Gastroenterology Research and Practice

Gastroenterology Research and Practice / 2016 / Article

Research Article | Open Access

Volume 2016 |Article ID 2968106 | https://doi.org/10.1155/2016/2968106

Peng Su, Shiwang Wen, Yuefeng Zhang, Yong Li, Yanzhao Xu, Yonggang Zhu, Huilai Lv, Fan Zhang, Mingbo Wang, Ziqiang Tian, "Identification of the Key Genes and Pathways in Esophageal Carcinoma", Gastroenterology Research and Practice, vol. 2016, Article ID 2968106, 11 pages, 2016. https://doi.org/10.1155/2016/2968106

Identification of the Key Genes and Pathways in Esophageal Carcinoma

Academic Editor: Robert Odze
Received28 Mar 2016
Revised01 Jul 2016
Accepted11 Jul 2016
Published12 Oct 2016

Abstract

Objective. Esophageal carcinoma (EC) is a frequently common malignancy of gastrointestinal cancer in the world. This study aims to screen key genes and pathways in EC and elucidate the mechanism of it. Methods. 5 microarray datasets of EC were downloaded from Gene Expression Omnibus. Differentially expressed genes (DEGs) were screened by bioinformatics analysis. Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and protein-protein interaction (PPI) network construction were performed to obtain the biological roles of DEGs in EC. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify the expression level of DEGs in EC. Results. A total of 1955 genes were filtered as DEGs in EC. The upregulated genes were significantly enriched in cell cycle and the downregulated genes significantly enriched in Endocytosis. PPI network displayed CDK4 and CCT3 were hub proteins in the network. The expression level of 8 dysregulated DEGs including CDK4, CCT3, THSD4, SIM2, MYBL2, CENPF, CDCA3, and CDKN3 was validated in EC compared to adjacent nontumor tissues and the results were matched with the microarray analysis. Conclusion. The significantly DEGs including CDK4, CCT3, THSD4, and SIM2 may play key roles in tumorigenesis and development of EC involved in cell cycle and Endocytosis.

1. Introduction

Esophageal carcinoma (EC) is the sixth leading cause of cancer mortality in males and the ninth leading cause of cancer mortality in females in 2012 worldwide [1]. The highest incident rates of EC are found in Eastern Asia, Southern Africa, and Eastern Africa and the lowest incidence rate of EC is found in Western Africa [1]. Esophageal carcinoma is usually 3 to 4 times more common among men than women. The 5-year overall survival ranges from 15% to 25% [2]. In China, it is predicted that EC is the fourth leading cause of cancer deaths in males and females after lung and bronchus, stomach, and liver in 2015 [3].

EC is classified as esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) according to histological type and ESCC is the predominant histological type of EC in the world [2]. It is reported that tobacco consumption, alcohol consumption, and low intake of fruits and vegetables are major risk factors for ESCC [4]. Overweight, obesity, gastroesophagus reflux disease (GERD), and Barrett’s esophagus increase incidence risk of EAC [1, 5].

In addition to the above-mentioned environmental factors, abnormal expression of miRNA and genes and methylation of genes and SNPs are associated with EC tumorigenesis and development. miR-219-1 rs107822G > A polymorphism might significantly decrease ESCC risk through changing individual susceptibility to Chinese Kazakhs [5]. The cases carrying the GG variant homozygote have a significant 2.81-fold increased risk of EC [6]. miR-330-3p promotes cell growth, cell migration, and invasion and inhibits cisplatin-induced apoptosis in ESCC cells via suppression of PDCD4 expression [7]. miR-199a-5p downregulation contributes to enhancing EC cell proliferation through upregulation of mitogen-activated protein kinase kinase kinase-11 [8]. DACT2 is frequently methylated in human esophageal cancer; methylated DATC2 accelerates esophageal cancer development by activating Wnt signaling [9]. RUNX3 methylation is associated with an increased risk, progression, and poor survival in EC [10].

Currently, the molecular mechanism of EC was unclear. In this study, we used bioinformatics methods to analyze the mRNA expression data of EC, which were available on the GEO database, to identify key genes and pathways in EC, aiming to provide valuable information for further pathogenesis mechanism elucidation and provide ground work for therapeutic targets identification for EC.

2. Materials and Methods

2.1. Expression Profile Microarray

Gene expression profiles data were downloaded from the Gene Expression Omnibus (GEO) data repository (http://www.ncbi.nlm.nih.gov/geo/). The datasets of patients receiving preoperative treatment before oesophagectomy and cell lines receiving drug stimulus were excluded. Total of 5 mRNA expression datasets of EC tissues/cell lines comprising GSE53625, GSE33810, GSE17351, GSE9982, and GSE12737 were included in our study.

2.2. Identification of DEGs

The raw data of the mRNA expression profiles were downloaded and analyzed by R language software [11]. Background correction, quartile data normalization, and probe summarization were applied for the original data. The limma [12] method in Bioconductor (http://www.bioconductor.org/) was used to identify genes which were differentially expressed between EC and normal controls; the significance of DEGs was calculated by t-test and was represented by value. To reduce the risk of false positives, values were adjusted for multiple testing using the Benjamini-Hochberg False Discovery Rate (FDR) method. The corrected value was represented by FDR [13]. FDR < 0.05 were considered as the cutoff values for DEG screening.

2.3. Gene Ontology Analysis

GO is a useful tool for collecting a large number of gene annotation terms [14]. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) [15], is bioinformatics resources consisting of an integrated biological knowledgebase and analytic tools aimed at systematically extracting biological functional annotation from large gene/protein lists, such as being derived from high-throughput genomic experiments. To gain the in-depth understanding of the biological functions of DEGs, DAVID tool was used to obtain the enriched GO terms of DEGs based on the hypergeometric distribution to compute values, which were corrected by the Benjamini and Hochberg FDR method for multiple hypothesis testing. FDR < 0.05 was set as the threshold value.

2.4. KEGG Enrichment Pathways

KEGG is a database resource for understanding functions of genes list from molecular level [16]. GeneCoDis3 is a valuable tool to functionally interpret results from experimental techniques in genomics [17]. This web-based application integrates different sources of information for finding groups of genes with similar biological meaning. The enrichment analysis of GeneCoDis3 is essential in the interpretation of high-throughput experiments. In the study, GeneCoDis3 software was used to test the statistical enrichment of DEGs in KEGG pathways. was set as the threshold value.

2.5. PPI Interaction Network

The Biological General Repository for Interaction Datasets (BioGRID: http://thebiogrid.org/) is an open access archive of genetic and protein interactions that are curated from the primary biomedical literature for all major model organism species including budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe, and the model plant Arabidopsis thaliana. In a word, BioGRID is a depository for genetic and protein interactions based on experimental verification [18]. The top 10 upregulated genes and top 10 downregulated genes between EC and normal controls were subjected to BioGRID database to get the predicted PPIs of these DEGs. The PPIs were visualized in Cytoscape [17].

2.6. qRT-PCR Validation

Total RNA of fresh paired EC tumor and adjacent nontumor specimens were extracted using TRIzol reagent (Invitrogen, CA, USA). The SuperScript III Reverse Transcription Kit (Invitrogen, CA, USA) was used to synthesize the cDNA. qRT-PCR reactions were performed using Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) on the Applied Biosystems 7500 (Foster City, CA, USA). β-actin was used as internal control for mRNA detected. The relative expression of genes was calculated using the comparative Ct methods [19]. The PCR primers were used as shown in supplementary Table S3 in Supplementary Material available online at http://dx.doi.org/10.1155/2016/2968106.

3. Results

3.1. Identification of DEGs

Five mRNA expression profiles including 208 EC samples and 195 normal controls were downloaded and analyzed, as shown in Table 1. 208 EC samples comprised 207 squamous cell carcinoma samples and 1 adenocarcinoma sample. 1955 DEGs were identified in EC compared to normal control, including 919 upregulated and 1036 downregulated genes. The top 10 significantly upregulated and downregulated genes were listed in Table 2. The most significantly up- and downregulated genes were CDK4 and THSD4, respectively. The full list of DEGs in EC was shown in supplementary Table S1.


GEO IDPlatformCase : controlSample typeCountryTimeAuthor

GSE53625GPL18109 CBC Homo sapiens lncRNA + mRNA microarray V2.0179 : 179Esophageal squamous cell carcinomaChina2014Li et al. [42]
GSE33810GPL570 [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array2 : 1Esophageal squamous cell carcinomaHK2013Chen et al. [43]
GSE17351GPL570 [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array5 : 5Esophageal squamous cell carcinomaUSA2009Long et al. [44]
GSE9982GPL1928 CodeLink Human 20K ver4.120 : 2Esophageal squamous cancerJapan2006Shimokuni et al. [45]
GSE12737GPL7262 Human ORESTES NoMatch 4.8k v1.02 : 8Squamous cell & adenocarcinomaBrazil2009Mello et al. [46]

EC: esophageal carcinoma.

Gene IDGene symbolOfficial full nameFDR

Upregulated (top 10)
1019CDK4Cyclin-dependent kinase 40.0002252
4605MYBL2MYB protooncogene like 20.0002252
7203CCT3Chaperonin containing TCP1 subunit 30.0003378
83461CDCA3Cell division cycle associated 30.0004504
1033CDKN3Cyclin-dependent kinase inhibitor 30.0004504
1063CENPFCentromere protein F0.0004729
9156EXO1Exonuclease 10.0004729
79075DSCC1DNA replication and sister chromatid cohesion 10.0005405
4751NEK2NIMA related kinase 20.0005405
Downregulated (top 10)
79875THSD4Thrombospondin type 1 domain containing 40.0002252
79026AHNAKAHNAK nucleoprotein0.0004729
6493SIM2Single-minded family bHLH transcription factor 20.0004729
7881KCNAB1Potassium voltage-gated channel subfamily A member regulatory beta subunit 10.0005405
90865IL33Interleukin 330.0008812
55287TMEM40Transmembrane protein 400.0008812
966CD59CD59 molecule0.0015608
5121PCP4Purkinje cell protein 40.0015608
22885ABLIM3Actin binding LIM protein family member 30.0016629
3590IL11RAInterleukin 11 receptor subunit alpha0.0016629

EC: esophageal carcinoma; FDR: false discovery rate.
3.2. GO Analysis of DEGs

Following GO analyses for up- and downregulated DEGs, significant GO terms including biological process, cellular component, and molecular function were collected. For upregulated DEGs, cell cycle was the most significant enrichment of biological process; membrane-enclosed lumen was the highest enrichment of cellular component; nucleotide binding was the highest enrichment of molecular function, as shown in Table 3. For downregulated DEGs, response to wounding was the most significant enrichment of biological process; actin cytoskeleton was the highest enrichment of cellular component and cytoskeletal protein binding was the highest enrichment of molecular function, as shown in Table 4.


GO IDGO termCount-valueFDR

Biological process
GO:0007049Cell cycle1524.10E − 137.59E − 10
GO:0022402Cell cycle process1183.44E − 126.36E − 09
GO:0022403Cell cycle phase902.00E − 103.71E − 07
GO:0000278Mitotic cell cycle825.34E − 109.87E − 07
GO:0051301Cell division678.80E − 091.63E − 05
GO:0000279M phase706.27E − 081.16E − 04
GO:0000087M phase of mitotic cell cycle493.38E − 060.0062547
GO:0000280Nuclear division484.65E − 060.0086014
GO:0007067Mitosis484.65E − 060.0086014
GO:0048285Organelle fission496.41E − 060.011854
GO:0033554Cellular response to stress952.02E − 050.0373322
Cellular component
GO:0031974Membrane-enclosed lumen2761.12E − 101.65E − 07
GO:0043233Organelle lumen2702.41E − 103.56E − 07
GO:0043232Intracellular non-membrane-bounded organelle3598.74E − 101.29E − 06
GO:0043228Non-membrane-bounded organelle3598.74E − 101.29E − 06
GO:0070013Intracellular organelle lumen2594.54E − 096.71E − 06
GO:0031981Nuclear lumen2161.90E − 082.80E − 05
GO:0000775Chromosome, centromeric region362.52E − 083.72E − 05
GO:0005829Cytosol1921.36E − 060.0020016
GO:0015630Microtubule cytoskeleton924.62E − 060.0068255
GO:0000793Condensed chromosome326.75E − 060.009972
GO:0000779Condensed chromosome, centromeric region217.55E − 060.011151
GO:0044427Chromosomal part699.92E − 060.0146408
GO:0005635Nuclear envelope431.37E − 050.0202598
GO:0000777Condensed chromosome kinetochore191.48E − 050.0219025
GO:0005694Chromosome781.75E − 050.02589
GO:0000776Kinetochore222.72E − 050.0401619
Molecular function
GO:0000166Nucleotide binding3055.53E − 060.0090275
GO:0017076Purine nucleotide binding2665.55E − 060.0090714
GO:0030554Adenyl nucleotide binding2231.07E − 050.0175078
GO:0001883Purine nucleoside binding2251.49E − 050.0242774
GO:0032555Purine ribonucleotide binding2522.35E − 050.0383944
GO:0032553Ribonucleotide binding2522.35E − 050.0383944
GO:0001882Nucleoside binding2252.44E − 050.0398342

EC: esophageal carcinoma; FDR: false discovery rate.

GO IDGO termCount valueFDR

Biological process
GO:0009611Response to wounding651.98E − 083.57E − 05
GO:0042060Wound healing335.75E − 081.04E − 04
GO:0030097Hemopoiesis321.85E − 050.0334238
GO:0007167Enzyme linked receptor protein signaling pathway412.03E − 050.0365533
GO:0030036Actin cytoskeleton organization312.05E − 050.0370181
GO:0048534Hemopoietic or lymphoid organ development342.06E − 050.0372021
GO:0007155Cell adhesion692.10E − 050.0378896
GO:0042692Muscle cell differentiation212.14E − 050.0386651
GO:0022610Biological adhesion692.19E − 050.0394751
GO:0007178Transmembrane receptor protein serine/threonine kinase signaling pathway192.53E − 050.0456886
Cellular component
GO:0015629Actin cytoskeleton365.84E − 060.008305
GO:0005794Golgi apparatus837.36E − 060.0104637
GO:0005856Cytoskeleton1181.23E − 050.0175254
Molecular function
GO:0008092Cytoskeletal protein binding598.55E − 070.0013403

EC: esophageal carcinoma; FDR: false discovery rate.
3.3. KEGG Enrichment Pathways of DEGs

Following KEGG enrichment analysis for DEGs, significant KEGG terms were collected. The pathways enriched by 919 upregulated DEGs were mainly related to cell cycle, RNA transport, and p53 signaling pathway (Table 5). 1036 downregulated DEGs were significantly enriched in Endocytosis, focal adhesion, and vascular smooth muscle contraction, as shown in Table 6.


KEGG IDKEGG termsCountFDRGenes

hsa04110Cell cycle197.86E − 08CDK6, CCNE2, CCNB2, FZR1, CCNA2, CDC7, YWHAQ, MCM7, CCNE1, CDK4, E2F5, CCNB1, MAD2L1, CDC25B, MCM6, BUB1, RBL1, MCM2, CDK1
hsa03013RNA transport201.09E − 07RAN, EIF3H, NUP43, UBE2I, NUP133, MAGOHB, POP5, THOC5, CLNS1A, NUP205, GEMIN6, NUP93, NUP62, SUMO1, EIF2S2, NUP153, RANGAP1, NUP160, RPP25, DDX20
hsa04115p53 signaling pathway52.90E − 06CCNE2, CCNB2, CCNE1, CCNB1, CDK1
hsa04914Progesterone-mediated oocyte maturation81.42E − 05CCNB2, FZR1, CCNA2, CCNB1, MAD2L1, CDC25B, BUB1, CDK1
hsa03050Proteasome91.56E − 05PSMD7, SHFM1, PSMD3, PSMA5, PSMB1, PSMB3, PSMA3, PSMD4, PSMA7
hsa03040Spliceosome151.66E − 05SNRPC, SRSF9, XAB2, MAGOHB, NAA38, BUD31, SNRPF, NHP2L1, SRSF3, PQBP1, USP39, SNRNP40, SNRPD1, SNRPD2, SF3B2
hsa03030DNA replication84.24E − 05RNASEH2A, RNASEH1, MCM7, POLE2, MCM6, RNASEH2C, MCM2, RFC4
hsa03008Ribosome biogenesis in eukaryotes114.37E − 05UTP18, RAN, UTP15, NOP56, DKC1, POP5, FBL, NHP2L1, TCOF1, GNL3L, RPP25
hsa03440Homologous recombination74.37E − 05SHFM1, MRE11A, RAD54B, XRCC2, RAD54L, BLM, TOP3A
hsa04114Oocyte meiosis88.96E − 05CCNE2, CCNB2, YWHAQ, CCNE1, CCNB1, MAD2L1, BUB1, CDK1
hsa05162Measles40.0001531CDK6, CCNE2, CCNE1, CDK4
hsa05222Small cell lung cancer40.0001531CDK6, CCNE2, CCNE1, CDK4
hsa05200Pathways in cancer230.0001815VEGFB, CDK6, MTOR, FH, CCNE2, LEF1, BIRC5, CCNE1, CDK4, TCEB1, MSH6, EGF, FZD2, TFG, CKS1B, TRAF4, HSP90AA1, TRAF3, PPARG, HSP90AB1, FGF12, PIAS4, STK4
hsa00510N-Glycan biosynthesis80.000312RFT1, ALG10, RPN2, ALG10B, ALG1, MOGS, ALG5, B4GALT2

EC: esophageal carcinoma; FDR: false discovery rate.

KEGG IDKEGG termsCountFDRGenes

hsa04144Endocytosis235.22E − 06STAMBP, RAB11FIP5, SH3KBP1, KIT, FOLR2, F2R, TGFBR2, VPS4B, SH3GLB1, CHMP5, CXCR2, PDGFRA, CLTB, FOLR1, STAM2, ARAP2, DAB2, EEA1, PDCD6IP, RAB11FIP2, CBL, EPN3, VPS37B
hsa04510Focal adhesion190.000354ITGA1, ZYX, LAMB2, MYLK, IGF1, CCND2, ITGA2, RAP1A, PDGFRA, ITGA5, TNXB, VWF, PIK3R1, JUN, COL6A2, BCL2, ROCK1, MYL12A, THBS3
hsa04270Vascular smooth muscle contraction140.000383JMJD7-PLA2G4B, MYLK, ADCY9, GNA13, PRKG1, ITPR2, PPP1R12B, GNAQ, MYH11, ACTG2, ROCK1, PLA2G2A, MRVI1, ITPR1
hsa00330Arginine and proline metabolism40.000425ALDH7A1, MAOB, GATM, MAOA
hsa04360Axon guidance150.000456EPHA1, ROBO1, SEMA4B, DPYSL2, ABLIM3, PPP3CC, NCK2, GNAI2, SEMA3F, PPP3CA, RGS3, NTN1, ROCK1, PPP3CB, EFNB2
hsa04020Calcium signaling pathway50.00046PPP3CC, ITPR2, PPP3CA, PPP3CB, ITPR1
hsa04662B cell receptor signaling pathway40.000508PPP3CC, JUN, PPP3CA, PPP3CB
hsa05014Amyotrophic lateral sclerosis 30.000583PPP3CC, PPP3CA, PPP3CB
hsa00340Histidine metabolism30.000583ALDH7A1, MAOB, MAOA
hsa04720Long-term potentiation60.000623PPP3CC, ITPR2, GNAQ, PPP3CA, PPP3CB, ITPR1
hsa04114Oocyte meiosis60.00068ADCY9, PPP3CC, ITPR2, PPP3CA, PPP3CB, ITPR1
hsa04730Long-term depression100.000701JMJD7-PLA2G4B, IGF1, GNA13, PRKG1, ITPR2, PPP2CB, GNAQ, GNAI2, PLA2G2A, ITPR1
hsa04141Protein processing in endoplasmic reticulum160.000709SEC63, UBE2J1, EIF2AK3, ATF6, CRYAB, UBE2D3, DNAJB2, SEC31B, MAN1A1, ERO1L, BCL2, HERPUD1, DNAJC3, UBQLN2, RAD23B, LMAN1
hsa04912GnRH signaling pathway120.000736JMJD7-PLA2G4B, MMP2, ADCY9, MAP3K3, HBEGF, ITPR2, MAPK7, GNAQ, MAP3K4, JUN, PLA2G2A, ITPR1
hsa00280Valine, leucine, and isoleucine degradation80.000738ALDH7A1, ACADM, HMGCS1, MUT, ABAT, ACADSB, ACAD8, AUH

EC: esophageal cancer; FDR: false discovery rate.
3.4. PPI Network Construction

Based on data from the BioGRID database, the PPI network was the top 10 upregulated and downregulated DEGs which were constructed by Cytoscape software (Figure 1). The network consisted of 451 nodes and 499 edges. In the PPI networks the nodes with high degree are defined as hub proteins. The most significant hub proteins in the PPI network were CDK4 (degree = 132) and CCT3 (degree = 127); as shown in Figure 1, the red circular nodes represent upregulated DEGs and green circular nodes represent downregulated DEGs, respectively.

3.5. qRT-PCR Validation of DEGs in EC Tissues

To validate the microarray analysis data, the expression of DEGs including CCT3, CDK4, MYBL2, CENPF, CDKN3, CDCA3, THSD4, and SIM2 was detected by qRT-PCR in 5 paired EC tumor and adjacent nontumor tissues. The 5 patients received surgery treatment in Fourth Hospital of Hebei Medical University. The histological type of 5 subjects was ESCC and the detailed information of subjects was shown in supplementary Table S2. As shown in Figures 2(a) and 2(b) the expression level of CCT3 and MYBL2 was significantly upregulated in ESCC. CDK4, CENPF, CDKN3, and CDCA3 had the upregulation tendency in ESCC (Figures 2(c)2(f)), respectively. SIM2 was significantly downregulated in ESCC (Figure 2(g)). THSD4 had the downregulation tendency in ESCC (Figure 2(h)). The qRT-PCR results were matched with the microarray analysis.

4. Discussion

CDK4 was identified as the most significantly upregulated gene in our microarray analysis and it had an upregulated tendency in EC tissues through the qRT-PCR validation. CDK4 was the hub protein and interacted with 132 genes in the regulatory network. CDK4 was significantly enriched in cell cycle, measles, small cell lung cancer, and pathways in cancer. CDK4 encodes cyclin-dependent kinase 4, a member of the Ser/Thr protein kinase family, which plays an important role in cell cycle G1 phase progression and G1/S transition. In our study, CDK1, CDK6, and CDK10 showed upregulation in EC. CDK1, CDK6, and CDK4 were significantly enriched in cell cycle pathway. CDK4 is overexpression in several cancer comprising of breast cancer, pancreas cancer, clear cell renal cell carcinoma, and colorectal cancer [2023]. Downregulation of MALAT1 (long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1) inhibits breast cancer cell proliferation and cell cycle progression in vitro and in vivo through miR-124 downregulation and CDK4 upregulation [20, 24]. Overexpression of cyclin D1/CDK4 is regulated by CEACAM6 and promotes cell proliferation in human pancreatic carcinoma [21]. CDK4 and CDK6 expression are decreased by miR-1 and contribute to inhibition of cell cycle progression and metastasis in clear cell renal cell carcinoma [22].

CCT3 was the top 3 upregulation DEGs in EC (Table 2). The qRT-PCR displayed that CCT3 was significantly upregulated in EC, which was in accordance with our microarray analysis (Figure 2). CCT3 interacted with 127 genes in the PPI network (Figure 1). CCT3 encodes chaperonin containing TCP1 subunit 3, a molecular chaperone, which is a member of the chaperonin containing TCP1 complex (CCT). In our study, CCT2, CCT4, CCT5, and CCT7 were upregulated in EC compared to normal controls, respectively. CCT3 depletion suppresses cell proliferation by inducing mitotic arrest at prometaphase and apoptosis eventually in HCC in vitro. Clinically, overexpression of CCT3 predicts poor prognosis in hepatocellular carcinoma patients after hepatectomy [25, 26]. CCT3 is significantly associated with carboplatin resistance in ovarian cancer patients after surgery treatment [27]. The proteomic-based study shows that patients with cholangiocarcinoma (CCA) which are positive for CCT3 and CCT3 might be potential biomarker for the diagnosis of CCA [28]. To our knowledge, this is the first report about CCT3 expressed status in EC and the biological function of upregulated CCT3 in EC needs further exploration.

THSD4 was the most downregulated DGE in EC through microarray analysis. The expression level of THSD4 had no significance in EC compared to normal controls but had the downregulated tendency in EC. THSD4 encodes thrombospondin type 1 domain containing 4. The methylated status of THSD4 shows positive correlation with short survival in glioblastoma patients and hypermethylation of THSD4 indicates poor survival [29]. The expression of THSD4 is regulated by GATA3 and mediates transformation of normal cells into breast cancer through deregulation of THSD4 [30]. The role of downregulated THSD4 in EC is unclear, and the investigation needs to be carried out in the future.

SIM2 was significantly downregulated in EC (Figure 2). SIM2 encodes single-minded family bHLH transcription factor 2. SIM2-s was dysregulated in glioma, prostate cancer, breast cancer, colorectal cancer, and ESCC [3135]. SIM2s is downregulated in human breast cancer samples and it suppresses tumor activity through decreased expression of matrix metalloprotease-3. In breast cancer, SIM2s is downregulated. It is a key regulator of mammary-ductal development. SIM2s inhibition is associated with cell invasive and EMT-like phenotype through regulating matrix metalloprotease-3 expression [34, 36] It is reported that SIM2s is downregulated in 70% ESCC tissues, which is consistent with our qRT-PCR verification [35]. SIM2 overexpression results in increase of drug- and radio-sensitivities in ESCC in vivo and in vitro and patients with high expression level of SIM2 are associated with favorable prognosis before chemotherapy [35]. It is suggested that SIM2 plays vital roles in EC onset and progression.

MYBL2, CENPF, CDKN3, and CDCA3 were upregulated in EC tissues (Figure 2). MYBL2 is frequently amplified in gastroesophageal cancer cell lines and Barrett’s adenocarcinoma [37, 38]. CENPF is frequently amplified in region around 1q32-q41 and is overexpressed in ESCC cell line [39]. CDKN3 is upregulated in 68.0% of the epithelial ovarian cancer samples and lung adenocarcinoma patients and is correlated with poor patient survival [40, 41]. CDCA3 expression status in EC was firstly reported in our study. The molecular mechanism of MYBL2, CENPF, CDKN3, and CDCA3 in EC is needed to be explored.

5. Conclusions

We identified 1955 DEGs comprising 919 upregulated genes and 1036 downregulated genes in EC. DEGs including CDK4, CCT3, THSD4, and SIM2 were verified in EC tissues through qRT-PCR. CDK4 and CCT3 were hub proteins in the PPI interaction network. We found that some genes including CDK4, CCT3, THSD4, and SIM2 may play essential roles in EC through cell cycle, RNA transport, Endocytosis, and focal adhesion signaling pathways. The genes could also be considered as potential candidate biomarkers for therapeutic targets for this malignancy. Furthermore, our study would shed light on the molecular mechanism underlying tumorigenesis of EC.

Competing Interests

All of the authors declare that they have no conflict of interests.

Acknowledgments

The work was supported by Major Medical Scientific Research Subject of Hebei Province (zd2013044).

Supplementary Materials

The expression level of 8 candidate genes with dysregulation in esophageal carcinoma were validated through qRT-PCR. The primers for amplification of 8 genes in qRT-PCR was shown in supplementary S3.

  1. Supplementary Material

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Copyright © 2016 Peng Su et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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