ARMCX Family Gene Expression Analysis and Potential...

Research ArticleARMCX Family Gene Expression Analysis and PotentialPrognostic Biomarkers for Prediction of Clinical Outcome inPatients with Gastric Carcinoma

TingAnWang,1HuaGe Zhong,1 YuZhou Qin,1WeiYuanWei,1 Zhao Li,1MingWei Huang ,1

and XiaoLing Luo 2

1Department of Gastrointestinal Surgery, Guangxi Medical University Cancer Hospital, Guangxi Clinical Research Center forColorectal Cancer, Nanning 530021, Guangxi Zhuang Autonomous Region, China2Guangxi Medical University Cancer Hospital, Nanning 530021, Guangxi Zhuang Autonomous Region, China

Correspondence should be addressed to MingWei Huang; [email protected] and XiaoLing Luo; [email protected]

Received 12 February 2020; Accepted 20 May 2020; Published 30 June 2020

Academic Editor: Ji-Fu Wei

Copyright © 2020 TingAn Wang 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.

Armadillo gene subfamily members (ARMCX1-6) are well-known to regulate protein-protein interaction involved in nucleartransport, cellular connection, and transcription activation. Moreover, ARMCX signals on cell pathways also implicated incarcinogenesis and tumor progression. However, little is known about the associations of the ARMCX subfamily members withgastric carcinoma. This study investigated the prognostic value of ARMCX subfamily mRNA expression levels with theprognosis of gastric carcinoma (GC). We retrieved the data of a total of 351 GC patients from TCGA database. Survival andgene set enrichment analyses were employed to explore the predictive value and underlying mechanism of ARMCX genes inGC. The multivariate survival analysis revealed that individually low expressions of ARMCX1 (adjusted P = 0:006, HR = 0:620,CI = 0:440 − 0:874) and ARMCX2 (adjusted P = 0:005, HR = 0:610, 95%CI = 0:432 – 0:861) were related to preferable overallsurvival (OS). The joint-effects analysis shown that combinations of low level expression of ARMCX1 and ARMCX2 werecorrelated with favorable OS (adjusted P = 0:003, HR = 0:563, 95%CI = 0:384 – 0:825). ARMCX1 and ARMCX2 were implicatedin WNT and NF-kappaB pathways, and biological processes including cell cycle, apoptosis, RNA modification, DNA replication,and damage response. Our results suggest that mRNA expression levels of ARMCX subfamily are potential prognostic markersof GC.

1. Introduction

Gastric carcinoma, one common type of malignant tumors, isthe fifth highest incidence and the second highest mortalityafter lung cancer worldwide [1]. Each year, more than300,000 newly diagnosed cases and about 260,000 people diein China. The poor prognosis is due to a high incidence ofadvanced disease, high recurrence rate, high metastasis, andabnormal gene expression. In addition, despite great advancesin the surgery and chemotherapy technology, the death rateremains high [2]. Therefore, new strategies to improve diag-nosis and prognosis of gastric cancer are shortly needed.

The armadillo genes are clustered on the X chromosome,also known as X-linked (ARMCX or ALEX). In 1989, it was

first discovered in the segment polarity gene armadillo inDrosophila [3, 4]. Since then, more andmore related proteinshave been identified and classified as armadillo repeat family.The common feature of these proteins is an amino acidsequence (arm repeats) approximately 42 residues, identifiedas 6-13 repeat units in all members of the family [5, 6], andeach repeat domain consists of three helices, designated asH1, H2, and H3 [7–9].

The armadillo domain protein has the functions of cellcontact and cytoskeletal-related protein and signal transmis-sion by producing and transmitting signals that affect geneexpression [5, 9]. Studies have revealed that armadillo repeatproteins regulate protein interactions through multiple bind-ing domains such as nuclear transport, transcriptional

HindawiBioMed Research InternationalVolume 2020, Article ID 3575038, 16 pageshttps://doi.org/10.1155/2020/3575038

https://orcid.org/0000-0002-8899-4366

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https://doi.org/10.1155/2020/3575038

activation, and cell connectivity [10]. For example, bioinfor-maticsanalysisshowsthatARMCX1,ARMCX2,andARMCX3are encoded by an single exon, containing some ARM repeatdomains, a DUF634 (domain 634 function unknown) and anN-terminal transmembrane domain [11–13].

Recent studies have shown a strong implication of differ-ent members of the Armcx1-6/Armc10 family in humantumorigenesis [14–16]. For instance, some members of theArmcx cluster can be regulated through the WNT signalingpathway by interacting with transcription factors of the

E-cadherin and T cytokine/lymphoid enhancement factor(TCF/LEF) families [17, 18], which is also implicated incarcinogenesis and tumor progression [19–21].

Although the ARMCX family plays an important rolein many biological processes including cell adhesion,tumorigenesis, and embryogenesis [22]. However, the rela-tionship between ARMCX genes and gastric cancer ispoorly understood. Therefore, in this study, we determinedthe associations between expression levels of ARMCXgenes and clinical outcomes of GC prognosis, with the

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Figure 1: Matrix graphs of the relative expression levels of ARMCX genes in multiple normal tissues were determined with the GTEx Portal.ARMCX5 was expressed at a medium level (e), whereas the other ARMCX genes (ARMCX1, ARMCX2, ARMCX3, ARMCX4, and ARMCX6)were expressed at low levels (a-d, f).

2 BioMed Research International

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3BioMed Research International

aim of providing insightful information regarding ARMCXgenes as a novel prognostic biomarker for GC patients.

2. Material and Methods

2.1. Data Source and Patient Information. First, we identifiedthe genes differentially expressed between normal gastrictissue and primary tumors of the ARMCX family using anonline database (http://merav.wi.mit.edu/; accessed Sept 25,2019). Then, we obtained mRNA expression levels ofARMCX1, ARMCX2, ARMCX3, ARMCX4, ARMCX5, andARMCX6 by using The Cancer Genome Atlas (TCGA,http://tcga-data.nci.nih.gov/tcga) and OncoLnc website(http://www.oncolnc.org/; accessed Sept 25, 2019) [23].

We downloaded the clinical information of 415 gastriccancer patients from UCSC Xena (http://xena.ucsc.edu/,accessed Sept 25, 2019), including age, gender, tumor stage,survival time, and survival status. Next, a total of 351 caseswere included for follow-up analysis after excluding the caseswith missing medical data and 0-day survival time.

2.2. Characteristics of Gene Expression Levels. The high-expression and low-expression groups of ARMCX geneswere distinguished according to the median of each gene.The relative expression levels of ARMCX genes in multiplenormal tissues were determined with the Genotype-Tissue Expression Portal (http://www.gtexportal.org/home/,accessed Sept 25, 2019) [24].The analysis of ARMCX mRNAexpression between primary gastric cancer tissue and adja-cent normal tissue was done by Gene Expression ProfilingInteractive Analysis (GEPIA, http://gepia.cancer-pku.cn/,accessed Sept 25, 2019) [25].

2.3. Bioinformatics Characteristic of ARMCX Genes. Genefunction enrichment analysis of ARMCX genes wasperformed to disclose the biological processes and signalpathways using the Database for Annotation and Enrich-ment KOBAS 3.0 (http://kobas.cbi.pku.edu.cn/index.php).The analysis included biological processes and molecularfunction, but no results for the ARMCX family wereobtained. GeneMANIA was employed to reveal the gene-gene and protein-protein interactions of ARMCX family(http://www.genemania.org/, accessed Sept 26, 2019) [26,27]. Additionally, the relationship among ARMCX1,ARMCX2, ARMCX3, ARMCX4, ARMCX5, and ARMCX6was evaluated using Pearson’s correlation coefficient. Resultswith a P value < 0.001 were considered to be statisticallysignificant.

2.4. Survival Analysis. According to the database, the 351 GCpatients were, respectively, divided into low- and high-expression groups for survival analysis. Overall survival(OS) and median survival time (MST) were used to assessthe prognosis of patients with gastric cancer, to evaluate thecorrelation of ARMCX member mRNAs with patient sur-vival by Kaplan-Meier estimator with a log-rank test. Therelative risk of survival in gastric cancer patients was assessedby calculating the hazard ratio (HR) and 95% confidenceinterval (CI).

2.5. Joint-Effects Analysis. By analyzing the TCGA data, theresults have shown that only ARMCX1 and ARMCX2 hadstatistical significance. The combination of ARMCX1 andARMCX2 was investigated by joint-effects analysis. Thecombination included group 1 (low ARMCX1 and lowARMCX2 expression), group 2 (low ARMCX1 and high

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Figure 2: Boxplots of ARMCX family gene levels in primary gastric tumors and adjacent tissues from databases. ARMCX1, ARMCX2, andARMCX4 were lowly expressed in primary gastric tumors (a, b, d). ARMCX5 was less expressed in normal gastric tissues (e). The expressionlevels of ARMCX3 and ARMCX6 have no significant difference between gastric tumors and normal gastric tissues (c, f). ∗P < 0:05.


http://merav.wi.mit.edu/;

http://tcga-data.nci.nih.gov/tcga

http://www.oncolnc.org/;

http://xena.ucsc.edu/

http://www.gtexportal.org/home/

http://gepia.cancer-pku.cn/

http://kobas.cbi.pku.edu.cn/index.php

http://www.genemania.org/

ARMCX2 expression, High ARMCX1 and low ARMCX2expression), and group 3 (high ARMCX1 and high ARMCX2expression). In addition, according to the results of TCGAdatabase, age and tumor stage were adjusted in the Coxproportional hazards regression model.

2.6. Nomogram Model. Due to the clinical characteristics andrisk score, a nomogram prediction model was constructed toevaluate the individual prognosis. Furthermore, the probableutility of the ARMCX family in predicting clinical grade wasevaluated. In terms of clinical data and survival analysis, age,tumor stage, and ARMCX expression level were includedin the risk model after Cox proportional risk regressionmodel adjustment. Scores for each factor could be

counted, and 1-year, 5-year, and 10-year survival rates alsocan be calculated [28].

2.7. Gene Set Enrichment Analysis. In order to explore thedifference in biological functions and pathways in the sur-vival of GC between low- and high-ARMCX gene expressiongroups, the potential mechanism in the molecular signaturedatabase (MSigDB) of c2 (c2.all.v6.1. Symbols) and c5(c5.all.v6.1. Symbols) was studied by GSEA (http://software.broadinstitute.org/gsea/index.jsp, accessed Sept 27, 2019)[29–31].The nominal P value < 0.05 and the false discoveryrate (FDR) <0.25 for the enriched gene sets in GSEA werestatistically significant.

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http://software.broadinstitute.org/gsea/index.jsp

http://software.broadinstitute.org/gsea/index.jsp

2.8. Statistical Analysis. Survival analysis was carried out byKaplan-Meier and the log-rank test to calculate MSTs andP values. The crude or adjusted HR and 95% CI were calcu-lated using the Cox proportional risk regression model forunivariate and multivariate survival analyses. The BenjaminiHochberg procedure was employed for multiple tests of FDRin GSEA to control [31–33], and P < 0:05 was consideredstatistically significant. GraphPad Prism v.6.0 (La Jolla, CA)was used to draw vertical scatter plots and survival curves.SPSS software v.22.0 (IBM, Chicago, IL, USA) was employedfor statistical analysis.

3. Results

3.1. ARMCX mRNA Expression Analysis. In human normalstomach tissue, ARMCX5 was expressed at a medium level(Figure 1(e), whereas the other ARMCX genes (ARMCX1,ARMCX2, ARMCX3, ARMCX4, and ARMCX6) wereexpressed at low levels (Figures 1(a)-1(d) and 1(f)), com-pared with other normal tissues. Box plots of ARMCX1-6genes were downloaded from GEPIA as shown in Figure 2.ARMCX1, ARMCX2, and ARMCX4 were lowly expressedin primary gastric tumors and has a high expression in

Table 1: Clinical data characteristic of 351 GC patients.

Items Cases (total n = 351) No. of events (%) MST (days) Crude P Crude HR (95% CI)

Age

≥60 239 108 (45.2) 7660.017

Ref.

<60 106 35 (33.0%) 1811 0.629 (0.429-0.923)

Missing 6

Gender

Male 236 100 (42.4%) 869

0.184

Ref.

Female 125 44 (35.2%) 1043 0.787 (0.552-1.122)

Missing 0

Tumor stage

i 47 11 (23.4%) 2197

<0.001

0.260 (0.126-0.537)

ii 109 34 (31.2%) 1686 0.424 (0.247-0.728)

iii 147 69 (46.9%) 779 0.643 (0.397-1.042)

iv 35

Missing 13 22 (62.9%) 476 Ref.

Abbreviations: MST: median survival time; Ref.: reference; HR: hazard ratio; 95% CI: 95% confidence interval.

Table 2: Univariate and multivariate survival analyses of the ARMCX family.

Items Cases (total n = 351) No. of events (%) MST (days) Crude P Crude HR (95% CI) Adjusted P Adjusted HR (95% CI)

ARMCX1

Low 175 61 (34.6%) 12940.016

0.667 (0.479-0.929)0.006

0.620 (0.440-0.874)

High 176 83 (47.6%) 766 Ref. Ref.

ARMCX2

Low 176 58 (33.0%) 16860.012

0.655 (0.469-0.915)0.005

0.610 (0.432-0.861)

High 175 86 (49.1%) 762 Ref. Ref.

ARMCX3

Low 176 66 (37.5%) 10950.366

Ref.0.690

Ref.

High 175 78 (44.6%) 794 1.163 (0.838-1.616) 1.072 (0.763-1.506)

ARMCX4

Lo 176 70 (39.8%) 10950.570

Ref.0.220

Ref.

High 175 74 (42.3%) 801 1.099 (0.793-1.525) 1.238 (0.880-1.741)

ARMCX5

Low 176 76 (43.2%) 7280.271

Ref.0.507

Ref.

High 175 68 (38.9%) 1153 0832 (0.599-1.155) 0.891 (0.634-1.253)

ARMCX6

Low 176 73 (41.5%) 11530.786

Ref.0.929

Ref.

High 175 71 (40.6%) 874 0.956 (0.689-1.326) 1.016 (0.722-1.428)

Abbreviations: MST: median survival time; Ref.: reference; HR: hazard ratio; 95% CI: 95% confidence interval; Notes: Adjusted P, adjustment for age andtumor stage.


normal gastric tissues. However, conversely, ARMCX5 wasless expressed in normal gastric tissues than in primary gas-tric tumors. The expression levels of ARMCX3 andARMCX6 have no significant difference between gastrictumors and normal gastric tissues.

3.2. Bioinformatics and Functional Annotation Analyses ofthe ARMCX Genes. Enrichment and functional analyses byKOBAS revealed that ARMCX genes were significantlyenriched in ubiquitin ligase complex and the process ofprotein modification (Figure 3(a)). However, we have not

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Figure 4: Univariate curves of OS of ARMCX family genes in GC. The lower expression levels of ARMCX1 and ARMCX2 were significantlyassociated with satisfactory OS results (a, b). The expression of ARMCX3, ARMCX4, ARMCX5, and ARMCX6 mRNA did not havesignificant prognostic value for OS (c, d).


found any associations of the ARMCX family using KyotoEncyclopedia of Genes and Genomes (KEGG) and Databasefor Annotation, Visualization, and Integrated Discovery(DAVID) analyses. By analyzing gene-gene and protein-protein interaction networks, we confirmed that the ARMCXfamily had strong protein homology and coexpression atboth gene and protein levels, as shown in Figure 3(b).

3.3. Correlation Analysis Value Assessment of the ARMCXFamily. Coexpression analyses of individual ARMCX geneswere analyzed using Pearson’s correlation coefficient. Theexpression level of ARMCX1, ARMCX2, ARMCX3, andARMCX6 was correlated with each other. Furthermore, therewas no significant correlation between the expressions ofARMCX4 and ARMCX5, but both the expressions ofARMCX4 and ARMCX5 all related to the other membersof the ARMCX family (∗∗P < 0:01; Figure 3(c)).

3.4. Clinical Characteristics of GC Patients. There were 351GC patients who had prognosis information included inthe current study; UCSC Xena dataset is shown inTable 1. The univariable survival analysis revealed thatage and tumor stage were correlated with MST in combi-nation with clinical data (P = 0:017 and P < 0:001, respec-tively), and preliminary stage was significantly correlatedwith favorable MST (2197 days, P < 0:001, HR = 0:260,95%CI = 0:126 – 0:537). On the other hand, gender wasnot associated with MST.

3.5. Survival Analysis of the ARMCX Gene Family. Survivalanalysis is shown in Table 2 and Figure 4. Due to the ageand tumor stage that were related with MST, both age andtumor stage were analyzed using the multivariate Coxproportional risk regression model. In univariate survivalanalysis, lower expression levels of ARMCX1 and ARMCX2were significantly associated with satisfactory OS results(log-rank P = 0:016, HR = 0:667, 95%CI = 0:479 – 0:929;log-rank P = 0:013, HR = 0:655, 95%CI = 0:469 – 0:915,respectively; Figures 4(a) and 4(b)). The expression ofARMCX3, ARMCX4, ARMCX5, and ARMCX6 mRNAdid not have a significant prognostic value for OS (log-rank P = 0:367, 0.570, 0.271, and 0.786, respectively;Figures 4(c)-4(f)).

3.6. Joint-Effects Analysis of ARMC1 and ARMCX2. Based onthe findings in the multivariate survival analysis, ARMCX1and ARMCX2 were associated with a significantly differentsurvival. A joint-effects analysis was employed to furtherdetermine the combined effects in prognostic prediction ofARMCX1 and ARMCX2 (grouped as summarized inTable 3). The combination of ARMCX1 and ARMCX2included group 1, group 2 and group 3, and results areshown in Table 4. Group 1 had the longest MST of 1686days (adjusted P = 0:003), while group 3 had the shortestMST of 762 days (adjusted P = 0:012). Kaplan–Meier sur-vival analyses of ARMCX1 and ARMCX2 are shown inFigure 5. Low expression levels of ARMCX1 andARMCX2 in group 1 were significantly correlated withbetter clinical outcome. In group 3, high expression of

ARMCX1 and ARMCX2 was correlated with poor OS(log-rank P = 0:007).

3.7. Nomogram Model. Nomogram risk scoring includesage, tumor stage, and the expression level of ARMCX1and ARMCX2 to calculate 1-year, 5-year, and 10-yearrelated survival rates. The higher total points, the lowersurvival rate, and the results substantiated that highexpression levels of ARMCX1 and ARMCX2, age of thepatient (>60 years old), and advanced tumor stage estab-lished a prognostic feature that conduced to the highestrisk for poor OS (Figure 6).

3.8. Gene Set Enrichment Analysis. In order to furtherexplore the underlying mechanisms of ARMCX genesin GC prognosis, we used the PAAD genome-wideRNA sequencing dataset for GSEA. GSEA results ofthe c2 reference gene set revealed that a low ARMCX1expression was involved in the WNT signaling pathway,regulation of cell metastasis (Figure 7(a)) and cell cyclebiological processes (Figures 7(b)-7(h)), and poor sur-vival of lung cancer (Figure 7(i)). Also, the enrichmentof c5 indicates that low ARMCX1 is also involved incell division (Figure 8(c)), cell cycle (Figures 8(a) and8(b)), gene silencing (Figure 8(d)), RNA modification(Figure 8(i)), and NF-kappaB signaling pathway(Figures 8(g) and 8(h)). GSEA results of c2 enrichmentsreveal that the low expression of ARMCX2 was correlated tothe cell cycle biological process (Figures 9(a), 9(b), and 9(f)),regulation of apoptosis (Figure 9(h)), DNA replication(Figure 9(c)) and damage response (Figure 9(g)), and E2F,WNT, and NF-kappaB signaling pathways (Figures 9(d),9(e), and 9(e)), whereas the c5 enrichments suggest thatlow ARMCX2 expression is involved in the biological processof cell division (Figure 10(d)), cell cycle (Figures 10(b), 10(c),and 10(i)), apoptosis (Figure 10(e)), gene silencing(Figure 10(g)), DNA damage checkpoint (Figure 10(f)), andthe NF-kappaB signaling pathway (Figure 10(a)). Moreover,the remaining results of this study can be seen in Supplemen-tary Tables 1 and 1.

4. Discussion

In our present study, we elucidated the associationsbetween the expression levels of ARMCX 1-6 genes withthe prognosis of GC patients. Our research disclosed thatARMCX 1 and ARMCX 2 contribute significantly to OS,but ARMCX 3-6 show no significant association with

Table 3: Grouping information for joint-effects analysis.

Group Combinations

1 Low ARMCX1+low ARMCX2

2Low ARMCX1+high ARMCX2High ARMCX1+low ARMCX2

3 High ARMCX1+high ARMCX2

Abbreviations: ARMCX: arm protein lost in epithelial cancers, Xchromosome.


Table 4: Joint-effects analysis of the combination of ARMCX1 and ARMCX2.

Items Cases (total n = 351) No. of events (%) MST (days) Crude P Crude HR (95% CI) Adjusted P Adjusted HR (95% CI)

Group 1 143 46 (32.2%) 1686 0.007 0.600 (0.414-0.870) 0.003 0.563 (0.384-0.825)

Group 2 65 27 (41.5%) 766 0.549 0.873 (0.559-1.362) 0.506 0.854 (0.537-1.358)

Group 3 143 71 (49.7%) 762 0.025 Ref. 0.012 Ref.

Abbreviations: MST: median survival time; Ref.: reference; HR: hazard ratio; 95% CI: 95% confidence interval; Notes: Adjusted P, adjustment for age and tumorstage.

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1−year survival0.95 0.9 0.85 0.8 0.75 0.7 0.6 0.5

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5−year survival0.8 0.75 0.7 0.6 0.5

10−year survival0.75 0.7 0.6 0.5

Figure 6: Nomogram for predicting the 1-, 3-, 5-, and 10-year events (death) with risk scores and clinical parameters. The high expressionlevels of ARMCX1 and ARMCX2, age of the patient (>60 years old), and advanced tumor stage established a prognostic feature that conducedto the highest risk for poor OS.


OS. Thus, the expression levels of ARMCX 1 and ARMCX2 both alone and in combination may serve as potentialbiomarkers of GC.

In 1989, the armadillo family proteins were first discov-ered in the polar gene fragment of Drosophila [3]. Subse-quently, more and more proteins containing arm repeatshave been analyzed and sequenced. Armadillo repeats con-taining x-chain (ARMCX 1-6) are involved in many biologi-cal processes, such as mediating protein-protein interactions

and intervening in cell assembly, nuclear transport, and tran-scriptional activation [34]. Many studies have demonstratedthat ARMCX is associated with the risk and prognosis of sev-eral diseases. For instance, the ARMCX family plays animportant role in embryogenesis and tumorigenesis [22].Scholars have found that some members of the ARMCXprotein family (Armcx1-3) were underexpressed in severalcancers of epithelial origin, including the lung, prostate,colon, and pancreatic [11].

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(i)

Figure 7: GSEA results of low ARMCX1 expressed in GC patients, using gene set c2. The low ARMCX1 expression was involved in the WNTsignaling pathway, regulation of cell metastasis (a) and cell cycle biological processes (b-h), and poor survival of lung cancer (i).Abbreviations: FDR: false discovery rate; GSEA: gene set enrichment analysis; KEGG: Kyoto Encyclopedia of Genes and Genomes; NES:normalized enrichment score; NOM: nominal.


ARMCX1, ARMCX2, and ARMCX3 are located inthe chromosome region xq21.33-q22.2, respectively. Theiramino N-terminal region has a transmembrane domain,indicating that these proteins may be located in themembrane structure of cells. ARMCX3 has been foundto be a complete membrane protein of the mitochon-drial outer membrane, which functions by interactingwith transcription regulator Sox10 [12]. In addition,ARMCX4, ARMCX5, and ARMCX6 were located in

chromosome regions xq22.1, xq22.1-q22.3, and xq21.33-q22.3, respectively. Studies have shown that ARMCX5can be activated by binding to the oncogene ZnF217[35] and ARMCX6 upexpressed at least 2-fold in periph-eral blood monocytes of rheumatoid arthritis patientscompared to those identified using oligonucleotide array[36]. Moreover, regardless of their function in other dis-eases, they are associated with tumorigenesis and wereinitially described as presumed tumor suppressors [11].

NES=2.04NOM P<0.001 FDR q=0.013


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(i)

Figure 8: GSEA results of lowARMCX1 expressed in GC patients, using gene set c5. The low ARMCX1 is also involved in cell division (c), cellcycle (a, b), gene silencing (d), RNA modification (i), and the NF-kappaB signaling pathway (g, h). Abbreviations: FDR: false discovery rate;GSEA: gene set enrichment analysis; KEGG: Kyoto Encyclopedia of Genes and Genomes; NES: normalized enrichment score; NOM: nominal.


Here, we downloaded and analyzed data fromGEOonlinedatabase to determine the potential relationship betweenARMCXmRNA expression and clinical outcomes of patientswith gastric cancer. We observed significant differences in theexpression of ARMCX1, ARMCX2, ARMCX4, andARMCX5between primary tumors and adjacent normal tissues, withoutARMCX3 and ARMCX6. More importantly, ARMCX1 andARMCX2 are more highly expressed in adjacent normal

tissues than in tumor tissues, leading to better OS inpatients with gastric cancer, although the mechanism ofaction needs further clarification.

In addition, a comprehensive survival analysis of thecurrent prognostic characteristics of ARMCX was performedby establish a nomogram, and stratified joint-effects survivalanalysis was conducted to explore its potential application.The results indicated that high ARMCX expression was an

NES=2.09NOM P<0.002 FDR q=0.005

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(i)

Figure 9: GSEA results of low ARMCX2 expressed in GC patients, using gene set c2. The low expression of ARMCX2 was correlated to thecell cycle biological process (a, b, and f), regulation of apoptosis (h), DNA replication (c) and damage response (g), and the E2F, WNT, andNF-kappaB signaling pathways (d, i, and e). Abbreviations: FDR: false discovery rate; GSEA: gene set enrichment analysis; KEGG: KyotoEncyclopedia of Genes and Genomes; NES: normalized enrichment score; NOM: nominal.


independent risk factor as a prognostic characteristic forpatients with gastric cancer, and the relevant risk score couldbe used as a prognostic indicator. Nomogram, composed ofrisk score and other clinical information such as age andtumor stage, is an important prognostic risk assessmentsystem for gastric cancer.

To explore the underlying mechanism of ARMCX genesin gastric cancer prognosis, we used a genome-wide RNA

sequencing dataset in GSEA. The NF-kappaB, E2F andWNT signaling pathways, the cell cycle, and gene silencingwere significantly enriched in the ARMCX1 and ARMCX2low-expression groups.

It is well established that, as members of the armadillo(Arm) family, β-catenin and adenomatous polyposis coli(APC) are important components of the WNT signalingpathway. Moreover, WNT signaling plays an important

NES=1.91NOM P=0.006 FDR q=0.015

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KINASE_ACTIVITY

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(i)

Figure 10: GSEA results of low ARMCX2 expressed in GC patients, using gene set c5. The low ARMCX2 expression is involved in biologicalprocess of cell division (d), cell cycle (c, i), apoptosis (e), gene silencing (g), DNA damage checkpoint (f), and the NF-kappaB signalingpathway (a). Abbreviations: FDR: false discovery rate; GSEA: gene set enrichment analysis; KEGG: Kyoto Encyclopedia of Genes andGenomes; NES: normalized enrichment score; NOM: nominal.


role in a variety of biological processes, such as tumori-genesis, embryonic development, and stem cell mainte-nance [37–39]. β-Catenin, which is a multifunctionalprotein, plays an essential role in a variety of biologicalresponses. For instance, in the WNT signaling pathway,β-catenin works by interact with E-cadherin and TCF/LEFtranscription factors, respectively [40, 41]. APC can regu-late the WNT signaling pathway by synergistically actingwith casein kinases 1, glycogen synthase kinase-3b, andAXIN to induce degradation of β-catenin [37, 42, 43].On the basis of GSEA results, we deduced that bothARMCX1 and ARMCX2 were involved in the pathwayand biological processes that are associated with the prog-ress and treatment of gastric cancer and may serve as aGC prognostic marker. Once these results are verified,ARMCX1 and ARMCX2 may be used as biomarkers incombination with other clinical factors to facilitate theselection of diagnosis and treatment decisions for GCand to benefit patients with better clinical outcomes.

Although significant results have been achieved in thecurrent study, there are still some deficiencies to be con-sidered. First, the results of this study were obtained froma single cohort in the TCGA database, and its demo-graphic characteristics may not be representative of allpatient groups. Therefore, genetic changes may have devi-ation and require further validation in other GC groups.Second, the clinical information from TCGA was incom-plete. Therefore, we cannot conduct a comprehensivestratification analysis in the Cox proportional risk regres-sion model including all layers. Third, the mechanismbetween the above ARMCX and WNT signaling pathwaysaffecting the clinical prognosis of GC still needs to bedetermined.

5. Conclusion

Our present study has determined that ARMCX has apotential prognostic value for gastric cancer and may haveclinical application value. In addition, further basicresearch is needed to clarify the specific mechanisms ofARMCX in GC.

Data Availability

The datasets used and/or analyzed during the current studyare available from the corresponding author on reasonablerequest and TCGA data base.

Ethical Approval

This article does not contain any studies with human partic-ipants or animals performed by any of the authors. Since alldatasets included in the present study were downloaded fromTCGA, additional approval by an Ethics Committee was notneeded. The procedures were in accordance with the Helsinkideclaration of 1964 and its later amendments.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ Contributions

TW, YQ, and XL conceived and designed the study. ZH,WW, and ZL interpreted the data and performed the analy-sis. TW wrote the article, and MH approved the final versionof the manuscript. All authors approved the final version ofthe manuscript.

Acknowledgments

We sincerely appreciate TCGA (https://cancergenome.nih.gov/) and UCSC Xena (http://xena.ucsc.edu/) for sharingthe GC data and for Dr Liucheng Wu for his advice withthe language consulting of the manuscript. The study wassupported by the National Natural Science Foundation ofGuangXi (No. 2019GXNSFBA185019) and National NaturalScience Foundation of China (No. 81760521).

Supplementary Materials

Supplementary 1. Supplementary Table 1: GSEA KEGG(c2.all.v6.2.symbols.gmt) results of ARMCX1 and ARMCX2.

Supplementary 2. Supplementary Table 2: GSEA GO(c5.all.v6.2.symbols.gmt) functional analysis results ofARMCX1 and ARMCX2.

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