Microbiota Small RNAs in Inflammatory Bowel Disease

Abstract

MiRNAs are a class of potential gene regulators of critical importance in Inflammatory Bowel Disease (IBD). This review aims to present the connection between gut microbiota, probiotics administration and microRNA (miRNA) expression in IBD. It also brings into question cross-kingdom RNAi (RNA interference). Not only that gut host cells garden the intestinal microbiome via miRNA, but also strong evidence supports the idea that different species of bacteria have an impact on the intestinal immune response by modulating miRNA expression. Cross-kingdom RNAi refers to RNA silencing signals that travel between two unrelated, interacting organisms. RNAs communication between prokaryotes and eukaryotes (bacteria and nematodes) via RNAs transfer has been proved. Some authors also support the idea that non-coding RNAs are being transferred by bacterial pathogens to the host cells as part of the intracellular infection process. Further studies are required in order to clarify whether the mechanism by which bacteria modulate miRNA expression concerns RNAs transfer. These findings may lead to a different approach to IBD therapy in the future. © 2016, Romanian Society of Gastroenterology. All rights reserved.

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J Gastrointestin Liver Dis, December 2016 Vol. 25 No 4: 509-516
1) Biochemistry Department,
Victor Babes University of
Medicine and Pharmacy,
Timisoara;
2) Department of Functional
Genomics, Proteomics and
Experimental Pathology,
Prof. Dr. I. Chiricuta Oncology
Institute, Cluj-Napoca,
Romania
Address for correspondence:
Andrei Anghel
Biochemistry Department,
Victor Babes University of
Medicine and Pharmacy from
Timisoara,
Piata Eimie Murgu Nr.2,
Timisoara 300041, Romania
biochim@um.ro
Received: 08.07.2016
Accepted: 11.10.2016
Microbiota Small RNAs in Inammatory Bowel Disease
Anca T. Filip1, Ovidiu Balacescu2, Catalin Marian1, Andrei Anghel1
INTRODUCTION
MicroRNAs (miRNAs) are
single-strande d non-cod ing
RNA mol e cu le s of 1 9 -2 5
nucleotides discovered in all
eukaryotic cells except fungi.
MiRNAs are transcribed from
intronic, intergenic or exonic
DNA as pri-miRNA transcripts.
Before being exported in the
REVIEW
ABSTRACT
MiRNAs are a class of potential gene regulators of critical importance in Inammatory Bowel Disease (IBD).
is review aims to present the connection between gut microbiota, probiotics administration and microRNA
(miRNA) expression in IBD. It also brings into question cross-kingdom RNAi (RNA interference). Not only
that gut host cells garden the intestinal microbiome via miRNA, but also strong evidence supports the idea
that dierent species of bacteria have an impact on the intestinal immune response by modulating miRNA
expression. Cross-kingdom RNAi refers to RNA silencing signals that travel between two unrelated, interacting
organisms. RNAs communication between prokaryotes and eukaryotes (bacteria and nematodes) via RNAs
transfer has been proved. Some authors also support the idea that non-coding RNAs are being transferred by
bacterial pathogens to the host cells as part of the intracellular infection process. Further studies are required
in order to clarify whether the mechanism by which bacteria modulate miRNA expression concerns RNAs
transfer. ese ndings may lead to a dierent approach to IBD therapy in the future.
Key words: bacteria – gastrointestinal microbiome – microRNAs – probiotics – RNA interference.
Abbreviations: AChE: Acetylcholinesterase; AIEC: Adherent-invasive E coli; ATF: Activating transcription
factor; Bcl-2: B-cell lymphoma 2; BMDC: Bone marrow-derived dendritic cells; C elegans: Caenorhabditis
elegans; CCL: Chemokine C-C motif ligand; CD: Crohn’s disease; CDC42: Cell division control protein
42 homolog; CXCL: Chemokine (C-X-C motif) ligand; DC: Dendritic cells; E Coli: Escherichia coli; EcN:
E coli Nissle; EPEC: Entheropathogenic E coli; FOXO3: Forkhead box protein O3; GF: Germ-free; IBD:
Inammatory bowel disease; IECs: Intestinal epithelial cells; IGLC: Immunoglobulin Lambda Constant
Region; IkB: Inhibitor of NF-Kb; IL: Interleukin; IRGM: Immunity-related GTPase family M protein; L del:
Lactobacillus delbrueckii; LGG: Lactobacillus rhamnosus GG; MAPK: Mitogen-activated protein kinases;
miRNA: MicroRNA; mRNA: Messenger RNA; MyD88: Myeloid dierentiation primary response gene 88;
NF-kB: Nuclear factor kappa B; NOD2: Nucleotide-binding oligomerization domain-containing protein 2;
PAR: Partitioning defective protein; RhoB: Ras Homolog Family Member B; RISC: RNA induced silencing
complex; RNAi: RNA interference; SHH: Sonic hedgehog (gene); SPF: Specic-pathogen-free; SRNA: Small
RNA; STAT3: Signal transducer and activator of transcription 3; TGF: Transforming growth factor; 17: T
helper 17 cells; TJ: Tight junction; TLR: Toll like receptor; TNF: Tumor necrosis factor; UC: Ulcerative colitis;
Xcv: Xanthomonas campestris pv. Vesicatoria; ZO-2: Zonula occludens-2.
Available from: http://www.jgld.ro/wp/archive/y2016/n4/a13
DOI: http://dx.doi.org/10.15403/jgld.2014.1121.254.lip
cytoplasm, the pri-miRNA is processed into a 70-base pair
stem loop precursor miRNA (pre-miRNA). Once in the
cytoplasm, the endonuclease Dicer cleaves the pre-miRNA
into a small double-stranded RNA duplex, and then a single
strand is loaded into the RNA-induced silencing complex
(RISC) containing an Argonaute protein, the catalytically
active RNase, forming a mature miRNA complex capable of
silencing mRNA via 3’untranslated region of target mRNA.
mRNA transcripts targeted by miRNAs are either silenced
if the base-pair match is imperfect or degraded if there is an
identical base-pair match. According to this fact, each miRNA

510 Filip et al
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can target hundreds of mRNAs and a single mRNA may have
multiple 3’ untranslated region binding sites allowing targeting
by multiple miRNAs [1, 2].
Inflammatory bowel diseases (IBD) include Crohn’s
disease and ulcerative colitis. Inammatory bowel diseases
are chronic, progressive diseases characterized by aberrant
immune responses to environmental and gut microbial
triggers in possibly genetically susceptible hosts [3]. MiRNA
is recognized to play an essential role in the development
of the innate and adaptive immune system. ere is strong
evidence that miRNAs are a class of potential gene regulators
of critical importance in IBD, as shown in Table I. It has been
also demonstrated that miRNAs have the possibility to be
used as biomarkers and therapeutic targets, especially because
miRNA expression changes according to the status of tissue
inammation in IBD patients [4, 5].
Clinical, genetic, and experimental data support the role
of gut microbes in initiating and perpetuating intestinal
inammation in IBD [3, 6]. Four broad mechanisms explain the
complex relationship between the commensal microbiota and
IBD: (i) dysbiosis of conventional microbiota; (ii) induction of
intestinal inammation by pathogens and commensal bacteria;
(iii) host genetic defects; and (iv) defective host immune-
regulation [3]. Probiotic therapies modify disease symptoms by
Tab le I. Implication of miRNAs in the pathogenesis of inammatory bowel diseases
miRNA mRNA/ pathway target Roles Ref.
miR- 30C ATG5 Downregulates autophagy [8]
miR-130a ATG16L1 Down regulates autophagy [8]
miR-142-3p ATG16L1 Targets immune globulin gene IGLC [9]
miR-93 ATG16L1 Prevent autophagy-dependent eradication of intracellular
bacteria.
[10]
miR-106B ATG16L1 Prevent autophagy-dependent eradication of intracellular
bacteria
[10,
11]
miR-196 IRGM Downregulates protective variant of IRGM (c313C) [12]
miR-29 IL-12p40 (direct target)
IL-12p19 (indirect target
via reduction of ATF2)
Contributes to elevated IL-23, therefore contributes to
pathogenesis in CD
[13]
miR-10a IL-12/IL-23p40 Contribute to elevated IL-23, therefore contributes to
pathogenesis in CD
[14,
15]
miR-124 STAT3 Results in proinammatory response in UC pediatric
pacients
[16]
miR-21 RhoB CDC42 Downregulation of miR-21 protects against inammation
and tissue injury.
[17]
miR-146b siah2 Improves intestinal injury by activating nuclear factor-κB
and improving epithelial barrier function.
[18]
miR-192 
miR-495 
miR-512 
miR-671 
NOD 2 Downregulate NOD2 expression, suppress NF-κB activity,
and inhibit interleukin-8 and CXCL3 messenger RNA
expression.
[19]
miR-200b TGFb Inhibits epithelial mesenchymal transition and promots
proliferation of IECs.
Ameliorates intestinal brosis TGF β1 induced
[20]
miR-126 IkBa Down-regulates an inhibitor of NF-kB signaling pathway [21]
miR-7 CD98 expression Regulates the expression of CD98 (which is up-regulated
in CD)
[22]
miR-155 17 pathway Loss of miR-155 results in decreases in T helper type 1/
type17, CD11b+, and CD11c+ cells, which correlated with
reduced clinical scores and severity of disease.
[23]
miR-141 CXCL12β Downregulates CXCL12β-mediated leukocyte migration [24]
miR-150 c-Myb C-Myb regulates Bcl-2, an anti-apoptotic protein. [25]
miR-132 AChE Potentiates the cholinergic blockade of inammatory
reactions.
[26]
FOXO3a Suppresses the level of IκBα leading to enhanced NF-κB
signaling.
[27]
miR-122 NOD 2 Inhibits the activation of NF-κB. [28]
miR-146a NUMB SHH signaling Amplies inammatory responses. [29]
miR-223 FOXO3a Suppresses the level of IκBα leading to enhanced NF-κB
signaling.
[27,30]

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favourably altering the bacterial composition, immune status,
and inammation. e rationale for administering strains
of live ‘‘benecial’’ bacteria for IBD is based largely on the
premise of dysbiosis [7]. However, it is not yet known whether
dysbiosis is a cause or a consequence in IBD. Nevertheless, it
is well known that certain bacteria and strains have protective
roles against inammation in IBD, while others are aggressive.
IMPLICATION OF SMALL RNAs IN IBD
Microbiota dependent miRNAs in IBD
The gastrointestinal tract houses a complex bacterial
ecosystem consisting of 1014 cells in humans; about 10
fold greater than the number of human cells. e intestinal
microbiome consists mainly of obligates anaerobes such as
Lactobacillus, Bacteroides, Bidobacterium, Eubacterium and
Clostridium. Facultative anaerobes such as E. coli are also
present [31].
Dysbiosis confers clear susceptibility to IBD. Not only that
the mucosal immune system is inuenced by its microbial
environment, but there is also growing evidence that the
immune system shapes the composition of the intestinal
microbiome. ere is a bidirectional interaction which achieves
homeostasis. ere is strong evidence that host cells garden the
intestinal microbiome, a fact that is critical for health (Fig. 1).
e gardening can be aected by genetic variation, diet, stress,
acquired immune decits [32]. A mechanism by which host
cells communicate with bacteria involves miRNAs. Host cells
send miRNAs which enter bacteria and regulate bacterial gene
expression and growth. MicroRNA mediates inter-species gene
regulation which facilitates host control of the gut microbiota
[33]. Such stranded miRNAs are normal constituents of murine
and human feces [33].
It is also known that microbiota modulates expression of the
host genes. ere is a correlation between microbial diversity
and the host transcriptome, as shown when expression proles
of germ-free (GF) mice were dierent from specic-pathogen-
free (SPF) mice [34]. Using miRNA arrays, comparative
proling of miRNA expression between GF mice and SPF mice
revealed 9 miRNAs that were dierentially expressed. is
suggests that the microbiota modulates the host microRNA
expression, which could in turn regulate the expression of
hundreds of host genes [35]. Moreover, when investigating
the murine caecal miRNA expression signature, 334 miRNAs
were detectable in the caecum of GF and conventional mice,
and 16 of them were dierentially expressed between the two
groups. Again, this study supports the idea that gut bacteria
may have an impact on the intestinal gene regulation at the
post-transcriptional level by modulating miRNA [36].
Previously it was shown that a number of intestinal barrier
genes are post-transcriptionally regulated in Dicer knock-out
mice, and therefore, they may depend on miRNAs [37]. ese
genes may also be targets of microbiota-dependent miRNAs.
Taking this data into consideration, the authors support the
hypothesis that microbiota can impact the intestinal barrier via
miRNAs expression modulation. Also, microbiota-dependent
miRNAs may inuence gastrointestinal disease, especially at
the immune response level. ey found that miR-455, which
was up-regulated in GF mice, targets hsf1, a gene involved in
inhibiting the production of pro-inammatory cytokines and
induction cell adhesion molecule [36].
Another study revealed that microbiota interferes with
the microRNA response upon oral Listeria monocytogenes
infection [38]. Comparing miRNA proles of conventional
and GF mice during infection with Listeria showed that 5 of
6 miRNAs that seem to increase upon Listeria infection were
dependent on the presence of microbiota.
As presented above, miR-10a is down-regulated in IBD,
and it targets IL-12/IL-23p40 [14]. Also, there is increasing
evidence that dysbiosis is implicated in the etiology and
pathogenesis of IBD [31]. Xue et al. [14] compared the miR-
10a expression between the GF mice and SPF mice. It resulted
in decreased expression under SPF conditions compared
to that in GF mice. Moreover, bone marrow-derived DCs
(BMDC) were stimulated with lysates of E coli and A4 bacteria.
ey have shown that BMDC miR-10a expression was down-
Fig. 1. e role of microbiota dependent miRNAs in Inammatory Bowel Diseases (IBD). e
bidirectional interaction between microbiome and host cells, mediated by miRNAs.

512 Filip et al
J Gastrointestin Liver Dis, December 2016 Vol. 25 No 4: 509-516
regulated by treatment with E coli and A4 bacteria. us,
microora down-regulates miR-10a allowing for expression of
IL-12 and IL-23, a fact which might be important in promoting
intestinal immune homeostasis by increasing a protective
immunity under steady-state or it could contribute to the
progression of intestinal inammation under inammatory
conditions [14].
Similar work was done referring to miR-107. Firstly, it was
proven that the inamed intestine of mice with colitis has lower
levels of miR-107 expression and higher levels of IL-23p19;
thus more IL-23 produced as compared with normal mice.
Secondly, recolonization of the GF mice with normal ora
resulted in decreased intestinal miR-107 expression. Moreover,
stimulating BMDC with lysates of E coli and A4 bacteria led to
down-regulation of miR-107 expression. Certain evidence was
presented suggesting that the mechanism by which commensal
bacteria negatively regulates miR-107 expression is through
interaction of TLR-TLR ligands in a MyD88 and NF-kB
dependent manner. It is still unknown whether this is the sole
or the predominant pathway negatively regulating miR-107 in
vivo or if there might be other pathways involved. e same
study demonstrated that miR-107 specically down-regulates
IL-23p19 expression, gene with an expression that seems
increased in the inamed intestinal tissues of mice with colitis.
Previously it was demonstrated that miR-10a targets IL-23p40,
the other subunit of IL-23 [25]. erefore, miR-10a and miR-
107 could mediate microbiota stimulation of host responses
to negatively regulate IL-23. IL-23 production could set up a
basic inammatory environment or contribute to progression
of chronic intestinal inammation [39, 40].
A high prevalence of the adherent-invasive E. coli (AIEC)
has been shown in the ileal mucosa of CD patients. Adherent-
invasive E. coli adhere and invade human intestinal epithelial
cells (IECs), survive and replicate within macrophages, and
produce a proinammatory response with high secretion of
cytokines and chemokines. us, AIEC are able to exacerbate
intestinal inammation, especially in a genetically susceptible
mouse model. Recent studies found one mechanism by which
the host is able to restrain the intracellular replication of AIEC,
through autophagy. Regarding this aspect, one study was able
to prove that AIEC infection up-regulated the levels of miR-
30C and miR-130a in T84 cells and in mouse enterocytes.
Up-regulation of these miRNAs seemed to reduce the levels of
ATG5 and ATG16L, genes known to be involved in autophagic
pathway. is way, through up-regulation of miRNAs, AIEC
is responsible for reducing expression of proteins required
for autophagy and autophagy response in intestinal epithelial
cells, thus conducting to AIEC replication and an increased
inammatory response [8].
Microbiota small RNAs
Similar to eukaryotic cells, myriad of bacterial species
harbor small RNAs (sRNAs) that regulate gene expression.
Bacterial sRNAs are an emerging class of small regulatory
RNAs, 40–500 nt in length, that bind to their mRNA or protein
target [41]. Interaction between sRNAs and their specic target
can result in positive or negative regulation (Fig. 2). Positive
regulation refers to the process where sRNA bind and alter
the secondary structure of the mRNA leading to unmasking
of a ribosome binding site, the rst step in the initiation of
Fig. 2. Interaction between bacteria sRNAs and their mRNAs specic target from the colon cell.
Aer their transcription and export from bacteria, sRNAs enter into the colon cells and based on
their seed region complementarities bind to their mRNA targets, producing a positive or negative
regulation. rough positive regulation, sRNAs can stabilize mRNAs, unmasking the ribosome
binding site, and facilitates gene expression. rough negative regulation, sRNAs leads to altering
the function of mRNAs through mRNA degradation and/or their translational inhibition.

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translation. ere are also studies that demonstrate that sRNA
can stabilize mRNA intermediates and full-length mRNAs.
Negative regulation refers to the process where sRNA bind to
target mRNAs and this interaction leads to the destabilization
and subsequent degradation of the mRNA, or it can only inhibit
translation by binding to ribosome-binding sites. In addition,
sRNA can negatively regulate gene expression by binding
and altering the function of post-transcriptionally regulatory
proteins. Also, having multiple copies of sRNAs may lead to
regulation of expression of the sRNAs themselves [42].
Concerning eukaryotic cells, the seed region is a conserved
heptametrical sequence which is mostly situated at positions
2-8 from the miRNA 5´-end. Complementarity between the
seed region of a microRNA and the 3’-UTR of its target mRNA
is the key determinant in recognition. erefore, the functional
importance of the seed region complementarity as the major
determinant of miRNA targeting is well established. However,
major role in miRNA-mRNA interaction is attributed to the
argonaute protein, too. Interaction between the miRNA seed
region and argonaute proteins specically pre-organize and
optimize the conformation of these bases for base pairing with
its mRNA target sequence [43].
In prokaryotic cells, the hybridization between sRNAs and
their targets are usually dependent on a core interaction of
six to eight contiguous bases pairs. Small RNAs are thought
to hybridize to well-accessible regions such as hairpin loops
or single strand sequence. However, little is known about the
mechanisms involved in sRNA-mRNA interaction and further
studies are expected to gradually complete our understanding
of sRNA–mRNA target interactions [44, 45].
RNA interference (RNAi) refers to non-coding regulatory
RNAs that silence genes with complementary sequences.
Cross-kingdom RNAi refers to RNA silencing signals that
travel between two, oen unrelated, interacting organisms.
Small RNAs mediate communication between a wide range of
species such as humans and Plasmodium falciparum, plants and
nematodes, fungal pathogens and plants, plants and insects,
plants and microbes and bacteria and nematodes. ere are
numerous studies regarding mammals-parasite and plant-
pathogen interaction; however, cross kingdom RNAi does
not only refer to eukaryotic cells from dierent organisms
sRNA communication, but it also refers to sRNA transfer from
prokaryotes to eukaryotes and from viruses to eukaryotic cells
[46, 47].
Liu et al. [48] proved that E. coli endogenous sRNAs, OxyS
and DsrA could regulate gene expression and physiological
conditions of C. elegans. In addition, other bacteria such as
Bacillus mycoides might also utilize its noncoding RNAs to
interfere with gene expression in C. elegans. e relevant
‘C. elegans-targeting sequences’ in both OxyS and DsrA are
distinct from the ‘E. coli functional segments’ of these RNAs,
suggesting that the ‘C. elegans-targeting’ eects of these RNAs
are probably secondary adaptations. is study supports cross-
kingdom RNAi between bacteria and nematodes, and between
prokaryotes and eukaryotes, respectively.
It has been shown that the sRNAome of intracellular
bacteria is dierent between in vitro conditions and during
infection. RNA-seq was used to determine the sRNAome of
Salmonella, Yersinia pestis, Mycobacterium tuberculosis, Listeria
monocytogenes and the results showed dierent expression of
sRNAs between in vitro conditions and in vivo infection models
[49]. Knocking out of specic sRNAs in Brucella abortus
resulted in a signicant decrease in intracellular survival, and
in the plant pathogen Xanthomonas campestris pv vesicatoria
(Xcv) resulted in decreased disease symptoms in infected
plants. Similarly, a signicant reduction in the lesion diameter
was observed in immature pears upon depletion of the sRNAs
from the plant pathogen Erwinia amylovora. erefore, the
implication of specic sRNA in virulence in pathogens is
evident. e real mechanism by which these sRNAs confers
virulence in pathogens is not entirely known yet. However,
according to the authors, all these observations taken together
support the tantalizing idea of some non-coding RNAs being
transferred by bacterial pathogens to the host cell as part of
the intracellular infection process.
MiRNA IMPLICATION IN PROBIOTICS’
ADMINISTRATION
Administration of beneficial bacteria-probiotics is a
potential therapeutic option for IBD. Certain mechanisms by
which probiotics have benecial eects consist in competitive
exclusion, release of antimicrobial factors, which suppress
pathogen growth, enhancement of barrier function and
modication of the intestinal immune response [31]. e
exact processes by which probiotics have these eects are still
unknown. Certainly, they aect signaling pathways that lead
to up-regulation of expression of some proteins involved in
tight junction stability, mucin secretion, defense enhancement.
ey also modulate expression of pro- and anti-inammatory
molecules and promote IgA secretion [31]. Also, oral
administration of the supernatant of some probiotic bacteria
lead to repression of mRNA expression of pro-inammatory
molecules [43, 31]. Another mechanism by which probiotics
ameliorate the inammatory response in mice was proposed
in 2004. One study showed that DNA from probiotic bacteria
could limit epithelial pro-inammatory responses in vitro as
well as in vivo [50].
Another study which supports the idea that probiotics
modulate the immune system in the gastrointestinal tract was
performed on piglets [51]. ey were fed with Enterococcus
faecium. e results consisted in up-regulation of miR-423-5p
and down-regulation of the immune globulin gene IGLC, a
target gene of miR-423-5p (Fig. 3).
One more step concerning the mechanism by which
probiotics inuence the immune response in IBD was made
by discovering the eect of heat-inactivated Lactobacillus
rhamnosus GG (LGG) and Lactobacillus delbrueckii subsp.
bulgaricus (L.del) in human monocyte-derived dendritic cells
(DCs). Amongst the changes in the expression of TLR4 and
signaling factors such as p38, MAPK and IkB at transcription
level, LGG induced a significant down-regulatory effect
on miR-146a expression, a negative regulator of immune
response, and up-regulatory eect on miR-155 [52]. In IBD,
overexpression of miR-146a leads to down-regulation of
NUMB, considered its target, and as a consequence to the
activation of Sonic hedgehog (SHH) signaling, including
inammatory genes such as IL-12, TNF-α, IL-6, CCL-5, and

514 Filip et al
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CXCL-9 [29]. erefore, down-regulation of miR-146a might
be benec in IBD, by decreasing SHH signaling and decreasing
expression of some inammatory genes. On the other hand,
miR-155 was up-regulated and, on the contrary, loss of miR-155
correlated with reduced clinical scores and severity of IBD [23].
e previous year, Zhao et al. published a study concerning
the eects of the probiotic Lactobacillus rhamnosus GG (LGG)
in alcoholic liver disease [53]. Chronic ethanol exposure
increased the intestinal miR122a expression, which decreased
occludin (OCLN) expression leading to increased intestinal
permeability. e study concluded that LGGs supplementation
functions in intestinal integrity by inhibition of miR122a,
leading to OCLN restoration. Also, miR-122a was found to be
deregulated in Crohn’s disease [28]. It might be interesting to
nd out whether LGGs supplementation has the same eects
in Crohn’s disease, too.
Even before that, when comparing miRNA prole of
epithelial T84 cells with those of T84 cells that have been
co-incubated with E. coli Nissle 1912 (EcN) and entero-
pathogenic E. coli strain E2348/69 (EPEC) separately, the
results were supportive. e study focused on miR-203,
miR-483-3p, and miR-595 which were down regulated by
EcN compared to normal and up-regulated by EPEC. E.
coli Nissle has been known for a long time as a probiotic
bacterium that has been used to treat inammatory intestinal
disorders. It resulted that, following infection with EcN,
expression of mRNAs for the TJ scaolding protein ZO-2
and for the regulatory proteins PAR-3 and PAR-6 were found
to be up-regulated. us, EcN strengthens barrier integrity
by up-regulating essential tight junction components.
Interestingly, the miRNAs that are down-regulated by this
probiotic and up-regulated by the entero-pathogenic E. coli,
target important regulatory and scaolding proteins of the TJ
complex such as ZO-2, PAR-3, and PAR-6 [54]. It seems that
probiotic EcN down-regulates miRNAs that target important
mRNAs, coding for proteins involved in strengthening the
barrier integrity.
CONCLUSIONS
In summary, there are benec bacteria that regulate the
miRNAs known to be dysregulated in IBD, and there are
bacteria that have negative eects on the miRNAs known
to be involved in the IBD pathogenesis, thus leading to a
pro-inammatory status. is could explain why dysbiosis is
linked to IBD.
The mechanism by which bacteria modulates miRNA
expression is not yet known. Supposing that there could indeed
be an action of bacterial sRNA in eukaryotic cells, the seed
region for targeting mRNAs in prokaryotes and eukaryotes
contains 6 to 8 nucleotides in both of them. We might assume
that there is a possibility that bacteria modulate host genes
by sending sRNAs into eukaryotic cells, sRNAs which might
interact with eukaryotic mRNA or even miRNA. In any case,
this is a novel eld of research and numerous studies are needed
to truly understand the entire mechanism by which microbiota
modulate miRNA expression.
ese ndings may lead to a dierent approach in IBD
therapy, based on the administration of probiotics, or even
bacteria derived sRNAs. Understanding the mechanisms
involved may allow us to genetically modify probiotics in order
to amplify their benecial eects.
Conicts of interest: e authors declare that there is no conict of
interest with regard to this work.
Authors’ contribution: A.T.F.: study concept and design, literature
search and selection, data interpretation; conceived and draed the
manuscript; O.B.: data interpretation, gures and manuscript revision;
C.M.: data interpretation, critical comments and manuscript revision;
Fig. 3. e positive role of probiotics, inducing anti-inammatory and immune responses in IBD,
based on miRNAs-mRNAs modulation.

Microbiota Small RNAs in inammatory bowel disease 515
J Gastrointestin Liver Dis, December 2016 Vol. 25 No 4: 509-516
A.A.: study concept and design, literature search and manuscript
revision for important intellectual content. All authors read and agreed
with the nal version of the manuscript.
Acknowledgements: O.B. is supported in part by an ANCS POC-A1-
A1.2.3-G-2015 grant mechanism, project number P_40_318/2016.
REFERENCES
1. Maudet C, Mano M, Eulalio A. MicroRNAs in the interaction between
host and bacterial pathogens. FEBS Lett 2014; 588: 4140-4147. doi:
10.1016/j.febslet.2014.08.002
2. Kalla R, Ventham NT, Kennedy NA, et al. MicroRNAs: newplayers in
IBD. Gut 2015; 64: 504-517. doi: 10.1136/gutjnl-2014-307891
3. Sartor RB, Mazmanian SK. Intestinal Microbes in Inammatory Bowel
Diseases. Am J Gastroenterol Suppl 2012; 1:15– 21; doi: 10.1038/
ajgsup.2012.4
4. Chen WX, Ren LH, Shi RH. Implication of miRNAs for inammatory
bowel disease treatment: Systematic review. World J Gastrointest
Pathophysiol 2014; 5: 63-70. doi: 10.4291/wjgp.v5.i2.63
5. Chapman CG , Pekow J. e emerging role of miRNAs in inammatory
bowel disease: a review. erap Adv Gastroenterol 2015; 8: 4-22. doi:
10.1177/1756283X14547360
6. Dalal SR, Chang EB. e microbial basis of inammatory bowel diseases.
J Clin Invest. 2014; 124: 4190–4196. doi: 10.1172/JCI72330
7. Tamboli CP, Neut C, Desreumaux P, Colombel JF. Dysbiosis in
inflammatory bowel disease. Gut 2004; 53: 1–4. doi: 10.1136/
gut.53.1.1
8. Nguyen HT, Dalmasso G, Müller S, Carrière J, Seibold F, Darfeuille-
Michaud A. Crohn’s disease–associated adherent invasive Escherichia
coli modulate levels of micrornas in intestinal epithelial cells to reduce
autophagy. Gastroenterology 2014; 146: 508–519. doi: 10.1053/j.
gastro.2013.10.021
9. Zhai Z, Wu F, Dong F, et al. Human autophagy gene ATG16L1 is
post-transcriptionally regulated by MIR142-3p. Autophagy 2014; 10:
468–479. doi: 10.4161/auto.27553
10. Lu C, Chen J, Xu HG, et al. MIR106B and MIR93 prevent removal
of bacteria from epithelial cells by disrupting ATG16L1-mediated
autophagy. Gastroenterology 2014; 146: 188-199. doi: 10.1053/j.
gastro.2013.09.006
11. Zhai Z, Wu F, C hua ng AY, Kwon JH. miR-106b ne tunes ATG16L1
expression and autophagic activity in intestinal epithelial HCT116
cells. Inflamm Bowel Dis 2013; 19: 2295-2301. doi: 10.1097/
MIB.0b013e31829e71cf
12. Brest P, Lapaquette P, Souidi M, et al. A synonymous variant in IRGM
alters a binding site for miR-196 and causes deregulation of IRGM-
dependent xenophagy in Crohn’s disease. Nat Genet 2011; 43: 242-245.
doi: 10.1038/ng.762
13. Brain O, Owens BM, Pichulik T, et al. The intracellular sensor
NOD2 induces microRNA-29 expression in human dendritic cells
to limit IL-23 release. Immunity 2013; 39: 521–536. doi: 10.1016/j.
immuni.2013.08.035
14. Xue X, Feng T, Yao S, et al. Microbiota downregulates dendritic cell
expression of miR-10a, which targets IL-12/IL-23p40. J Immunol 2011;
187: 5879–5886. doi: 10.4049/jimmunol.1100535
15. Wu W, He C , Liu C, et al. miR-10a inhibits dendritic cell activation and
1/17 cell immune responses in IBD. Gut 2015; 64: 1755-1764. doi:
10.1136/gutjnl-2014-307980
16. Koukos G, Polytarchou C, Kaplan JL, et al. MicroRNA-124 regulates
STAT3 expression and is downregulated in colon tissues of pediatric
patients with ulcerative colitis. Gastroenterology 2013; 145: 842–852.
e2. doi: 10.1053/j.gastro.2013.07.001
17. Shi C, Liang Y, Yang J, et al. MicroRNA-21 knockout improve the
survival rate in DSS induced fatal colitis through protecting against
inammation and tissue injury. PLoS One 2013; 8: e66814. doi: 10.1371/
journal.pone.0066814
18. Nata T, Fujiya M, Ueno N, et al. MicroRNA-146b improves intestinal injury
in mouse colitis by activating nuclear factor-κB and improving epithelial
barrier function. J Gene Med 2013; 15: 249-260. doi: 10.1002/jgm.2717
19. Chuang AY, Chuang JC, Zhai Z, Wu F, Kwon JH. NOD2expression is
regulated by microRNAs in colonic epithelial HCT116 cells. Inamm
Bowel Dis 2014; 20: 126-135. doi: 10.1097/01.MIB.0000436954.70596.9b
20. Chen Y, Ge W, Xu L, et al. MiR-200b is involved in intestinal brosis
of Crohn’s disease. Int J Mol Med 2012; 29: 601-606. doi: 10.3892/
ijmm.2012.894
21. Feng X, Wang H, Ye S, et al. Up-regulation of microRNA-126 may
contribute to pathogenesis of ulcerative colitis via regulating NF-
kappaB inhibitor IkBa. PLoS One 2012; 7: e52782. doi: 10.1371/journal.
pone.0052782
22. Nguyen HT, Dalmasso G, Yan Y, et al. MicroRNA-7 Modulates CD98
expression during intestinal epithelial cell dierentiation. J Biol Chem
2010; 285: 1479-1489. doi: 10.1074/jbc.M109.057141
23. Singh UP, Murphy AE, Enos RT, et al. miR-155 deciency protects mice
from experimental colitis by reducing T helper type 1/type 17 responses.
Immunology 2014; 143: 478–489. doi: 10.1111/imm.12328
24. Huang Z, Shi T, Zhou Q, et al. miR-141Regulates colonic leukocytic
trafficking by targeting CXCL12β during murine colitis and
human Crohn’s disease. Gut 2014; 63: 1247-1257. doi: 10.1136/
gutjnl-2012-304213
25. Bian Z, Li L, Cui J, et al. Role of miR-150-targetingc-Myb in
colonicepithelial disruption during dextran sulphate sodium-induced
murine experimental colitis and human ulcerative colitis. J Path ol 2011;
225: 544-553. doi: 10.1002/path.2907
26. Maharshak N, Shenhar-Tsarfaty S, Aroyo N, et al. MicroRNA-132
modulates cholinergic signaling and inflammation in human
inammatory bowel disease. Inamm Bowel Dis 2013; 19: 1346-1353.
doi: 10.1097/MIB.0b013e318281f47d
27 . Kim HY, Kwon HY, Ha i HT, et al. MicroRNA-132 and microRNA-223
control positive feedback circuit by regulating FOXO3a in inammatory
bowel disease. J Gastroenterol Hepatol 2016. doi: 10.1111/jgh.13321
28. Chen Y, Wan g C, Liu Y, et al. miR-122targetsNOD2 to decrease intestinal
epithelial cell injury in Crohn’s disease. Biochem Biophys Res Commun
2013; 438: 133-139. doi: 10.1016/j.bbrc.2013.07.040
29. Ghorpade DS, Sinha AY, Holla S, Singh V, Balaji KN. NOD2-nitric
oxide-responsive microRNA-146a activates sonic hedgehog signaling to
orchestrate inammatory responses in murine model of inammatory
bowel disease. J Biol Chem 2013; 288: 33037-33048. doi: 10.1074/jbc.
M113.492496
30. Wang H, Zhang S, Yu Q, et al. Circulating MicroRNA223 is a New
Biomarker for Inammatory Bowel Disease. Medicine (Baltimore).
2016; 95(5): e2703. doi: 10.1097/MD.0000000000002703
31. Mikov MM, Stojancevic MP, Bojic GM. Probiotics as a Promising
Treatment for Inammatory Bowel Disease. Hospital Pharmacology
2014; 1: 52-60
32. Jacobs JP, Braun J. Immune and genetic gardening of the intestinal
microbiome. FEBS Lett 2014; 588: 4102-4111. doi: 10.1016/j.
febslet.2014.02.052

516 Filip et al
J Gastrointestin Liver Dis, December 2016 Vol. 25 No 4: 509-516
33. Liu S, da Cunha AP, Rezende RM, et al. e Host Shapes the Gut
Microbiota via Fecal MicroRNA. Cell Host Microbe 2016; 19: 32-43.
doi: 10.1016/j.chom.2015.12.005
34. El Aidy S, Derrien M,Merrield CA, et al. Gut bacteria–host metabolic
interplay during conventionalisation of the mouse germfree colon. e
ISME J 2013; 7: 743–755. doi: 10.1038/ismej.2012
35. D almasso G, Nguyen HTT, Yan Y et al. Microbiota modulate host gene
expression via microRNAs. PLOS One 2011; 6: e19293 doi: 10.1371/
journal.pone.019293
36. Singh N, Shirdel EA, Waldron L, Zhang RH, Jurisica I, Comelli EM.
e murine caecal microRNA signature depends on the presence of the
endogenous microbiota. Int J Biol Sci 2012; 8: 171-186. doi: 10.7150/
ijbs.8.171
37. McKenna LB, Schug J, Vourekas A, et al. MicroRNAs control
intestinal epithelial differentiation, architecture, and barrier
function. Gastroenterology 2010; 139: 1654-1664.e1. doi: 10.1053/j.
gastro.2010.07.040
38. Archambaud C, Sismeiro O, Toedling J, et al. e Intestinal Microbiota
Interferes with the microRNA Response upon Oral Listeria Infection.
MBio 2013; 4: e00707-13. doi: 10.1128/mBio.00707-13
39. Xue X, Cao AT, Cao X, et al. Downregulation of MicroRNA-107
in intestinal CD11c+ myeloid cells in response to microbiota and
proinflammatory cytokines increases IL-23p19 expression. Eur J
Immunol 2014; 44: 673-682. doi: 10.1002/eji.201343717
40. Masotti A. Interplays between gut microbiota and gene expression
regulation by miRNAs. Front Cell Infect Microbiol 2012; 2: 137. doi:
10.3389/fcimb.2012.00137
41. Gurung M, Moon DC, Choi CW, et al. Staphylococcus aureus produces
membrane-derived vesicles that induce host cell death. PLoS One 2011;
6: e27958. doi: 10.1371/journal.pone.0027958
42. Caswell CC, Oglesby-Sherrouse AG, Murphy ER. Sibling rivalry:
related bacterial small RNAs and their redundant and non-redundant
roles. Front Cell Infect Microbiol 2014; 4: 151. doi: 10.3389/
fcimb.2014.00151
43. Lambert NJ, Gu SG, Zahler AM. e conformation of microRNA
seed regions in native microRNPs is prearranged for presentation to
mRNA targets. Nucleic Acids Res 2011; 39: 4827-4835. doi: 10.1093/
nar/gkr077
44. Cao Y, Wu J, Liu Q, et al. sRNATarBase: A comprehensive database of
bacterial sRNA targets veried by experiments. RNA 2010; 16: 2051-
2057. doi: 10.1261/rna.2193110
45. Harris JF, Micheva-Viteva S, Li N, Hong-Geller E. Small RNA-mediated
regulation of host–pathogen interactions. Virulence 2013; 4: 785–795.
doi: 10.4161/viru.26119
46. Weiberg A, Bellinger M, Jin H. Conversations between kingdoms:
small RNAs. Curr Opin Biotechnol 2015; 32: 207-215. doi: 10.1016/j.
copbio.2014.12.025
47. Knip M, Constantin ME, ordal-Christensen H. Trans-kingdom
cross-talk: small RNAs on the move. PLoS Genet 2014; 10: e1004602.
doi: 10.1371/journal.pgen.1004602
48. Liu H, Wang X, Wang HD, et al. Escherichia coli noncoding RNAs can
aect gene expression and physiology of Caenorhabditis elegans. Nat
Commun 2012; 3: 1073. doi: 10.1038/ncomms2071
49. Ortega ÁD, Quereda JJ, Pucciarelli MG, García-del Portillo F. Non-coding
RNA regulation in pathogenic bacteria located inside eukaryotic cells.
Front Cell Infect Microbiol 2014; 4: 162. doi: 10.3389/fcimb.2014.00162
50. Jijon H, Backer J, Diaz H, et al. DNA from probiotic bacteria modulates
murine and human epithelial and immune function. Gastroenterology
2004; 126: 1358-1373. doi: 10.1053/j.gastro.2004.02.003
51. Kreuzer-Redmer S, Bekurtz JC, Arends D, et al. Feeding of Enterococcus
faecium NCIMB 10415 Leads to Intestinal miRNA-423-5p-Induced
Regulation of Immune-Relevant Genes. Appl Environ Microbiol 2016;
82: 2263-2269. doi: 10.1128/AEM.04044-15
52. Giahi L, Aumueller E, Elmadfa I, Haslberger AG. Regulation of TLR4,
p38MAPkinase, IκB and miRNAs by inactivatedstrains of lactobacilli
in humandendriticcells. Benef Microbes 2012; 3: 91-98. doi: 10.3920/
BM2011.0052
53. Zhao H, Zhao C, Dong Y, et al. Inhibition of miR122a by Lactobacillus
rhamnosus GG culture supernatant increases intestinal occludin
expression and protects mice from alcoholic liver disease. Toxicol Lett
2015; 234: 194-200. doi: 10.1016/j.toxlet.2015.03.002
54. Veltman K, Hummel S, Cichon C, Sonnenborn U, Schmidt MA.
Identication of specic miRNAs targeting proteins of the apical
junctional complex that simulate the probiotic eect of E. coli Nissle
1917 on T84 epithelial cells. Int J Biochem Cell Biol 2012; 44: 341-349.
doi: 10.1016/j.biocel.2011.11.006