Abstract
Lipid peroxides (LOOHs) abound in processed food and have been
implicated in the pathology of diverse diseases including gut,
cardiovascular, and cancer diseases. Recently, RNA Sequencing (RNA-seq)
has been widely used to profile gene expression. To characterize gene
expression and pathway dysregulation upon exposure to peroxidized
linoleic acid, we incubated intestinal epithelial cells (Caco-2) with
100 µM of 13-hydroperoxyoctadecadienoic acid (13-HPODE) or linoleic
acid (LA) for 24 h. Total RNA was extracted for library preparation and
Illumina HiSeq sequencing. We identified 3094 differentially expressed
genes (DEGs) in 13-HPODE-treated cells and 2862 DEGs in LA-treated
cells relative to untreated cells. We show that 13-HPODE enhanced lipid
metabolic pathways, including steroid hormone biosynthesis, PPAR
signaling, and bile secretion, which alter lipid uptake and transport.
13-HPODE and LA treatments promoted detoxification mechanisms including
cytochrome-P450. Conversely, both treatments suppressed oxidative
phosphorylation. We also show that both treatments may promote
absorptive cell differentiation and reduce proliferation by suppressing
pathways involved in the cell cycle, DNA synthesis/repair and
ribosomes, and enhancing focal adhesion. A qRT-PCR analysis of
representative DEGs validated the RNA-seq analysis. This study provides
insights into mechanisms by which 13-HPODE alters cellular processes
and its possible involvement in mitochondrial dysfunction-related
disorders and proposes potential therapeutic strategies to treat
LOOH-related pathologies.
Keywords: lipid peroxidation, gene expression, metabolism
1. Introduction
Dietary lipids, including vegetable oils, contain different quantities
of the most common dietary polyunsaturated fatty acid (PUFA), linoleic
acid (LA), in the form of triglycerides, which are hydrolyzed by bile
and lipases. This process releases large amounts of free fatty acids
(FFAs), often in millimolar quantities, that are absorbed by intestinal
cells [[32]1]. Depending on processing (e.g., deep frying), the
ingested lipids may contain varying quantities of peroxidized PUFAs and
their decomposition products [[33]2]. Dietary lipid peroxides (LOOHs)
are broken down in the gut, resulting in the production of other lipid
peroxidation products, such as epoxy ketones, and the release of
peroxidized fatty acids (FAs) that reach the enterocytes, where they
get absorbed [[34]3]. It has been demonstrated that dietary LOOHs from
overheated oils contribute to the presence of peroxidized FAs in the
lipoproteins [[35]4], which indicates that even though dietary LOOHs
undergo a set of enzymatic digestion, peroxidized FAs reach the
intestine and get absorbed.
LOOHs have been strongly linked to disorders of the digestive system,
including inflammatory bowel disease (IBD) and malignancy [[36]5].
Studies have demonstrated that while LOOH is efficiently absorbed by
the fully differentiated (Dif) intestinal epithelial cells and
transported in the lymph, it is poorly taken up by undifferentiated
cells [[37]3,[38]6]. In addition, it has been demonstrated that the
uptake of oxidized FAs by Dif Caco-2 cells is dependent on the presence
of brush borders and is comparable to the uptake of unoxidized FAs in
Dif Caco-2 cells [[39]3]. 13-hydroperoxyoctadecadienoic acid (13-HPODE)
is decomposed by cells rapidly into aldehydes, including
4-hydroxynonenal (4-HNE) and oxononanoic acid (ONA), which are
cytotoxic and cause the generation of reactive oxygen species (ROS).
Previous studies have shown that LOOH caused oxidative stress and the
loss of cellular integrity in the intestinal epithelium [[40]7].
Peroxidized fat consumption has also been reported to cause
pro-inflammatory changes in the intestine [[41]8,[42]9].
Iron/ascorbate-mediated production of LOOH in Caco-2 cells has been
shown to result in activation of NF-κB, which regulates inflammatory
processes [[43]10]. Dietary LOOHs have also been implicated in
cardiovascular disease [[44]3,[45]11]. Previous studies also showed
that peroxidized fat was carried in the chylomicrons [[46]12], which
was correlated with the peroxidized fat content in the diet
[[47]13]—these are believed to be re-packaged and distributed in the
lipoproteins. The presence of peroxidized fat in the chylomicrons has
been shown to increase the atherogenicity of dietary cholesterol
[[48]14] and promote the absorption of cholesterol [[49]15].
The Caco-2 cell line is a human intestinal epithelial cell line that is
derived from human colorectal adenocarcinoma [[50]16]. Under certain
cultivation conditions, Caco-2 cells differentiate into a cell
monolayer that possesses absorptive features and brush borders
resembling human enterocytes. This cell line has been widely used in
studies involving cellular uptake, transport, and metabolism of drugs
and food molecules, including lipids [[51]3,[52]15,[53]17]. Using the
Caco-2 cell line to study lipid transport and metabolism by intestinal
epithelium is less challenging than using in vivo animal models
[[54]18]. Previous research has revealed that sub-cytotoxic levels of
LOOH cause significant injury and mitogenic changes to Caco-2 cells,
whereas higher concentrations of LOOH promote cell death
[[55]19,[56]20]. LOOHs have also been suggested to induce redox
imbalance and disruption of intestinal epithelial turnover [[57]21].
Accordingly, the response of Caco-2 cells to LOOH depends on the amount
of LOOH to which intestinal cells are exposed. In addition, LOOH
reduced cell membrane fluidity and increased permeability in intestinal
epithelial cells [[58]22]; the latter of which is a reported effect in
patients with IBD [[59]23]. Changes in membrane fluidity and dynamics
could affect several cellular processes [[60]24], including lipid
absorption by enterocytes and secretion into lacteals [[61]25]. In
addition, LOOH appeared to induce DNA damage as well [[62]7].
A recently published study demonstrated similar results between Caco-2
cells treated with 13-HPODE and mice fed with 13-HPODE [[63]26], which
makes treatment of Caco-2 cells with 13-HPODE a good model of dietary
LOOH intake. Thus, in this study, we used Caco-2 cells to investigate
the effects of 13-HPODE, the most common dietary lipid peroxide, on the
metabolic processes, cellular pathways, and phenotype of the intestinal
epithelium. We generated gene expression profiles using RNA-seq to gain
insights into how Caco-2 cells respond to 13-HPODE. We used these data
to identify molecular mechanisms that may explain the contribution of
lipid peroxidation to health conditions and their potential role in gut
pathology. Here, we conducted RNA sequencing of Caco-2 cells treated
with a specific LOOH, 13-HPODE, a lipoxidase-derived product from LA,
since LA is the most abundant dietary PUFA [[64]27]. Bioinformatic
analyses of the generated RNA-seq data provided valuable information on
dysregulated genes and disrupted pathways in Caco-2 cells in response
to 13-HPODE, the most common dietary LOOH. We compared the results
obtained from 13-HPODE-treated cells with the gene expression profile
of Caco-2 cells treated with LA, which represented non-peroxidized
lipids. Treatment of Caco-2 cells with LA mimics the intake of
vegetable oils such as soybean and canola oils and nuts as LA is the
most common omega-6 PUFA in vegetable oils and in the Western diet
[[65]28]. In addition, comparable results were seen when intestinal
culture cells were treated with pure linoleic acid or lipase-digested
sesame oil [[66]29]. Validation of RNA-seq results was carried out
using qRT-PCR analysis, which showed consistent results with our
RNA-seq data. In future works, these investigations could provide
potential therapeutic strategies to treat diseases associated with the
consumption of peroxidized linoleic acid.
2. Materials and Methods
2.1. Cell Culture
Caco-2 cells were purchased from American Type Culture Collection
(ATCC) (Rockville, MD, USA). Cells were cultured in Dulbecco’s modified
Eagle’s medium (DMEM; Invitrogen, Carlsbad, USA) supplemented with 15%
fetal bovine serum (FBS; Invitrogen, Carlsbad, USA), 2 mM L-glutamine
(Invitrogen, Carlsbad, CA, USA), and 1% penicillin-streptomycin
(Invitrogen, Carlsbad, CA, USA). After attaining confluence, cells were
cultured in the same medium supplemented with 7.5% FBS and the same
concentration of other constituents. Confluent cells were trypsinized
using 0.25% Trypsin-EDTA solution (Thermo Fisher Scientific, Waltham,
MA, USA). Caco-2 cells were seeded in 6-well plates and experiments
were carried out on fully differentiated cells (Dif; Day-14).
2.2. Preparation of Lipid Peroxide
Stock solution of linoleic acid (LA) (Sigma #W338001-25G, St. Louis,
MO, USA) was prepared in ethanol, and LA (200 μM) in phosphate-buffered
saline (PBS; Invitrogen, Carlsbad, CA, USA) was prepared for LA
treatment of cells. 13-hydroperoxyoctadecadienoic acid (13-HPODE) was
freshly prepared in PBS as previously described [[67]9,[68]14,[69]30].
Briefly, LA (200 μM) in PBS (pH 7.4) was oxidized with the addition of
10 U soybean lipoxygenase (Sigma #L6632-1MU), which can be easily
miscible with the medium. Conjugated diene formation during oxidation
was monitored by scanning the absorption between 200 nm and 300 nm
using a spectrophotometer (Uvikon XL, Biotek Instruments, El Cajon, CA,
USA), using PBS as the reference. The conversion of linoleic acid into
its oxidized form was observed as an increase in the optical density at
234 nm. Peroxide content was determined using a leucomethylene blue
(LMB) assay [[70]31]. The whole solution of the freshly prepared
13-HPODE was filter-sterilized to reduce the risk of contamination and
used for the cell culture experiments within 2 h of preparation to
minimize spontaneous peroxide decomposition.
2.3. Treatment of Cells with 13-HPODE/LA
As previously described [[71]26], Dif Caco-2 cells were starved in
serum-free medium for 3 h prior to treatment. Cells were treated with
100 µM of either 13-HPODE or LA for 24 h. Untreated control cells were
maintained with equal amounts of PBS to match the treatments. After 24
h incubation, cells were rinsed with PBS and harvested into TRIzol for
total RNA isolation. Experiments were run in triplicate.
2.4. Total RNA Extraction and Quantification
Cells were lysed directly in the 6-well culture plates using TRIzol
reagent (Invitrogen, 15596026). Cell lysate was transferred to tubes
and chloroform was added. The samples were vortexed and incubated at
room temperature for 3 min then centrifuged at 12,400× g for 15 min.
The upper aqueous phase, containing RNA, was transferred into fresh
tubes. Isopropyl alcohol was added to the samples and centrifuged for
10 min at 12,000× g to precipitate RNA from the aqueous phase. Total
RNA was washed with ethanol at 7500× g for 5 min and air-dried for 2–3
min. RNA was resuspended in RNase-free water, then sample
concentration, purity, and quality were determined using a NanoDrop
spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), which
showed absorbance ratios of 1.8–2 at 260 nm and 280 nm. Any
co-extracted DNA was removed from RNA samples using the TURBO DNA-free
kit (Invitrogen, AM1907, Carlsbad, CA, USA), following the
manufacturer’s instructions.
2.5. RNA-seq Library Preparation and Sequencing
We isolated mRNA from total RNA samples using the NEBNext Poly(A) mRNA
Magnetic Isolation Module (New England Biolabs, E7490S, Ipswich, MA,
USA) with an approximate input of 500 ng of total RNA per sample.
RNA-seq libraries were prepared from mRNA samples using the NEBNext
Ultra II Directional RNA Library Prep kit for Illumina (New England
Biolabs, E7760S) according to the manufacturer’s instructions,
including NEBNext Sample Purification Bead cleanups to remove
unincorporated primers and adapters. Samples were fragmented for 15 min
at 94 °C to achieve a target fragment size of 200 base pairs (bp).
Indexed adaptors for Illumina sequencing (New England Biolabs, E7710,
E7730) were ligated to libraries through 8 cycles of PCR. Library
quality was assessed using High Sensitivity D1000 reagents (Agilent
Technologies, 5067-5585, Santa Clara, CA, USA) on a TapeStation 2200
instrument (Agilent Technologies, Santa Clara, CA, USA). Library
concentrations were determined using the NEBNext Library Quant Kit for
Illumina (New England Biolabs, E7630S) following the manufacturer’s
instructions. Three dilutions of each library were prepared (1:1000,
1:10,000, 1:100,000) and plated in triplicate on a 96-well qPCR plate
along with manufacturer-supplied standards (20 µL reactions).
Concentration data were used to ensure equimolar pooling across
libraries for multiplexing. The final library pool was checked for
quality using the High Sensitivity D1000 ScreenTape assay, which showed
good quality with a maximum peak size of 337 bp ([72]Figure S1), and
sent to GENEWIZ (South Plainfield, NJ, USA) for sequencing (HiSeq4000 2
× 150 bp). The number of sequencing reads ranged between 48 and 79
million reads with a mean quality score > 37.
2.6. Sequence Data Processing
The following pipeline was used to analyze the paired-end sequencing
reads. We used FastQC (version v0.11.7) to check the quality of reads,
which were all of good quality scores > 30, and the presence of
adapters or overrepresented sequences [[73]32]. Trimmomatic (version
0.36) was used to remove adapters and poor-quality bases [[74]33].
Hisat2 (version 2.1.0) was used to align the reads to a human reference
genome (Ensembl/Genome Reference Consortium Human Build 38, GRCh38)
[[75]34]; all samples showed overall read alignment rates > 90%. Then,
FeatureCounts (version 1.5.0) was run to count the number of fragments
mapped to a specific gene/exon [[76]35]. We used DESeq2 (version
1.30.0), an R-package which uses negative binomial distribution to
model read counts, to identify differentially expressed genes (DEGs;
adjusted p < 0.05) between different groups [[77]36]. Gene symbols were
used from the Ensembl database.
2.7. Enrichment Analyses
Differentially expressed genes were evaluated further using gene
ontology and enrichment analyses using the enrichGO function of the
clusterProfiler (version 3.18.0) R package [[78]37], and Generally
Applicable Gene-set Enrichment (GAGE, version 2.40.0; also R package)
[[79]38], respectively. Gene ontology (GO) analysis was carried out by
running the enrichGO function on the list of DEGs (adjusted p < 0.05)
(from DESeq2) in the treated cell group to identify enriched GO terms
including biological processes, molecular functions, and cellular
components. Pathway and gene set enrichment analyses (GSEA) were
conducted by running the gage function on the list of DEGs and their
log2 fold change scores. This generated a list of dysregulated Kyoto
Encyclopedia of Genes and Genomes (KEGG) pathways, their p values, and
their direction (downregulated or upregulated).
2.8. Validation via qRT-PCR
RNA-seq results were technically confirmed via qRT-PCR using the same
RNA samples used for the RNA-seq study. Fourteen representative DEGs
were chosen to perform qRT-PCR for 13-HPODE-treated cells and eleven
DEGs for LA-treated cells. SsoAdvanced^TM Universal SYBR^® Green
Supermix (BioRad, 1725271), 2.5 μM of primer (forward and reverse
each), and 0.1 ng of cDNA was used in a 10 μL PCR reaction. The
protocol was set as 95 °C for 30 s (initial denaturation), and 40
cycles of 95 °C for 15 s (denaturation) and 60 °C for 30 s
(annealing/extension). GAPDH was used as a housekeeping gene. Relative
mRNA expression levels were determined using the
[MATH: ΔΔ :MATH]
Ct method. A t-test was used to determine the statistical differences
between the treated and untreated groups and the data were presented as
means ± SD. Primer sequences are provided in [80]Table S1.
3. Results
We incubated Dif Caco-2 cells with 100 µM of 13-HPODE or LA for 24 h.
We chose these concentrations as the proximal intestine is exposed to
millimolar concentrations of FA and our preliminary results showed
little or no cytotoxicity [[81]3,[82]15]. The control (untreated) group
was maintained in PBS. Experiments were run in triplicate (details are
explained in the [83]Section 2). Following RNA extraction, processing,
and sequencing, differential gene expression and enrichment analyses
were carried out. Principal component analysis showed that replicate
samples demonstrated similarity in gene expression with respect to
treatment, and there was good separation between untreated and treated
groups ([84]Figure S2). The log ratio vs. mean average (MA) plots
([85]Figure S3) show that differentially expressed genes (DEGs; red
dots) with a large mean expression across untreated and treated cell
groups call for significance.
3.1. 13-HPODE-Treated Caco-2 Cells
3.1.1. Differential Gene Expression
Using DESeq2, we identified 3094 DEGs (adjusted p < 0.05) between
13-HPODE-treated cells and untreated cells ([86]Table S2); 1692 genes
were downregulated and 1402 genes were upregulated in 13-HPODE-treated
cells relative to untreated cells. Among the upregulated genes were
genes involved in lipid metabolism such as PLIN2, FABP1, CPT1A, and
PCK1, which are involved in PPAR signaling, which on one hand has shown
to exert anti-inflammatory effects in obesity, diabetes, and
cardiovascular disease [[87]39], but on the other hand, could have both
anti-proliferative and carcinogenic effects [[88]40]. CPT1A and ACADVL,
which are involved in mitochondrial beta-oxidation of fatty acids, and
PDK4 and PCK1, which have a role in lipid and glucose metabolism, were
upregulated. Induction of these genes may promote both fat metabolism
and gluconeogenesis. Genes involved in stress response, such as CREB3L3
and NDRG1, were also induced. Although we observed reduced expression
of glutathione peroxidases GPX1 and GPX7, there was upregulation of
GCLC, which is essential for glutathione (GSH) synthesis, which might
indicate increased GSH contents. Reduced glutathione peroxidase
activity has been linked with colon cancer [[89]41], cardiovascular
disease [[90]42], obesity, and insulin resistance [[91]43]. We also
observed upregulation of some nuclear factor erythroid 2-related factor
2 (Nrf2) target genes that play a role in the antioxidant defense
systems such as HMOX1, CAT, UGT2B4, and TXNRD1. Several solute carrier
(SLC) transporters were upregulated, such as SLC26A3, a chloride
transporter; SLC38A4, an amino acid transporter; and SLC5A3, a
myo-inositol transporter, indicating changes in substrate transport
across the cell membrane. In addition, SLC transporters have been
implicated in various diseases, including IBD and metabolic diseases
[[92]44,[93]45]. Other upregulated genes included COL7A1, which forms
fibrils between epithelial cell basement membrane and extracellular
matrix, and PDZK1, which regulates epithelial cell surface proteins and
is involved in cholesterol metabolism. Among the downregulated genes
were ODC1 and POLD2, which are important for polyamine synthesis and
DNA replication, respectively. GPCPD1, which is involved in
glycerophospholipid biosynthesis, and PTGES2, which is involved in
prostaglandin E synthesis, were also downregulated. 13-HPODE treatment
also caused the reduction of NOX1, a NADPH oxidase that generates ROS,
as well as DKK1, which is an inhibitor of Wnt signaling [[94]46].
[95]Figure 1 shows the top 50 DEGs in 13-HPODE-treated Caco-2 cells
compared to untreated cells.
Figure 1.
[96]Figure 1
[97]Open in a new tab
Differential gene expression between 13-HPODE-treated cells and
untreated control cells. Heatmap shows the top 50 differentially
expressed genes (DEGs; adjusted p < 0.05) in 13-HPODE-treated Caco-2
cells (14D.H1, 14D.H2, and 14D.H3) compared to untreated control cells
(14D.C1, 14D.C2, and 14D.C3). Green, upregulated; red, downregulated.
3.1.2. Gene Ontology
Gene ontology analysis was performed using the enrichGO function of the
clusterProfiler R-package. The results revealed the enrichment of
diverse biological processes (adjusted p < 0.05) involved in
translation, ribosome biogenesis, RNA processing, response to hypoxia
and oxidative stress, mitochondrial translation, and gene expression.
Purine nucleoside monophosphate, alcohol, and amino acid metabolic
processes ([98]Figure 2), as well as carbohydrate and lipid metabolic
processes (not shown), were also enriched due to 13-HPODE treatment.
Figure 2.
[99]Figure 2
[100]Open in a new tab
Biological process enrichment upon treating Caco-2 cells with 13-HPODE.
Top enriched gene ontology (GO) biological processes in
13-HPODE-treated differentiated Caco-2 cells relative to untreated
control cells (adjusted p < 0.05).
Among the enriched molecular functions (adjusted p < 0.05) in Caco-2
cells treated with 13-HPODE were ATPase, oxidoreductase, antioxidant,
electron transfer, RNA polymerase I, and helicase activities.
Additionally, coenzyme, carboxylic acid, heat shock protein, and
cadherin and chaperone binding functions were enriched ([101]Figure
S4).
The enriched cellular components (adjusted p < 0.05) included the
mitochondrial inner membrane, matrix, ribosome, and protein complex, as
well as ribosomal subunits, the spliceosomal complex, focal adhesion,
and the cell-substrate adherens junction ([102]Figure S5). The brush
border and apical plasma membrane were also enriched (not shown).
3.1.3. Pathway Enrichment Analysis
We used the gage R-package to perform pathway enrichment analysis
([103]Table 1). Among the top upregulated KEGG pathways (p < 0.05) in
13-HPODE-treated cells, relative to untreated cells, were steroid
hormone biosynthesis, bile secretion, and protein digestion and
absorption. We also observed upregulation of importantly relevant
pathways such as metabolism of xenobiotics by cytochrome P450, focal
adhesion, and PPAR signaling, but with slightly higher p values.
Table 1.
Pathway enrichment analysis in 13-HPODE-treated Caco-2 cells. Pathway
enrichment demonstrates upregulated and downregulated Kyoto
Encyclopedia of Genes and Genomes (KEGG) pathways in 13-HPODE-treated
Caco-2 differentiated cells relative to untreated cells.
KEGG Pathway (Upregulated) p-Value
[104]I00140 Steroid hormone biosynthesis 0.018
[105]I04976 Bile secretion 0.029
[106]I04974 Protein digestion and absorption 0.029
[107]I00980 Metabolism of xenobiotics by cytochrome P450 0.062
[108]I04510 Focal adhesion 0.069
[109]I03320 PPAR signaling pathway 0.199
KEGG Pathway (Downregulated) p-Value
[110]I03040—Spliceosome 0.0001
[111]I00240—Pyrimidine metabolism 0.0004
[112]I03008—Ribosome biogenesis in eukaryotes 0.0005
[113]I03013—RNA transport 0.002
[114]I00190—Oxidative phosphorylation 0.003
[115]I04110—Cell cycle 0.004
[116]I00230—Purine metabolism 0.006
[117]I03420—Nucleotide excision repair 0.009
[118]I03020—RNA polymerase 0.009
[119]I03050—Proteasome 0.014
[120]I03030—DNA replication 0.018
[121]I04120—Ubiquitin-mediated proteolysis 0.030
[122]I03410—Base excision repair 0.031
[123]I03010—Ribosome 0.034
[124]Open in a new tab
Among the downregulated pathways (p < 0.05) observed for
13-HPODE-treated cells, relative to untreated cells, were spliceosome,
RNA transport, ribosome biogenesis, and RNA polymerase pathways, as
well as purine and pyrimidine metabolism. Oxidative stress can cause
base modification and DNA damage [[125]47]. Pathways of cell cycle, DNA
replication, and excision repair appeared to be suppressed as well. The
proteasomal pathway involved in proteolytic degradation of
intracellular protein was also downregulated. The results also showed a
reduction in the oxidative phosphorylation pathway.
3.2. LA-Treated Caco-2 Cells
3.2.1. Differential Gene Expression
We identified 2862 DEGs (adjusted p < 0.05) in LA-treated cells
compared to untreated cells ([126]Table S3); 1179 genes were
upregulated and 1683 genes were downregulated in LA-treated cells. As
in 13-HPODE-treated cells, PDZK1 was induced. TTN, which has been
reported as a key component of intestinal epithelial brush borders
[[127]48], was also upregulated. Among the downregulated genes in
LA-treated cells were RGCC, a cell cycle regulator; CYTOR, a long
non-coding RNA that enhances proliferation; and CEACAM6, which is a
cell adhesion molecule that promotes tumor progression. HMGCS1,
involved in cholesterol biosynthesis; DDX47, involved in spliceosome
and ribosomal RNA processing; EIF5A, a translation elongation factor;
and ODC1 were suppressed as well. Reduction in the expression of these
genes may indicate reduced cell proliferation upon LA treatment. Genes
involved in the regulation of metabolic pathways were also
downregulated, such as INSIG1, which regulates lipid synthesis and
glucose homeostasis, and the RPIA gene, involved in carbohydrate
metabolism. In addition, genes that are involved in the regulation of
cellular fate and processes were suppressed, such as JUN, a
transcription factor that regulates gene expression; DUSP4, dual
specificity phosphatase of mitogen-activated protein kinase; and DKK1.
Other downregulated genes included PI3, a peptidase inhibitor
(antimicrobial); SLC2A1, a glucose transporter; and SLC20A1, a
phosphate transporter [[128]46]. [129]Figure 3 illustrates the top 50
DEGs in LA-treated Dif Caco-2 cells compared to untreated cells.
Figure 3.
[130]Figure 3
[131]Open in a new tab
Differential gene expression between linoleic acid (LA)-treated cells
and untreated control cells. Heatmap shows the top 50 DEGs (adjusted p
< 0.05) in LA-treated Caco-2 cells (14D.LA1, 14D.LA2, and 14D.LA3)
compared to untreated cells (14D.C1, 14D.C2, and 14D.C3). Green,
upregulated; red, downregulated.
3.2.2. Gene Ontology
Among the enriched biological processes in LA-treated cells relative to
untreated cells were ribosome biogenesis; rRNA and ncRNA processing;
small molecule, amino acid, and coenzyme metabolic processes;
ribonucleoprotein complex assembly; and mitochondrial translation and
gene expression. In addition, the response to decreased oxygen levels,
purine nucleotide biosynthesis, DNA duplex unwinding, anion transport,
and carbohydrate metabolic processes were enriched in LA-treated cells
([132]Figure 4).
Figure 4.
[133]Figure 4
[134]Open in a new tab
Biological process enrichment upon treating Caco-2 cells with LA. Top
enriched GO biological processes in LA-treated differentiated Caco-2
cells relative to untreated cells (adjusted p < 0.05).
Molecular functions enriched in LA-treated Dif Caco-2 cells included
organic acid transmembrane transporter activity, helicase, ligase, RNA
polymerase, and ATPase activities. Moreover, ribonucleoprotein complex,
cadherin, ATPase, coenzyme, organic acid, cell adhesion molecule, and
RNA binding were also enriched ([135]Figure S6).
Among the enriched cellular components in LA-treated cells relative to
untreated cells were preribosome, and organellar and mitochondrial
ribosomes. Mitochondrial outer/inner membrane and matrix, cellular and
organellar outer membrane, and envelop lumen components were also
enriched. In addition, complexes such as proteasome, spliceosome, and
endopeptidase complexes were enriched in LA-treated cells, as well as
focal adhesion and adherens junctions ([136]Figure S7). The brush
border and apical plasma membrane were also enriched (not shown).
3.2.3. Pathway Enrichment Analysis
Linoleic acid treatment of Dif Caco-2 caused upregulation (p < 0.05) of
metabolic processes including retinol, xenobiotic, and drug metabolism
by cytochrome P450, relative to untreated cells. The protein digestion
and absorption pathway was also upregulated.
Among the pathways downregulated (p < 0.05) in LA-treated cells,
relative to untreated cells, were ribosome biogenesis, spliceosome, RNA
transport, and RNA polymerase pathways. As in 13-HPODE-treated cells,
purine and pyrimidine metabolism, oxidative phosphorylation,
proteasome, cell cycle, and excision repair pathways were also
suppressed in LA-treated cells. Toll-like receptor signaling, which
activates innate immunity, was downregulated ([137]Table 2).
Table 2.
Pathway enrichment analysis in LA-treated Caco-2 cells. Pathway
enrichment demonstrates upregulated and downregulated KEGG pathways in
LA-treated Caco-2 differentiated cells relative to untreated cells.
KEGG Pathway (Upregulated) p-Value
[138]I00830—Retinol metabolism 0.010
[139]I04080—Neuroactive ligand-receptor interaction 0.017
[140]I00980—Metabolism of xenobiotics by cytochrome P450 0.017
[141]I00982—Drug metabolism—cytochrome P450 0.039
[142]I04974—Protein digestion and absorption 0.047
KEGG Pathway (Downregulated) p-Value
[143]I03008—Ribosome biogenesis in eukaryotes 0.0001
[144]I03040—Spliceosome 0.0004
[145]I03013—RNA transport 0.0007
[146]I00240—Pyrimidine metabolism 0.0009
[147]I00190—Oxidative phosphorylation 0.016
[148]I03020—RNA polymerase 0.017
[149]I03050—Proteasome 0.020
[150]I00230—Purine metabolism 0.037
[151]I04110—Cell cycle 0.038
[152]I03420—Nucleotide excision repair 0.043
[153]I04620—Toll-like receptor signaling pathway 0.045
[154]Open in a new tab
3.3. Differential Gene Expression between 13-HPODE-Treated and LA-Treated
Cells
We identified 291 DEGs (adjusted p < 0.05) between 13-HPODE-treated and
LA-treated cells ([155]Figure S8; [156]Table S4). We found that genes
involved in PPAR signaling showed higher expression levels in
13-HPODE-treated cells compared to LA-treated cells. Among those genes
were CPT1A, which is involved in FA oxidation; PLIN2, which coats lipid
droplets; ACSL5 and FABP1, which are involved in FA transport; HMGCS2,
which is involved in ketogenesis; and PCK1. Upregulation of the latter
gene and PDK4 could promote gluconeogenesis, as mentioned earlier.
These results might suggest that 13-HPODE is a more potent activator of
PPAR signaling than LA, although both oxidized and unoxidized LA have
been shown in previous studies to activate PPARs [[157]30,[158]49].
This also indicates a significant effect of 13-HPODE on glucose and
lipid metabolism and transport as PPAR signaling has been shown to
regulate metabolic processes including FA and glucose metabolism, as
well as cell proliferation and differentiation [[159]50,[160]51].
Additional genes involved in FA oxidation, including SLC25A20 and
ACADVL, were upregulated in 13-HPODE-treated cells compared to
LA-treated cells. On the other hand, G6PD, which plays a role in FA and
cholesterol biosynthesis, also showed higher expression in
13-HPODE-treated cells relative to LA-treated cells. Lower expression
of APOH, which has an atheroprotective role by inhibiting the uptake of
oxidized low density lipoprotein (LDL) [[161]52], was observed in
13-HPODE-treated cells relative to LA-treated cells. We observed
increases in the expression of genes involved in defense mechanisms in
13-HPODE-treated relative to LA-treated cells. Among these were
CREB3L3; ABCG2, which is a xenobiotic transporter that extrudes toxins
from cells; and CYP2B6 and AKR1B1, which metabolize xenobiotics and
aldehydes.
We observed increased expression of genes that play a role in
detoxification in LA-treated cells relative to 13-HPODE-treated cells.
Among these were EPHX2, which is involved in xenobiotic metabolism;
ADH6, which metabolizes alcohols and lipid peroxidation products; and
ALDH6A1, which plays a role in protective detoxification of aldehydes.
Sucrase-isomaltase gene, SI, which is involved in dietary carbohydrate
digestion and is expressed in the intestinal brush border, showed
upregulated expression in LA-treated cells relative to 13-HPODE-treated
cells. Interestingly, cell adhesion molecules CEACAM1/M6/M5, which are
considered biomarkers of tumor progression and metastasis, showed low
expression levels in LA-treated cells relative to 13-HPODE-treated
cells.
3.4. Validation of RNA-Seq Results
RNA-seq results were verified via qRT-PCR. Different gene sets were
selected for validation in each treatment according to the differential
gene expression and pathway analysis in the treated groups relative to
the untreated group. Fourteen representative DEGs were chosen to
perform qRT-PCR for 13-HPODE-treated cells and eleven DEGs for
LA-treated cells. Genes involved in PPAR signaling, as well as
mitochondrial beta oxidation, such as PLIN2, FABP1, and CPT1A, were
upregulated in 13-HPODE-treated cells relative to untreated cells
([162]Figure 5a,c). PDK4 and PCK1 genes, which play a role in the
regulation of lipid and glucose metabolism, were also upregulated in
13-HPODE-treated cells, suggesting enhanced gluconeogenesis in these
cells relative to untreated cells ([163]Figure 5d,e). CREB3L3,
inflammatory response gene, and BAAT, involved in bile acid synthesis,
were upregulated by 13-HPODE compared to untreated cells ([164]Figure
5f,g). 13-HPODE treatment was associated with increased expression of
COL7A1, a collagen type VII alpha 1 chain, and VIL1 genes ([165]Figure
5h,i), involved in focal adhesion and the brush border cytoskeleton,
respectively, which might indicate enhanced cell differentiation.
Aldo/keto reductase AKR1C2, which is involved in steroid hormone and
bile acid synthesis, was induced by 13-HPODE ([166]Figure 5j). CYP2B6
was also upregulated in 13-HPODE-treated cells ([167]Figure 5k).
13-HPODE reduced the expression of NADPH oxidase NOX1 ([168]Figure 5l).
Other genes with reduced expression when treated with 13-HPODE
treatment included DKK1 and RPP40, which are involved in Wnt signaling
and rRNA processing, respectively ([169]Figure 5m,n).
Figure 5.
[170]Figure 5
[171]Figure 5
[172]Open in a new tab
Quantitative Real-time PCR validation of RNA-seq results in
13-HPODE-treated cells (a–n). qRT-PCR of representative DEGs from
RNA-seq data. Relative mRNA expression of genes is presented in
13-HPODE-treated (HPODE) Caco-2 cells with statistical significance of
* p < 0.05 compared to untreated (control) cells. Results were
normalized to GAPDH.
The relative expression of PCK1 and DKK1 in LA-treated cells was
similar to that of 13-HPODE-treated cells when both were compared to
untreated cells ([173]Figure 6a,b). In addition, CYP2C9, a cytochrome
P450 mono-oxygenase; UGT2B4, a detoxifying UDP glucuronosyltransferase;
and COL7A1 were upregulated in LA-treated cells relative to untreated
cells ([174]Figure 6c–e). Reduced expression of RGCC and ODC1 could be
attributable to reduced proliferative potential and enhanced
differentiation ([175]Figure 6f,g). LA treatment led to downregulation
of INSIG1 and TOMM5 genes, which might affect metabolic processes and
mitochondrial function ([176]Figure 6h,i). DUSP4, as well as cell
adhesion molecule CEACAM6, was reduced in LA-treated cells as well,
which could be a protective response against tumor progression
([177]Figure 6j,k). These results are consistent with the RNA-seq data.
Figure 6.
[178]Figure 6
[179]Figure 6
[180]Open in a new tab
Quantitative Real-time PCR validation of RNA-seq results in LA-treated
cells (a–k). qRT-PCR of representative DEGs from RNA-seq data. Relative
mRNA expression of genes is presented in LA-treated (LA) Caco-2 cells
with statistical significance of * p < 0.05 compared to untreated
(control) cells. Results were normalized to GAPDH.
4. Discussion
LA, an essential PUFA consumed through the human diet, is an important
cellular component. It is a precursor of lipid peroxides including
hydroxyoctadecadienoic acid (HODE), the reduced form of 13-HPODE
[[181]53]. LOOHs have been linked to several pathological conditions
including cardiovascular disease, degenerative disease, and malignancy
[[182]54]. Diet is a major source of LOOH, and dietary peroxides are
absorbed, transported, and incorporated into lipoproteins that carry
these lipids into cells and tissues. We used mRNA sequencing to
investigate how the most common dietary LOOH, 13-HPODE, modulates the
gene expression profile in Dif Caco-2 cells, the results of which could
provide insights into changes in the physiological processes of cells
and the mechanisms by which 13-HPODE may contribute to disease.
Treating Caco-2 cells with 13-HPODE, a peroxidized LA, significantly
alters metabolic and signaling pathways, as well as different cellular
processes.
In the current study, we have demonstrated that 13-HPODE increased the
expression of aldo/keto reductases, such as AKR1C2, that are essential
for steroid hormone synthesis. Studies have reported the ability of the
intestine to synthesize steroid hormones [[183]55,[184]56]. Steroid
hormones have been shown in a previous study to play a role in
maintaining the intestinal epithelial barrier [[185]57]; on the other
hand, they may not only cause inhibition of T-cell response [[186]55],
but may also promote immune function [[187]58], which might contribute
to inflammatory disease. It has been reported previously that oxidized
LA can induce steroid hormone synthesis in rat and human adrenal cells
[[188]59,[189]60]. Upregulated aldo/keto reductases might indicate that
13-HPODE could be converted to aldehydic products in intestinal
epithelial cells and promote the detoxification of reactive carbonyl
species and the reduction of 4-HNE, which may enhance adaptive response
[[190]61]. In addition, oxidized LA metabolites have been linked to
obesity and shown to induce synthesis of steroids [[191]62]. Oxidized
LA may possess bile acid activity and the ability to form mixed
micelles with fat, which increases cholesterol solubilization and
absorption [[192]15]. Although bile secretion occurs in the liver, our
pathway analysis showed that this pathway was enriched in Caco-2 cells
treated with 13-HPODE. We found that the BAAT gene, which is
responsible for bile acid conjugation that enhances fat solubility, was
upregulated in 13-HPODE-treated cells. This could propose another
mechanism by which 13-HPODE increases lipid solubilization. A previous
study demonstrated that bile secretion in response to a high-fat diet
could injure the intestinal epithelium and that secondary bile acids
produced by intestinal flora may induce tumor formation [[193]63]; this
might be studied as an indirect mechanism by which LOOH could
contribute to malignancy. On the other hand, the BAAT gene was not
differentially expressed in LA-treated cells relative to untreated
cells. It is also worth mentioning that both 13-HPODE and LA treatments
enhanced the expression of APOB, apolipoprotein B, which is a major
component of chylomicron and LDL, along with A1CF, a complementation
factor of ApoB mRNA editing enzyme complex essential for the generation
of ApoB48, which promotes dietary fat absorption [[194]64].
Although upregulation of the PPAR signaling pathway upon treating cells
with 13-HPODE had a slightly higher p-value of 0.1, several downstream
genes involved in fatty acid and lipid homeostasis were among the top
differentially upregulated genes (adjusted p < 0.05) in
13-HPODE-treated cells relative to untreated cells. Among those were
PLIN2, which forms the lipid droplet coat, and FABP1, which may promote
cholesterol uptake and protect against oxidative stress caused by LOOH
[[195]65]. HMGCS2, which is involved in ketogenesis and has been found
to promote cellular differentiation of Caco-2 cells [[196]66], was also
upregulated. CPT1A and PCK1 were also among the differentially
upregulated genes of PPAR signaling which may indicate promoted fatty
acid oxidation, as well as gluconeogenesis. As oxidized LA is
considered a ligand that activates PPAR signaling, which plays multiple
roles ranging between metabolism, anti-inflammatory, and anti- and
pro-carcinogenic effects, PPAR signaling should be considered a
powerful form of crosstalk between 13-HPODE and peroxidized
lipid-related diseases and should be studied further. Moreover,
CREB3L3, a transcription factor that works with PPAR
[MATH: α :MATH]
in an auto-loop manner [[197]67] and is involved in unfolded stress
response and lipid metabolism, was upregulated in 13-HPODE-treated
cells. Although we observed upregulation of several genes involved in
PPAR signaling and FA oxidation, including PLIN2, PCK1, and CPT1A in
LA-treated cells, the expression of these genes appeared to be higher
in 13-HPODE-treated cells compared to LA-treated cells. This suggests
that 13-HPODE may have a more powerful effect on PPAR signaling and FA
oxidation than non-peroxidized LA. We also observed higher expression
of G6PD, required for FA and cholesterol biosynthesis, and a lower
expression of atheroprotective APOH in 13-HPODE-treated cells compared
to LA-treated cells, which could propose a mechanism by which LOOHs
contribute to the development of cardiovascular disease.
Our results indicate that 13-HPODE may enhance detoxification by
cytochrome P450 enzymes, which play a role in the metabolism of
ingested drugs and toxins, and in the synthesis of steroid hormones,
bile acids, and some other fats [[198]68,[199]69]. Upregulated
CYP1B1/2C9/2B6 may be a protective response against 13-HPODE treatment
as it is considered a harmful metabolite of oxidized LA. in addition to
aldo/keto reductases’ function in steroid hormone synthesis, they are
also involved in xenobiotic metabolism by cytochrome P450, as well as
bile acid synthesis and transport [[200]70,[201]71]. In the current
study, LA also induced cytochrome P450 enzymes, including CYP2C9.
Moreover, LA treatment resulted in higher expression of alcohol and
aldehyde dehydrogenases ADH6 and ALDH6A1 in LA-treated cells compared
to 13-HPODE-treated cells, which could indicate a protective effect of
LA against lipid peroxidation products, including aldehydes. This may
also suggest that LA could undergo oxidation in the intestinal cells to
which the cells respond by enhancing the detoxification mechanism.
As 13-HPODE comes in contact with the cell membrane, we could expect
oxidation of vitamin E (Tocopherol; TP) and the formation of tocopheryl
quinone (TQ), which is anti-androgenic [[202]72] and has an apoptotic
effect that has been reported to inhibit colon cancer cell growth
[[203]73]. Moreover, since we observed that 13-HPODE caused
downregulation of glutathione peroxidases GPX1 and GPX7 and
upregulation of the catalytic subunit of glutamate-cysteine ligase,
GCLC, essential for glutathione (GSH) synthesis, the cells could
accumulate GSH. The latter has previously shown to convert the
tocopheroxyl radical back to TP to maintain its scavenging effect on
reactive oxygen species (ROS) via oxidation of vitamin E, thus
preventing further lipid oxidation and inhibiting cell proliferation
[[204]74]. TP regeneration has been also linked to thioredoxin
reductase [[205]75], which was upregulated in 13-HPODE-treated cells.
TP has been shown to reduce chemokines in human aortic endothelial
cells (HAECs) [[206]76], and has been shown to reduce expression of the
chemokine CXCL1 [[207]77]. In our study, we observed downregulation of
CXCL1, CXCL8 (IL-8), and CCL20 chemokines in 13-HPODE-treated cells. It
has been suggested that tocopheryl hydroquinone (TQH) is a more
powerful antioxidant than TQ [[208]78,[209]79]. In our study,
upregulation of cytochrome p450 oxidoreductase (POR gene) [[210]78],
which is reported to catalyze the formation of TQH, was observed in
13-HPODE-treated cells. TQ has been shown to induce apoptosis via
activation of the caspase 3 cascade and to reduce anti-apoptotic Bcl-2
[[211]80]. In this context, we observed upregulation of CASP3 and a
number of Bcl family members that have apoptotic activity, such as BMF,
BCL2L11, and BNIP5 (this gene has not been studied yet), and
downregulation of the anti-apoptotic BAG1 gene in 13-HPODE cells. This
is consistent with a recently published study [[212]26] which
demonstrated reduced cell viability on annexin V staining of Caco-2
cells treated with 100 μM 13-HPODE.
Although we did not observe expression changes in members of activator
protein-1 (AP-1) or NFE2L2 (Nrf2), we did observe upregulation of some
target genes that play a role in the antioxidant defense systems,
including HMOX1, CAT, UGT2B4, and TXNRD1, as well as downregulation of
SOD1 in 13-HPODE-treated cells. This indicates that a degree of the
antioxidant protective response was enhanced by 13-HPODE. Moreover, we
observed increased expression of FOXO3 and FOXO4 transcription factors,
by which 13-HPODE could modulate insulin signaling and the antioxidant
response and could trigger apoptosis [[213]81]. In addition to CAT,
another FOXO-regulated gene, CDKN1A (p21), which inhibits cell
proliferation in response to DNA damage, was also upregulated in
13-HPODE-treated cells.
13-HPODE treatment caused the downregulation of events that have been
reported to be reduced during Caco-2 cell differentiation [[214]82].
Among the pathways downregulated due to 13-HPODE treatment were RNA
transport, spliceosome, and translation machinery. In parallel with
this, events of the cell cycle, DNA replication and repair, and protein
degradation, including the proteasomal pathway, were also suppressed.
Under culture conditions, Caco-2 cells undergo extensive genetic
reprogramming during differentiation and lose their tumorigenic
phenotype [[215]16]. This genetic reprogramming includes downregulation
of genes involved in cell cycle progression, DNA replication/repair, as
well as genes involved in RNA splicing/transport and protein
degradation, which indicates reduced proliferation; this was reported
by Mariadason et al. [[216]82]. Hence, downregulation of these events
upon treating the cells with 13-HPODE or LA, as we observed in the
current study, may suggest a further reduction in the proliferative
potential and a shift towards differentiation. Among downregulated
genes in both 13-HPODE-treated and LA-treated cells were POLD2, MCM7,
and PCNA, which are involved in DNA replication; CDC25A, which is
important for cell cycle progression; and RPP40 and EIF5A, which are
involved in RNA processing and translation. On the other hand, tumor
suppressor genes including APC, KLF4, and FOXO4 were upregulated in
13-HPODE-treated cells. In LA-treated cells, we observed downregulation
of the proto-oncogene, JUN, and upregulation of the tumor suppressor
genes KLF7 and FOXO4. Interestingly, LA treatment caused a reduction in
the expression of cell adhesion molecules CEACAM1/M5/M6, which are
considered biomarkers of tumor progression [[217]83]; this may also
indicate reduced proliferation. LOOH-derived oxidative stress can cause
base modification and DNA damage [[218]47] due to the formation of DNA
adducts via interaction between nucleotides and malondialdehyde (MDA),
hence the protective response of DNA synthesis reduction was required
to prevent further DNA damage by LOOH end-products [[219]84]. Despite
the various studies on LOOH-induced DNA damage, the mechanism by which
LOOHs reduce DNA synthesis needs to be further investigated.
In support of shifting towards differentiation, the focal adhesion
pathway, which is involved in cell motility, differentiation, and other
processes, was enhanced (p-value 0.06) in 13-HPODE-treated cells
relative to untreated cells. Among the related induced genes was
COL7A1. In the intestine, this pathway plays a role in epithelial
barrier homeostasis and repair and tight junction organization
[[220]85]. This may indicate enhanced cellular differentiation, which
promotes the development of brush borders, as evidenced by the
upregulated VIL1 gene in 13-HPODE-treated cells. In addition to the
upregulation of VIL1, the sucrase-isomaltase gene, SI, which is another
marker of intestinal differentiation [[221]86] showed elevated
expression in LA-treated cells compared to 13-HPODE-treated cells,
indicating enhanced cellular differentiation. Furthermore, we observed
upregulation of retinol metabolism in LA-treated cells, relative to
untreated cells, which has been shown to play a role in intestinal
epithelial cell processes including growth and differentiation
[[222]87]. In addition, UGT2B4, a detoxifying enzyme that plays a role
in the retinol metabolic pathway, was detected and upregulated in
LA-treated cells.
Our results showed a reduction in the oxidative phosphorylation pathway
in both 13-HPODE- and LA-treated cells relative to untreated cells.
Disrupted oxidative phosphorylation has been previously reported in
mice fed with oxidized linoleic acid [[223]88]. Moreover, mitochondrial
components including membranes and matrix were among the top enriched
GO terms in both 13-HPODE- and LA-treated cells, relative to untreated
cells, which indicates the significant effect of 13-HPODE and LA on
mitochondrial function. The TOMM5 gene, which is a translocase of the
outer mitochondrial membrane, was downregulated in both treatments. It
has been reported that PUFAs can cause a change in mitochondrial
membrane composition and organization that leads to increased
production of ROS, which in turn causes peroxidation of membrane
phospholipids and mitochondrial dysfunction [[224]89].
Toll-like receptor (TLR) signaling was downregulated in LA-treated
Caco-2 cells relative to untreated cells, which supports the previously
reported inhibitory effect of PUFAs on TLR activation and inflammatory
gene expression [[225]90]. Specifically, n-3 PUFAs appeared to be more
powerful inhibitors of TLR signaling than n-6 PUFAs [[226]91]. There
was a relative similarity in the data results between LA- and
13-HPODE-treated cells, suggesting the possibility of LA being oxidized
in the intestinal epithelium, which has been previously reported
[[227]92] ([228]Table 3).
Table 3.
Comparative differential gene expression. Comparison of differential
expression (↑ upregulation or ↓ downregulation) of selected genes in
the two treatment cell groups relative to the untreated cell group.
Gene 13-HPODE-Treated LA-Treated
DKK1 ↓↓ ^3 ↓↓
CPT1A ↑↑ ^3 ↑ ^2
PLIN2 ↑↑ ↑
ODC1 ↓ ^2 ↓
CREB3L3 ↑↑ ↑
FABP1 ↑↑ ↑
PDK4 ↑↑ ↑
PCK1 ↑↑ ↑
COL7A1 ↑↑ ↑
NOX1 ↓ ↓
AKR1C2 ↑↑ ↑
SOS1 ↑ ↑
BAAT ↑ n ^1
CYP2B6 ↑↑ ↑
CYP2C9 ↑ ↑
INSIG1 n ↓
DDIT4 ↑ ↑
DUSP4 ↓ ↓↓
UGT2B4 ↑ ↑
TXNRD1 ↑ n
HMOX1 ↑ n
GSTP1 ↓ n
CXCL1 ↓ ↓
GPX1 ↓ ↓
FOXO4 ↑ ↑
CEACAM6 ↓ ↓↓
POLD2 ↓ ↓
ADH6 n ↑
[229]Open in a new tab
^1 n represents not differentially expressed (adjusted p > 0.05). ^2
Single arrow (↑, ↓) represents differentially expressed (DE; adjusted p
< 0.05). ^3 Double arrows (↑↑, ↓↓) represent DE with more pronounced
effect.
5. Conclusions
The results of our transcriptomic profiling of Caco-2 cells carried out
under standard in vitro conditions shed light on the response of
intestinal epithelial cells to 13-HPODE or LA in terms of gene
expression and pathway enrichment. The results presented in this study
suggest that the most common dietary peroxidized lipid, 13-HPODE, may,
on the one hand, enhance bile acid conjugation, which alters lipid
uptake and might contribute to intestinal injury. On the other hand,
13-HPODE may affect multiple pathways of lipid metabolism including
steroid hormone synthesis, which might affect intestinal barrier and
immune function; and PPAR signaling, which alters fatty acid and
glucose metabolism, energy production, and mitochondrial function
leading to alteration of gut physiology. In addition, 13-HPODE as well
as LA may have the ability to promote the antioxidant response and
detoxification by cytochrome P450 in intestinal epithelial cells.
Moreover, both treatments could reduce proliferative potential and
could play a role in the absorptive cell differentiation and survival
fates as they might suppress pathways involved in the cell cycle, DNA
synthesis/repair, and enhance focal adhesion pathways. While similar
effects could be seen in liver cells, it is conceivable that dietary
peroxidized LA could have an impairment effect on energy production and
lipid storage. In conclusion, this in vitro study using Caco-2 cells
provides insights into the physiological changes that might occur in
the intestinal epithelium upon exposure to 13-HPODE and the possible
mechanisms by which it contributes to disease. Future directions of
this research include studying the response of Caco-2 cells to LOOH
using an experimental setting that simulates in vivo intestinal
environment, such as the INFOGEST method [[230]93].
Acknowledgments