In Silico Identification of Common and Specific Signatures in Coronary Heart Diseases

EXPERIMENTAL AND THERAPEUTIC MEDICINE 20: 3595-3614, 2020

In silico identification of common and specific signatures in coronary heart diseases

ZHIJIA YANG1, HAIFANG MA2 and WEI LIU3

1The Third Department of Cardiovascular Medicine, Handan Central Hospital; 2The First Department of Cardiovascular Medicine, Affiliated Hospital of Hebei University of Technology, Handan, Hebei 056002; 3The First Department of Cardiovascular Medicine, Handan Central Hospital, Handan, Hebei 056001, P.R. China

Received January 3, 2020; Accepted April 15, 2020

DOI: 10.3892/etm.2020.9121

Abstract. Coronary heart disease (CHD) is on the increase in Introduction developing countries, where lifestyle choices such as smoking, bad diet, and no exercise contribute and increase the incidence Coronary heart disease (CHD), also known as coronary artery of high blood pressure and high cholesterol levels to cause disease (CAD) is one of a group of diseases of the heart blood CHD. Through utilization of a biomarker-based approach for vessels affecting millions of individuals worldwide. According developing interventions, the aim of the study was to identify to the center for disease control (CDC) reports, each death out differentially expressed genes (DEGs) and their association of four is related to heart diseases, leading to approximately and impact on various bio-targets. The microarray datasets of 610,000 mortalities annually worldwide (1). Among the heart both healthy and CHD patients were analyzed to identify the diseases, CAD is the most common, responsible for the death DEGs and their interactions using Gene Ontology, PANTHER, of 370,000 individuals annually worldwide (1). CAD occurs Reactome, and STRING (for the possible associated genes when the elasticity of arteries, as well as vein and vessel with multiple targets). Our data mining approach suggests that smoothing, become plaque in the inner wall, making them the DEGs were associated with molecular functions, including rigid and narrowed. This condition restricts the blood flow to protein binding (75%) and catalytic activity (56%); biological the heart muscle, leading to oxygen starvation. The condition processes such as cellular process (83%), biological regulation of plaque rupture leads to the heart failure or cardiac death (2). (57%), and metabolic process (44%); and cellular components Recently, there has been an increase in the incidence of such as cell (65%) and organelle (58%); as well as other asso- CHD (also known as ischemic heart disease) in China (3). In ciations including apoptosis, inflammatory, cell development addition, CHD has become the most common reason for death and metabolic pathways. The molecular functions were further in middle and high-income countries (4). According to the data analyzed, and protein binding in particular was analyzed using report by NHANES, CHD prevalence was higher in males network analysis to determine whether there was a clear asso- than females across all ages (7.4 v/s 5.3%, respectively) (3). ciation with CHD and disease. The ingenuity pathway analysis The American Heart Association explains ‘The important revealed pathways related to cell cholesterol biosynthesis, the difference between sex and pathology’, clinical presentation immune system including cytokinin signaling, in which, the and outcomes in CHD patients (5). Thus it is crucial to pay understanding of DEGs is crucial to predict the advancement attention to sex disparities and subsequently to personalize of preventive strategies. Results of the present study showed treatment (6). Patients with CHD are also susceptible to more that, there is a need to validate the top DEGs to rule out their complicated clinical problems. Currently, the diagnosis and molecular mechanism in heart failure caused by CHD. therapy of CHD is rare and costly as compared to coronary angiography, which is the most popular clinical management option (7). CHD is one of the leading causes of death, and markedly affects the immunity of the body, making it an economic burden worldwide (8,9). This is a complex disease involving multiple mechanisms and influenced by many Correspondence to: Dr Wei Liu, The First Department of risk factors, including physical activity, genetics, diet, and Cardiovascular Medicine, Handan Central Hospital, 15 Zhonghua smoking (10,11). Recently, a genome-wide association study North Street, Handan, Hebei 056001, P.R. China (GWAS) identified many candidate loci associated with CHD E-mail: [email protected] and myocardial infection (MI) (12-14). Although genetics Key words: coronary heart disease, microarray, differentially play an important role, accounting for approximately 50% of expressed genes CHD heritability, the exact mechanism and causative agent of CHD are not yet revealed clearly (15-17). In this regard, it is important to understand and address the candidate genome association in developing CHD. 3596 YANG et al: GENOMIC APPROACHES FOR CORONARY HEART DISEASES Gene ontology: Molecular function Molecular binding polymerase II regulatory region ase activator activity Nucleotide binding Nucleotide binding Nucleotide binding binding Sequence-specific DNA Rna polymerase II regulatory region Cyclic-nucleotide phosphodiesterase activity Protein binding Peroxidase activity 14-3-3 Protein binding Protein binding Nucleic acid binding activator activity, Transcriptional sequence-specific DNA binding sequence-specific DNA Nucleotide binding GTP DNA RNA polymerase II transcription RNA regulatory region sequence-specific binding Cytokine activity Protein binding Epidermal growth factor Epidermal growth factor Transcription factor activity, Transcription Nicotinate-nucleotide diphosphorylase Virus receptor activity Virus Receptor activity Phosphoprotein phosphatase activity Gene ontology: Cellular component Nucleus Nucleus Nucleus Extracellular space Nucleus Cytoplasm Mitochondrion Cytoplasm Nucleus Nucleus Intracellular Plasma membrane Nucleus Nucleus

Intracellular Extracellular region Cytoplasm Extracellular region Extracellular region Nucleus Extracellular region Cytoplasm Extracellular region Nucleus activity activity -templated -templated e e MAPK DNA DNA MAPK CHD CHD polymerase II promoter . Gene ontology: Biological function Biological Cas Cas RNA CHD sequence-specific DNA binding sequence-specific DNA Regulation of cell growth Regulation of cell growth Regulation of cell growth Protein dephosphorylation Transcription, Transcription, Negative regulation of transcription Regulation of heart rate Apoptotic process Immune response Prostaglandin biosynthetic process Keratinocyte development Response to hypoxia Regulation of cytokine production Activation of from RNA polymerase II promoter from RNA polymerase II promoter from RNA receptor binding receptor binding response to virus by host (carboxylating) activity from Negative regulation of transcription MAPK MAPK Angiogenesis Apoptotic process Negative regulation of transcription Vitamin metabolic process Vitamin Positive regulation of defense Inactivation of

Transcription, Transcription,

Gene symbol NR4A2 LOC102724428 HBEGF CXCL8 BCL2A1 DDIT4 EREG G0S2 DUSP2 RGS1 PTGS2 TREM1 KLF3 SSH2 CD83 SGK1 PDE4D SGK1 CXCR4 NAMPT FOSL2 SGK1 CREBRF CSRNP1

LogFC

ID Table I. Up - and down regulated genes with associated function in associated with genes regulated down - and I. Up Table A, Upregulation 3.963 11725632 2.98 11716771 3.593 11719898 6.035 11718841 2.91 11761272 2.917 11743972 4.2 11732719 6.925 11744219 3.385 11721695 4.1 11742765 5.573 11724037 2.647 11746721 3.44 11759749 1.885 11744850 2.982 11743000 3.478 11715931 2.157 11724447 3.272 11737750 2.058 11728190 2.268 11743110 2.103 11763170 3.143 11733698 2.245 11744932 2.265 11757721 EXPERIMENTAL AND THERAPEUTIC MEDICINE 20: 3595-3614, 2020 3597 binding DNA Gene ontology: Molecular function Binding binding binding polymerase II regulatory region polymerase II core promoter Sequence-specific Prostaglandin-D synthase activity Transcriptional activator activity, activator activity, Transcriptional activity phosphodiesterase Cyclic-nucleotide Protein binding Guanyl-nucleotide exchange factor activity Transmembrane signaling Transmembrane Phosphoprotein phosphatase activity receptor activity Virus Protein kinase inhibitor activity receptor protein tyrosine Transmembrane kinase adaptor activity receptor activity Virus Phosphoprotein phosphatase activity factor activity, Transcription binding sequence-specific DNA Phosphoprotein phosphatase activity Phosphoprotein phosphatase activity Receptor activity (Carboxylating) activity proximal region sequence-specific binding Protein binding DNA DNA RNA RNA DNA Nicotinate-nucleotide diphosphorylase Transcription cofactor activity Transcription Virus receptor activity Virus Signal transducer activity Protease binding Gene ontology: Cellular component

Chromatin Extracellular region Nucleus Nucleus Cytoplasm Nucleus Extracellular region Cytosol Integral component Nucleus Nucleus Cytoplasm Intracellular Nucleus Cytoplasm Nucleus Nucleus Nucleus Nucleus of plasma membrane Intracellular Extracellular region Nucleus Cytoplasm Plasma membrane Nucleus -templated DNA activity MAPK Gene ontology: Biological function polymerase II pomoter polymerase II promoter RNA RNA Negative regulation of transcription Prostaglandin biosynthetic process Double-strand break repair Positive regulation of Regulation of heart rate Multicellular organismal development Multicellular organismal response to virus by host Endocytosis Signal transduction Signal transduction Activation of Negative regulation of transcription Inactivation of MAPK activity Activation of MAPK activity Response to hypoxia Cellular glucose homeostasis Activation of MAPK activity Protein dephosphorylation Negative regulation of transcription polymerase II promoter from RNA Inactivation of MAPK activity Protein dephosphorylation via homologous recombination from endothelial cell proliferation from Regulation of transcription, Cell fate determination Vitamin metabolic process Vitamin Neutrophil apoptotic process B-1 B cell homeostasis

Gene symbol DUSP1 CXCR4 SOCS3 PIK3R1 CXCR4 PTPRE TSC22D3 DUSP1 PTP4A1 PHC3 PTGDS CD69 TNFAIP3 TAGAP CXCR4 NABP1 NAMPT JUNB NR4A1 ZFP36 BTG1 PDE4D C9ORF72 MCL1 HCAR2 /// HCAR3

2.552 2.465 2.567 2.195 2.54 1.75 1.69 3.005 1.79

Log Table I. Continued. Table ID 11715766 11728191 11719218 11739540 11728189 11743596 11717830 11752993 11717897 1.468 11752039 2.1 11756587 2.607 11723679 2.723 11718939 2.2 11736467 2.712 11739094 1.535 11763367 2.857 11743111 2.002 11715673 2.812 11717994 2.008 11715691 2.29 11733022 1.738 11724446 2.052 11737176 1.405 11715487 2.275 11722615 3598 YANG et al: GENOMIC APPROACHES FOR CORONARY HEART DISEASES Gene ontology: Molecular function Opsonin Binding Receptor activity corepressor activity Transcription polymerase II regulatory region RNA binding sequence-specific DNA polymerase II core promoter RNA binding proximal region sequence-specific DNA polymerase II regulatory RNA binding region sequence-specific DNA Peroxidase activity Protein serine polymerase II regulatory RNA binding region sequence-specific DNA receptor activity polymerase II core promoter RNA binding proximal region sequence-specific DNA Complement component C5a binding Actin binding Nucleotide binding Ubiquitin-protein transferase activity factor activity, Transcription binding sequence-specific DNA Binding Actin binding Cytokine activity Phosphoprotein phosphatase activity DNA binding DNA Nucleic acid binding Transmembrane signaling Transmembrane

Gene ontology: Cellular component Nucleoplasm Extracellular region Nucleus Nucleus Nuclear chromosome Nucleus Nucleus Protein phosphatase complex type 2A Nucleus Nucleus Cytosol Ruffle Mitochondrion Cul3-RING ubiquitin ligase complex Nucleus Cytoplasm Cytoskeleton Extracellular region Nucleus Cytoplasm Nucleus Nucleus catabolic process,

RNA Gene ontology: Biological function Response to yeast Positive regulation of defense response to virus by host Negative regulation of transcription polymerase II promoter from RNA Keratinocyte development Angiogenesis DNA-templated Transcription, Prostaglandin biosynthetic process Response to reactive oxygen species Negative regulation of transcription polymerase II promoter from RNA Immune system process DNA-templated Transcription, Activation of MAPK activity Actin cortical patch assembly m Nuclear-transcribed deadenylation-dependent decay Long-chain fatty acid metabolic process Protein ubiquitination Negative regulation of transcription polymerase II promoter from RNA DNA-templated Transcription, Regulation of smooth muscle contraction Positive regulation of acute inflammatory response Inactivation of MAPK activity cell differentiation Hematopoietic progenitor

Gene symbol TREM1 RLIM FOSL2 JUN ZBTB21 PTGS2 CYCS NR4A2 /// FCGR2A FCGR2C MAFB C5AR1 WIPF1 ZFP36L1 ACSL1 KBTBD2 TSC22D3 BZW1 CNN1 OSM DUSP1 ZBTB24 CLEC7A

2.093 1.505 1.667 3.535 1.69 4.715 1.37 3.285 1.28 2.755 1.655 1.838 1.505 1.375 1.895 1.688 1.497 1.2 3.808 1.39 Log Table I. Continued. Table ID 11719862 11734799 11763169 11718394 11749652 11724038 11753803 11725631 11751242 11721629 11733355 11759628 11726889 11750700 11716602 11751415 11744775 11747736 11727757 11758730 1.312 11744810 1.357 11737147 EXPERIMENTAL AND THERAPEUTIC MEDICINE 20: 3595-3614, 2020 3599 Gene ontology: Molecular function Magnesium ion binding Atpase activator activity Protein binding cofactor activity Transcription Core promoter sequence-specific binding DNA Signal transducer activity Nucleotide binding Ferroxidase activity Cytokine activity Peptidyl-prolyl cis-trans isomerase activity Ubiquitin-specific protease activity Receptor binding N-acetylgalactosamine 4-O-sulfotransferase activity Nucleotide binding polymerase II regulatory region RNA binding sequence-specific DNA proximal promoter core II polymerase RNA binding region sequence-specific DNA proximal promoter core II polymerase RNA binding region sequence-specific DNA Magnesium ion binding Protein binding Protein binding Protein binding proximal promoter core II polymerase RNA binding region sequence-specific DNA Protein binding Protein kinase activity Nucleotide binding DNA binding DNA

Gene ontology: Cellular component Cytoplasm Nucleus Nucleus Nucleus Nucleus Plasma membrane Golgi membrane Cell Extracellular region Nucleus Cytoplasm Extracellular region Golgi membrane Cytoplasm Nucleus Nucleus Nucleus Nucleus Nucleoplasm Cytoplasm Nucleus Nucleus Nucleus Intracellular Nucleus catabolic process,

RNA Gene ontology: Biological function Protein dephosphorylation Protein folding Response to protozoan DNA-templated Transcription, Glucose metabolic process Neutrophil apoptotic process Immune response Iron ion transport Response to molecule of bacterial origin Protein polyubiquitination Angiogenesis Negative regulation of transcription polymerase II promoter from RNA m Nuclear-transcribed deadenylation-dependent decay Chondrocyte development Activation of MAPK activity Protein import into nucleus, translocation Conditioned taste aversion from Transcription RNA polymerase II promoter Release of cytochrome c from mitochondria Regulation of cell adhesion Transport from Transcription RNA polymerase II promoter Protein phosphorylation Response to organic substance Response to organic Intracellular protein transport

Gene symbol PPM1B DNAJB1 IER3 NFKBIZ CREM HCAR2 /// HCAR3 GBP2 FTH1 CXCL2 PPIL2 OTULIN IL1B ZFP36L2 CHST11 RIPK2 BCL6 FOS CEBPD S100P PMAIP1 CYTIP RAB11FIP1 CEBPD RMND5A TRIB1 ARL4A

1.662 1.248 2.98 2.262 1.92 1.355 1.695 1.438 5.325 1.337 1.328 4.9 1.18 1.4 1.272 1.815 1.75 1.58 1.585 2.365 1.252 1.395 1.27 1.67 2.357 1.558 Log Table I. Continued. Table ID 11719713 11715445 11720062 11757513 11758522 11724835 11762406 11752577 11744128 11760678 11727569 11719916 11715817 11743434 11724236 11720745 11749291 11764029 11718347 11724509 11734690 11736782 11764030 11743344 11716048 11756387 3600 YANG et al: GENOMIC APPROACHES FOR CORONARY HEART DISEASES Gene ontology: Molecular function Protein binding Cytokine activity Receptor binding Opsonin binding polymerase II core promoter RNA binding sequence-specific DNA Protein binding Nucleic acid binding polymerase II core promoter RNA proximal region sequence-specific binding DNA binding sequence-specific DNA Calcium ion binding polymerase II core promoter RNA proximal region sequence-specific binding DNA Gtpase activator activity Microtubule motor activity polymerase II core promoter RNA proximal region sequence-specific binding DNA Cytokine activity Nucleotide binding Catalytic activity Nucleotide binding polymerase II core promoter RNA proximal region sequence-specific binding DNA Poly(A)-specific ribonuclease activity Gene ontology: Cellular component Nucleus Extracellular region Endoplasmic reticulum Nuclear chromosome Endoplasmic reticulum Intracellular Nuclear chromosome Nucleus Nucleus Extracellular region Dynein complex Nucleus Extracellular region Nucleus Chromatin Nuclear chromosome Cytoplasmic mrna processing body Nucleoplasm Gene ontology: Biological function Release of cytochrome c from mitochondria Immune system process Protein retention in ER lumen Chromatin silencing at rdna Translation DNA-templated Transcription, Angiogenesis Posit regulatory region Transcription polymerase II promoter RNA Cytosolic calcium ion homeostasis DNA-templated Transcription, Cellular defense response Positive regulation of gtpase activity Microtubule-based movement Negative regulation of transcription polymerase II promoter from RNA Response to molecule of bacterial origin mrna catabolic process, Nuclear-transcribed deadenylation-dependent decay Metabolic process G1 Angiogenesis Negative regulation of transcription polymerase II promoter from RNA Response to yeast

Gene symbol PMAIP1 VSTM1 ANKRD13C CLEC7A H3F3B SSR1 ZNF267 JUN NSG1 Nucleus PVALB MAFB GNLY SIPA1L2 DNHD1 MXD1 CXCL2 EIF4A1 CDADC1 PLK3 JUN SCARNA9L CNOT1

2.298 1.782 1.348 1.333 1.325 1.2 1.36 4.04 1.88 2.138 1.068 1.542 1.03 1.498 4.107 1.777 1.252 1.87 3.16 1.04 1.12 Log Table I. Continued. Table ID 11724510 11732665 11759780 11737148 11758593 11763972 11727523 11718395 -2.47 11727032 tive regulation of transcription from 11718061 11721630 11763755 11757924 11732859 11750016 11744127 11763556 11745466 11756358 11718397 11763954 11730655 EXPERIMENTAL AND THERAPEUTIC MEDICINE 20: 3595-3614, 2020 3601 Gene ontology: Molecular function Protein binding Nucleotide binding region regulatory II polymerase RNA binding sequence-specific DNA Peroxidase activity region regulatory II polymerase RNA binding sequence-specific DNA ion channel activity Voltage-gated Nucleotide binding signaling Transmembrane receptor activity activity, Transferase transferring acyl groups Ubiquitin-protein transferase activity polymerase II core RNA promoter proximal region sequence- binding specific DNA cofactor activity Transcription Nucleotide binding Nucleic acid binding polymerase II core promoter RNA proximal region sequence-specific binding DNA Nucleotide binding Protein binding Phosphoprotein phosphatase activity Protein binding Hydrolase activity Ubiquitin-protein transferase activity Nucleotide binding Receptor activity signaling Transmembrane receptor activity Gene ontology: Mitochondrion Nucleus Nucleus Nucleus Plasma membrane Cytoplasm Membrane Nucleus Nucleus Extracellular region Immunological synapse Nucleoplasm Nucleus Cytoplasm Nucleus Nucleus Nucleus Extracellular space Ubiquitin ligase complex Cytoplasm Plasma membrane Cytoplasm Intracellular Intracellular Cellular component Gene ontology: Biological function Apoptotic process Negative regulation of transcription polymerase II promoter from RNA Prostaglandin biosynthetic process Negative regulation of transcription polymerase II promoter from RNA Transport Immune system process Metabolic process Epithelial cell maturation DNA-templated Transcription, MAPK casCHDe Mesoderm formation Regulation of translation Conditioned taste aversion Protein phosphorylation Nucleosome assembly Protein dephosphorylation development Multicellular organismal Nitrogen compound metabolic process Ubiquitin-dependent protein catabolic process Cellular protein modification process Retinoid metabolic process Immune system process Intracellular protein transport Intracellular protein transport

Gene symbol G0S2 ARL4A NFIL3 PTGS2 NR4A2 KCNK7 ARL4A /// FCGR2A FCGR2C TMEM68 FEM1B MAFB NRG1 PRKAR1A ANKHD1 FOS PIM3 NAP1L5 ANKRD44 PTP4A1 BTBD7 VNN3 UBR1 CDKL3 STRA6 /// FCGR2A FCGR2C

4.195 1.132 2.372 4.27 1.64 1 1.617 0.978 0.987 1.285 2.205 1.2 2.535 1.45 1.54 1.3 1.04 0.98 1.285 1.525 1.88 1.413 1.05 1.052 0.958 Log Table I. Continued. Table ID 11754074 11739230 11743010 11724036 11747474 11740892 11733140 11724769 11760192 11734988 11757798 11740347 11753791 11725114 11734659 11716071 11720612 11755987 11717895 11725199 11746681 11720726 11760818 11733686 11724768 3602 YANG et al: GENOMIC APPROACHES FOR CORONARY HEART DISEASES Gene ontology: Molecular function Protein binding protein kinase inhibitor activity Structural molecule activity Binding Protein binding Protein binding Nucleotide binding Receptor binding Protein binding Protein binding Protein kinase C binding Receptor binding Nucleic acid binding Methyltransferase activity Phosphoprotein phosphatase activity Nucleotide binding Nucleotide binding Ubiquitin-protein transferase activity Signal transducer activity factor activity, Transcription binding sequence-specific DNA polymerase II regulαtory region RNA binding sequence-specific DNA factor activity, Transcription binding sequence-specific DNA Nucleus Nucleus Nucleus Gene ontology: Cellular component Golgi membrane Cytoplasm Spindle pole Cytoplasm Cytoplasm Chromosome, centromeric region Golgi membrane Cell Nucleus Golgi membrane Intracellular Nucleus Nucleus Extracellular region Chromosome, centromeric region Cul3-RING ubiquitin ligase complex Cytosol Nucleus

Gene ontology: Biological function Nucleosome assembly Negative regulation of transcription polymerase II promoter from RNA Negative regulation of transcription from polymerase II promoter RNA Immune system process Regulation of cell growth G2 Neuron migration Mitotic cytokinesis Carbohydrate metabolic process metabolic process DNA Positive regulation of receptor recycling Cell redox homeostasis Negative regulation of transcription polymerase II promoter from RNA Positive regulation of receptor recycling DNA-templated Transcription, Methylation Protein dephosphorylation G1 Mitotic cell cycle Protein ubiquitination Chemotaxis Negative regulation of transcription polymerase II promoter from RNA Gene NAP1L5 ARID5B ID1 symbol HLA-DRB5 CISH NUMA1 ASPM CEP55 HMMR MKI67 NSG1 KLHL35 TXNDC9 DACT1 NSG1 ZNF2 METTL18 DLGAP5 TYMS PLGLB1 /// BUB1 KBTBD6 CCR9 ID3 PLGLB2

0.932 0.975 2.692 -3.328 -2.185 -2.075 -1.783 -2.223 -1.885 -1.542 -2.188 -1.588 -1.417 -1.752 -1.55 -1.775 -1.407 -1.55 -1.83 -1.498 -1.518 -1.732 -1.465 -1.623 Log Table I. Continued. Table ID 11748516 11718927 11716195 Β, Down regulation 11720657 11724843 11762593 11742832 11758261 11758089 11721145 11743423 11736674 11759710 11735385 11748198 11732363 11741074 11722367 11751805 11764270 11747230 11723209 11732390 11716666 EXPERIMENTAL AND THERAPEUTIC MEDICINE 20: 3595-3614, 2020 3603 Gene ontology: Molecular function Magnesium ion binding Actin binding Protein binding Protein binding G-protein coupled receptor binding polymerase II regulatory region RNA binding sequence-specific DNA Ubiquitin-protein transferase activity Phosphoprotein phosphatase activity Ubiquitin-protein transferase activity Phosphoprotein phosphatase activity Signal transducer activity Nucleotide binding Magnesium ion binding N-acetyltransferase activity, Lysine acting on acetyl phosphate as donor Ribonucleoside-diphosphate reductase acceptor as disulfide thioredoxin activity Single-stranded DNA endodeoxyribonuclease activity Nucleic acid binding Nucleotide binding Ribonucleoside-diphosphate thioredoxin reductase activity, disulfide as acceptor Protein binding Guanyl-nucleotide exchange factor activity Gene ontology: Cellular component telomeric region damage recognition DNA Cytoplasm Cytoplasm Nucleus Spindle pole Extracellular region Nucleus Intracellular Nucleus Nucleus Nucleus Membrane Cytoplasm Nucleus Plasma membrane Cytoplasm Chromatin Nucleus Extracellular region Nuclear chromosome, Intracellular Cytoplasm Nucleus Cytoplasm Nucleus Gene ontology: Biological function defense response to virus by host Protein dephosphorylation Actin filament organization G2 Mitotic cell cycle Generation of precursor metabolites and energy Regulation of transcription, DNA-templated Signal transduction Regulation of cyclin-dependent protein serine Protein polyubiquitination Protein dephosphorylation Transport G1 Response to biotic stimulus Protein dephosphorylation Mitotic cell cycle G1 Nucleotide-excision repair, DNA-templated Transcription, Positive regulation of G1 Protein folding to membrane Protein targeting

Gene symbol

/// PLGLB2 PSPH TMSB15A CCNB2 CDC20 POMC ZNF691 TRIM13 CDKN3 DTL DLGAP5 TM2D2 GULP1 TYMS LOC100507547 /// PSPH ESCO2 RRM2 /// PLGLB1 PLGLA DCLRE1C ZNF780A DDX58 RRM2 CDC37L1 RAB3IP

-1.705 -1.307 -1.518 -1.375 -1.71 -1.4 -2.403 -1.295 -1.245 -1.44 -1.502 -1.272 -1.782 -1.245 -1.165 -1.148 -1.48 -1.73 -1.635 -1.97 -1.62 -1.762 -2.118 -1.47 Log Table I. Continued. Table ID 11763252 11720240 11716793 11717163 11716427 11755958 11724022 11730821 11726302 11744793 11718599 11760734 11718058 11734748 11730796 11727968 PRRT1 11758219 11755381 11762018 11734263 11733149 11754360 11758872 11728830 3604 YANG et al: GENOMIC APPROACHES FOR CORONARY HEART DISEASES Gene ontology: Molecular function Protein binding polymerase II transcription RNA sequence-specific factor activity, binding DNA Structural molecule activity polymerase II core promoter RNA proximal region sequence-specific binding DNA Protein binding Receptor binding Protein binding Protein binding Receptor activity Nucleic acid binding binding DNA Binding Nucleotide binding Phosphoprotein phosphatase activity Peptidyl-prolyl cis-trans isomerase activity Protein binding Protein binding Nucleic acid binding Protein binding Gtpase activator activity Nucleotide binding Nucleic acid binding Protein binding Phosphoprotein phosphatase activity Cytoskeletal protein binding Actin binding Gene ontology: Cellular component

Nucleoplasm Nucleus Cytoplasm Intracellular Ruffle Extracellular region Plasma membrane Extracellular space Intracellular Nucleus Nucleus Nucleus Nucleus Cytoplasm Nucleolus Cytoplasm Intracellular Nucleus Membrane Intracellular Intracellular Intracellular Cytoplasm Stress fiber Gene ontology: Biological function from RNA polymerase II promoter from RNA response to virus by host filament depolymerization Protein folding Negative regulation of transcription MAPK casCHDe DNA-templated Transcription, Establishment of epithelial cell polarity Apoptotic process Protein homooligomerization Adaptive immune response Cell cycle Positive regulation of defense damage checkpoint DNA Mitotic spindle elongation Regulation of cyclin-dependent protein serine Protein peptidyl-prolyl isomerization Immune system process Signal transduction Mitotic cell cycle DNA-templated Transcription, Keratinocyte development Regulation of cyclin-dependent protein serine Negative regulation of actin

Gene symbol DNAJB4 ZNF174 NEFL ZNF680 FRMD4B TNFSF14 KCTD6 SIT1 TACSTD2 ZNF681 CENPBD1 INTS7 KIF23 CDKN3 TTC9C SH3RF3 WDR5B THAP2 TNFAIP8L2 DEPDC1B UBE2C TMEM60 ZNF566 EXPH5 CDC25A EPB41L4A LIMA1

-1.37 -1.157 -1.195 -1.218 -1.177 -1.055 -1.183 -1.397 -1.218 -1.38 -1.667 -1.865 -1.09 -1.18 -1.312 -1.2 -1.825 -1.765 -1.728 -1.32 -1.055 -1.227 -1.455 -1.255 -1.165 -1.278 -1.147 Log Table I. Continued. Table ID 11759287 1172130009 11756778 11740504 11758615 11738883 11726443 11727112 11717301 11734218 11728339 11764234 11721932 11753763 11721190 11721418 11733069 11730969 11719780 11758125 11733695 11756100 11732295 11724984 11726757 11727604 11716226 EXPERIMENTAL AND THERAPEUTIC MEDICINE 20: 3595-3614, 2020 3605 Gene ontology: Molecular function Transcriptional activator activity, activator activity, Transcriptional polymerase II distal enhancer RNA sequence-specific binding Catalytic activity Patched binding Sh3 Chromatin binding Protein binding Signal transducer activity Protein binding Gtpase activator activity Ubiquitin-protein transferase activity Signal transducer activity Protein binding binding DNA Signal transducer activity Nucleotide binding binding DNA binding RNA Protein binding polymerase II transcription factor RNA binding sequence-specific DNA activity, Signal transducer activity Holocytochrome-c synthase activity Protein binding Signal transducer activity Protein binding Protein binding Nucleic acid binding Glucosamine-6-phosphate deaminase activity Gene ontology: Cellular component

Nucleus Nucleus Spindle pole Cytoplasm Nucleus Cytoplasm Plasma membrane Cytoplasm Nucleus Acrosomal vesicle Nucleus Cytoplasm Nucleus Chromosome, centromeric region Plasma membrane Mitochondrion Chromosome, centromeric region Extracellular region Prp19 complex Intracellular Cytoplasm Mitochondrion Endosome Nucleus Intracellular Nucleus Gene ontology: Biological function polymerase II promoter from RNA Transcription, DNA-templated Transcription, In utero embryonic development G1 Signal transduction repair DNA Kidney development Signal transduction Mitochondrial transport DNA-templated Transcription, Mitotic cytokinesis Ovarian follicle development Blood vessel remodeling Cell cycle Establishment of mitotic spindle orientation Signal transduction long-chain fatty acid metabolic process Very processing RNA Spliceosomal complex assembly Protein homooligomerization Negative regulation of transcription Blood vessel remodeling Metabolic process endocytosis Receptor-mediated Fibroblast growth factor receptor signaling pathway HCG8 /// ZNRD1-AS1 methylation RNA DNA-templated Transcription, Carbohydrate metabolic process

Gene symbol FUBP1 ALKBH1 CCNB1 GRB10 EYA3 CYS1 GPR19 KIF1BP MSL3P1 RACGAP1 FANCF CCR2 SAC3D1 CENPA GPR18 SLC27A2 WDHD1 PLGLB2 CRNKL1 KCTD21 ZNF202 CCR2 HCCS SOWAHD ACKR4 SHCBP1 BCDIN3D ZNF436 GNPDA2

-1.105 -1.087 -1.115 -1.218 -1.133 -1.188 -1.258 -1.82 -1.15 -0.975 -1.44 -1.062 -1.278 -1.192 -1.292 -1.118 -0.968 -1.598 -1.282 -1.198 -1.37 -1.567 -1.388 -1.292 -1.05 -1.113 -0.968 -1.655 -1.005 -1.425 Log Table I. Continued. Table ID 11760155 11725833 11723939 11722160 11759488 11739828 11740637 11717629 11753965 11758149 11732175 11750856 11757036 11758529 11732544 11718213 11725709 11739531 11745077 11730590 11727196 11731676 11721473 11737395 11737108 11728404 11760278 11728360 11743686 11762571 3606 YANG et al: GENOMIC APPROACHES FOR CORONARY HEART DISEASES

MicroRNAs (miRNA) are small noncoding RNAs with a length of 22-25 nucleotide and which play a key role in the regulation of gene expression and have implications in many human disorders (18), including many biological processes such as cell differentiation, proliferation and apoptosis (19-21). To the best of the knowledge of the authors, the association pattern of miRNAs to CHD is lacking, leading to demand for specific CHD patients. Although relevant research has been undertaken to address DEGs associated with CHD, DEGs have only been used to check the expression pattern in case of CHD. In this study, we addressed the possible association of genes with CHD, which may be useful for the diagnosis and treatment of this disease in the near future. Additionally, analysis of gene expression data and network analysis were performed to gain a better understanding of CHD for the identification of differentially expressed genes (DEGs), biomarkers and therapeutic target options.

Materials and methods

Data availability. To identify key genes for the development Figure 1. Designing of CHD data set for DEGs study. of CHD biomarkers, we used gene expression datasets of 4 angiographically proven patients who were being treated for more than 3 months or from group-1 (n=100) compared to healthy control (n=50). This dataset was downloaded Results and Discussion from the GEO module of National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/geo/query/acc. Screening of differentially expressed common genes from cgi?acc=GSE56885). Microarray gene expression profiles microarray data sets. Atherosclerosis is one of the leading were downloaded and further analyzed for the identification causes of cardiovascular diseases such as CHD (26). of DEGs. In this dataset, GSM1370681 and GSM1370682 Understanding of the key players in expression, regulation represent the replicate samples of healthy individuals and and function, of GWAS CHD genes will provide the options GSM1370683 to GSM1370686 of four patients as baseline to treat this disease, leading to further developments of associated with CAD. novel therapeutic interventions (27). In this study, the first compressive investigation was conducted to identify the Differential Expression Analysis (DEG). Using the default expression profile of collected microarray data sets of CHD. parameters, WEGO 2.0, and GEO2R (https://www.ncbi.nlm. The dataset of two controls in replicate and four baseline nih.gov/geo/geo2r/) were used to analyze the GEO series (22). test samples were used. We report an overall expression The Benjamini and Hochberg false discovery rate method and function of genes associated with different biological was utilized to adjust the P-values. NCBI-generated annota- processes, which may lead to CHD during pathological tions were employed to display the DEG list by comparing conditions. The overall study design is shown in Fig. 1, which the overall common gene expression pattern as compared to presents CHD data of Homo sapiens from the GEO database, the control. On the basis of this analysis, possible associa- with four series of test samples and two control study sets. tions related to CHD were reported. Although inappropriate First, we used WEGO to visualize the GO annotations and the to consider the data for analysis on inter-datasets, the average percentage of genetic association of different functions in cells value of LogFC for all four datasets was assessed to represent to address the possible association with CHD. the expression level. A total of 52,998 genes sharing different functions such as Gene ontology (GO) analysis. The major bioinformatics tool cellular (18,476), biological (17,307) and molecular (17,215) GO was used as an initiative to understand the function of functions were identified. Out of those, the highest gene asso- genes and gene products of Homo sapiens. The PANTHER ciation to cell, cell part, organelle, organelle part, membrane, (Protein ANalysis THrough Evolutionary Relationships) clas- binding, cellular process, biological regulation, and metabolic sification database (23) was used to perform the GO analysis, were topmost in the metadata of the CHD-associated data set and the pathway analysis was performed using Reactome (24). (Fig. 2). The different GO representing the 0-90% range of gene expression as compared to control data set is shown in Fig. 3. Protein-Protein Interaction (PPI) network construction The principal findings of this study confirm the association analysis. An online freely available software package, of immune system, inflammation, and apoptosis as media- STRING, was utilized to establish the PPI network (25), and tors in the development of CHD. The impact of the immune all the cut-off points were combined to analyze the topology system plays a key role in the development of heart failure. A property of networks. Gene edges of >15 degrees were defined transcriptomic study reported the sustained activation of the as hub genes. adoptive immune system which may be a contributing factor EXPERIMENTAL AND THERAPEUTIC MEDICINE 20: 3595-3614, 2020 3607

Figure 2. Up- and downregulated gene association comparing all the genetic data set.

in the progression of CHD (28). Another report suggests that family and subfamily, molecular function, biological process, the imbalance in inflammatory and anti-inflammatory cyto- and pathway (31). In the dataset analyzed, the two significant kines may lead to the onset of extensive fibrosis (29). changes in molecular function were protein binding (75%) and catalytic activity (56%), followed by molecular regulator, From the GEO database, accession GSE56885 of CSD molecular transducer activity, structural activity, transcription patients, who were being treated for more than 3 months was regulator activity, and transporter activity (Fig. 4A). In terms selected. From the included patients, two controls in replicate of the biological process, the three most significant classes and four overall test samples were used to consolidate data of CHD were cellular process (83%), biological regulation refining. The GEO2R was used to analyze the control and (57%), and metabolic process (44%) (Fig. 4B). Additionally, test data series by normalizing the microarray data for high in terms of cellular components, another two more significant quality. DEGs with different fold change confirm their crucial components are cell (65%) and organelle (58%), which were role in CHD (Table I). found to be associated with CHD (Fig. 4C). Many other target- associated DEGs were involved in the biological process, In our study, many immune response processes were signifi- molecular function and cellular components. cantly changed and DEGs are associated with the metabolic process, which is associated with CHD. The CHDs of the Analyzed potential DEGs of the CHD data set shows protein innate immune system were largely mediated through neutro- classes distributed among transcription factor (24%), enzyme phils and monocyte, and macrophages (30), to contribute to the modulator (20%), nucleic acid binding (18%), and signaling process of the chronic inflammation process. molecules (18%) (Fig. 5A). The DEGs mainly associated with CHD key pathways showed the significance are inflammation Functional enrichment and unified DEG analysis.To precisely mediated by chemokine and cytokine signaling pathway (11%), understand the gene changes during CHD, the DEGs GO was CCKR signaling map (11%), gonadotropin-releasing hormone performed using the online PANTHER database for high- receptor pathway (8%), apoptosis signaling pathway (6%), and throughput analysis to classify the proteins and their genes into p53 pathway (5%) (Fig. 5B). This result was consistent with 3608 YANG et al: GENOMIC APPROACHES FOR CORONARY HEART DISEASES

Figure 3. GO analysis of the microarray CHD data set. The x-axis shows selected GO terms, and y-axis is the percentage of the gene association from selected data set.

Figure 4. Analyzed Gene ontology (GO) of DEGs in CHD. Enriched GO terms in the (A) molecular function class, (B) biological process class, and (C) cellular component class of common DEGs. EXPERIMENTAL AND THERAPEUTIC MEDICINE 20: 3595-3614, 2020 3609

Figure 5. Analyzed protein class and pathways of DEGs in CHD. (A) The proteins of common DEGs were classified according to function. (B) Significantly enriched pathways of common DEGs.

Figure 6. The CHD-associated data set PPI showing both gene interaction and binding properties. 3610 YANG et al: GENOMIC APPROACHES FOR CORONARY HEART DISEASES

Table II. Pathway enrichment and reactome selected for CHD associated pathways.

Client Client text Client Client Homo-sapiens text box Client text Client text box input text box text box Reactome REFLIST Input box input box input (fold input input pathways (20996) (212) (expected) (over/under) enrichment) (raw P-value) (FDR)

PI3K events in ERBB4 signaling (R-HSA-1250342) 9 4 0.09 + 44.02 6.47E-06 3.54E-03 PI3K events in ERBB2 signaling (R-HSA-1963642) 13 4 0.13 + 30.47 2.09E-05 6.53E-03 ERBB2 activates PTK6 Signaling (R-HSA-8847993) 11 3 0.11 + 27.01 3.30E-04 4.53E-02 Chemokine receptors bind chemokines (R-HSA-380108) 48 6 0.48 + 12.38 1.61E-05 5.88E-03 Interleukin-10 signaling (R-HSA-6783783) 45 5 0.45 + 11 1.40E-04 2.79E-02 Interleukin-4 and Interleukin-13 signaling (R-HSA-6785807) 111 12 1.12 + 10.71 3.96E-09 8.68E-06 Peptide ligand-binding receptors (R-HSA-375276) 186 9 1.88 + 4.79 1.58E-04 2.66E-02 G alpha (i) signalling events (R-HSA-418594) 392 15 3.96 + 3.79 1.59E-05 6.98E-03 Signaling by Interleukins (R-HSA-449147) 449 17 4.53 + 3.75 4.90E-06 3.58E-03 Class A/1 (Rhodopsin- like receptors) (R-HSA-373076) 321 12 3.24 + 3.7 1.40E-04 2.56E-02 Cytokine signaling in Immune system (R-HSA-1280215) 669 23 6.76 + 3.4 4.96E-07 5.43E-04 Generic transcription Pathway (R-HSA-212436) 1,094 26 11.05 + 2.35 6.61E-05 1.45E-02 RNA polymerase II transcription (R-HSA-73857) 1,216 28 12.28 + 2.28 5.19E-05 1.26E-02 Gene expression (transcription) (R-HSA-74160) 1,351 29 13.64 + 2.13 1.95E-04 3.05E-02 Immune system (R-HSA-168256) 2,035 41 20.55 + 2 2.09E-05 5.73E-03 Signal transduction (R-HSA-162582) 2,667 46 26.93 + 1.71 2.66E-04 3.89E-02

GO analysis, confirming the classes of proteins associated likely to serve as the marker selected in the development of with CHD. Many genes associated with inflammatory roles, CHD interventions. The P-value indicates the Fisher's exact and a previous study showed a conserved signature of dilated test (37) or Binomial statistic in which the probability is the cardiomyopathy (DCM) plays an important role in cell survival number of genes observed in this category occurred by chance promotion during end-stage of heart failure (32). In the present (randomly), as determined by the reference list (Table II). study, we also revealed the expression pattern of apoptotic or inflammatory genes (Fig. 4) (33,34). PPI analysis. To address the PPI of the CHD dataset in this study, STRING online suits was used to address the possible Pathway analysis. To address the overview of data insight into interaction of protein of CHD associated DEGs. A total of the pathways, which are associated and connected for CHD 112 nodes, 257 edges, 4.59 average node edge, 0.387 average development (35), we analyzed 164 DEGs involved in different clustering coefficient, 77 expected edge number, and <1.0e-16 functional pathways compared to reference and expected PPI enrichment value were observed, and shown the network genes for those pathways. A total of 13 pathways were found was significantly interacted than expected. Previous studies to be associated with signaling-, immune-, and transcription- investigated the rare variants through targeted expression related pathways (36). Genes were confirmed in the uploaded profiling across CHD relevant tissues from appropriate list over the expected one (number in the list divided by the cases and controls (38,39). The PPI indicates the interaction expected number). If >1, it indicated that the category is over- of genes associated with multiple genes for outcome. In the represented in the experiment. Conversely, the category is present study, we identified 422 GO for biological process, under-represented if <1. In the future, overexpressed genes are 31 GO for molecular function, 12 GO for cellular component, EXPERIMENTAL AND THERAPEUTIC MEDICINE 20: 3595-3614, 2020 3611

Table III. Protein-protein interaction network of CHD associated genes.

A, Biological process (GO).