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This article summarizes recent findings on the role of microRNAs (miRNAs) in biological processes associated with the regulation of chronic inflammation and autoimmunity. miRNAs are small non-coding RNA molecules that have been recently emerged as a new class of modulators of gene expression at the post-transcriptional level. MiRNAs bind to complementary sequences of specific targets of messengers RNA, which can interfere with protein synthesis. We reviewed studies that evaluated the expression patterns of miRNAs in different autoimmune diseases, especially in celiac disease (CD). CD is a chronic enteropathy triggered by gluten proteins, characterized by altered immune responses in genetically susceptible individuals that results in damage to the bowel mucosa. CD has a high prevalence and an effective treatment by a specific diet ("gluten free diet"). Genetic factors confer susceptibility but do not explain the whole disease, suggesting that environmental factors do playa relevant role in the development of the condition.The evaluation of the potential role of miRNA is of particular interest in CD given that these epigenetic mechanisms in the pathogenesis of autoimmune and inflammatory diseases have been recently described. Improving our understanding of miRNAs in CD will contribute to clarify the role of altered epigenetic regulation in the development and course of this disease.
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REVIEW
MicroRNAs: An epigenetic tool to study celiac disease
Karla A. Bascuñán-Gamboa1, Magdalena Araya-Quezada2 and Francisco Pérez-Bravo1
1Laboratory of Nutrigenomics. Department of Nutrition. Faculty of Medicine. University of Chile. Santiago, Chile.
2Laboratory of Immunogenetics. Institute of Nutrition and Food Technology (INTA). University of Chile. Santiago, Chile
1130-0108/2014/106/5/325-333
Revista española de enfeRmedades digestivas
CopyRight © 2014 aRán ediCiones, s. l. Rev esp enfeRm dig (Madrid
Vol. 106, N.º 5, pp. 325-333, 2014
REVIEW
ABSTRACT
This article summarizes recent findings on the role of
microRNAs (miRNAs) in biological processes associated with the
regulation of chronic inflammation and autoimmunity. miRNAs
are small non-coding RNA molecules that have been recently
emerged as a new class of modulators of gene expression at
the post-transcriptional level. MiRNAs bind to complementary
sequences of specific targets of messengers RNA, which can
interfere with protein synthesis. We reviewed studies that
evaluated the expression patterns of miRNAs in different
autoimmune diseases, especially in celiac disease (CD). CD is a
chronic enteropathy triggered by gluten proteins, characterized
by altered immune responses in genetically susceptible individuals
that results in damage to the bowel mucosa. CD has a high
prevalence and an effective treatment by a specific diet (“gluten
free diet”). Genetic factors confer susceptibility but do not explain
the whole disease, suggesting that environmental factors do play
a relevant role in the development of the condition.
The evaluation of the potential role of miRNA is of particular
interest in CD given that these epigenetic mechanisms in the
pathogenesis of autoimmune and inflammatory diseases have been
recently described. Improving our understanding of miRNAs in CD
will contribute to clarify the role of altered epigenetic regulation in
the development and course of this disease.
Key words: Celiac disease. miRNAs. Epigenetics. Autoimmunity.
ABBREVIATIONS
ADN: Deoxyribonucleic acid.
AID: Autoimmune diseases.
CD: Celiac disease.
DM1: Diabetes mellitus type 1.
GWAS: Genome-wide association study.
HLA: Human leucocyte antigen.
IBD: Inflammatory bowel disease.
IgA: Immunoglobulin A.
IL-1β: Interleukin 1β.
INF γ: Interferon gamma.
ME: Multiple sclerosis.
miRNAs: MicroRNAs.
mRNA: Messenger RNA.
NFκB: Nuclear factor kappa B.
Pre-miRNA: Premature miRNA.
Pri-miRNA: Primary miRNA.
RNA: Ribonucleic acid.
RNAase: RNA polymerase.
SLE: Systemic lupus erythematosus.
SNP: Single nucleotide polymorphism.
T reg: Regulatory T cells.
TG2: Tissue tranglutaminase 2.
TNF α: Tumor necrosis factor-α.
tTG: Tissue anti-transglutaminase.
UC: Ulcerative colitis.
UTR: Untranslated region.
INTRODUCTION
Celiac disease (CD) is a gluten-sensitive enteropathy
mediated by the immune system and considered one of
the more complex genetic diseases. The rate of concord-
ance in monozygotic twins is about 75 % (1,2). The hap-
lotypes HLA (human leucocyte antigen) DQ2/DQ8 con-
fer the highest estimated heritability reported so far (close
Received: 26-11-2013
Accepted: 10-03-2014
Correspondence: Francisco Pérez Bravo. Laboratory of Nutrigenomics.
Department of Nutrition. Faculty of Medicine. University of Chile. Avenida
Independencia 1027, Independencia. Santiago, Chile
e-mail: fperez@med.uchile.cl
Bascuñán-Gamboa KA, Araya-Quezada M, Pérez-Bravo F .
MicroRNAs: An epigenetic tool to study celiac disease. Rev Esp
Enferm Dig 2014;106:325-333.
326 K. A. BASCUÑÁN-GAMBOA ET AL. Rev esp enfeRm Dig (maDRiD)
Rev esp enfeRm Dig 2014; 106 (5): 325-333
to 35 %) (3). Exposure to gliadin, a constitutive gluten
protein is the main factor associated with the onset of
symptoms of CD, triggering an altered immune response
in patients carrying the risk HLA haplotypes. However,
presence of these HLA haplotypes is a necessary but not
sufficient condition for the appearance of CD. In fact,
approximately 30 to 40 % of healthy subjects carry HLA
alleles of risk and only about 1 % of the population devel-
ops the disease (4,5). As for genetic factors not associ-
ated with HLA, several genome-wide association studies
(GWAS) have described several susceptibility loci, each
of which is associated with risk of developing CD (6), but
as a whole they explain only a small proportion of the risk
of CD. An interesting field related to this pathology is the
role of epigenetic regulation mechanisms, an area that has
been scarcely investigated until now.
The microRNAs (miRNAs) are small (between 20 to 25
nucleotides) non-coding ribonucleic acid (RNAs) that regu-
late gene expression through the canonical base pairing of
complementary sequences in the 3’-untranslated region
(UTR) of the target messenger RNA (mRNA) (7). Since its
identification in 1993 (8) it has been demonstrated miRNAs
are important in both physiological and pathological condi-
tions (9). They are involved in the regulation of gene expres-
sion in a variety of biological processes, such as autoimmune
disorders (10) and the development and function of mature
immune cells (11,12). In this article we will discuss the basic
concepts of CD and review the current evidence on the par-
ticipation that miRNAs may have in the modulation of gene
expression in autoimmunity, with special emphasis on CD.
CELIAC DISEASE
In the last few decades the concept of CD has substan-
tially changed. The use of plasma anti-endomysial and
anti-tissue transglutaminase (tTG) antibodies allowed an
active search of affected individuals among less sympto-
matic individuals. Historically, the disease was diagnosed
on the basis of small bowel biopsies showing intense
mucosal damage. It was considered a gastrointestinal
disease mainly affecting children, with a low frequency
in adults. However, studies in the last few decades have
revealed that CD affects equally to adults, being even more
frequent than in pediatric population (13,14).
Clinical pediatric presentations of CD typically appear
during the first years of life, after the introduction of gluten
in the diet. Among these, the most frequent is the so-called
“classic” clinical presentation, characterized by prominent
digestive disorders, especially diarrhea with malabsorption
syndrome, weight loss, and nutritional deficiencies (15). In
the adult, CD frequently appears with less gastrointestinal
symptoms, absence of malabsorption syndrome and less
intense flattening of the villi in the small intestinal epithe-
lium. In “atypical” presentations extra-intestinal symptoms
and associated diseases (usually autoimmune diseases) are
the more prominent features leading to diagnosis, a fact
that for a long time made difficult their identification and
diagnosis (16,17). A study that evaluated differences in
clinical manifestations at the time of diagnosis in a group
of patients diagnosed during childhood versus adulthood,
showed that in the former classic forms of the disease
clearly predominated, with a high positivity of serologi-
cal markers and flattening of the mucosa, which helped
reaching diagnosis in shorter times. In contrast, in patients
diagnosed at adult ages, atypical forms were more frequent
and histological lesions more variable and less intense;
also, associated autoimmune diseases (AID) were frequent
and diagnosis was delayed (18). Currently, several preva-
lence data come from studies based on the serum levels
of antibodies IgA- anti-tTG (IgA-tTG). When CD diag-
nosis is confirmed by small intestinal biopsy in seroposi-
tive individuals, the frequency of CD decreases, but is still
much greater than previously known, ranging from 1:70
to 1:500 inhabitants (19). This may be due to the fact that
these antibodies identify individuals affected mainly by
the autoimmune components of the disease, which often
coincides with less intense digestive symptoms.
Serological diagnosis of CD, i.e., diagnosis based solely
on positive serological tests, is not currently accepted. An
exceptional situation is recognized by the European Soci-
ety for Pediatric Gastroenterology, Hepatology, and Nutri-
tion (ESPGHAN) (20); they indicate that in small children,
shown the classical presentation, are intensely symptomatic
and their IgA-tTG levels is greater than 100 U/ml, the small
intestinal biopsy is not necessary to diagnose the condition.
In all other cases, the small bowel biopsy continues being
required to confirm the diagnosis of CD (21).
Thus, today we understand that CD is a frequent disease
affecting approximately 1 % of the population (22), both
children and adults, that can appear in any time in life, and
that has a relevant genetic and autoimmune component
(22). Currently, one of the main challenges for physicians
and researchers is to understand the interactions between
genetic factors, the environment and the immune system,
such that we can understand the huge clinical variability.
In relation to genetics, the failure to explain the disease
completely has led to study the entire genome and, on the
other hand, epigenetic is receiving increasing attention as
an interesting field that may shed light in this complex situ-
ation. It is for this reason we will be review this topic, giv-
ing especial attention to miRNAs, an interesting epigenetic
mechanism appears as an important tool for understanding
gene regulation and their alterations, which are proving to
be involved in several autoimmune diseases and might be
involved in the onset and course of CD.
Physiopathology
CD is a chronic autoimmune-mediated enteropathy
which develops in the small intestine triggered by gluten
Vol. 106, N.º 5, 2014 microRNAs: AN EPIGENETIC TOOL TO STUDY CELIAC DISEASE 327
Rev esp enfeRm Dig 2014; 106 (5): 325-333
proteins present in wheat, rye, and barley in genetically
susceptible individuals. As a result, damage develops in the
small intestinal mucosa. Their typical histological features
are an increase in intraepithelial lymphocytes, flattening
of the intestinal villi, crypt hyperplasia and an important
infiltration of inflammatory cells in the lamina propria.
A gluten-free diet usually results in a rapid recovery of
the intestinal mucosa which is accompanied by a signifi-
cant improvement in the absorption of several nutrients.
To date, this dietary treatment is an efficacious treatment
for a large majority of patients, who show evident clinical
improvement within a few weeks (23).
In CD, CD4 T cells play a major role in the initiation
and organization of the altered immune response (24). A
model proposed to understand the pathogenesis of CD
involves luminal, epithelial and mucosal events, includ-
ing the activation of T lymphocytes (25). Among luminal
events, gluten digestion is the primary feature. Gluten is
digested to peptides in the intestinal lumen; due to lack
of prolyl endopeptidases in the intestinal villi, gastric and
pancreatic secretions, after initial digestion residual (rela-
tively large) gluten peptides rich in proline and glutamine
may remain for long periods of time in the lumen, favoring
the contact of these peptides and the epithelium. However,
in 99 % of individuals, including those that carry HLA-
DQ2 and HLA-DQ8 (the susceptibility alleles for CD),
this does not represent a problem or an increased risk for
CD (25).
Routes through which gluten passes through the epi-
thelium are not fully understood yet, but there is some
evidence suggesting that it could occur via paracellular
transport, passage through transepithelial transport (trans-
cytosis) (26), or by protrusions of dendritic cells that sense
the luminal content. Partially-digested gluten peptides
reach then the antigen-presenting cells present in the sub-
epithelial region of the small intestine. An acute infection
or inflammatory states of the small intestinal mucosa of
different origins would set the conditions for mucosal T
cells response to gluten peptides. With a Th1 type response
established in the mucosa, gluten peptides preferentially
binds to HLA-DQ2 or HLA-DQ8 and activates T-cells,
starting the production of Th1 cytokines. Then, release of
IFN-γ and other cytokines perpetuate this response, alter-
ing intestinal permeability and leading to the activation
and release of metalloproteinases that will be responsible
for the collapse of the small intestinal architecture (27).
Genetic
CD is a polygenic disorder that involves HLA and non-
HLA genes. The best known risk factors are encoded in
the HLA class II molecules, HLA DQ2 and HLA DQ8,
known as susceptibility factors. About 90 and 10 % of
individuals with CD are carriers of these heterodimers,
respectively (28). Deamidated gliadin peptides have a
high binding affinity for HLA DQ2 and DQ8 molecules,
explaining the immunogenicity of gluten in susceptible
individuals (29). Tissue transglutaminase (TG2) is a
highly ubiquitous enzyme that deamidates gluten pep-
tides after crossing the intestinal epithelium (30). This
process introduces a negative charge in a favorable posi-
tion, eliciting the attachment of gluten peptides to HLA
DQ2 and DQ8 molecules and as a consequence, increas-
ing gluten-specific CD4 T cells.
In order to improve our understanding of the genet-
ics of no-HLA genes in CD, genes have been exten-
sively assessed through genome-wide association studies
(GWAS). This technique provides a general approach that
permits identifying genes and pathways involved in a par-
ticular phenotype. The first GWAS on CD was carried out
in England (2007) and included 778 patients with CD and
1,422 controls (31). Results showed that, in addition to
HLA regions, 13 regions in the genome were associated
with CD. The majority of the identified regions contained
genes that control immune responses; of them it is worth
mentioning locus IL-2 and IL-21 in chromosome 4q27.
IL-2 is a critical cytokine for T cells homeostasis and func-
tion, and IL-21 is a new member of the superfamily of
type 1 cytokines that regulates many other immune cells.
These finding suggested, for the first time the potential
role of IL-2 and IL-21 in the pathogenesis of CD (31).
The same GWAS study also revealed the phenomenon
of pleiotrophy, indicating that genetic variants associated
with CD are also involved with other immunity-related
diseases (32). A second GWAS study, which included more
than 4,500 patients with CD and near 11000 controls from
four different populations (England, Italy, Finland and the
Netherlands) identified 13 new genome regions associated
with CD; the majority of the identified genes were found
to have immune functions and an important regulatory role
in the selection of the T-cells in the thymus, for example
THEMIS/PTPRK (33,34).
Studies on genetics of AID have been focused on genes
encoding proteins, aimed at finding a single nucleotide
polymorphism (SNP) that alters both the sequence of pro-
teins and their function. However, one of the more surpris-
ing findings indicates that only three (MMEL1, SH2B3 and
IRAQ1) of the 57 SNPs identified affect protein sequences
in celiac individuals (34). Several SNPs have been identi-
fied in intronic and intergenic regions of the genome, sug-
gesting that the transcriptional regulation of these genes
could be affected by SNPs of risk for CD.
There are several ways through which epigenetic
mechanisms could exert an effect on gene transcription;
for example, by altering or creating binding sites for tran-
scription factors or by modifying binding sites of protein
complexes that regulate chromatin, translating into an
epigenetic effect on the methylation of DNA and his-
tones modification (35,36). Also, the association of CD
with genes that affect 3’ UTR sequences could lead to a
decreased stability or increased degradation of the respec-
328 K. A. BASCUÑÁN-GAMBOA ET AL. Rev esp enfeRm Dig (maDRiD)
Rev esp enfeRm Dig 2014; 106 (5): 325-333
tive mRNA. On the other hand, they could promote the
inhibition of protein translation to alter binding sites of
RNAs or affect binding sites to miRNAs (6).
EPIGENETICS
Epigenetics refers to inheritable changes of genes func-
tion, which do not imply a change in the deoxyribonucleic
acid (DNA) sequence (37). Heritability implies that an epi-
genetic marker has the ability to persist during develop-
ment and that is potentially transmitted from generation to
generation (38). The main role of epigenetics is gene regu-
lation. This is a key process in: a) Modulation of individual
genes and their activity; b) regulation of groups of genes
that are functional in each specific cell type and in the
development and differentiation of cell types; and c) the
cellular metabolic plasticity, a characteristic that confers
an adaptation capacity to exogenous changes. There are
different epigenetic mechanisms regulating gene expres-
sion, either activating or suppressing it. Examples include
DNA methylation, histone modification, the positioning
of nucleosomes and miRNAs. It is important to note that
these mechanisms may act jointly in the regulation of gene
expression (39). Epigenetic is rising as a powerful tool to
the study of AIDs, conditions in which there is a strong
genetic component but where genetics does not fully
explain the disease.
CD has a high prevalence and genetic factors explain
many but not all cases. HLA-DQ2/DQ8 risk alleles
are the major and best known susceptibility factors.
Research on non-HLA genes has not been successful
and continues under study. In this sense, epigenetics
appears as an important tool that may contribute to the
understanding of the pathogenesis of CD. In the follow-
ing sections, we reviewed the current evidence regard-
ing miRNAs, one of the four best known mechanisms
used in epigenetics. New evidence is appearing referred
mostly to AID others than CD, such as rheumatoid
arthritis, psoriasis, type 1 diabetes (DM1), systemic
lupus erythematosus (SLE), multiple sclerosis (MS)
and inflammatory bowel disease (IBD). In all them gene
expression of several miRNAs has been assessed and
changes have been demonstrated.
microRNAs
miRNAs are a class of endogenous single-stranded
RNAs, small and non-coding RNA that regulate gene
expression through the control of stability and transla-
tion of the mRNA (40-42). Some of the biological pro-
cesses regulated by miRNAs include cell differentiation,
proliferation, apoptosis and cell cycle control (42-44).
The transcription of genes of a primary miRNA (pri-
miRNA) is carried out initially by the RNA polymerase
(RNAase) II or III in the nucleus. Then, a microproces-
sor complex, composed of the protein Drosha and their
associated proteins (DGCR8/Pasha) process the pri-
miRNA precursor to a mother strand (of approximately
70 nucleotides) generating a premature miRNA (pre-
miRNA) (45-47). Pre-miRNAs are exported to the cyto-
plasm where they are split into a 21 nucleotide miRNA
double strand by the enzyme RNAse III Dicer, together
with its associated protein TRBP (transactivator RNA
binding protein). Then, one strand is included within
the RNA-induced silencing complex (RSIC) guiding
this complex to the non-translatable 3’ region of the
sequences of the target mRNA, inducing the degrada-
tion of the mRNA by suppressing the expression of the
protein (48,49) (Fig. 1).
MiRNAs bind to their mRNA targets through matching
bases of RNA-RNA. The sequence of union of an miRNA
to the target mRNA is generally known as the Element
of Recognition of miRNA (ERM) (50). The mechanism
used by miRNAs to silence the mRNA remains unclear.
However, it is now known that a complementary strand
of miRNA of at least 6 nucleotides is sufficient to carry
out the post-translational regulation associated with the
miRNAs. A single miRNA could act on several hundreds
of target mRNAs and each mRNA can be the target for
many miRNAs. For this reason, several degrees of interac-
tions have been proposed, including degradation of pro-
teins, inhibition of the elongation of translation, the early
termination of translation, or inhibition of the initiation of
translation (51). If the matching degree between miRNA
and the target mRNA is partial, the translation of the target
mRNA is repressed, without affecting the level of mRNA.
However, in cases where there is a perfect or extensive
matching degree, the target mRNA is deadenilated and
destabilized by endonucleotidic excision and therefore pro-
teins synthesis stops by degradation of the target mRNA
in the cell (52).
miRNAs IN THE CONTROL
OF AUTOIMMUNITY
Since the discovery of the miRNAs, several studies have
shown that they could play a role in AIDs (10,53). It is well
known that miRNAs are involved in the development of
immune cells and in controlling of their functions. To date,
miRNA have been associated with various pathological
conditions of the immune system. Recent studies reveal
that the miRNAs regulate not only the development of
innate or adaptive immunity cells but also the critical bal-
ance of this response (54).
The altered miRNAs expression in some inflamma-
tory diseases and AID has been extensively studied, as
they could affect key transcripts in the development of
these conditions (55). It has been described that miR-155
and miR-146 are overexpressed in synovial fibroblasts of
Vol. 106, N.º 5, 2014 microRNAs: AN EPIGENETIC TOOL TO STUDY CELIAC DISEASE 329
Rev esp enfeRm Dig 2014; 106 (5): 325-333
patients with rheumatoid arthritis compared with healthy
subjects. The expression of miR-155 is increased by pro-
inflammatory molecules such as tumor necrosis factor-
alpha (TNFα) and interleukin-1beta (IL-1β), determining
an inhibitory effect on the expression of metalloproteins
in synovial fibroblasts (56). On the other hand, miR-146,
whose function is inhibiting the nuclear factor Kappa B
(NF-κB), is up regulated by pro-inflammatory cytokines
(57,58).
In patients with psoriasis, miR-21 and miR-146a have
been reported to be overexpressed in skin samples while
the expression of miR-125b was diminished in comparison
with controls (59). There are a few studies of miRNAs
expression in DM1; they suggest that regulatory T cells
(T regs) function is influenced by changes in the expres-
sion of specific miRNAs. In T regs of diabetic patients
there was increased expression of miR-510 and decreased
expression of miR-342 and miR-191, miRNAs with a still
unknown function. Other studies have shown that miRNAs
could have a cytokines mediated cytotoxic effect on pan-
creatic β cells. When IL-1B and TNF-α induce miR-21,
miR-34a and miR146a expression in pancreatic islet cells,
the increased production of pro-inflammatory cytokines
would lead to β-cell failure (60,61).
The majority of genes associated with SLE contain at
least one target site for miRNAs. For example, miR-146a
is a negative regulator of Toll-like receptors and its expres-
sion is decreased in patients with SLE. Also miR-146a, a
negative regulator of the signaling pathway of INF type I,
acts regulating the interferon regulatory factor-5 (IRF-5)
and signal transducers and activators of transcription pro-
tein (STAT-1). Therefore, a decreased expression of miR-
146a in peripheral blood mononuclear cells could contrib-
ute to the increase in the production of IFN type 1 in SLE
(62). Another miRNA regulating T and B cell function is
miR-155, whose up-regulation in T and B lymphocytes
could lead to abnormal activation of B cells and to the
abnormal development of inflammatory T cells in patients
with SLE (63,64).
MS is another autoimmune disorder in which miRNAs
are thought to be involved in pathogenesis. A recent study
showed that miR-326 plays a key role regulating the differ-
entiation of Th-17 cells, because of its role inhibiting Est-1,
a negative regulator of differentiation in these cells; miR-
326 was found to be significantly up regulated in patients
with MS in remission or relapsed, causing an increase in
the number of Th-17 cells and severe symptoms of the
disease (65). Other miRNAs reported in MS are miR-34a
and miR-155; it is proposed that they could be involved in
the active phase of the disease because of their action on
CD47 cells; miR-155 is a membrane protein that allows
recognition of proteins and avoids phagocytosis by spe-
CITOPLASM
microRNAs
DNA
Methylation
Protein
expression
Modification
of histones
TRANSCRIPTION
Pri-miRNA
Pre-miRNA
Pre-miRNA
miRNA duplex
miRNA mature
NUCLEUS
Maduration
RISC Complex
Blocking translation Degradation of RNAm target
Gppp
AAAA
AAAA
RNAm target Gppp
AAAA
RNAm target
DGCR8
Drosha
TR8
Dicer
Exportin 5
Ran-GTP
Gppp
Fig. 1. Epigenetics and biogenesis of miRNAs. The epigenetic regulation of gene expression can be conducted through different mechanisms, activating
or repressing gene expression. The expression of protein resulting from the action of these mechanisms would ultimately translate into in the observed
phenotype. miRNAs are translated from the genome, being processed in the nucleus and exported to the cytoplasm. miRNA are loaded into the RISC
complex and can bind to specific sequences of mRNA to repress the protein translation.
330 K. A. BASCUÑÁN-GAMBOA ET AL. Rev esp enfeRm Dig (maDRiD)
Rev esp enfeRm Dig 2014; 106 (5): 325-333
cialized cells. Macrophages with low levels of CD47 act
independently from this inhibitory control signal, causing
increased phagocytosis of myelin in MS. Mir-155 also pro-
motes the development of Th-1 and Th-17 inflammatory
cells (66).
In IBD, Wu et al. (67) in 2008 were the first to report
the miRNAs expression in samples of colonic mucosa of
IBD patients. They identified 11 miRNAs differentially
expressed in ulcerative colitis (UC) in comparison with
controls subjects, showing an inverse relationship between
the chemokine called macrophage inflammatory protein
(MIP 2) –previously implicated in IBD (68)– and miR-192.
Similarly, Bian et al. (69) found a significant overexpres-
sion of miR-150 in inflamed colonic mucosa of patients
with UC; they established an inverse correlation between
miR150 and its mRNA target, c-Myb, a proto-oncogene
that is involved in apoptotic processes (70). On the other
hand, a recent study on Crohn’s disease, showed that a
silent mutation in the reading frame of the IRGM gene (a
gene required for autophagy of intracellular bacteria), acts
as a functional variant by altering the site target of union
for miR196. This finding showed that the IRGM protec-
tive variant possesses a target site for mir196, union that
usually results in a decrease in the amount of IRGM pro-
teins in cellular inflamed epithelial cells. This observation
provides the first evidence that a silent mutation may be
clinically significant (71).
miRNAs IN CELIAC DISEASE
Despite the growing number of studies about the role
of miRNAs in AIDs, data about CD are scarce. A recent
study highlighted the importance of the miRNAs in the
differentiation and function of intestinal epithelium of
mice. Authors quantified the expression profile of the total
miRNAs present in the intestinal mucosa and determined
the contribution of miRNAs to intestinal homeostasis. The
study identified 453 families of miRNAs, mmu-miR-192
being the most highly expressed in both the small and large
intestine. In Dicer-deficient mice, the intestinal epithelium
was disorganized with a decreased numbers of goblet cells
and a significant increase of crypts apoptosis in jejunum
and colon, in addition with accelerated cell migration in
jejunum. Moreover, the function of intestinal barrier was
altered resulting in bowel inflammation with infiltration of
lymphocytes and neutrophils; the authors concluded that
Dicer protein possesses a vital role in the differentiation
and function of the intestinal epithelium (43).
Capuano et al., studied small intestinal biopsies of
celiac children and found that about 20 % of the miR-
NAs expression tested were different when compared
with control children, regardless of whether the disease
was active or not. The study of celiac patients showed high
levels of miR-449 expression and an inverse association
between the overexpression of miR449a, NOTCH1 sign-
aling (crucial in maintaining homeostasis of the intestine
by controlling cell proliferation and differentiation) and
the production of goblet cells, two features considered
characteristic of the small intestine of celiac patients (72).
A more recent report analyzed the expression of miRNA
in duodenal mucosa of a group of untreated adult celiac
patients (with classic presentation or iron-deficiency ane-
mia), a group of treated patients (with or without remission
of the duodenal mucosal lesion) and control subjects with-
out CD. Authors reported a deregulation of seven miR-
NAs (miR-31-5p, miR-192-3p, miR-194-5p, miR-551a,
miR-551b-5p, miR-638, and miR-1290) in patients with
different clinical phenotypes compared to subjects without
CD. These 7 miRNAs were then measured in duodenal
fibroblasts obtained from patients and incubated with glia-
din peptides (13 and 33mer). The group of miR-192/194
(involved in remodeling of the extra-cellular matrix) was
found deregulated in CD, with variations among the dif-
ferent clinical presentations. After stimulation with gliadin
peptides, expression of miR-192-3p was found decreased
in fibroblasts derived from celiac patients, whereas its
expression remained unchanged in fibroblasts of control
subjects. The authors concluded that analysis of miRNAs
deserves special consideration, especially as a potential
tool to control treatment and management of CD (73).
It is important to note that studies of miRNAs in CD
have been conducted mainly in intestinal epithelial cells.
The regulation of gene expression in the intestinal epi-
thelium is complex and controlled by different signaling
pathways that regulate the balance between proliferation
and differentiation, processes altered in pathologies such
as CD (74). It would be important to determine the regu-
lation of miRNAs in cells of the immune system, either
locally or systemically, to understand how these molecules
would be participating in the pathogenesis of the disease.
Studies in this area are needed to determine the relevance
of miRNAs expression in the functioning of the immune
system in CD (Fig. 2).
CONCLUSIONS
miRNAs are important factors in the differentiation and
function of the intestinal epithelium, and they have a rel-
evant role in regulating gene expression in physiological
and pathological conditions, including inflammatory and
autoimmune disorders. Given that at present the molecular
determinants underlying the pathogenesis of CD remain
unclear, is interesting to investigate miRNA profiles and
function in these patients. Current evidence on the interac-
tion between the miRNAs and the spectrum of autoimmune
pathologies opens a new approach to the study of CD. At
present, the available evidence in celiac patients, with dif-
ferent clinical characteristics, shows an altered expression
pattern of miRNA when compared with healthy controls.
This suggests that miRNAs deserve further study, as they
Vol. 106, N.º 5, 2014 microRNAs: AN EPIGENETIC TOOL TO STUDY CELIAC DISEASE 331
Rev esp enfeRm Dig 2014; 106 (5): 325-333
may represent potential biomarkers that could help distin-
guishing patients with different clinical profiles. Moreover,
specific miRNAs profiles could contribute to the individu-
alized management of a patient, transforming the wide-
spread clinical approach to a custom one. Finally, miRNAs
could contribute to elucidate how epigenetic alterations
participate in the development and course of CD.
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... In contrast, miR-21-3p did not (start to) normalize after start of the GFD in the PreventCD cohort. Previously, Bascuñań et al. also reported that miR-21 levels in circulation did not return to normal levels after start of the GFD (37). The observations that miR-21 levels are already elevated more than two years before detection of positive TGA and diagnosis raises the question whether this miRNA is correlated with the development of CeD or rather reflects intrinsic differences between CeD and controls that are independent of the (intestinal) inflammation and intestinal damage. ...
Article
Full-text available
Background & Aims Celiac disease (CeD), an immune-mediated disease with enteropathy triggered by gluten, affects ~1% of the general European population. Currently, there are no biomarkers to predict CeD development. MicroRNAs (miRNAs) are short RNAs involved in post-transcriptional gene regulation, and certain disease- and stage-specific miRNA profiles have been found previously. We aimed to investigate whether circulating miRNAs can predict the development of CeD. Methods Using next-generation miRNA-sequencing, we determined miRNAs in >200 serum samples from 53 participants of the PreventCD study, of whom 33 developed CeD during follow-up. Following study inclusion at 3 months of age, samples were drawn at predefined ages, diagnosis (first anti-transglutaminase antibody (TGA) positivity or diagnostic biopsy) and after the start of a gluten-free diet (GFD). This allowed identification of circulating miRNAs that are deregulated before TGA positivity. For validation of the biomarkers for CeD and GFD response, two additional cohorts were included in subsequent meta-analyses. Additionally, miRNAs were measured in duodenal biopsies in a case-control cohort. Results 53 circulating miRNAs were increased (27) or decreased (26) in CeD versus controls. We assessed specific trends in these individual miRNAs in the PreventCD cohort by grouping the pre-diagnostic samples of the CeD patients (all had negative TGA) by how close to seroconversion (first sample positive TGA) the samples were taken. 8/53 miRNAs differed significantly between controls and samples taken <1 year before TGA positivity: miR-21-3p, miR-374a-5p, 144-3p, miR-500a-3p, miR-486-3p let-7d-3p, let-7e-5p and miR-3605-3p. 6/26 downregulated miRNAs reconstituted upon GFD, including miR-150-5p/-3p, whereas no upregulated miRNAs were downregulated upon GFD. 15/53 biomarker candidates also differed between CeD biopsies and controls, with a concordant direction, indicating that these circulating miRNAs might originate from the intestine. Conclusions We identified 53 circulating miRNAs that are potential early biomarkers for CeD, of which several can be detected more than a year before TGA positivity and some start to normalize upon GFD.
... Moreover, certain microRNA profiles could contribute to individualized patient management, shifting clinical approach from general to personalized. Eventually microRNAs could elucidate the way in which epigenetic alterations contribute to the onset and evolution of CD (22,23) (Table 1). ...
Article
Full-text available
Celiac disease (CD) is a systemic condition of autoimmune origin, affecting genetically predisposed individuals who at some point lose tolerance towards dietary gluten. Prevalence in the general population is 0.5 - 1%, with a higher frequency in women. The most important environmental factor for CD is ingestion of specific gluten peptides. It triggers a sequence of molecular events, involving the intestinal permeability and the immune system, which ends in damage of the intestinal mucosa. A number of studies have demonstrated the correlation between the intestinal microbiota and celiac disease. MicroRNAs through their regulatory role on gene expression have been implicated in the pathogenesis of CD and suggested as potential biomarkers. In the pediatric and adult population, CD displays different clusters of clinical symptoms. Persistent diarrhea, abdominal pain and involuntary weight loss are the classic symptoms of CD. In the majority of cases diagnosis relies on the combination of serum autoantibodies (anti-transglutaminase and anti-endomisium IgA) and duodenal biopsy showing villous atrophy, crypt hyperplasia and intraepithelial lymphocytes. Observance of a lifelong gluten-free diet, which interrupts the immune response to gluten peptides, is the only effective treatment of CD.
... Several miRNAs are involved in the activation, differentiation, and functioning of immune cells by affecting the translation of key transcripts [6]. Studied mostly in cancer and other autoimmune pathologies, they now emerge as potentially relevant factors in CD [7]. Modified expression of miRNA-146a, miRNA-155, miRNA-21, and miRNA-125b has been reported in autoimmune and inflammatory diseases. ...
Article
Full-text available
Background The role of microRNAs (miRNAs) in celiac disease (CD) is unclear. Aims We evaluated inflammation-related miRNA-146a, miRNA-155, miRNA-21, and miRNA-125b expression in peripheral blood and intestinal mucosa of CD adults. Methods Thirty patients with CD were included: patients with active CD on a gluten-containing diet (CD-active, n = 10), patients on a gluten-free diet (for at least 1 year), and patients with negative blood antibodies (CD-inactivePE, n = 10). In addition, ten healthy volunteers formed the comparison/control group. MiRNA expression was measured in duodenal biopsies from patients (CD-inactiveMU, n = 10) after in vitro exposure to PT gliadin and 33-mer peptide. MiRNAs expression was measured in plasma and in peripheral blood mononuclear cells (PBMCs) and monocytes, before and after in vitro exposure to native gliadin (gliadinN). Results Expression levels of miRNA-146a, miRNA-155, and miRNA-21 in PBMCs, miRNA-155 in monocytes and miRNA-155, miRNA-21, and miRNA-125b in plasma were elevated in both groups of celiac patients. After in vitro exposure with gliadinN, miRNA-146a and miRNA-155 expression markedly increased in PBMCs and monocytes, while miRNA-155 and miRNA-21 increased in the CD-active group. MiRNAs expression in intestinal mucosa did not change. MiRNA-146a and miRNA-155 expression showed high sensitivity and specificity for the presence of CD, irrespective of the current dietary treatment. Conclusions Selected inflammation-related miRNAs expression is elevated in the peripheral blood of celiac. This suggests their participation in the immune processes underlying the pathology. Their similar response in active and inactive CD suggests that they should be further evaluated, as potential diagnostic biomarkers for CD.
... Mean platelet volume (MPV) has been recognized as a marker of inflammation in various diseases such as ulcerative colitis, acute pancreatitis and myocardial infraction etc. [103][104][105]. Purnak et al. have shown significantly higher MPV in CeD compared to healthy subjects and significant decrease in MPV in those showing good compliance to GFD compared to non-compliant individuals [106]. ...
Article
Full-text available
Once thought to be uncommon, celiac disease has now become a common disease globally. While avoidance of the gluten-containing diet is the only effective treatment so far, many new targets are being explored for the development of new drugs for its treatment. The endpoints of therapy include not only reversal of symptoms, normalization of immunological abnormalities and healing of mucosa, but also maintenance of remission of the disease by strict adherence of the gluten-free diet (GFD). There is no single gold standard test for the diagnosis of celiac disease and the diagnosis is based on the presence of a combination of characteristics including the presence of a celiac-specific antibody (anti-tissue transglutaminase antibody, anti-endomysial antibody or anti-deamidated gliadin peptide antibody) and demonstration of villous abnormalities. While the demonstration of enteropathy is an important criterion for a definite diagnosis of celiac disease, it requires endoscopic examination which is perceived as an invasive procedure. The capability of prediction of enteropathy by the presence of the high titer of anti-tissue transglutaminase antibody led to an option of making a diagnosis even without obtaining mucosal biopsies. While present day diagnostic tests are great, they, however, have certain limitations. Therefore, there is a need for biomarkers for screening of patients, prediction of enteropathy, and monitoring of patients for adherence of the gluten-free diet. Efforts are now being made to explore various biomarkers which reflect different changes that occur in the intestinal mucosa using modern day tools including transcriptomics, proteomics, and metabolomics. In the present review, we have discussed comprehensively the pros and cons of available biomarkers and also summarized the current status of emerging biomarkers for the screening, diagnosis, and monitoring of celiac disease.
... A few studies have mainly focused on intestinal epithelium, in which the regulation of gene expression is complex and controlled by different signaling pathways that regulate the balance between proliferation and differentiation of epithelial cells, processes that are altered in pathologies such as celiac disease. 43 In patients affected by celiac disease, these processes and the expression of specific miRNAs (i.e., miR-212, miR-192 and miR-122a) are altered, suggesting that miRNAs could be involved in the pathogenesis of intestinal barrier dysfunction and be associated with 44 These molecules may have a role in disease characterization, for celiac disease diagnosis or as predictors of gFD outcome. Moreover, starting from these dysregulated miRNAs, we investigated the putative miRNA targets by using simple computational approaches in order to classify them by gene ontology and pathway analysis. ...
Chapter
MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression at post-transcriptional level and play a key role in the pathogenesis of gastrointestinal and other pediatric diseases. Previous studies have revealed that miRNAs are dysregulated in intestinal biopsies of patients affected by intestinal diseases. These miRNAs belong to several important biological processes, such as Wnt signaling, cell proliferation and differentiation and adherens junction pathways. miRNAs are also present in extracellular spaces, packaged into various membrane-bound vesicles or associated with RNA-binding proteins. Circulating miRNAs are highly stable and can act as intercellular messengers, affecting many physiological processes. miRNAs circulating in body fluids have generated a strong interest as potential use as clinical biomarkers. In fact, their remarkable stability and the relative ease of detection make circulating miRNAs ideal tools for rapid and non-invasive diagnosis. Therefore, we have outlined recent insights about the origin, functions and diagnostic potential of extracellular miRNAs by focusing on the feasibility of using different sampling sources for the development of non-invasive pediatric diagnostics. We will also discuss specific bioinformatics tools and databases for circulating miRNAs used for the identification and discovery of novel diagnostic biomarkers and for investigating the functional role of miRNAs in intestinal and other pediatric diseases.
... CD is characterized by an immunological reaction against the TG2 (transglutaminase type 2) enzyme triggered by the ingestion of gluten peptides from wheat and related cereals in genetically predisposed individuals (2,5,6). This autoimmune reaction induces a duodenal damage characterized by villous atrophy, intraepithelial lymphocytosis, infiltration of inflammatory cells in the lamina propria and crypt hyperplasia (7,8). Defects in regulation of gene expression through microRNAs (miRNAs) could be responsible of the changes in intestinal permeability and intestinal immune system (2), suggesting ...
Article
Full-text available
Celiac disease is an enteropathy induced by ingestion of gluten triggering an immune response in genetically predisposed individuals. MiRNAs are small non-coding RNAs that have a role as regulators of gene expression at the post transcriptional level. The aim of this study is to evaluate the possibility of using circulating miRNAs as non-invasive biomarkers in pediatric patients with celiac disease. In addition, we examine the effect of a gluten-free diet on the expression of these miRNAs in serum of CD patients. The expression pattern of miR-21 and miR-31 was estimated in serum of 25 untreated CD patients (recently diagnosed), 25 treated CD patients (on gluten-free diet) and 20 healthy controls using qRT-PCR. Our results demonstrated the significant up-regulation of microRNA-21 in the untreated celiac patients in comparison with the treated group and healthy controls. Moreover, miR-31 expression was significantly under-expressed in the untreated celiac patients in comparison with the treated group and healthy controls. Furthermore, the results showed that miR-21 expression level was significantly positively correlated with the tTG IgA auto-antibodies. In conclusion, circulating miRNA-21 and miRNA-31 could serve as potential non-invasive biomarkers for pediatric CD patients.
... In fact, growing evidences are constantly accumulating for the pivotal role of epigenetics in driving autoimmunity. It is not only a loss of tolerance [33], since abnormal epigenetic modifications can present biomarker for ADs and be involved in multiple ADs pathogenic progression [34], CD not excluded [35,36]. Interestingly, most recently the microbiota memory was suggested to govern diseases by epigenetic regulation [37]. ...
Article
Celiac disease is an autoimmune condition of the small intestine caused by prolamins in genetically susceptible individuals evoked by multiple environmental factors. The pathological luminal intricate eco-events produce multiple signals that irradiate the entire body, resulting in a plethora of extra-intestinal manifestations. Nutrients, dysbiosis, dysbiotic components and their mobilome, post-translational modification of naive proteins, inter-enterocyte's tight junction dysfunction resulting in a leaky gut, microbial lateral genetic transfer of virulent genes, the sensing network of the enteric nervous systems and the ensuing pro-inflammatory messengers are mutually orchestrating the autoimmune interplay. Genetic-environmental-luminal events-mucosal changes are driving centrifugally the remote organs autoimmunity, establishing extra-intestinal multi organ injury. Exploring the underlying intestinal eco-events, the sensing and the delivery pathways and mechanisms that induce the peripheral tissues’ damages might unravel new therapeutical strategies to prevent and help the gluten affected patients.
Article
Introduction Celiac disease is an autoimmune enteropathy triggered by ingestion of gluten. While presenting many similarities with other autoimmune diseases, celiac disease is unique in that the external trigger, gluten, and the genetic background necessary for disease development (HLA DQ2/DQ8) are well described. The prevalence of celiac disease is dramatically increasing over the years and new epidemiologic data show changes regarding age of onset and symptoms. A better understanding of celiac disease pathogenesis is fundamental to highlight the reasons of this rise of celiac diagnoses. Areas covered In this review we describe celiac disease pathogenesis by dissecting all the components necessary to lose tolerance to gluten (ingestion of gluten, genetic predisposition, loss of barrier function and immune response). Additionally, we also highlight the role that microbiome plays in celiac disease as well as new proposed therapies and experimental tools. Expert opinion Prevalence of autoimmune diseases is increasing around the world. As a result, modern society is strongly impacted by a social and economic burden. Given the unique characteristics of celiac disease, a better understanding of its pathogenesis and the factors that contribute to it may shed light on other autoimmune diseases for which external trigger and genetic background are not known.
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