or amputation due to peripheral vascular significantly enriched GO terms for molecular functions were disease. nucleotide binding (GO:0000166, P=8.24‑19) and nucleic acid binding (GO:0003676, P=1.94‑07), for biological processes were Raw data processing. All 27 sample files were downloaded signal transduction (GO:0007165, P=5.26‑08) and regulation of from the GEO database and were reanalyzed using R soft- transcription, DNA‑dependent (GO:0006355, P=9.19‑06), and for ware (version 3.1.1; http:// www.r‑project.org/). The Affy cellular component were (GO:0005737, P=8.98‑25) package was applied to read the probe set data from the CEL and nucleus (GO:0005634, P=1.91‑23; Fig. 1). files (12). Robust Multiarray Averaging was used to normalize the original data. Following standardization, a total of 54,675 Significant pathways. KEGG pathway enrichment analysis was probe set IDs' expression levels in different samples were performed to further evaluate the biological significance of the obtained. DEGs. The most significant pathway in our KEGG analysis was Pathways in cancer (P=0.000336681). Furthermore, Melanoma Screening and annotation of the DEGs. The limma package (P=0.000336681) and Regulation of actin cytoskeleton in the R software was used to compare the expression levels of (P=0.00165229) were revealed to be highly enriched. The top 15 the probe sets between the two groups (13). The threshold was enriched KEGG pathways of the DEGs are listed in Table IV. set as P<0.05 or a fold change >1.5. The annotate package was used to annotate the DEGs. PPI network construction and visualization. By analyzing the identified 1,329 DEGs using Cytoscape and the reactome Enrichment analysis of DEGs. GeneCodis online tools plugin, 330 genes (node) and 796 gene‑gene interactions (edge) (http://genecodis2.dacya.ucm.es/) were used to annotate were identified. The result was visualized in Cytoscape and and analyze the DEGs (14,15). The annotation and analysis the majority of the nodes were located within one network. were predominantly focussed on the molecular function, To modify the PPI network, the sizes of the nodes were set the biological process and the cellular component of Gene according to their interaction density with the other nodes. The Ontology (GO) enrichment and Kyoto Encyclopedia of Genes node color of the upregulated DEGs were made red and those and Genomes (KEGG) pathway analysis. The threshold was downregulated were made blue (Fig. 2). The more that one gene set as P<0.05. interacts with the other genes, the larger the node was and the more central this gene occurs within the network. The genes, Protein‑protein interaction (PPI) network analysis. Cytoscape GNG4, MAPK8 and PIK3R2 were the three predominantly (version 3.1.1; The Cytoscape Consortium, San Diego, CA, upregulated genes, while EP300, CREB1 and PIK3CB were the USA) and reactome plugin were applied to analyze the predominantly downregulated genes in the PPI network. The DEGs (16), and to construct and visualize the PPI network. details of the nodes are listed in Table V. 1748 MOLECULAR MEDICINE REPORTS 13: 1746-1764, 2016

Table I. Baseline clinical characteristics of AMI patients with or without recurrent events following primary AMI, who under- went whole‑genome blood gene expression microarray analysis.

Variable Event (n=5) No event (n=22) P‑value

Age, years 51 (41‑53) 56.5 (48‑65) 0.110 Gender, male, n (%) 3 (60) 13 (59) 0.972 Body mass index, kg/m2 36.5 (25.0‑46.5) 31.5 (22.9‑48.4) 0.140 Cardiovascular risk factor score 5 (4‑6) 4 (1‑6) 0.266 Cardiovascular history, n (%) Arterial hypertension, n (%) 5 (100) 13 (59) 0.080 Smoking, n (%) 2 (40) 16 (73) 0.161 Diabetes mellitus, n (%) 0 (0) 3 (14) 0.381 Family history of coronary artery disease, n (%) 5 (100) 12 (55) 0.057 Lipid profile, mg/dl Total cholesterol, mg/dl 128 (110‑219) 191 (126‑325 0.190 Low density lipoprotein cholesterol, mg/dl 65 (41‑152) 115 (70‑254) 0.169 HDL cholesterol, mg/dl 31 (25‑47) 38 (25‑72) 0.165 Medication Statin therapy, n (%) 3 (60) 7 (33) 0.271 Aspirin, n (%) 4 (80) 12 (55) 0.296 ACE inhibitor, n (%) 1 (20) 4 (19) 0.961 Beta blocker, n (%) 4 (80) 5 (22) 0.014 Severity of AMI Ejection fraction, % 55 (43‑65) 57 (35‑71) 0.240 Troponin, ng/ml 2.24 (0.11‑9.51) 0.47 (0.04‑16.43) 0.142 STEMI, n (%) 2 (40) 7 (32) 0.726

AMI, acute myocardial infarction.

Table II. Top 10 upregulated genes.

Mean of intensity ‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ Gene Fold change Recurrence No recurrence P‑value Official gene name

LRRC18 1.58 21.38 13.50 1.14‑04 Leucine rich repeat containing 18 IRAK1BP1 1.77 90.64 51.19 6.19‑04 Interleukin‑1 ‑associated kinase 1 binding protein 1 MGAT4A 1.57 385.69 245.89 6.00‑03 Mannosyl (α‑1,3‑)‑ β‑1, 4‑N‑acetylglucosaminyltransferase, isozyme A BZW2 1.74 921.80 529.93 9.33‑03 Basic and W2 domains 2 LOC152586 1.64 19.38 11.84 1.25‑02 MGAT4 family, member D ADTRP 1.78 272.08 153.19 1.46‑02 Androgen‑dependent TFPI‑regulating protein SMC1B 1.78 24.76 13.94 2.02‑02 Structural maintenance of chr 1B LOC283788 1.53 147.03 96.39 2.39‑02 Hypothetical protein LOC283788 CLDN12 1.52 37.24 24.58 2.56‑02 Claudin 12 LEF1‑AS1 1.79 74.60 41.72 3.32‑02 LEF1 antisense RNA 1

Genes with a fold change >1.5 are shown. Data are sorted by P‑value. LIAO et al: DIFFERENTIAL GENE EXPRESSION ANALYSIS AND NETWORK CONSTRUCTION 1749

Table III. Top 10 downregulated genes.

Mean of intensity ‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ Gene Fold change Recurrence No recurrence P‑value Official gene name

HIST1H2AC 1.57 2086.82 3273.74 2.50‑03 Histone cluster 1, H2ac NEXN 1.65 199.36 329.40 4.46‑03 Nexilin (F actin binding protein) SIRPB2 1.76 102.91 181.12 4.77‑03 Signal‑regulatory protein β2 TUBB1 1.56 462.45 719.52 5.44‑03 Tubulin β1 HIST1H2BD 1.56 254.17 397.28 5.81‑03 Histone cluster 1, H2bd TSPAN2 1.76 142.35 250.81 6.02‑03 Tetraspanin 2 GUCY1A3 1.55 12.14 18.80 6.57‑03 1, soluble, α 3 FAR2 1.77 129.96 230.38 7.84‑03 Fatty acyl CoA reductase 2 STON2 1.66 91.55 151.83 9.06‑03 Nexilin (F actin binding protein) DLEU2 1.62 63.13 102.52 1.11‑02 Deleted in lymphocytic leukemia 2

Genes with a fold change >1.5 are shown. Data are sorted by P‑value.

A B

C

Figure 1. GO enrichment. (A) Molecular functions, (B) biological processes and (C) cellular components for the differentially expressed genes. The order of GO enrichment terms were sorted by P‑value. GO, .

Discussion different regions and hospitals. There are divergences between different area and different grades of hospitals (17), conserva- Cardiovascular events are important in the prevention and tive and aggressive strategies are being used, not to mention the treatment of cardiovascular diseases. When it occurs in patients circumstances vary among individuals, efficient and effective with risk factors, the heart function must be re‑evaluated, personalized evaluation and treatment are urged (18). Previous and the prevention and treatment strategy must be adjusted. research has revealed that whole‑genome sequencing can be For patients who had experienced cardiovascular events, the used in cardiovascular disease risk‑prediction algorithms, prevention and treatment strategies are not uniform between to more accurately forecast whether patients will develop 1750 MOLECULAR MEDICINE REPORTS 13: 1746-1764, 2016

Table IV. Top 15 enriched KEGG pathways of differentially expressed genes.

KEGG ID KEGG pathway Gene no. P‑value hsa05200 Pathways in cancer 12 3.37-04 hsa05218 Melanoma 12 3.37-04 hsa04810 Regulation of actin cytoskeleton 9 1.65-03 hsa05215 Prostate cancer 11 1.77-03 hsa04010 MAPK signaling pathway 23 2.04-03 hsa04120 Ubiquitin mediated proteolysis 14 6.34-03 hsa04660 receptor signaling pathway 4 7.80-03 hsa05340 Primary immunodeficiency 4 7.80-03 hsa05214 Glioma 7 8.34-03 hsa05222 Small cell lung cancer 10 9.31-03 hsa04510 Focal adhesion 6 1.68-02 hsa04144 Endocytosis 16 1.98-02 hsa03030 DNA replication 4 2.07-02 hsa03430 Mismatch repair 4 2.07-02 hsa04115 signaling pathway 8 2.28-02

KEGG, Kyoto Encyclopedia of Genes and Genomes.

disease (19). However, there remains a lack of research about functions were nucleotide binding and nucleic acid binding, microarray profiling in recurrent cardiovascular events. The for biological processes were signal transduction and regu- present study performed a microarray profiling of peripheral lation of transcription (DNA‑dependent), and for cellular blood samples from patients with AMI, downloaded from the component were cytoplasm and nucleus. The GO terms GEO database, to focus on the DEGs of those with or without mentioned above are basic and vital to the biological and recurrent cardiovascular events 18 months following AMI. pathological process. Fibroblast receptor R is an integrated suite of software facilities for data signaling pathway (GO:0008543; P=0.00151494), blood manipulation, calculation and graphical display. Using R soft- coagulation (GO:0007596; P=0.00166723) and ware and certain packages, the present study identified the (GO:0007155; P=00170222) were also significantly enriched DEGs between patients with AMI, with or without recurrent in GO biological process. Ronca et al (23) reported that fibro- cardiovascular disease. A total of 1,329 genes were identified blast growth factor receptor‑1 gene knockout impairs cardiac and 1,023 were upregulated in recurrent group compared with and haematopoietic development in murine embryonic stem the no recurrent group, while 306 of them were downregulated. cells, and the fibroblast growth factor receptor is required for The genes with the most significant P‑value and fold change cardiomyocyte differentiation. Yukawa et al (24) demonstrated >1.5 in the up and downregulated DEGs are listed in Tables II that impaired fibroblast growth factor receptor gene would and III. Among them, TUBB1 (tubulin β1, class VI; P=0.00544; suppress the growth of vascular smooth muscle. As for blood fold change=1.56) encodes a member of the β tubulin protein coagulation and cell adhesion, which are associated with the family, and this protein is specifically expressed in platelets formation and breaking off of thrombosis, they are important and megakaryocytes, and may be involved in proplatelet in both primary and recurrent cardiovascular events. production and platelet release. Previous research revealed In KEGG pathway analysis, regulation of actin cytoskel- that the prevalence of TUBB1 was higher among healthy eton is significantly enriched. Actin cytoskeleton is involved individuals compared with patients with cardiovascular in the inward remodeling process associated with cytoskeletal disease (20). This may be associated with the TUBB1 function modifications. It is also involved in reducing the passive of suppressing microtubule dynamics, fragmenting microtu- diameter of resistance vessels, which are the vascular compo- bules and inhibiting cell division (21). Although there is little nents of the circulatory system, and exert a preponderant role previous research about other significant genes involved in in the regulation of blood flow and the modulation of blood cardiovascular diseases, the method in the present study may pressure (25). Therefore, the regulation of actin cytoskeleton be the initial and alternative way to explore the pathological may have profound consequences on the incidence of cardio- mechanism of recurrent cardiovascular events. vascular events. To further investigate the roles of the DEGs identified The results from PPI network analysis of the top 10 up and in the pathological mechanism of recurrent cardiovascular downregulated DEGs revealed the significant nodes, including events, GO enrichment analysis and KEGG pathway analysis GNG4, MAPK8, PIK3R2, EP300, CREB1 and PIK3CB. was used. GO is widely used as the tool for the organization MAPK8 is one member of the MAPK family, which has vast and functional annotation of molecular aspect (22). It was implications in signaling and crosstalk with other signaling revealed that the significantly enriched GO terms for molecular networks. The MAPK signal pathway is highly associated LIAO et al: DIFFERENTIAL GENE EXPRESSION ANALYSIS AND NETWORK CONSTRUCTION 1751

Figure 2. Protein‑protein interaction network visualization of differentially expressed genes. The nodes in red represent the upregulated genes and those in blue represent the downregulated genes. The larger size nodes interact more with other nodes. The type of edges represents the interaction between the nodes, → to indicate activating/catalyzing, ‑| to indicate inhibition, ‑ for functional interactions extracted from complexes or inputs and --‑ for predicted functional interactions.

with mitochondria, the power houses of the cell, which provide The results from the present study suggested that DEGs >80% of ATP for normal cardiomyocyte function and have a exist between patients with AMI, with and without recurrent crucial role in cell death (26). EP300 is the node with the most cardiovascular events. These genes are involved in different interactions with other nodes in the PPI network, and previous GO enrichment terms and signaling pathways, from which research revealed that it is associated with arterial stiffness insights into the pathological processes of recurrent events prior to hypertension, increased pulse pressure, and struc- can be obtained. Several genes, including TUBB1, GNG4, tural vessel wall changes (27). CREB1, also termed CREB, MAPK8, PIK3R2, EP300 and CREB1, with or without phosphorylation induced by the prostacyclin/IP pathway may previous research, may provide potential candidates for suppress cardiac fibrosis, which is a consequence of numerous distinguishing the susceptibility to recurrent cardiovascular cardiovascular diseases, and contributes to impaired ventric- events in the future. Therefore, the present research may ular function (28). The PPI results suggested that MAPK8, provide important references for the prevention and treatment EP300 and CREB1 may be important in the development of strategies in patients with primary cardiovascular events. recurrent cardiovascular events. Nevertheless, the genes and the associated GO enrichment 1752 MOLECULAR MEDICINE REPORTS 13: 1746-1764, 2016 Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Down Down Down Down Down Down Down Down Down Down Down Down Down Regulation 5 2 7 2 7 8 7 9 4 1 8 5 1 6 1 9 5 6 11 11 11 11 56 10 16 14 10 17 16 15 12 10 17 Outdegree 9 4 9 5 4 8 8 0 0 1 5 4 4 5 8 1 6 3 1 9 11 11 11 11 13 25 12 10 13 10 Indegree 0 11 11 11 11 11 18 81 22 21 18 18 18 18 18 17 17 16 16 16 16 15 14 14 13 13 12 12 12 12 22 17 12 26 13 7 Degree 0 0 0 0 0.000341 0.0020355 0.0016332 0.00112437 0.00123254 0.03886829 0.00373044 0.00881333 0.00045679 0.00044752 0.00044752 0.00084908 0.00039501 0.00042396 0.00003574 0.00046983 0.00051894 0.00190278 0.00101008 0.00086181 0.00064179 0.00076915 0.00050967 0.00000927 0.00031838 0.00275468 0.00123249 0.00246082 0.00358728 Betweenness centrality

/ ) ) β 11 (Gq class) 11 4 γ α drosophila polypeptide β Description lymphoma 3 / E1A binding protein p300 E1A Guanine nucleotide binding protein (), Mitogen ‑ activated 8 Phosphoinositide ‑ 3 kinase, regulatory subunit 2 (p85 1 responsive element binding protein 1 CAMP Phosphoinositide ‑ 3 kinase, catalytic, Histone cluster 1, H2bc Histone cluster 1, H2bk Histone cluster 1, H2bj Histone cluster 2, H2be Histone cluster 1, H2bd Anaphase promoting complex subunit 10 (dihydrotestosterone receptor; testicular feminization; spinal and bulbar muscular atrophy; kennedy disease) ‑ dependent kinase 4 Histone cluster 1, H2ac member S H2B histone family, SMAD specific E3 ubiquitin protein ligase 1 Poly (A) binding protein, cytoplasmic 1 Ring finger protein 1 associated factor X Fms ‑ related tyrosine kinase 1 (vascular endothelial growth factor vascular permeability factor receptor) S ‑ phase kinase associated protein 2 (p45) Chromobox homolog 8 (Pc class homolog, Drosophila) box binding protein (TBP) ‑ TATA polymerase II, RNA TAF1 250 kDa associated factor, B ‑ cell CLL Guanine nucleotide binding protein (G protein), Guanine nucleotide binding protein (G protein), q polypeptide Polyamine ‑ modulated factor 1 Chromobox homolog 2 (Pc class homolog, WWE domain containing 1 and UBA HECT, (K)‑specific demethylase 6B Lysine Ubiquitin protein ligase E3A (human papilloma virus E6 ‑ associated Ubiquitin protein ligase E3A

Table V. Network features of differentially expressed genes included in protein ‑ interaction network sorted by degree. Network features of differentially V. Table Gene EP300 GNG4 MAPK8 PIK3R2 CREB1 PIK3CB HIST1H2BC HIST1H2BK HIST1H2BJ HIST2H2BE HIST1H2BD ANAPC10 AR CDK4 HIST1H2AC H2BFS SMURF1 PABPC1 RING1 MAX FLT1 SKP2 CBX8 TAF1 BCL3 GNA11 GNAQ PMF1 CBX2 HUWE1 KDM6B UBE3A LIAO et al: DIFFERENTIAL GENE EXPRESSION ANALYSIS AND NETWORK CONSTRUCTION 1753 Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Down Down Down Down Down Down Down Regulation 5 1 5 3 9 6 8 2 2 7 9 6 3 0 8 8 6 9 3 4 3 5 2 1 3 0 6 2 6 8 3 3 Outdegree 6 8 2 4 2 8 8 3 1 4 7 2 2 3 0 6 5 6 4 6 7 5 8 2 6 2 6 5 10 Indegree 9 9 9 9 9 9 8 8 8 8 8 8 8 9 8 6 11 10 11 11 11 11 10 10 10 10 10 10 10 10 10 10 10 10 2 Degree 0 0 0 0 0 0 0 0 0.000139 0.0020102 0.0000556 0.0000556 0.00167253 0.00010193 0.00350998 0.00000927 0.00000927 0.00200163 0.00015167 0.00017607 0.00296725 0.00004973 0.00004973 0.00137496 0.00140147 0.00038457 0.00096904 0.00166231 0.00081239 0.00189305 0.00008649 0.00008649 Betweenness centrality ) Drosophila )

‑ urogastrone) Drosophila β ) )

Description 9 6 S. cerevisiae S. cerevisiae ‑ 1) binding protein α α conjugating E2W (putative) Ubiquitin ‑ conjugating enzyme E2W protein, Angelman syndrome) Fibroblast growth factor receptor 1 (fms ‑ related tyrosine kinase 2, syndrome) Pfeiffer Polyhomeotic homolog 1 ( SMAD family member 9 Cytoskeleton associated protein 5 Notch homolog 1, translocation ‑ associated ( F ‑ box protein 3 Ubiquitin ‑ conjugating enzyme E2Q (putative) 1 Kinesin heavy chain member 2A domain containing 3 HECT Ankyrin repeat and SOCS box ‑ containing 3 Epidermal growth factor ( binding protein 2 GATA SA Vpr (HIV domain containing linker protein 1 CAP ‑ GLY binding protein S1, serine ‑ rich domain RNA Glypican 3 Eukaryotic 3, subunit B Ribosomal protein S28 Ribosomal protein S15a Promyelocytic leukemia Jun D proto ‑ oncogene NSL1, MIND kinetochore complex component, homolog ( member D Ras homolog gene family, Heparan sulfate proteoglycan 2 SPC24, NDC80 kinetochore complex component, homolog ( CD4 molecule coactivator 1 ‑ like growth factor 1 (somatomedin C)

Table V. Continued. V. Table Gene FGFR1 PHC1 SMAD9 CKAP5 NOTCH1 FBXO3 UBE2Q1 KIF2A UBE2W HECTD3 ASB3 EGF GATA2 RPSA VPRBP CLIP1 RNPS1 GPC3 EIF3B RPS28 RPS15A PML ITGA9 , JUND NSL1 RHOD HSPG2 SPC24 CD4 NCOA1 IGF1 ITGA6 Integrin, 1754 MOLECULAR MEDICINE REPORTS 13: 1746-1764, 2016 Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Down Down Down Down Down Regulation

3 6 5 4 1 2 4 8 3 0 2 5 0 2 7 5 0 5 0 6 0 1 4 5 0 1 0 7 0 6 6 7 0 6 Outdegree

4 1 4 7 6 4 0 5 8 5 2 7 5 0 2 7 2 7 0 6 5 2 1 5 0 0 1 0 0 Indegree 7 8 2 6 8 8 8 8 8 8 8 7 7 7 7 7 7 7 7 7 7 6 6 6 6 6 7 6 8 7 7 6 7 7 6 6 Degree 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.0009675 0.0002709 0.0002085 0.0001058 0.0001529 0.00021578 0.00000185 0.00038457 0.00023013 0.00004633 0.00007058 0.00010657 0.00012974 0.00126029 Betweenness centrality ) C. elegans )

β

S. cerevisiae 2 subunit α polypeptide homolog, Drosophila) α α Description

1 α α

1 ) 4 α α E. coli lymphoma 2 / ‑ like 1 (with EGF repeat domains) β ‑ related protein complex 2, Ribosomal protein S14 Rho ‑ related BTB domain containing 1 Replication factor C (activator 1) 4, 37 kDa Kinetochore associated 1 Centromere protein T RuvB ‑ like 2 ( Interleukin 2 receptor, MAD1 mitotic arrest deficient‑like 1 (yeast) , epsilon 1 Ras protein‑specific guanine nucleotide‑releasing factor 2 Fibroblast growth factor 7 (keratinocyte factor) SIX 5 Structural maintenance of 1B (K)‑specific demethylase 1 Lysine CD3e molecule, epsilon (CD3 ‑ TCR complex) Platelet ‑ derived growth factor , type IV, homolog, E. coli) ( RAD51 homolog (RecA B ‑ cell CLL decay factor ( Smg ‑ 7 homolog, nonsense mediated mRNA Fibroblast growth factor 5 Opa interacting protein 5 Ribosomal protein L14 A Small nuclear ribonucleoprotein polypeptide Collagen, type XVII, Fibroblast growth factor 4 (heparin secretory transforming protein 1, Kaposi sarcoma oncogene) LIM and senescent cell ‑ like domains 1 Collagen, type XII, Retinoblastoma ‑ like 1 (p107) Chromobox homolog 5 (HP1 type II, Protein kinase, cAMP ‑ dependent, regulatory, Splicing factor 3b, subunit 3, 130 kDa Adaptor Immunoglobulin lambda variable 1 ‑ 44

Table V. Continued. V. Table Gene RPS14 RHOBTB1 RFC4 KNTC1 CENPT RUVBL2 IL2RA MAD1L1 PLCE1 RASGRF2 FGF7 SIX5 SMC1B KDM1A CD3E PDGFA COL4A4 RAD51 BCL2 SMG7 FGF5 OIP5 RPL14 SNRPA COL17A1 FGF4 LIMS1 ITGBL1 Integrin, COL12A1 RBL1 CBX5 PRKAR2B SF3B3 AP2A2 ‑ 44 IGLV1 LIAO et al: DIFFERENTIAL GENE EXPRESSION ANALYSIS AND NETWORK CONSTRUCTION 1755 Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Down Down Down Down Down Down Down Regulation 5 1 6 1 0 0 2 3 5 1 4 4 0 4 0 2 0 3 3 1 4 4 3 1 2 2 0 3 3 2 2 5 0 Outdegree 0 0 4 5 3 0 4 1 1 5 0 4 2 4 1 1 3 0 0 1 3 2 2 3 0 0 1 1 5 Indegree 5 4 3 6 5 5 5 5 5 2 5 5 5 5 5 4 4 4 4 4 4 4 4 4 4 4 4 4 3 3 3 3 4 2 6 5 Degree 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.0000139 0.00117688 0.00003707 0.00034009 0.00468901 0.00107495 0.00002008 0.00061315 0.00014364 0.00003243 0.00103422 0.00003707 0.00047261 0.00076297 0.00007105 0.00000463 0.00003398 0.00045515 Betweenness centrality

1A subunit 1A α α Q type, /

Description 1 polypeptide α ‑ associated factor 1 ) α glucocorticoid regulated kinase 1 / ( specific extinguisher 1) Cyclin ‑ dependent kinase 9 , Proline, glutamic acid and leucine rich protein 1 TNF receptor Cartilage associated protein Nibrin Calcium channel, voltage ‑ dependent, P Class II, major histocompatibility complex, transactivator Interferon regulatory factor 1 Frizzled homolog 1 (Drosophila) Apolipoprotein A ‑ I Zeta ‑ chain (TCR) associated protein kinase 70kDa Dynein, cytoplasmic 2, light intermediate chain 1 Oxysterol binding protein ‑ like 1A Replication factor C (activator 1) 3, 38kDa clade H (heat shock protein 47), Serpin peptidase inhibitor, member 1, (collagen binding protein 1) Recombinant Heterogeneous nuclear ribonucleoprotein M Phosphoinositide ‑ 3 kinase, class synthetase Alanyl ‑ tRNA Frizzled homolog 8 (Drosophila) Myosin IC member RAS oncogene family RAB11A, Major histocompatibility complex, class II, DO A) Insulin ‑ like growth factor 2 (somatomedin Low density lipoprotein receptor (familial hypercholesterolemia) Enolase 1, ( protein 280A Rho GTPase activating protein 22 binding protein 1, 127kDa Damage‑specific DNA Excision repair cross‑complementing rodent deficiency, complementation groUp 8 dependent, regulatory, type I, Protein kinase, cAMP ‑ dependent, regulatory, α

Table V. Continued. V. Table Gene CDK9 CSNK2A1 PELP1 TRAF1 VIM CRTAP NBN CACNA1A CIITA IRF1 FZD1 SGK1 Serum APOA1 ZAP70 DYNC2LI1 OSBPL1A RFC3 SERPINH1 HNRNPM PIK3C3 PRKAR1A AARS FZD8 MYO1C RAB11A HLA ‑ DOA IGF2 LDLR ENO1 ZNF280A ARHGAP22 DDB1 ERCC8 1756 MOLECULAR MEDICINE REPORTS 13: 1746-1764, 2016 Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Regulation 0 0 2 0 0 2 3 1 1 2 0 2 2 2 3 0 0 1 0 1 0 0 2 2 1 1 0 3 1 0 0 2 2 Outdegree 3 3 1 3 3 1 0 2 2 1 3 1 1 1 0 3 3 2 3 2 3 3 1 1 2 2 3 0 2 3 3 1 1 Indegree 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 Degree 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.0000278 0.0000556 0.00011429 0.00012047 0.00000927 0.00040774 0.00009267 0.00000927 0.00001853 0.00006487 0.00009267 0.00104252 0.00012371 0.00045407 0.00003707 Betweenness centrality

2 subunit β Description light polypeptide gene enhancer in κ WASL interacting protein family, member 2 interacting protein family, WASL / ‑ ski sarcoma viral oncogene homolog (avian) ‑ lymphocyte activation 1) Vitamin D (1,25 ‑ dihydroxyvitamin D3) receptor Vitamin directed), delta 2, regulatory subunit 50kDa Polymerase (DNA Calcium channel, voltage ‑ dependent, Patched homolog 1 (Drosophila) Secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T Kinesin family member 5B domain, ankyrin repeat and PH domain 3 with RhoGAP ArfGAP V Epidermal growth factor receptor pathway substrate 15 ‑ like 1 FK506 binding protein 1A, 12kDa kinase, 90kDa, polypeptide 4 BH3 interacting domain death agonist ionotropic, N ‑ methyl D aspartate 2A Glutamate receptor, Neurofilament, light polypeptide 68kDa Ankyrin 2, neuronal homolog (Drosophila) Timeless Staphylococcal nuclease and tudor domain containing 1 Ribosomal protein S6 kinase, 90kDa, polypeptide 5 Ribosomal protein S6 kinase, 90kDa, polypeptide 2 M2 polypeptide Ribonucleotide reductase M2 B (TP53 inducible) G protein ‑ coUpled receptor 4 ‑ like regulator CASP8 and FADD (embryonic lethal, abnormal vision, Drosophila) ‑ like 1 ELAV (Hu antigen R) Inhibitor of B ‑ cells, kinase epsilon Paired box 5 receptor 1 Tachykinin cell line derived transforming sequence ‑ like MCF.2 Neuropilin 2 binding protein 2 XPA domain containing oxidoreductase WW Erbb2 interacting protein

Table V. Continued. V. Table Gene VDR POLD2 CACNB2 PTCH1 SPP1 KIF5B ARAP3 SKI EPS15L1 FKBP1A RPS6KA4 BID GRIN2A NEFL ANK2 TIMELESS SND1 RPS6KA5 RPS6KA2 RRM2 RRM2B GPR4 WIPF2 WAS CFLAR ELAVL1 IKBKE ERBB2IP PAX5 TACR1 MCF2L NRP2 XAB2 WWOX LIAO et al: DIFFERENTIAL GENE EXPRESSION ANALYSIS AND NETWORK CONSTRUCTION 1757 Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Down Down Down Down Down Down Down Down Down Down Regulation

1 3 1 2 3 0 1 2 0 1 2 0 2 0 0 1 1 0 1 2 1 0 1 2 1 0 0 2 2 1 2 2 Outdegree

2 0 2 1 0 2 1 0 2 1 0 2 0 2 2 1 1 2 1 0 1 2 1 0 1 2 2 0 0 1 0 0 Indegree 3 3 3 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Degree 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 0.00010116 0.00042627 0.00003398 0.00001853 0.00037994 0.00000927 0.00000927 0.00001853 Betweenness centrality related subfamily, ‑ related subfamily,

1 (muscle) α ‑ cells 5, tonicity responsive Description ‑ associated NFKB activator 1,3 ‑ galactosyltransferase polypeptide 6 β Gal β member 1 ‑ cell activation GTPase activating protein TRAF family member vasopressin receptor 1A Arginine NCK interacting protein with SH3 domain (never in mitosis gene a) ‑ related kinase 6 NIMA cyclin ‑ dependent kinase 19 T Nuclear factor of activated Heparan sulfate 2 ‑ O sulfotransferase 1 Rho GTPase activating protein 18 acidic coiled ‑ coil containing protein 1 Transforming, 17 protein D52 ‑ like 1 Tumor Aldolase C, fructose ‑ bisphosphate Fibroblast growth factor binding protein 1 ionotropic, kainate 1 Glutamate receptor, Prostaglandin E receptor 4 (subtype EP4) Thioredoxin reductase 2 Forkhead box L1 MAX dimerization protein 1 ‑ like enhancer of split 2 (E(sp1) homolog, Drosophila) Neuronal cell adhesion molecule Potassium voltage ‑ gated channel, shaker Ubiquitin ‑ activating enzyme E1 domain containing 1 specific, HMG‑box) factor 7 (T‑cell Transcription WD repeat domain 77 Kinesin family member 23 nicotinic, receptor, Cholinergic Aquaporin 2 (collecting duct) Male germ cell ‑ associated kinase Secreted frizzled ‑ related protein 4 ‑ like 5 Chitinase 3 ‑ like 1 (cartilage glycoprotein 39) Centrosomal protein 290kDa smooth muscle Myosin, heavy chain 11, β T

Table V. Continued. V. Table Gene TANK AVPR1A NCKIPSD NEK6 CDK19 UDP ‑ Gal: B3GALT6 NFAT5 HS2ST1 ARHGAP18 TACC1 SNX17 TPD52L1 ALDOC FGFBP1 GRIK1 PTGER4 TXNRD2 FOXL1 MXD1 TLE2 NRCAM KCNAB1 UBA5 TCF7 WDR77 KIF23 CHRNA1 AQP2 MAK SFRP4 TAGAP DPYSL5 CHI3L1 CEP290 MYH11 1758 MOLECULAR MEDICINE REPORTS 13: 1746-1764, 2016 Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Down Down Down Down Down Down Down Down Regulation

1 1 2 1 0 2 0 1 0 2 0 0 0 0 2 2 0 0 2 1 1 0 0 2 2 1 0 0 0 2 1 Outdegree

1 2 2 1 2 0 2 2 2 2 0 0 2 2 0 1 1 2 2 0 0 1 2 2 2 0 1 Indegree 2 1 2 2 0 2 1 2 2 0 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Degree 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0.00114908 0.00007413 0.00000927 0.00015136 0.00000927 0.66666667 0.00042627 0.66666667 Betweenness centrality

α 110kDa β ) α calcium exchanger), member 1 Description /

β N ‑ sulfotransferase (heparan glucosaminyl) 1 / lymphoma 10 / ‑ cell co ‑ stimulator Zinc finger protein 341 Solute carrier family 8 (sodium A, pappalysin 1 Pregnancy ‑ associated plasma protein type 3 Inositol 1,4,5 ‑ triphosphate receptor, Glia maturation factor, Solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 4 Catenin (cadherin ‑ associated protein), delta 2 (neural plakophilin ‑ related arm repeat protein) ADAM metallopeptidase domain 12 (meltrin Interleukin 6 signal transducer (gp130, oncostatin M receptor) Insulin ‑ like growth factor binding protein 6 General transcription factor IIIC, polypeptide 2, putative tumor sUppressor 1 Leucine zipper, Glutathione S ‑ transferase omega 2 domain protein 2 Neuronal PAS Rho GTPase activating protein 26 Disabled homolog 2, mitogen ‑ responsive phosphoprotein (Drosophila) initiation factor 4E family member 2 complex 1 Regulatory associated protein of MTOR, RAS protein activator like 2 Fibroblast growth factor 14 Glucoside xylosyltransferase 2 Bridging integrator 1 Presenilin 2 (Alzheimer disease 4) Regulator of G ‑ protein signaling 18 (formerly 2C), magnesium ‑ dependent 1A Cytochrome P450, family 2, subfamily E, polypeptide 1 B ‑ cell CLL Inducible T 5 ‑ hydroxytryptamine (serotonin) receptor 1B 5 ‑ hydroxytryptamine (serotonin) receptor 1D musculoaponeurotic fibrosarcoma oncogene homolog (avian) V‑ Kin of IRRE like (Drosophila)

Table V. Continued. V. Table Gene ZNF341 DES NDST1 N ‑ deacetylase SLC8A1 PAPPA ITPR3 GMFB SLC25A4 CTNND2 ADAM12 IL6ST IGFBP6 GTF3C2 LZTS1 GSTO2 NPAS2 ARHGAP26 DAB2 EIF4E2 RPTOR RASAL2 FGF14 GXYLT2 BIN1 PSEN2 RGS18 PPM1A KIRREL CYP2E1 BCL10 ICOS HTR1B HTR1D MAF LIAO et al: DIFFERENTIAL GENE EXPRESSION ANALYSIS AND NETWORK CONSTRUCTION 1759 Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Down Down Down Down Down Down Regulation

0 0 0 0 2 1 0 0 0 0 1 0 1 1 1 0 0 1 0 Outdegree

1 0 0 1 1 1 1 0 1 0 0 0 1 1 0 1 Indegree 1 1 2 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Degree 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Betweenness centrality ‑ )

γ C. elegans , coactivator γ )

related subfamily, ‑ related subfamily,

2 (neuronal) β S. cerevisiae Description fructose ‑ 2,6 biphosphatase 4 / ‑ activated receptor ‑ associated protein like 1 ‑ propeller repeat containing 2 β SUppressor of Ty 3 homolog ( Ty SUppressor of Carbamoyl ‑ phosphate synthetase 2, aspartate transcarbamylase, and synthase II CTP Programmed cell death 11 binding motif protein 28 RNA Carboxypeptidase D Dipeptidyl ‑ peptidase 4 (CD26, complexing protein 2) Neurocalcin delta Zinc finger protein 169 GABA(A) receptor Peroxisome proliferator related 1 G ‑ protein signaling modulator 1 (AGS3 like, Mitochondrial ribosomal protein L14 Mitochondrial ribosomal protein L4 repeat containing 6A Trinucleotide Rap guanine nucleotide exchange factor (GEF) 2 Potassium voltage ‑ gated channel, shaker member 1 (episodic ataxia with myokymia) Platelet ‑ activating factor acetylhydrolase, isoform Ib, subunit 29kDa G ‑ protein coUpled, 13 receptor P2Y, Purinergic zinc finger domain containing 2A GATA BCL2 ‑ associated athanogene 6 6 ‑ phosphofructo 2 kinase W2 domains 2 Basic leucine zipper and nicotinic, receptor, Cholinergic synthetase Seryl ‑ tRNA YY1 binding protein RING1 and Adenylate cyclase 5 Eukaryotic translation initiation factor 5A

Table V. Continued. V. Table Gene SUPT3H CHAD Chondroadherin CAD CTPS2 PDCD11 RBM28 EIF5A CPD DPP4 NCALD ZNF169 GABARAPL1 TECPR2 Tectonin PPRC1 GPSM1 MRPL14 MRPL4 TNRC6A RAPGEF2 KCNA1 PAFAH1B3 AIRE P2RY13 GATAD2A BAG6 PFKFB4 BZW2 CHRNB2 SARS RYBP ADCY5 1760 MOLECULAR MEDICINE REPORTS 13: 1746-1764, 2016 Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Down Down Down Down Down Down Down Down Regulation

1 0 0 0 0 1 1 1 0 1 0 0 0 0 1 0 0 0 1 0 0 1 0 0 0 1 0 Outdegree

0 1 1 1 1 0 0 0 1 0 1 1 1 1 0 1 1 1 0 1 1 0 1 1 1 0 1 Indegree 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Degree 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Betweenness centrality ) S. cerevisiae lysine ‑ rich protein 1 / 80kDa α Description light polypeptide gene enhancer in B ‑ cells, κ Galactose ‑ 1 phosphate uridylyltransferase Stonin 2 Diacylglycerol kinase, Plexin A4 Myosin, light chain kinase Myogenic factor 6 (herculin) MDN1, midasin homolog (yeast) Amnionless homolog (mouse) Dedicator of cytokinesis 9 Pyruvate dehydrogenase kinase, isozyme 2 MOB kinase activator 1B Salvador homolog 1 (Drosophila) phosphoinositide ‑ 3 kinase, regulatory subunit 6 Inositol monophosphatase domain containing 1 member 2 Nuclear receptor subfamily 2, groUp F, Jumonji domain containing 1C Importin 5 Peptidylprolyl isomerase (cyclophilin) ‑ like 2 A, polypeptide 1 Cytochrome P450, family 1, subfamily Elongation protein 2 homolog ( kinase complex ‑ associated protein Bromodomain containing 9 Receptor (G protein ‑ coUpled) activity modifying 3 Nucleosome assembly protein 1 ‑ like 4 type 3 Neurotrophic tyrosine kinase, receptor, Chemokine (C ‑ X C motif) ligand 5 NDRG family member 2 Sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3F Runt ‑ related transcription factor 2 , palmitoylated 2 (MAGUK p55 subfamily member 2) Solute carrier family 40 (iron ‑ regulated transporter), member 1 Splicing regulatory glutamine member 2 Inhibitor of growth family, Inhibitor of

Table V. Continued. V. Table Gene GALT STON2 DGKA PLXNA4 MYLK MYF6 MDN1 AMN DOCK9 PDK2 MOB1B SAV1 PIK3R6 IMPAD1 NR2F2 JMJD1C IPO5 PPIL2 CYP1A1 ELP2 IKBKAP BRD9 RAMP3 NAP1L4 NTRK3 CXCL5 NDRG2 SEMA3F RUNX2 MPP2 SLC40A1 SREK1 ING2 LIAO et al: DIFFERENTIAL GENE EXPRESSION ANALYSIS AND NETWORK CONSTRUCTION 1761 Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Down Down Down Down Down Down Down Down Down Regulation

0 0 0 1 0 0 0 0 0 1 1 1 0 1 0 0 0 1 1 0 1 0 1 0 1 0 0 Outdegree

1 1 1 0 1 1 1 1 1 0 0 0 1 0 1 1 1 0 0 1 0 1 0 1 1 1 Indegree 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 Degree 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Betweenness centrality ) γ , 2 γ Description ‑ 2,3 sialyltransferase 6 ‑ 2,3 sialyltransferase 3 α α 1 β ‑ galactoside ‑ galactoside β β Akirin 2 Cadherin 6, type 2, K ‑ cadherin (fetal kidney) Cadherin 7, type 2 member 1 Potassium voltage ‑ gated channel, Shaw related subfamily, protein 173 Transmembrane Zinc finger protein 775 Deltex 3 homolog (Drosophila) Eukaryotic translation initiation factor 4 with SH3 domain, ankyrin repeat and PH domain 1 ArfGAP Zinc finger protein 133 Sperm associated antigen 9 member 4 Solute carrier family 4, sodium bicarbonate cotransporter, member 7 Solute carrier family 4, sodium bicarbonate cotransporter, MYB binding protein (P160) 1a synthetase 2, mitochondrial tRNA Tryptophanyl Potassium inwardly ‑ rectifying channel, subfamily J, member 2 Diacylglycerol kinase, eta Angiopoietin ‑ like 4 Mitogen ‑ activated protein kinase associated 1 family interacting protein 1 (class I) RAB11 Indian hedgehog homolog (Drosophila) Solute carrier family 2 (facilitated glucose transporter), member 1 ADAM metallopeptidase domain 9 (meltrin Phosphatidylinositol glycan anchor biosynthesis, class B Solute carrier family 7 (cationic transporter, y+ system), member 8 Abelson helper integration site 1 member 2 Discoidin domain receptor family, Melanocortin 3 receptor Thioredoxin 2 Prospero homeobox 1 Nucleic acid binding protein 1 Phospholipase C ‑ like 2

Table V. Continued. V. Table Gene AKIRIN2 CDH6 CDH7 KCNC1 TMEM173 ZNF775 DTX3 EIF4G2 ASAP1 ZNF133 SPAG9 SLC4A4 SLC4A7 MYBBP1A WARS2 KCNJ2 DGKH ANGPTL4 MAPKAP1 RAB11FIP1 IHH SLC2A1 ADAM9 ST3GAL6 ST3 ST3GAL3 ST3 PIGB SLC7A8 AHI1 DDR2 MC3R TXN2 PROX1 NABP1 TUBB1 Tubulin, PLCL2 1762 MOLECULAR MEDICINE REPORTS 13: 1746-1764, 2016 Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Down Down Down Down Down Down Down Down Down Down Regulation

0 0 0 0 1 0 0 0 0 1 1 0 0 1 0 0 0 0 0 1 1 0 0 Outdegree

1 1 1 1 0 1 1 1 0 0 1 1 0 1 1 1 1 1 0 0 1 1 Indegree 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Degree 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Betweenness centrality subunit β threonine kinase 1 / Description ‑ like transcription regulator 1 ‑ related protein complex 4, epsilon 1 subunit Caspase recruitment domain family, member 14 Caspase recruitment domain family, 2 Zinc finger protein 77 Zinc finger and BTB domain containing 25 Adaptor ADAM metallopeptidase domain 15 Dynein, axonemal, heavy chain 1 synthetase, Phenylalanyl ‑ tRNA Insulin ‑ like 3 (Leydig cell) type 2, member 45 receptor, Taste Prolactin Ca++ transporting, cardiac muscle, fast twitch 1 ATPase, Eyes absent homolog 4 (Drosophila) Bone morphogenetic protein 6 kinase interacting serine MAP (SII), 2 A Transcription l (3) mbt ‑ like 1 with SH3 domain, ankyrin repeat and PH domain 2 ArfGAP FYVE domain containing 16 Zinc finger, Hexamethylene bis ‑ acetamide inducible 1 (never in mitosis gene a) ‑ related kinase 3 NIMA DEAD (Asp ‑ Glu Ala Asp) box polypeptide 56 production factor 1 homolog 3 Syntaxin 7 amidinotransferase) Glycine amidinotransferase (L ‑ arginine:glycine Enhancer of zeste homolog 1 (Drosophila) rich interactive domain 2 AT Jumonji, Naked cuticle homolog 2 (Drosophila) Zinc finger protein 569 Complement component 1, q subcomponent, C chain Dihydropyrimidinase Leucine ‑ zipper domain containing 1 A factor Willebrand Von Peroxisomal biogenesis factor 14 Elongation factor RNA polymerase II Elongation factor RNA

Table V. Continued. V. Table Gene CARD14 JDP2 ZNF77 ZBTB25 AP4E1 ADAM15 DNAH1 FARSB INSL3 TAS2R45 PRL ATP2A1 EYA4 BMP6 MKNK1 TCEA2 L3MBTL1 ASAP2 ZFYVE16 HEXIM1 NEK3 DDX56 RPF1 STX3 STX7 GATM ELL EZH1 JARID2 NKD2 ZNF569 C1QC DPYS LZTR1 VWA1 PEX14 LIAO et al: DIFFERENTIAL GENE EXPRESSION ANALYSIS AND NETWORK CONSTRUCTION 1763 up Up Up Up Up Up Down Down Regulation

0 0 0 0 1 0 Outdegree

1 1 1 1 0 Indegree 1 1 1 1 1 1 1 1 Degree 0 0 0 0 0 0 0 0 Betweenness centrality ) S. pombe

Description Peroxisomal biogenesis factor 3 urokinase Plasminogen activator, Pleiomorphic adenoma gene ‑ like 1 Low density lipoprotein receptor adaptor protein 1 domain protein 4 Neuronal PAS 1 homolog ( RCD1 required for cell differentiation Zinc finger protein 337 Bromodomain adjacent to zinc finger domain, 1A

Table V. Continued. V. Table Gene PEX3 PLAU PLAGL1 LDLRAP1 NPAS4 BAZ1A RQCD1 ZNF337 Up, upregulated; Down, downregulated. 1764 MOLECULAR MEDICINE REPORTS 13: 1746-1764, 2016 terms and pathways identified here require further investiga- 11. Suresh R, Li X, Chiriac A, Goel K, Terzic A, Perez‑Terzic C and Nelson TJ: Transcriptome from circulating cells suggests tion and confirmation. dysregulated pathways associated with long‑term recurrent In conclusion, the present study revealed the underlying events following first‑time myocardial infarction. J Mol Cell molecular differences between patients with AMI, with and Cardiol 74: 13‑21, 2014. 12. Gautier L, Cope L, Bolstad BM and Irizarry RA: Affy‑analysis of without recurrent cardiovascular events, including DEGs, Affymetrix GeneChip data at the probe level. Bioinformatics 20: their biological function, signaling pathways and key genes 307‑315, 2004. in the PPI network, which may contribute to the prevention 13. Smyth GK, Michaud J and Scott HS: Use of within‑array replicate spots for assessing differential expression in microarray of recurrent events and personalized treatment for primary experiments. Bioinformatics 21: 2067‑2075, 2005. cardiovascular events. Further functional studies may provide 14. Nogales‑Cadenas R, Carmona‑Saez P, Vazquez M, Vicente C, additional insights into the role of the DEGs in the pathological Yang X, Tirado F, Carazo JM and Pascual‑Montano A: GeneCodis: Interpreting gene lists through enrichment analysis process of recurrent cardiovascular events. and integration of diverse biological information. Nucleic Acids Res 37: W317‑W322, 2009. Acknowledgements 15. Carmona‑Saez P, Chagoyen M, Tirado F, Carazo JM and Pascual‑Montano A: GENECODIS: A web‑based tool for finding significant concurrent annotations in gene lists. Genome Biol 8: The present research was supported by a grant from the R3, 2007. National Natural Science Foundation of China (no. 81173166). 16. Saito R, Smoot ME, Ono K, Ruscheinski J, Wang PL, Lotia S, Pico AR, Bader GD and Ideker T: A travel guide to cytoscape plugins. Nat Methods 9: 1069‑1076, 2012. References 17. Mortensen MB and Falk E: Real‑life evaluation of European and American high‑risk strategies for primary prevention of cardio- 1. Go AS, Mozaffarian D, Roger VL, Benjamin EJ, Berry JD, vascular disease in patients with first myocardial infarction. BMJ Blaha MJ, Dai S, Ford ES, Fox CS, Franco S, et al: Heart disease Open 4: e005991, 2014. and stroke statistics‑2014 update: A report from the American 18. Montecucco F, Carbone F, Dini FL, Fiuza M, Pinto FJ, heart association. Circulation 129: e28‑e292, 2014. Martelli A, Palombo D, Sambuceti G, Mach F and De Caterina R: 2. Goff DC Jr, Lloyd‑Jones DM, Bennett G, Coady S, Implementation strategies of systems medicine in clinical D'Agostino RB, Gibbons R, Greenland P, Lackland DT, Levy D, research and home care for cardiovascular disease patients. 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