Ecotoxicology and Environmental Safety 224 (2021) 112637

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Whole transcriptome-based ceRNA network analysis revealed ochratoxin

A-induced compromised intestinal tight junction proteins through WNT/
Ca2+ signaling pathway
Xue Yang a, b, c, d, 1, Yanan Gao a, b, c, d, 1, Shengnan Huang a, b, c, d, Chuanyou Su a, b, c, d,
Jiaqi Wang a, b, c, d, Nan Zheng a, b, c, d, *
a
Key Laboratory of Quality & Safety Control for Milk and Dairy Products of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of
Agricultural Sciences, Beijing 100193, China
b
Laboratory of Quality and Safety Risk Assessment for Dairy Products of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of
Agricultural Sciences, Beijing 100193, China
c
Milk and Dairy Product Inspection Center of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing
100193, China
d
State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China

A R T I C L E I N F O A B S T R A C T

Edited by: Dr Yong Liang Ochratoxin A (OTA) is a widespread environmental pollutant that is a threat to humans and livestock and re­
mains a global concern to public health. It has negative effects on both humans and animals that are in a
Keywords: continuously exposed environment. The compromised intestinal barrier caused by OTA has aroused widespread
Ochratoxin A concern. This study aimed to investigate the mechanism of OTA-induced tight junction (TJ) protein damage and
Whole transcriptome
the relevant components of the intestinal barrier through in vivo whole transcriptome analysis combined with in
CeRNA regulatory network
vitro functional verification. Bioinformatics analysis in OTA-treated Balb/c mice demonstrated that regulated TJ
Tight junction proteins
Intestinal barrier protein related mRNAs were perturbed, and activated the WNT/Ca2+ signaling pathway possibly regulated by
key lncRNAs and miRNAs. Competing endogenous RNA (ceRNA) network analysis revealed that lncRNA Zeb1
regulated FZD4 binding with WNT5a to release Ca2+ by targeting miR-1258-x and reduced the expression of TJ
proteins, thus damaging the function of the intestinal barrier. An in vitro experiment with Caco-2 cells verified
that an increase in Ca2+ level was involved in OTA-induced decreases in the expression of TJ proteins. Taken
together, these results will help to identify targets in the intestinal barrier that are compromised by OTA, and will
provide the basis for preventing the associated hazard and risk.

1. Introduction as a widespread environmental contaminant, OTA is found to exist in the

The fungal toxin ochratoxin A (OTA) is a commonly found envi­
ronmental contaminant that is produced by certain strains of fungi
belonging to Aspergillus and Penicillium, for example, P. verrucosum, P.
carbonarius, A. niger, and A. ochraceus (Creppy, 2002). Recently, the high
pollution rate of OTA in agricultural products (natural grains and food
products) has caused a significant threat to the food industry worldwide
(Tang et al., 2019; Wei et al., 2020). Various environments, including
abnormal weather, improper harvesting, and storage, could increase the
levels of OTA in crops (Jaksic et al., 2020; Raiola et al., 2015). Moreover,
middle environment of dust carried by raw materials, workers, and
processing sites in the process of food production (Fog Nielsen, 2003;
Halstensen et al., 2004). In addition, because of its nephrotoxicity,
hepatotoxicity, and carcinogenicity, OTA has been classified as a 2B
carcinogen by the International Agency for Research on Cancer (Akbari
et al., 2017; Marin et al., 2019). A provisional tolerable weekly intake of
100 ng/kg.bw/week for OTA has been established by the Joint
FAO/WHO Expert Committee on Food Additives (Bondy and Pestka,
2000). In addition to nephrotoxicity and hepatotoxicity, the intestinal
toxicity of OTA has attracted more and more attention (Wang et al.,

* Corresponding author at: Key Laboratory of Quality & Safety Control for Milk and Dairy Products of Ministry of Agriculture and Rural Affairs, Institute of Animal
Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
E-mail address: zhengnan@caas.cn (N. Zheng).
1
These authors contributed equally.

https://doi.org/10.1016/j.ecoenv.2021.112637
Received 22 April 2021; Received in revised form 19 July 2021; Accepted 11 August 2021
Available online 20 August 2021
0147-6513/© 2021 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
X. Yang et al.
2018; Ying et al., 2019). There is an urgent need to find the mechanism
of intestinal toxicity induced by OTA.
The intestinal barrier is the first barrier exposed to food pollutants, of
which mycotoxins are among the most universal contaminants (Gal­
arza-Seeber et al., 2016; Gambacorta et al., 2016). Tight junction (TJ)
proteins, which could block the intercellular space and prevent bacteria
and toxins from entering the blood circulation, are of great significance
in maintaining the intestinal barrier function (Al-Sadi et al., 2011;
Marchiando et al., 2011; Yu et al., 2010). They are composed of several
multi-protein complexes, including transmembrane proteins (tricellulin,
occludin, claudins, and junction adhesion molecules), and cytoplasmic
scaffold proteins zona occludens (ZOs) (Steed et al., 2010). Evidence
from previous studies suggested that the impairment of TJ proteins may
be closely related to various intestinal pathogenesis (Arrieta et al., 2006;
Turner, 2009). By using in vitro experiments in intestinal Caco-2 cells,
OTA was found to induce intestinal barrier impairment through
increasing its permeability and decreasing the expression of TJ proteins
(Gao et al., 2017; Maresca et al., 2008; Peng et al., 2019). OTA exposure
down-regulated the expression of TJ proteins claudin-1, claudin-3, and
claudin-4 in Caco-2 cells (Alizadeh et al., 2019). Studies have suggested
that OTA decreased the levels of TJ proteins in IPEC-J2 cells (Wang
et al., 2018). The nontoxic concentration of OTA aggravates the intes­
tinal barrier dysfunction of IPEC-J2 cells, accompanied by claudin-3 and
claudin-4 disruption and decreases in expression (Ying et al., 2019). In
addition, OTA significantly decreased the mRNA expression of ZO-1,
occludin, claudin-c, f, 7a,7b, 11,12, 15a,15b in the intestinal of Juve­
nile Grass Carp (Liu et al., 2020). Despite these effects, the mechanisms
underlying OTA-induced TJ protein disruption are still not clear, espe­
cially in vivo.
Based on mRNA sequencing, the whole transcriptome constructs two
types of library (small RNA library and ribosome-specific library), which
can analyze the data of four different RNAs: long non-coding (lnc)RNA,
circle (circ)RNA, micro (mi)RNA and mRNA, simultaneously (Zheng
et al., 2019). LncRNA is an RNA molecule with a length of more than 200
nucleotides that cannot encode proteins but is involved in transcrip­
tional regulation by binding with miRNA (Guttman et al., 2009; Wang
et al., 2019a; Yi et al., 2019). CircRNA is a class of conserved and
structurally stable non-coding (nc)RNA, which can act as a miRNA
sponge to regulate mRNA expression, cell migration, and invasion.
MiRNA is a small ncRNA molecule, which can silence RNA and regulate
the expression of post-transcriptional genes (Bartel, 2004). Through
miRNA and the competitive binding of three kinds of RNA (lncRNA,
circRNA, and mRNA), a competing endogenous RNA (ceRNA) network
can be established, which provides a way to find the key RNAs that affect
biological processes (Li et al., 2018; Taborda et al., 2017; Wang et al.,
2020; Jiang et al., 2015). In recent years, analyzing the whole tran­
scriptome has gradually become a trend for studying the mechanism of
mycotoxins (Wang et al., 2019b). It was used to explore the molecular
mechanism of the toxicological effect of zearalenone on porcine gran­
ulosa cells, but it has not indicated how ncRNA regulated the expression
of the cell cycle-related genes CDK1, CCNB1, and CHEK1 (Li et al.,
2020). To date, few examples of in vitro verification have been found in
whole transcriptome studies (Huang et al., 2019b).
In this study, the whole transcriptome combined with in vitro veri­
fication was performed to profile the mechanism underlying intestinal
toxicity in Balb/c mice exposed to OTA. We constructed a lncRNA-
miRNA-mRNA ceRNA network to reveal OTA compromised TJ pro­
teins through the activation of the WNT/Ca2+ signaling pathway.
Verification by Caco-2 cells in vitro showed that OTA increased the level
of Ca2+, inducing the decreased expression of TJ proteins. These results
may provide new evidence for studying the molecular basis of intestinal
barrier dysfunction caused by OTA.
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Ecotoxicology and Environmental Safety 224 (2021) 112637
2. Materials and methods
2.1. Materials
OTA powder was purchased from Pribolab (Qingdao, China), and
then dissolved in 1% dimethyl sulfoxide (DMSO) corn oil solution
(Sigma-Aldrich, St. Louis, MO, USA). The solution was freshly prepared
just before use. Mouse anti-occludin, mouse anti-ZO-1, and rabbit anti-
claudin-3 were purchased from Proteintech (Chicago, IL, USA). Rabbit
anti-claudin-4 was obtained from Thermo Scientific (Waltham, MA,
USA). Goat anti-rabbit/mouse IgG conjugated to horseradish peroxidase
and β-actin were from Bioss (Beijing, China). Mouse serum diamine
oxidase (DAO), D-lactic acid (D-lactate), and serum intestinal fatty acid
binding protein (I-FABP) enzyme-linked immunosorbent assay (ELISA)
kits were obtained from Sinogene Scientific (Beijing, China). Trizol re­
agent kit was obtained from Invitrogen (Carlsbad, CA, USA). Trypsin
(2.5%), antibiotics (100 units/mL penicillin, 100 μg/mL streptomycin),
Fluo-4 AM, BCA protein assay kit, Radio-Immunoprecipitation Assay
(RIPA) lysate, and phosphate buffer saline (PBS) were purchased from
Beyotime (Shanghai, China). Fetal bovine serum, nonessential amino
acids, and Dulbecco’s Modified Eagle’s Medium (DMEM) were from
Gibco (Carlsbad, CA, USA). Primers and prime script II 1st strand cDNA
synthesis kits were from Sangon Biotech (Shanghai, China). BAPTA-AM
(Ca2+ inhibitor) was obtained from Sigma-Aldrich (St. Louis, MO, USA)
and dissolved in DMEM.
2.2. Experimental animals
Thirty-six male healthy Balb/c mice in a good mental state were
purchased from Beijing Vital River (Beijing, China). The mice were SPF
clean grade aged 6–8 weeks, and the body weight was in the range of 20
± 2 g. They were raised in an SPF barrier system at the animal experi­
ment platform of Beijing Vital River Laboratory Animal Technology Co.,
Ltd. The whole feeding process strictly followed regulations on animal
feeding and management with the permission code P2020055. The
temperature of the feeding environment was controlled at 20–26 ◦ C and
humidity was within the range of 40–70% for 12 h of light and 12 h of
darkness per day.
2.3. Gavage experiment and sample collection in mice
After the mice were transferred to the barrier system, they were first
adapted to the environment for 7 days with sterile feed and water pro­
vided freely. Then the mice were randomly divided into three groups
with 12 mice in each group. A subacute treatment consisted of 1/5–1/20
of the half lethal dose (LD50) given each day for about 15–28 days
(OECD/OCDE 407, 2008). In the early stage of the experiment, ac­
cording to the acute oral toxicity experiment, the LD50 range of OTA was
estimated at 60 mg/kg b.w., which was estimated through a preliminary
acute oral toxicity experiment based on the standard of GB/T
21826–2008. With reference to the results of previous studies (Gao
et al., 2021) and OECD guides (OECD/OCDE 407, 2008), 1/20 of the
LD50 were selected as the dose value of intragastric administration. Each
mouse in the control group (CTL) was fed with normal saline per day,
and each mouse in the solvent control group (DMSO) was fed with 1.0%
DMSO corn oil solution. The concentration of OTA was 3 mg/kg b.w.
and each animal received a volume of 200 μL once a day at a fixed time
(9–10 AM) for 28 days. The mice drank and ate freely during intragastric
administration. Health status and abnormal activities were observed and
recorded every day. The body weight of mice was weighed and recorded
every 3 days. After the last intragastric administration, the mice were
fasted for 24 h. Then the mice were euthanized with carbon monoxide to
obtain the blood from the heart and intestinal jejunal tissue. The intes­
tinal contents were quickly rinsed with precooled PBS, frozen in liquid
nitrogen for 10 min, and stored at − 80 ◦ C.
X. Yang et al.
2.4. Caco-2 cell culture
Caco-2 cells were purchased from the American Type Culture
Collection (Manassas, VA, USA), and the cells were cultured as before
(Yang et al., 2019). When cell confluence reached 90%, they were
seeded in 96-well plates, and then Ca2+ levels were measured. After they
were cultured and differentiated in 6-well plates for 14 d, Caco-2 cells
undergo spontaneous differentiation resulting in polarization and for­
mation of the TJ proteins between adjacent cells. Then the differentiated
Caco-2 cells were used for TJ protein expression. In this study, the
concentration of OTA adopted was according to the study by Gao et al.
(2018). OTA (2000 μM) was acquired as stock by dissolving in methanol
and then diluted with DMEM to the concentration of the working solu­
tion (20 μM). After incubation with 20 μM OTA for 48 h, Caco-2 cells
were collected and processed for Ca2+ levels. For TJ protein expression,
the differentiated Caco-2 cells were pretreated with 10 μM BAPTA-AM
for 2 h before incubating with 20 μM OTA for 48 h.
2.5. Determination of blood biochemical indexes and data analysis
Whole blood was collected and coagulated after 1.5 h at 37 ◦ C
(without anticoagulant). Precipitated blood was placed in a centrifuge at
4 ◦ C for 10 min at 4000 rpm. The supernatant was taken as the serum,
transferred to a clean centrifuge tube, and packed into small parts for
standby. Serum biochemical indexes were determined by automatic
biochemical analyzer HITACH17080 (Hitachi, Ltd., Tokyo, Japan) and
ELISA kit, including DAO, D-lactate, and I-FABP. ELISA procedures were
followed according to kit instructions.
2.6. Observation of villous structure of jejunum in mice
Jejunal tissues were fixed with 4% paraformaldehyde for 36 h,
dehydrated with ethanol, removed with xylene, and embedded in
paraffin. Paraffin-embedded tissues were sectioned at 4 µm and stored at
room temperature. After hematoxylin-eosin (HE) staining, the sections
were examined by light microscope (eclipse CI; Nikon, Tokyo, Japan).
2.7. Goblet cell count in the jejunum
The number of intestinal jejunal goblet cells was counted by periodic
acid-Schiff (PAS) staining. The above sections were stained. Goblet cells
were purplish-red and the nuclei were blue under the microscope and
used for image acquisition and analysis. The jejunal tissue sections were
taken from six mice in each group, five villi were taken from each sec­
tion. The number of goblet cells was counted, and the average value was
calculated.
2.8. Western blot for TJ proteins expression
Jejunum tissue preserved at − 80 ◦ C was weighed and a proportion of
RIPA lysate (RIPA+ phenylmethylsulfonyl fluoride) was added (1
mg:10 μL). The tissue was ground for 40 s with a homogenizer, then
cracked on ice for 15 min, three times. After centrifugation at 4 ◦ C/
12,000 rpm for 15 min, intestinal proteins were obtained from the su­
pernatant. The differentiated Caco-2 cells collected above were used to
extract proteins by RIPA lysate.
The tissue obtained above and cell protein concentration was
determined by a BCA protein assay kit method. Then the protein was
denatured at 100 ◦ C for 10 min with 5 × loading buffer and stored at
− 20 ◦ C for western blot analysis, remaining denatured total protein was
stored at − 80 ◦ C. Western blot analysis was used to assess TJ protein
(claudin-3, claudin-4, occludin, and ZO-1) expression. Equal amounts of
protein were electrophoresed on sodium dodecyl sulfate (SDS)-poly­
acrylamide gels and electroblotted onto polyvinylidene difluoride
membranes. Western blot analysis in vivo and in vitro was performed as
described previously (Yang et al., 2019).
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Ecotoxicology and Environmental Safety 224 (2021) 112637
2.9. Immunofluorescence assay for TJ protein localization
Slides incubated with antibodies were placed in PBS, then shaken
and washed on the decolorizing shaker three times, 5 min each time.
After the slides were slightly dried, 4′ , 6-diamidin-2-phenylindol (DAPI)
staining solution was dripped into the circle and incubated at room
temperature without light for 10 min. The glass slides were placed in
PBS and then shaken and washed. An autofluorescence quenching agent
was added to the ring for 5 min, and slides were rinsed for 10 min with
running water. After slides were slightly dried, they were sealed with
anti-fluorescence quenching tablets. The sections were observed under a
fluorescence microscope and the images were collected. The nucleus
stained by DAPI is blue under UV excitation, and the positive expression
is red or green light labeled with corresponding fluorescein.
2.10. MiRNA sequence analysis
A trizol reagent kit was used to extract total RNA from mice jejunum
tissue according to the manufacturer’s protocol. The RNA was
sequenced using Illumina HiSeq Xten by Gene Denovo Biotechnology
Co. (Guangzhou, China). All of the clean tags were aligned with small
RNAs in the GeneBank (Release 209.0) and Rfam (Release 11.0) data­
bases to identify and remove rRNA, scRNA, snRNA, snoRNA, and tRNA.
To identify and remove rRNA, scRNA, snRNA, snoRNA, and tRNA, all
the clean tags were aligned with small RNAs in the GeneBank (Release
209.0) and Rfam (Release 11.0) databases. Differential expression (DE)
miRNA analysis was performed by edgeR software between two
different groups. We identified miRNAs with a fold change (FC) ≥ 2 and
P value < 0.05 as DEmiRNAs. Two software, Miranda (Version 3.3a) and
TargetScan (Version 7.0), were used to predict targets. The Gene
Ontology (GO) (http://www.geneontology.org/) and Kyoto Encyclo­
pedia of Genes and Genomes (KEGG) (https://www.kegg.jp) databases
were used for the functional annotation of target mRNAs (Kanehisa
et al., 2008). GO terms or pathways with P < 0.05 were defined as
significantly enriched GO terms or pathways.
2.11. The mRNA, lncRNA, and circRNA sequence analysis
Total RNA was obtained from mice jejunum tissue, RNA quality was
assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo
Alto, CA, USA) and checked using RNase-free agarose gel electropho­
resis. To obtain high quality clean reads, the reads were further filtered
by fastp (version 0.18.0). The short reads alignment tool Bowtie2
(version 2.2.8) was used for mapping reads to the rRNA database. The
coding ability of the new transcripts was predicted by two software, CPC
and CNCI. Differential expression analysis of mRNAs and ncRNAs was
determined using DESeq2 software between two different groups and by
edgeR between two samples. The mRNAs/ncRNAs with false discovery
rate (FDR) < 0.05/absolute FC ≥ 1.5 and P value < 0.05/FC ≥ 2 were
considered DEmRNAs/DEncRNAs, respectively. DEmRNAs were then
subjected to enrichment analysis of GO function and KEGG pathways.
GO and KEGG analysis was performed on DEncRNAs after the prediction
of target genes.
2.12. The ceRNA regulation analysis
Spearman Rank Correlation Coefficient (SRCC) was used to evaluate
the expression correlation between mRNA-miRNA or lncRNA-miRNA.
Pairs with SRCC < − 0.7 were chosen as co-expressed negative pairs,
both DEmRNA and DElncRNA were DEmiRNA target genes. All the co-
expression competitive triplets identified above were assembled into a
lncRNA-miRNA-mRNA network and visualized using Cytoscape soft­
ware (v3.6.0) (http://www.cytoscape.org/).
X. Yang et al.
2.13. Validation of mRNA, miRNA, and lncRNA expression by
quantitative reverse transcription polymerase chain reaction (RT-qPCR)
A Prime Script II 1st strand cDNA synthesis kit and synthesis of
miRNA first-strand cDNA (stem-loop method) were used to reverse
transcribe cDNAs. The RT primers of miRNA and the primer sequences
of all RNAs are shown in Table S1 and Table S2. The GAPDH gene was
chosen as an internal control for mRNA, lncRNA, and circRNA, U6 was
used for miRNA (Ma et al., 2018; Zhang et al., 2019). The relative
expression of genes was calculated by the 2− ∆∆Ct method, and the mean
and standard deviation of three biological replicates are shown as the
results.
2.14. Measurement of c(Ca2+)
Caco-2 cells were treated with 20 μM OTA for 48 h. The level of Ca2+
was measured by a Fluo-4AM kit. An appropriate amount of Fluo-4AM
stock solution was taken and diluted to 5 μM by PBS. Then Caco-2
cells were incubated with Fluo-4 AM for 30 min in the dark to load
the fluorescent probe. Fluorescence intensity before and after treatment
was detected with an excitation wavelength of 488 nm and an emission
wavelength of 520 nm.
2.15. Statistical analysis
GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA, USA)
was used for statistical analysis. One-way ANOVA and Tukey’s multiple
comparison were applied to test statistical differences between the
treatment groups and the CTL group. P value < 0.05 was considered
statistically significant.
Fig. 1. The effect of 3 mg/kg b.w. OTA on Balb/c mice jejunum structure. (a) Changes in the body weight of Balb/c mice after OTA treatment. (b) Effect of OTA
treatment on blood biochemical indexes level. (c) Effects of OTA on histological structures of jejunum villi. (d, e) Effect of OTA on villi length and crypt depth of mice
intestinal jejunum, V and C represent villus height and crypt depth, respectively. (f, g) The number of goblet cells in CTL, DMSO, and OTA groups. * P < 0.05
represents a significant difference, and ns represents no significant difference.
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Ecotoxicology and Environmental Safety 224 (2021) 112637
3. Results
3.1. OTA damaged intestinal barrier
Starting from the first day of intragastric administration, the body
weight of mice was weighed and recorded every 3 days. As depicted in
Fig. 1a, the average final body weight of mice was ranked as follows:
CTL>DMSO>OTA. The body weight of Balb/c mice decreased signifi­
cantly from the 7th day to the 29th day after OTA gavage (P < 0.05).
The indexes of impaired intestinal barrier function (DAO, D-lactate,
and I-FABP) were measured by ELISA. As the results showed, OTA ten­
ded to increase the level of DAO (P > 0.05), yet, the concentrate of D-
lactate and I-FABP were significantly increased by OTA (P < 0.05)
(Fig. 1b). At the end of the experiment, although there was no significant
difference in jejunal crypt depth between the CTL and OTA groups
(P > 0.05), the length of jejunum villi and the ratio of jejunal villi length
to crypt depth in the OTA group was significantly lower than in the CTL
group (P < 0.05) (Fig. 1c, d, e). In addition, the number of mice jejunal
goblet cells in the OTA group was significantly lower than in the CTL
groups (P < 0.05) (Fig. 1 f, g).
3.2. Effect of OTA on the expression and localization of intestinal TJ
proteins
As the western blot results showed, OTA significantly decreased
claudin-3, claudin-4, occludin, and ZO-1 protein expression levels in vivo
(P < 0.05) (Fig. 2a, b). To expound the effect of OTA on the TJ structure
in mice, the TJ proteins (claudin-3, occludin, and ZO-1) in jejunum
epithelial cells of different groups were labeled with fluorescence.
Fig. 2c showed the localization of TJ proteins in the jejunum epithelial
cells of the CTL and treatment groups. Compared with the CTL group,
the distribution of TJ proteins, especially for claudin-3, on the lateral
membrane of adjacent cells was significantly reduced in the OTA group,
X. Yang et al.
Fig. 2. The changes of TJ proteins induced by OTA. (a) Jejunum tissue protein (30 μg) was immunoblotted to detect TJ proteins, (b) Quantified analysis of TJ protein
expression. (c) Effect of OTA on the localization of intestinal TJ proteins in mice. Nuclei are indicated in blue, fluorescent-labeled TJ proteins are red. *P < 0.05
represents a significant difference.
and the cell gap was not clear.
3.3. OTA exposure changed mRNA expression
Whole transcriptome analysis was performed to understand the
molecule mechanism of OTA-induced intestinal toxicity. In general, 760
DEmRNAs were obtained under the screening conditions. These were
the sum of DEmRNAs in the three groups (CTL, DMSO, and OTA) after
comparison between every two groups (Fig. 3a). Venn analysis found
that the genes from the OTA/CTL and OTA/DMSO groups but not the
DMSO/CTL groups were the key DEmRNAs in OTA-treated samples
(Fig. 3b). As depicted by the Venn, 564 DEmRNAs were considered to be
the key genes in the OTA-treated group.
GO annotation demonstrated that terms in the lipid metabolism
process, extracellular region part, response to external stimulus, and
catalytic activity were enriched (Fig. 3c). KEGG analysis revealed that
5
Ecotoxicology and Environmental Safety 224 (2021) 112637
DEmRNAs were found to be enriched in the regulation of actin, focal
adhesion, PI3K-Akt signaling pathway, and WNT signaling pathway
(Fig. 3d). According to the KEGG key pathways, the key genes were
selected to predict protein-protein interactions (PPI) through STRING
analysis. As the PPI results depicted, the DEmRNAs CCND1, WNT5a,
FZD4, Axin2, Cacan1d, and Cav1 were target genes (Fig. 3e). The
STRING analysis indicated that these key genes were tightly related with
the WNT signaling pathway. Moreover, the key DEmRNAs Cav1 and
Cacan1d related to the Ca2+ signal cascade were also found in the PPI
network. Four DEmRNAs were randomly selected to determine relative
expression. As the RT-qPCR results depicted, CCND1 and CLDN8 were
down-regulated and OAS2 and LPAR2 were upregulated in the OTA
group compared with the CTL group (Fig. 3f). All these results were
consistent with RNA sequencing.
X. Yang et al. Ecotoxicology and Environmental Safety 224 (2021) 112637

Fig. 3. The overview of the DEmRNAs. (a) The number of DEmRNAs in different groups. (b) Venn analysis of the three groups. (c,d) The GO and KEGG annotation of
the target gene cluster. (e) PPI analysis of 43 key genes. The size of the node indicates the strength of the connectivity between them, the different colors shown in the
node represent different pathways. (f) The mRNA expression results (OTA/CTL) compared with RNA sequencing.

6
X. Yang et al.
3.4. OTA treatment altered miRNA expression
A total of 93 DEmiRNAs were identified by pairwise comparison
among the three groups (CTL, DMSO, and OTA) (Fig. 4a). Next, Venn
analysis was used to screen DEmiRNAs caused by OTA rather than
DMSO (Fig. 4b).
To understand the function of the DEmiRNAs, we performed the GO
and KEGG analysis of DEmiRNA target genes. The GO results showed
that the target genes mostly focused on biological process (BP), such as
response to external stimulus, growth, and regulation of cytokine pro­
duction (Fig. 4c). For KEGG analysis, circadian rhythm, WNT signaling
pathway, and MAPK signaling pathway were most enriched after OTA
treatment (Fig. 4d). According to the function prediction and target of
key DEmRNAs, miR-1258-x (target FZD4, Cav1, CCND1, Rasgrp3,
Foxp1), mmu-miR-1258–3p (target Plau, E2f5, Hspb1), mmu-miR-
122–5p (target WNT5a), miR-205-z (target Cav1, ccm2), mmu-miR-
1981–5p (target WNT5a), mmu-miR-1943–5p (target Igf2, Axin2, Pkd1),
and mmu-miR-146b-5p (target ccl8, Lgr6) were possibly key DEmiRNAs
related to intestinal barrier function especially for TJ proteins (Fig. 4e).
According to the results by RT-qPCR, mmu-miR-203b-3p, mmu-miR-
28c, mmu-miR-196a-5p, and mmu-miR-216b-5p were all consistent
with RNA sequencing results (Fig. 4f).
3.5. OTA exposure altered lncRNA expression
We identified 640 DElncRNAs in all the three groups (CTL, DMSO,
and OTA) after pairwise comparison (Fig. 5a). To obtain the DElncRNAs
induced by OTA, Venn analysis was performed to eliminate the influ­
ence of DMSO, OTA/CTL and OTA/DMSO but not DMSO/CTL were the
target DElncRNAs of OTA-treated samples (Fig. 5b).
Function prediction of the DElncRNAs obtained above was per­
formed on the annotation of their target mRNAs. For GO annotation,
most of the DElncRNAs were enriched in cellular component (CC) and
BP, shown in extracellular space, extracellular region, immune response,
and response to external stimulus (Fig. 5c). The DElncRNAs target bar­
plot shows that the cGMP-PKG signaling pathway, MAPK signaling
pathway, cAMP signaling pathway, and gap junction were found to be
enriched (Fig. 5d). After function prediction of target DEmRNAs, the
lncRNAs Zeb1, Stk36, Phkb, Prss23, Inpp5e, and Gm28588 were tightly
associated with intestinal TJ proteins (Fig. 5e). According to the RT-
qPCR results, the expression levels of Gm5614 and Srsf5 were
increased, yet Npr3 and Mirt2 were decreased after OTA treatment. All
these were consistent with RNA sequencing results (Fig. 5f).
3.6. The ceRNA regulation network of TJ proteins
Using the targets pairs DEmiRNA-DEmRNA and DElncRNA-
DEmRNA, ceRNA analysis was performed to obtain further insights
into the role of ncRNAs and mRNAs in intestinal TJ proteins. The rela­
tionship between DEmRNAs and DEncRNAs related to TJ proteins was
depicted in Fig. 6. The analysis of key DEmRNAs showed that the WNT/
Ca2+ signal cascade played a key role in damaging TJ proteins induced
by OTA. KEGG analysis indicated that FZD4, WNT5a, and Cav1 partic­
ipated in regulating the levels of Ca2+. Through ceRNA, the upstream
target DEmiRNAs and DElncRNAs involved in the regulation of Ca2+
signal was found. It was found that lncRNA Zeb1 regulated the expres­
sion of FZD4 binding with WNT5a to activate the Ca2+ signal cascade by
targeting miR-1258-x. Moreover, Cav1 and several lncRNAs combined
competitively with miR-205-z to regulate the release of Ca2+.
3.7. Verification that OTA induces TJ protein damage through the Ca2+
signal cascade in vitro
To further verify the effect of Ca2+ on the expression of TJ proteins,
we used an in vitro cell model. OTA increased the level of Ca2+ (Fig. 7a)
and decreased the expression level of TJ proteins in differentiated Caco-
7
Ecotoxicology and Environmental Safety 224 (2021) 112637
2 cells (Fig. 7b). When Ca2 + inhibitor BAPTA-AM was added, the
expression level of the TJ protein was significantly higher than that of
OTA alone (P < 0.05, Fig. 7c). All of the results suggested that the
change of Ca2+ level caused the change of TJ protein expression.
4. Discussion
As an environmental pollutant, OTA is a common mycotoxin in food
and feed that is almost impossible to avoid and can cause health issues
and economic loss (Chen et al., 2018; Lan et al., 2020). The intestine is
the first tract to interact with mycotoxins, and the intestinal toxicity of
mycotoxins has attracted attention. Many studies have identified
OTA-induced intestinal toxicity in vitro (Peng et al., 2019; Wang et al.,
2018), but there were few findings in vivo (Tong et al., 2020). Conse­
quently, in this study, we reported for the first time that mRNA and
ncRNA (lncRNA, miRNA, and circRNA) were related to OTA-induced TJ
protein disruption in the jejunum of Balb/c mice by using whole
transcriptome.
In general, our results have confirmed that OTA damaged the in­
testinal barrier function of Balb/c mice (Fig. 1). The concentrates of D-
lactate and I-FABP were significantly increased by OTA (P < 0.05). Once
the intestinal barrier injury occurred, a large amount of D-lactate and I-
FABP will be released into the blood, which can increase blood levels (Ji
et al., 2018). The epithelial monolayer along with the microstructure of
villi and crypts can control the entry of pathogens and exogenous sub­
stances (Sumigray et al., 2018). In the present study, we observed that
OTA injured the structure of villus and crypts, which was consistent with
Ruan’s results. The results indicated that the intestinal villi of ducks
became blunt, the epithelium exfoliated, the length of villi decreased,
and the depth of crypts was increased by OTA (Ruan et al., 2019). Mucin
is produced and secreted by goblet cells, OTA reduced the number of
goblet cells, suggesting that OTA impaired intestinal function through
decreasing goblet cells, which was consistent with previous studies
(Feng et al., 2019; Gao et al., 2017; Huang et al., 2019a; Wu et al., 2019).
TJ proteins are an important part of the intestinal barrier, and play a
barrier role in structure and function to prevent the paracellular
permeation of luminal substances (Tsukita et al., 2001; Gao et al., 2020).
In our study, TJ proteins deleteriously affected (expression and locali­
zation) by OTA, such as claudin-4, occludin, and ZO-1 (Fig. 2), which
was consistent with previous experimental results (Gao et al., 2017).
Compared with the control group, the protein expression of occludin and
TJ protein 1 in ducks fed with OTA were significantly decreased, and the
intestinal barrier was damaged (Ruan et al., 2019). Previous studies
have shown that OTA resulted in intestinal barrier dysfunction, though
the elaborate mechanism is still unknown. In the present study, a whole
transcriptome was first used to investigate the molecular mechanism of
OTA intestinal toxicity on Balb/c mice. Forty-three key genes were used
for PPI analysis (Fig. 3), most key genes were tightly shared in the WNT
signaling pathway. Yet, although Axin2, Lamc2, and Plau in the PPI
network did not belong to the WNT signaling pathway, they were also
WNT target genes (Tanigawa et al., 2011). The WNT signal is a chief
switch of self-renewal tissue homeostasis, responsible for sustaining
intestinal homeostasis, and intestinal epithelial cells (IECs) proliferation
and migration (Mah et al., 2016). WNT ligand binding to the FZD re­
ceptor can activate a variety of intracellular transduction molecules,
including β-catenin and Ca2+, which were activated through canonical
or non-canonical signaling pathways, respectively (Bhanot et al., 1996;
Slusarski et al., 1997). Additionally, researchers have shown that the
WNT signal was also related with the TJ protein (Xing et al., 2020; Yang
et al., 2021). In our study, Cacna1d and Cav1, found in the PPI network,
shared the release of Ca2+. We speculated that OTA regulated TJ protein
expression through the WNT/Ca2+ signaling pathway activated by the
binding of FZD4 and WNT5a. Wang et al. (2019c) showed that the
non-canonical WNT (WNT/Ca2+) signaling pathway mediated the
regulation of TJ proteins. Likewise, a previous study has also shown that
deoxynivalenol induced intestinal integrity damage and intestinal
X. Yang et al. Ecotoxicology and Environmental Safety 224 (2021) 112637

Fig. 4. The overview of the DEmiRNAs. (a) OTA changed the number of DEmiRNAs. (b) Venn analysis between different treatments of DEmiRNAs. (c,d) The GO and
KEGG annotation of the target genes. (e) Sankey diagram of key DEmiRNAs and target key DEmRNA network. (f) The DEmiRNA expression results (OTA/CTL)
compared with RNA sequencing.

8
X. Yang et al. Ecotoxicology and Environmental Safety 224 (2021) 112637

Fig. 5. The analysis of DElncRNAs. (a) The number of DElncRNAs after OTA treatment. (b) The Venn analysis of three comparison groups. (c,d) The GO annotation
and KEGG analysis of DElncRNA target mRNAs. (e) Sankey diagram of key DElncRNAs and the target key DEmRNA networks. (f) The DElncRNA expression results
(OTA/CTL) compared with RNA sequencing.

9
X. Yang et al. Ecotoxicology and Environmental Safety 224 (2021) 112637

Fig. 6. LncRNA-miRNA-mRNA ceRNA network of TJ protein in Balb/c mice. The ellipse represents mRNA, V shape represents miRNA, and the diamond repre­
sents lncRNA.

Fig. 7. The verification of Ca2+ in Caco-2 cells. (a) The changes of Ca2+ level after OTA treatment measured by calcium fluorescent probe Fluo-4 AM. (b,c) The effects
of OTA and BAPTA-AM on claudin-4/occludin expression by western blot analysis and quantification. Different letters (a-c) indicate significant differences
(P < 0.05). BA represents the BAPTA-AM.

10
X. Yang et al.
barrier dysfunction by activating the WNT β-catenin signal pathway
(Zhou et al., 2020).
MiRNA was considered to be a key element in maintaining and
improving intestinal barrier function (Lee et al., 2015). In our results,
miR-122, miR-146b, miR-203, and miR-1258 were found to regulate TJ
protein expression and intestinal barrier function (Fig. 4). Ye et al.
(2011) identified that miRNA-122 had a share in regulating intestinal
permeability. Another study indicated that miR-122a induced specific
mRNA degradation by binding to the nc region of occludin mRNA,
leading to occludin protein depletion (Zhang et al., 2017). MiR-146b has
been shown to regulate the growth and development of the intestinal
tract and IECs (Liao et al., 2013). This can improve the barrier function
of colonic midgut epithelium in dextran sodium sulfate treated mice
(Nata et al., 2013). MiRNA analysis revealed that miR-203 is one of the
differentially regulated miRNAs targeting TJ proteins (Veltman et al.,
2012). Compared with NCM460 normal colon epithelial cells, miR-1258
was down-regulated in tumor tissues and colorectal cancer cell lines and
regulated the expression of CCND1 (Zhang et al., 2018).
LncRNA also played a vital role in the intestinal barrier, especially
for constituent IECs (Jiang and Zhang, 2020). In our study, OTA induced
the changes of lncRNAs expression, including Zeb1, Phkb, Micu1, Mirt2,
Gm28588, and so on (Fig. 5). Micu1 maintained intestinal barrier
integrity by regulating oxidative stress and cell proliferation. After
Micu1 silencing, the integrity of the Caco-2 cell barrier was destroyed
with increased cell bypass permeability and the decreased expression of
TJ proteins (claudin-1, occludin, and ZO-1) (Leng et al., 2016). Mirt2 has
a protective effect on endotoxemia-induced mouse death by regulating
inflammation and recovering intestinal barrier function (Du et al.,
2017). In this literature, lncRNA Micu1 regulated the Mapk4, lncRNA
Mirt2 targeted to LPAR2, in which LPAR2 bore a role in the regulation of
the TJ protein (Shukla et al., 2020).
With circRNA/lncRNA as a decoy, miRNA as the center, and mRNA
as a target, there were two ceRNA regulation mechanisms, one for
circRNA-miRNA-mRNA and the other for lncRNA-miRNA-mRNA (Zhang
et al., 2019). In the present study, we did not find the key circRNA
related to TJ proteins and intestinal barrier (Fig S1), so we found the
related DElncRNAs and DEmiRNAs by constructing a
lncRNA-miRNA-mRNA ceRNA network (Fig. 6). Previous results showed
that the WNT signaling pathway played a key role in TJ protein
compromise induced by OTA. As the target gene of the WNT signal,
Axin2 was regulated by lncRNA Eya3, Tafld, C4a, and Tomm401 via
binding with mmu-miR-1981–5p and mmu-miR-1943–5p. CCND1, also
as the target gene of the WNT signal cascade, regulated by lncRNA phkb
targeted the miR-1258-x as a ceRNA, which was consistent with Zhang
et al. (2018). MiRNA lin-4-z, also found in the ceRNA network, con­
tained many lncRNA targeting sites, including Stk36, Trim65,
MSTRG.10317.1, Mir22hg, Ubxn8, MSTRG.12356.1, Ly6m, Gm43189,
MSTRG12259.3, and Rab11flip3. Lamc2 may be the downstream target
of all these DElncRNAs. Interestingly, Lamc2 was also the target of the
WNT signal, which was shown to be related to intestinal mucosal
inflammation (Coskun et al., 2017). As the ligand and receptor of the
WNT pathway, the combination of WNT5a and FZD4 will release Ca2+ to
activate the WNT/Ca2+ signal cascade reaction. Cav1, one of the classic
Ca2+ channels, has a role in the calcium-dependent signal transduction
pathway (Wheeler et al., 2012). It can target miR-205-z and miR-1258-x
by competing with lncRNA Gm28588, Tut4, and MSTRG.12902.1.
Ca2+ could be a common step in developing OTA-induced toxicity
(Ringot et al., 2006; Wang et al., 2018). Increasing the Ca2+ level was
related to the redistribution of occludin and ZO-1 from the intercellular
junctions of colonic epithelium, TJ protein disruption was in parallel to
the elevation of Ca2+ (Gangwar et al., 2017; Wang et al., 2018). In this
study, the results suggested the increase of Ca2+ level was involved in
decreasing the expression of the TJ protein (Fig. 7). Several studies have
demonstrated that the inhibitor of Ca2+ BAPTA-AM significantly
reversed the decrease of TEER, the increase of permeability, the decrease
of ZO-1 expression, and the interference of ZO-1 and occludin
11
Ecotoxicology and Environmental Safety 224 (2021) 112637
distribution induced by OTA (Wang et al., 2018).
Based on the in vivo whole transcriptome and in vitro verification, we
profiled an mRNA-miRNA-lncRNA ceRNA network to elucidate the
OTA-induced intestinal toxicity in Balb/c mice. This ceRNA network
revealed that OTA disrupted TJ proteins through the WNT/Ca2+
signaling pathway. We found that there were two upstream mecha­
nisms, (i) lncRNA Zeb1 regulates FZD4 binding with WNT5a to release
the Ca2+ by targeting miR-1258-x, and (ii) miRNA-1258-x regulated the
expression of Cav1, which controlled extracellular Ca2+ entry.
In conclusion, this study explored the molecular mechanism of OTA-
induced disruption of intestinal TJ proteins in Balb/c mice by using the
whole transcriptome combined with in vitro verification. OTA exposure
regulated the expression of mRNAs/ncRNAs in many aspects, resulting
in compromised TJ proteins and impairment of intestinal barrier func­
tion. The wide distribution of OTA exposure in the environment is a
serious threat to animals and human health. Our results will provide
strong data support for elucidating the intestinal toxicological mecha­
nism of OTA and reducing the threat induced by it. These results may
provide new evidence for improving our understanding of the hazards
and risks that OTA poses to public health and the environment.
CRediT authorship contribution statement
Xue Yang: Conceptualization, Methodology, Software, Investiga­
tion, Writing – original draft, Data curation, Writing – review & editing.
Gao Yanan: Conceptualization, Writing – review & editing, Validation.
Huang Shengnan: Investigation, Software. Su Chuanyou: Methodol­
ogy, Visualization. Zheng Nan: Conceptualization, Supervision, Project
administration, Writing – review & editing, Validation. Wang Jiaqi:
Conceptualization, Supervision.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This work was supported by the National Natural Science Foundation
of China (31972190), the Modern Agro-Industry Technology Research
System of the PR China (CARS-36); the Scientific Research Project for
Major Achievements of Agricultural Science and Technology Innovation
Program (CAAS-ZDXT2019004); and the Agricultural Science and
Technology Innovation Program (ASTIP-IAS12).
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.ecoenv.2021.112637.
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