ORIGINAL RESEARCH

published: 09 July 2021

doi: 10.3389/fgene.2021.697514

Integrated Analysis of Multiple

Microarray Studies to Identify Novel
Gene Signatures in Ulcerative Colitis
Zi-An Chen 1 , Yu-Feng Sun 1 , Quan-Xu Wang 1 , Hui-Hui Ma 1 , Zhi-Zhao Ma 2* and
Chuan-Jie Yang 1*
1
Department of Gastroenterology, The Second Hospital of Hebei Medical University, Shijiazhuang, China, 2 Department
of Neurosurgery, The Second Hospital of Hebei Medical University, Shijiazhuang, China

Background: Ulcerative colitis (UC) is a chronic, complicated, inflammatory disease

with an increasing incidence and prevalence worldwide. However, the intrinsic molecular
mechanisms underlying the pathogenesis of UC have not yet been fully elucidated.
Methods: All UC datasets published in the GEO database were analyzed and
Edited by:
Shulan Tian, summarized. Subsequently, the robust rank aggregation (RRA) method was used to
Mayo Clinic, United States identify differentially expressed genes (DEGs) between UC patients and controls. Gene
Reviewed by: functional annotation and PPI network analysis were performed to illustrate the potential
Espiridión Ramos-Martínez,
Universidad Nacional Autónoma functions of the DEGs. Some important functional modules from the protein-protein
de México, Mexico interaction (PPI) network were identified by molecular complex detection (MCODE),
Panwen Wang,
Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG), and
Mayo Clinic Arizona, United States
analyses were performed. The results of CytoHubba, a plug for integrated algorithm for
*Correspondence:
Zhi-Zhao Ma biomolecular interaction networks combined with RRA analysis, were used to identify
[email protected] the hub genes. Finally, a mouse model of UC was established by dextran sulfate sodium
Chuan-Jie Yang
[email protected] salt (DSS) solution to verify the expression of hub genes.
Results: A total of 6 datasets met the inclusion criteria (GSE38713, GSE59071,
Specialty section:
This article was submitted to GSE73661, GSE75214, GSE87466, GSE92415). The RRA integrated analysis revealed
Human and Medical Genomics, 208 significant DEGs (132 upregulated genes and 76 downregulated genes). After
a section of the journal
Frontiers in Genetics
constructing the PPI network by MCODE plug, modules with the top three scores were
Received: 19 April 2021
listed. The CytoHubba app and RRA identified six hub genes: LCN2, CXCL1, MMP3,
Accepted: 07 June 2021 IDO1, MMP1, and S100A8. We found through enrichment analysis that these functional
Published: 09 July 2021
modules and hub genes were mainly related to cytokine secretion, immune response,
Citation:
and cancer progression. With the mouse model, we found that the expression of all six
Chen Z-A, Sun Y-F, Wang Q-X,
Ma H-H, Ma Z-Z and Yang C-J (2021) hub genes in the UC group was higher than that in the control group (P < 0.05).
Integrated Analysis of Multiple
Microarray Studies to Identify Novel Conclusion: The hub genes analyzed by the RRA method are highly reliable. These
Gene Signatures in Ulcerative Colitis. findings improve the understanding of the molecular mechanisms in UC pathogenesis.
Front. Genet. 12:697514.
doi: 10.3389/fgene.2021.697514 Keywords: ulcerative colitis, robust rank aggregation, differentially expressed genes, GEO database, microarray

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Chen et al. RRA Analysis for Ulcerative Colitis

INTRODUCTION MATERIALS AND METHODS

Ulcerative colitis (UC) is a chronic, complicated, inflammatory Search Strategy for the UC Microarray
disease that affects the colonic mucosa and most commonly
Datasets
presents with abdominal pain, diarrhea, and blood in the
A total of 68 datasets were collected from the Gene
stools. Pathological characteristics include relapsing and
Expression Omnibus (GEO) Database1 by systematic
remitting mucosal inflammation, starting in the rectum
retrieval using the keywords: (“colitis, ulcerative”[MeSH
and sigmoid colon and extending continuously to proximal
Terms] OR ulcerative colitis [All Fields]) AND “Homo
segments of the colon or even the entire colon, which leads
sapiens”[porgn] AND (“Expression profiling by array”[Filter]
to permanent fibrosis and tissue damage (Ungaro et al.,
AND (“2010/01/01”[PDAT]: “2021/03/05”[PDAT])). The
2017). The incidence and prevalence of UC have been
inclusion criteria were as follows: (1) a dataset sample size > 30;
increasing worldwide. It usually has a long, chronic clinical
(2) the dataset included both cases and normal controls; (3) the
course, and UC patients are at increased risk of colorectal
sample source was “colon,” and (4) the differentially expressed
cancer. Thus, UC has become one of the critical threats
genes (DEGs) with | logFC| > 1.5 and adjusted P < 0.05 were
and challenges to human health (Kaplan and Ng, 2017;
identified from the dataset (Figure 1).
Ng et al., 2017).
At the present, the etiology of UC remains unclear. It
is believed that multifactorial pathogenesis plays a role in Identification of DEGs in UC
the occurrence and development of UC, and the factors We first downloaded the gene expression profiles from the GEO
involved include environmental and psychological factors, database for all the datasets included in the final analysis. The
dysregulated intestinal flora and immune responses, scaled expression values for each gene were averaged when
genetic predisposition, and epithelial barrier defects. multiple probes target the same gene. Second, PERL software
The colonic epithelium facilitates host-microorganism was used to extract the matrix file, and the Limma package
interactions to control mucosal immunity, coordinate in R software was used to perform quantile normalization
nutrient recycling and form a mucus barrier. Breakdown with the normalizeBetweenArrays function. Additionally, we
of the epithelial barrier is underlying in UC pathogenesis identified the DEGs for each dataset with the criteria of | logFC|
(Ungaro et al., 2017; Parikh et al., 2019). Routinely used > 1.5 and adjusted P < 0.05 for comparison with the RRA
indices in the clinical diagnosis and dynamic monitoring analysis results.
of UC include C reactive protein (CRP), erythrocyte
sedimentation rate (ESR), and fecal calprotectin, but these RRA Integrated Analysis
lack sensitivity and specificity in differentiating between
In the RRA analysis, DEGs (both upregulated and
UC and functional gut disorders (Brookes et al., 2018).
downregulated) were sorted for each dataset and ranked
Therefore, it is of great importance to further understand
according to their logFC using the Limma package of
the pathogenesis and regulation of UC at the molecular
R software. Then, all the DEGs were scored according
level, as well as the identification of key biomarkers
to the ranked list and aggregately analyzed using the
for UC.
RRA package of R software. The adjusted P-value in this
In recent years, several studies based on microarray
method reflects the probability of the highly ranked genes
technology have been published to identify effective biomarkers
in the datasets identified as DEGs. The criteria for the
in UC (Cheng et al., 2019, 2020; Chen Y. et al., 2020; Shi
identification of DEGs were set as | logFC| > 1.5 and adjusted
et al., 2020; Cao et al., 2021). However, differences in
P < 0.05.
measurement platforms, lab protocols, sample sizes, and
operators render gene expression levels incomparable. Based
on multiple microarray datasets, robust rank aggregation Functional Annotation
(RRA) is a method that integrates the results of differential Gene Ontology (GO) is a community-based bioinformatics
expression analysis, expanding the sample size and resource for annotating gene sets, which can be divided into
reducing the influence of different microarray platforms three parts: biological process (BP), cellular component (CC),
and imbalanced sample sizes (Kolde et al., 2012). To date, and molecular function (MF) (Dalmer and Clugston, 2019).
RRA has not been applied in microarray studies of UC. The Kyoto Encyclopedia of Genes and Genomes (KEGG)
Thus, this study involved a comprehensive evaluation of is a database covering various biological signaling pathways.
the published datasets. Based on the inclusion criteria, we GO enrichment and KEGG pathway analysis provide essential
screened and included six datasets and identified DEGs perspectives for bioinformatics analysis. Enrichment analysis
using RRA. A protein-protein interaction (PPI) network was for DEGs with the criteria of adjusted P < 0.05 was
constructed to analyze the hub genes, the gene modules, and performed using the Cluster Profile package of R software
the involved functions and pathways. Several additional (Yu et al., 2012).
biomarkers were identified that may contribute to the
diagnosis of UC, thus providing potential therapeutic targets
for patients. 1
https://www.ncbi.nlm.nih.gov/geo/

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Chen et al. RRA Analysis for Ulcerative Colitis

FIGURE 1 | Flowchart of dataset retrieval and inclusion.

Protein-Protein Interaction (PPI) Network (PFA). The other part was homogenized with TRIzol reagent
Analysis (Invitrogen, Carlsbad, CA, United States), immediately frozen in
liquid nitrogen, and stored at −80◦ C.
For the obtained DEGs, the String database2 was used
to construct the PPI network, with the parameter of
confidence > 0.4. Visualization of the PPI network was Real-Time Quantitative PCR (RT-qPCR)
performed by Cytoscape (v3.7.2) (Smoot et al., 2011), and Total RNA was extracted according to the manufacturer’s
molecular complex detection (MCODE) (a plugin in Cytoscape) instructions. For examination of mRNA expression, the RNA
(Bader and Hogue, 2003) was used to identify the functional was reverse transcribed into cDNA, followed by an examination
modules. Essential genes were identified by the plugin of of RT-qPCR using SYBR Mix (CWBIO, Beijing, China). β-actin
CytoHubba (Chin et al., 2014) and sorted by degree scores. The was indicated as internal controls. All samples were examined in
overlap of genes in the PPI network (degree score > 15) and triplicate for each specific gene. The primer sequences for PCR
the top 15 DEGs (upregulated or downregulated) in the RRA are listed in Supplementary Table 1.
analysis were determined as hub genes.
Pathological Examination
Establishment of UC Model The distal colons of the mice were fixed with 4% PFA
All animal experiments were performed according to and embedded in paraffin. Tissue sections were stained with
Institutional Animal Care and Use Committee (IACUC) hematoxylin-eosin (HE). According to the scoring criteria by
guidelines and were approved by the Ethics Committee at Hebei Dieleman et al. (1998), the intestinal damage level was evaluated
Medical University. under an optical microscope, as shown in Table 1. Three visual
Sixteen BALB/c mice (male, 6–8 weeks) were obtained from fields were randomly selected from each section, and the scores
Vital River and were reared in a specific pathogen-free (SPF) were averaged to determine the damage level of the colonic tissue.
environment. All mice were randomly divided into standard
control and experimental groups. The mice in the regular control
RESULTS
group were given normal drinking water. In contrast, the mice
in the experimental group were given a 3.5% DSS solution
(dextran sulfate sodium salt, MPbio, MW 36,000–50,000 Da, CA,
Characteristics of the Included
United States) for 7 days continuously to induce acute colitis Microarrays
and to create the UC animal model. After modeling, all mice According to the criteria above, a total of six datasets were
were given standard drinking water, kept for another 3 days, and included in the final analysis: GSE38713 (Planell et al., 2013),
then euthanized with CO2 . The colon tissue from each mouse GSE59071 (Vanhove et al., 2015), GSE73661 (Arijs et al., 2018),
was collected. One part was stored in 4% paraformaldehyde GSE75214 (Vancamelbeke et al., 2017), GSE87466 (Li et al., 2018),
and GSE92415. The flowchart of dataset retrieval, inclusion
2
https://www.string-db.org/ criteria, and exclusion criteria is shown in Figure 1. From the

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Chen et al. RRA Analysis for Ulcerative Colitis

TABLE 1 | Histological grading of colitis.

Inflammation Extent Regeneration Crypt damage Percent involvement Grade

None None Complete regeneration or normal tissue None None 0

Slight Mucosa Almost complete regeneration Basal 1/3 damaged 1–25% 1
Moderate Mucosa and submucosa Regeneration with crypt depletion Basal 2/3 damaged 26–50% 2
Severe Transmural Surface epithelium not intact Only surface epithelium intact 51–75% 3
No tissue repair Entire crypt and epithelium lost 76–100% 4

TABLE 2 | Characteristics of the included microarray datasets.

GSE ID Participants(control/UC) Analysis type Platform Year Tissues

GSE38713 13/22 Array GPL570 2012 Colon

GSE59071 11/97 Array GPL6244 2015 Colon
GSE73661 12/67 Array GPL6244 2016 Colon
GSE75214 11/97 Array GPL6244 2017 Colon
GSE87466 21/87 Array GPL13158 2018 Colon
GSE92415 21/162 Array GPL13158 2018 Colon

FIGURE 2 | Volcano maps of the six datasets. Red points represent upregulated genes, while green points represent downregulated genes. Black points indicate
genes with no significant difference.

six datasets, a total of 532 cases of UC (including 46 inactive homogeneity of the data met the requirements and could be
UC cases) were included in the experimental group, and 89 were included in the analysis. Then, the DEGs were identified in each
included in the standard control group. The characteristics of the dataset using the Limma package of R software, and the volcano
included microarray datasets are shown in Table 2. maps are shown in Figure 2.

Identification of DEGs in UC RRA Integrated Analysis

First, the datasets were standardized to correct batch differences A total of 208 DEGs (132 upregulated and 76 downregulated)
within the datasets (Supplementary Figure 1), showing that the were identified by RRA analysis, and the heatmap of the top 20

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Chen et al. RRA Analysis for Ulcerative Colitis

FIGURE 3 | Heatmap of the top 20 DEGs (upregulated or downregulated) identified in the RRA analysis. Red represents a relatively high expression of genes in
patients with UC. In contrast, green represents a relatively low expression of genes in patients with UC. The numbers in the heatmap represent logarithmic fold
change in each dataset calculated by R software.

DEGs (upregulated or downregulated) is shown in Figure 3. The the operator influence, thus improving the reliability of the
top 10 significant genes aberrantly expressed in UC included five conclusions. In this study, according to the criteria of | logFc|
upregulated genes [DUOX2 (P = 1.66E-18), SLC6A14 (P = 1.66E- > 1.5 and adjusted P < 0.05, 270, 272, 436, 272, 298, and 231
18), MMP3 (P = 6.32E-18), REG1A (P = 1.06E-16), REG1B DEGs were identified in each dataset. After excluding duplicates,
(P = 1.95E-15)], and five downregulated genes [AQP8 (P = 4.04E- 666 DEGs were identified, among which 79 common DEGs were
22), HMGCS2 (P = 1.89E-17), PCK1 (P = 1.06E-16), SLC26A2 identical in all datasets. The DEGs identified in the RRA analysis
(P = 2.15E-16), ABCG2 (P = 4.04E-16)]. The overall results from (n = 208) accounted for 40.82–71.69% in each dataset, indicating
RRA analysis are listed in Supplementary Table 2. that RRA integrated the results of the datasets, especially when
Compared with the single dataset analysis, the RRA integrated high-throughput sequencing data were collected from different
analysis significantly increased the sample size and reduced platforms covering different sets of the gene probes (Table 3).

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Chen et al. RRA Analysis for Ulcerative Colitis

Functional Annotation that these genes were mainly involved in cytokine-cytokine

We uploaded the 132 upregulated and 76 downregulated DEGs receptor interactions, viral protein interactions with cytokines
to perform GO (including biological process, molecular function, and cytokine receptors, chemokine signaling pathways, and IL-17
and cellular component) analysis and KEGG analysis. The results signaling pathways (Figure 7B).
indicated that the upregulated DEGs were particularly enriched GO enrichment analysis of module 2 revealed that the
in humoral immune response, in leukocyte migration, in DEGs were mainly related to the humoral immune response
response to lipopolysaccharide, in the secretory granule lumen, in (Figure 8A). The KEGG analysis revealed that these genes
the cytoplasmic vesicle lumen, in the vesicle lumen, in receptor- were mainly involved with Staphylococcus aureus infection, in
ligand activity, in cytokine activity, and cytokine receptor binding the NOD-like receptor signaling pathway, and transcriptional
and were the top three enriched terms, depending on the P-value misregulation in cancer (Figure 8B).
of the respective categories (Figure 4A). The downregulated GO enrichment analysis of module 3 suggested that the
DEGs were mainly enriched in anion transmembrane transport DEGs were mainly related to the regulation of complement
and inorganic anion transport based on the P-value (Figure 4B). activation, to the regulation of the protein activation cascade, and
The KEGG pathway enrichment analysis suggested the regulation of the humoral immune response (Figure 9A),
that the upregulated DEGs predominantly participated in and KEGG analysis revealed that these genes were mainly
inflammation-related pathways, including the cytokine-cytokine involved in complement and the coagulation cascade-related
receptor interaction and IL-17 signaling pathway, while the pathways (Figure 9B).
downregulated genes were mainly enriched in the bile secretion
pathway (Figures 5A,B). The Determination of Hub Genes
CytoHubba was used to identify the critical genes in the PPI
network and sort them by degree scores. Integrating the RRA
PPI Network Analysis and Identification analysis results, six hub genes were obtained, including LCN2,
of Hub Genes CXCL1, MMP3, IDO1, MMP1, and S100A8. All results are listed
A visual network of DEGs identified from the RRA analysis was in Supplementary Table 4.
constructed using the String (Search Tool for the Retrieval of
Interacting Genes database) website, and these were comprised
of 205 nodes and 880 edges. The network was then imported into Verification of Hub Genes in a Mouse UC
Cytoscape for subsequent genetic analysis (Figure 6A). Model
The top three modules with the highest scores were identified Finally, the expression of the six hub genes was verified in a
by MCODE (Figures 6B–D). Module 1 comprised CXCL6, mouse UC model. All mice were alive at the end of the experiment
CXCL8, CCL18, C3, HCAR3, CXCL, CXCL13, IL1β, NPY1R, (n = 8). UC was confirmed by the pathological examination of
MMP3, CCL20, MMP1, CXCR2, CCL2, CXCL1, CXCL10, the colonic tissue. The normal structure of the colonic tissue in
CXCL9, CXCL3, and CXCL11 with the seed gene MMP9; module UC mice was almost gone, with inflammatory cell infiltration into
2 contained DEFA5, DEFA6, REG3A, and REG1B with the seed the submucosa. Hence, the pathological score of the experimental
gene REG1A; and module 3 consisted of C2, C4BPA, CFI, CFB, group was significantly higher than that of the normal control
and CD55 with the seed gene C4BPB. The score of each module group (Figures 10A,B).
is shown in Supplementary Table 3. The expression of the six hub genes in colon tissue was
GO enrichment analysis of module 1 showed that the quantified by qPCR, showing that the expression was significantly
genes were mainly related to inflammatory cell migration and higher in the experimental group than in the normal control
cytokine activity (Figure 7A), and KEGG analysis revealed group (Figure 10C).

TABLE 3 | Comparison of DEGs identified by single dataset analysis and RRA integrated analysis.

GSE ID RRA analysis

Identical DEGs,n (%) Different DEGs,n (%)

GSE38713 138 (51.11) 132 (48.89)

GSE59071 130 (47.94) 142 (52.21)
GSE73661 178 (40.82) 258 (59.17)
GSE75214 195 (71.69) 77 (28.31)
GSE87466 178 (59.73) 120 (40.27)
GSE92415 156 (67.53) 75 (32.47)
Overall number of unique genesa 113 (16.97) 553 (83.03)
Common genes with significant differenceb 79 (100) 0 (0)
a The genes that all differentially expressed genes identified by six datasets.
b The genes that were commonly identified by six datasets.

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Chen et al. RRA Analysis for Ulcerative Colitis

FIGURE 4 | Gene Ontology (GO) analysis of DEGs. (A) Functional enrichment analysis of upregulated genes. (B) Functional enrichment analysis of downregulated
genes.

DISCUSSION remains unclear, as the genetic, environmental, and psychological

factors involved in the pathogenesis make interpreting its
UC is an intestinal inflammation disease with multiple causes; pathological mechanism and diagnosis difficult (Kaplan and Ng,
it is becoming increasingly common and is characterized by 2017; Ungaro et al., 2017). In this study, several published datasets
prolonged clinical courses and recurrent attacks. The etiology were combined for bioinformatics analysis.

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Chen et al. RRA Analysis for Ulcerative Colitis

FIGURE 5 | The Kyoto Encyclopedia of Gene and Genome (KEGG) pathway enrichment analysis for DEGs. (A) The functional enrichment analysis of upregulated
genes. (B) Functional enrichment analysis of downregulated genes.

GEO (see text footnote 1) is an international public expression levels incomparable. In recent years, several studies
repository for high-throughput microarray and next-generation based on microarray technology have been published to identify
sequence functional genomic datasets submitted by the research effective biomarkers in UC, most of which are prone to utilizing
community. Currently, it is the world’s largest public database intersecting genes from different microarrays to perform analyses
for storing gene expression data, so it was searched to identify (Cheng et al., 2019, 2020; Chen Y. et al., 2020; Shi et al., 2020;
relevant UC datasets. In total, six datasets were retrieved and Cao et al., 2021). As shown in Table 3, these methods can be
combined for RRA analysis, which identified 208 DEGs. There applied to fewer datasets (≤3 datasets) because more datasets
are several methods for the combination of multiple microarrays represent overly strict inclusion criteria, leading to fewer DEGs.
to perform bioinformatics analysis. Batch normalization can In addition, the results based on intersecting genes are prone
integrate different datasets. However, differences in measurement to be influenced by a single abnormal dataset. In contrast, RRA
platforms, lab protocols, sample sizes, and operators render gene analysis focuses on the ranking of each gene in each dataset.

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Chen et al. RRA Analysis for Ulcerative Colitis

FIGURE 6 | Visualization and module identification of the PPI network. (A) A total of 208 DEGs were mapped using Cytoscape software. Three modules of the PPI
networks were identified by the MCODE plug-in. (B) Module 1 comprised CXCL6, CXCL8, CCL18, C3, HCAR3, CXCL, CXCL13, IL1β, NPY1R, MMP3, CCL20,
MMP1, CXCR2, CCL2, CXCL1, CXCL10, CXCL9, CXCL3, and CXCL11 with the seed gene MMP9. (C) Module 2 contained DEFA5, DEFA6, REG3A, and REG1B
with the seed gene REG1A, and (D) module 3 consisted of C2, C4BPA, CFI, CFB, and CD55 with the seed gene C4BPB. The red points represent upregulated
genes, while the blue points represent downregulated genes.

With the assumption that each gene identified in each dataset is Sankarasubramanian et al., 2020; Yoon et al., 2020; de Carvalho
randomly arranged, the RRA compares the ranking of a randomly et al., 2021; Ye et al., 2021). We believe that the advantage of RRA
ordered list with the baseline case, and a higher gene rank is analysis is that more potential biomarkers can be found at one
associated with a lower P-value. To our knowledge, RRA was first time, which can provide clues for subsequent research. At the
used to integrate datasets in UC and has preliminarily proven same time, the data were collected from microarray analysis to
its reliability via our investigation in animal models. Notably, avoid the interference of human factors such as improper blind
there are dozens of ways to perform meta-analyses of different method. However, there are also some shortcomings, such as the
studies, and RRA is only one of them. We reviewed the current biomarkers may come from data overfitting, and the follow-up
studies on ulcerative colitis (Bopanna et al., 2017; Gubatan et al., validation of these biomarkers still needs follow-up experimental
2019; Szemes et al., 2019; Chen M. et al., 2020; Li et al., 2020; studies, clinical validation, and multi-center clinical trials.

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Chen et al. RRA Analysis for Ulcerative Colitis

FIGURE 7 | Functional enrichment analysis for the genes in module 1 (A) GO analysis for DEGs. (B) The KEGG analysis for DEGs.

PPI network analysis was performed for all DEGs. MCODE, used to identify multiple functional gene modules (Bader and
an algorithm that allows the automated prediction of protein Hogue, 2003). The top three modules with the highest scores
complexes from qualitative protein-protein interaction data, was were further analyzed, revealing that module 1 was mainly related

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Chen et al. RRA Analysis for Ulcerative Colitis

FIGURE 8 | Functional enrichment analysis for the genes in module 2 (A) GO analysis for DEGs. (B) The KEGG analysis for DEGs.

to inflammation, with the genes involved in the chemotaxis, while KEGG analysis showed that it was primarily involved in
aggregation, and cytokine activity of inflammatory cells, and Staphylococcus aureus infection. Studies have reported that the
these genes in module 1 were previously reported on in several severity of UC is related to the imbalanced intestinal flora in
bioinformatic analyses of UC. The genes in modules 2 and 3 patients. Intestinal antigens from intestinal bacteria and their
have been less frequently reported on in the literature; however, metabolites are common, with various antibodies produced in the
all genes were upregulated in UC. Module 2 comprised REG1A, intestinal immune response (Frehn et al., 2014; Jansen et al., 2016;
DEFA5, DEFA6, REG3A, and REG1B. The results from the Soontararak et al., 2019). Additionally, some studies have shown
GO analysis linked module 2 to various humoral responses, that the REG family proteins play a role in mucosal regeneration

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Chen et al. RRA Analysis for Ulcerative Colitis

FIGURE 9 | Functional enrichment analysis for the genes in module 3 (A) GO analysis for DEGs. (B) The KEGG analysis for DEGs.

in UC (Sekikawa et al., 2010; Tsuchida et al., 2017; Takasawa with a tailored defense reaction. MAMP recognition of microbial-
et al., 2018). Module 3 contained C4BPB and was mainly associated molecular patterns by intestinal epithelial cells (IECs)
related to the regulation of complement activation, while KEGG and appropriate immune responses are of significant importance
analysis revealed that it mainly participated in complement and for maintaining intestinal barrier function. Proper activation of
coagulation cascades. The complement system is part of the the intestinal complement system might play an essential role in
innate sensor and effector systems, such as the Toll-like receptors resolving chronic intestinal inflammation, while overactivation
(TLRs), which recognize and quickly systemically and locally and/or dysregulation might worsen intestinal inflammation.
respond to microbial-associated molecular patterns (MAMPs) Hence, how IECs, intestinal bacteria, and epithelial cells express

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Chen et al. RRA Analysis for Ulcerative Colitis

FIGURE 10 | Determination of hub genes in mouse UC model H&E staining of colon tissues from the control and dextran sulfate sodium-induced colitis model mice
(A) and histological lesion score of colon tissues (B) were performed. The expression of hub genes was examined by qPCR, and β-actin served as an internal
reference (C). **Indicates p < 0.05.

complement and interact in the long-term course of UC remains receptor 2 (CXCR2), and these two ligands act indirectly on
to be elucidated (Geremia et al., 2014; Sina et al., 2018). tumor angiogenesis by regulating the trafficking of leukocytes
Furthermore, the activation of the complement system may that produce angiogenic factors and a variety of inflammatory
promote UC-associated carcinogenesis (Ning et al., 2015). cytokines (Mantovani et al., 2010). Recently, studies have shown
The DEGs were screened using CytoHubba, a novel Cytoscape that the CXCL1/CXCR2 signaling pathway could regulate the
plugin for scoring and ranking nodes in a network through inflammatory response and promote tumor cell proliferation,
different algorithms to evaluate the importance of nodes and invasion, and transvascular metastasis, acting as essential
gene connectivity in a biological network. It provides eleven molecules in the progression of inflammation (Acharyya et al.,
topological analysis methods, among which degree is the most 2012). Studies have also shown that the blockage of CXCR2 in
commonly used (Chin et al., 2014). The integrated analysis of neutrophils by a selective inhibitor could significantly alleviate
the top 20 DEGs ranked by degree scores (≥ 15), and RRA the symptoms of DSS-induced colitis in mice and could suppress
identified six potential hub genes, LCN2, CXCL1, MMP3, IDO1, the production of proinflammatory cytokines. Hence, CXCR2 is a
MMP1, and S100A8. potential target for UC treatment (Zhu et al., 2020). LCN2 (Østvik
The C-X-C motif chemokine ligand 1 (CXCL1) has been et al., 2013; Stallhofer et al., 2015; Buisson et al., 2018; Zollner
implicated in the malignant behavior of solid and hematological et al., 2021) and S100A8 (Manolakis et al., 2011; Azramezani Kopi
neoplasms in combination with the C-X-C motif chemokine et al., 2019; Okada et al., 2019, 2020) are critical proinflammatory

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Chen et al. RRA Analysis for Ulcerative Colitis

cytokines that have been reported in recent studies as potential Cao et al., 2021). In contrast, the advantage of this research lies
molecular markers of UC in serum and stool samples. LCN2 in the larger dataset obtained by combining data from six
and S100A8 also have antimicrobial effects and may be GEO datasets, which increased the sample size and ensured
involved in the regulation of intestinal flora as antimicrobial the stability and relative reliability of the conclusions. On
peptides, which may be indirectly related to UC. Host and the other hand, the RRA method was used to reduce the
microbial tryptophan (Trp) metabolism have emerged as critical influences of the measurement platform, the sample size of
regulators in mucosal homeostasis. Indoleamine 2,3 dioxygenase- datasets, the experimental design, and other factors on the
1 (IDO1) is the first enzyme in Trp metabolism in the final results.
kynurenine (Kyn) pathway and is perhaps most relevant in the
context of homeostasis. Therefore, IDO1 may be an important
molecular marker (Sofia et al., 2018; Alvarado et al., 2019). CONCLUSION
Although the specific mechanisms of IDO1 remain obscure,
numerous studies have shown an increased expression of IDO1 In conclusion, six datasets were integrated for bioinformatics
in inflammatory bowel disease, infection, and diverticulosis analyses and identified three functional gene modules and six
(Ferdinande et al., 2008; Nikolaus et al., 2017; Vancamelbeke hub genes, the expression of which was confirmed in a mouse
et al., 2017). IDO1 has also been closely related to disease model of UC. These results will help to further explore the
remission, with genetic abnormalities or drug inhibition of IDO1 mechanisms related to the occurrence and development of UC
aggravating the disease (Ciorba et al., 2010; Gupta et al., 2012; and to provide potential targets for the detection and treatment
Nikolaus et al., 2017). of UC patients in the future.
In recent years, the association between UC and colon
cancer has attracted significant attention. CXCL1, LCN2,
S100A8, and IDO1 may play essential roles in cancer DATA AVAILABILITY STATEMENT
progression. Studies have shown that IDO1 could promote
colitis-associated tumorigenesis in mice. This approach revealed The original contributions presented in the study are included
a cell-autonomous mechanism by which IDO1 tryptophan in the article/Supplementary Material, further inquiries can be
catabolites (kynurenine and quinolinic acid) directly promote directed to the corresponding author/s.
cancer cell proliferation (Thaker et al., 2013). Further research
is needed regarding the related evolutionary mechanism of UC
and colon cancer. ETHICS STATEMENT
Matrix metalloproteinases (MMPs) belong to a family of
The animal study was reviewed and approved by The Ethics
zinc-dependent endopeptidases, which are mainly produced and
Committee at the Hebei Medical University.
secreted by connective tissue, endothelial cells, mononuclear
macrophages, neutrophils, and tumor cells. MMPs participate
in the degradation of ECM components (Karamanos et al.,
2021), with increased expression in the UC lesion area
AUTHOR CONTRIBUTIONS
(Schuppan and Hahn, 2000; Medina et al., 2003; Wang Z-AC collected the manuscripts and analyzed the data, analyzed
and Yan, 2006; Meijer et al., 2007). Additionally, genetic the conclusions, and drafted the manuscript. Y-FS reviewed
variations in MMPs may be associated with an increased the data and conclusions. Q-XW and H-HM contributed to
risk of UC differences in clinical symptoms (Morgan et al., writing. C-JY and Z-ZM presented the idea of this manuscript,
2011). Studies have shown that the overexpression of MMP- supported the funding, analyzed the conclusions, and drafted and
1 and MMP-3 play an important role in the pathogenesis revised the manuscript. All authors contributed to the article and
of steroid-dependent uncreative colitis (SDUC). The protein approved the submitted version.
expressions of MMP-1 and MMP-3 significantly increased in
the healing regions of colonic tissues in SDUC remission
patients but not in non-SDUC remission patients, suggesting FUNDING
that the overexpression of MMP-1 and MMP-3 are not
the only factors involved in the pathogenesis of UC but This study was funded by the Natural Science Foundation of
also are a critical feature in the steroid dependency in UC Hebei Province (Grant No. H2020206337).
(Wang and Qiu, 2010).
The expression of the six hub genes was confirmed in
the DSS-induced UC mouse model. In published studies, SUPPLEMENTARY MATERIAL
some scholars screened diagnostic biomarkers for UC from
the DEGs identified from a single dataset or an overlap The Supplementary Material for this article can be found
of two or three datasets via bioinformatics analyses (Cheng online at: https://www.frontiersin.org/articles/10.3389/fgene.
et al., 2019, 2020; Chen Y. et al., 2020; Shi et al., 2020; 2021.697514/full#supplementary-material

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Chen et al. RRA Analysis for Ulcerative Colitis

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inflammatory bowel disease. PLoS One 14:e0220522. doi: 10.1371/journal.pone. absence of any commercial or financial relationships that could be construed as a
0220522 potential conflict of interest.
Stallhofer, J., Friedrich, M., Konrad-Zerna, A., Wetzke, M., Lohse, P., Glas, J.,
et al. (2015). Lipocalin-2 is a disease activity marker in inflammatory bowel Copyright © 2021 Chen, Sun, Wang, Ma, Ma and Yang. This is an open-access
disease regulated by IL-17A, IL-22, and TNF-α and modulated by IL23R article distributed under the terms of the Creative Commons Attribution License
genotype status. . Inflamm. Bowel Dis. 21, 2327–2340. doi: 10.1097/MIB. (CC BY). The use, distribution or reproduction in other forums is permitted, provided
0000000000000515 the original author(s) and the copyright owner(s) are credited and that the original
Szemes, K., Soós, A., Hegyi, P., Farkas, N., Erős, A., Erőss, B., et al. (2019). publication in this journal is cited, in accordance with accepted academic practice. No
Comparable long-term outcomes of cyclosporine and infliximab in patients use, distribution or reproduction is permitted which does not comply with these terms.

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