MicroRNA-378 protects against intestinal ischemia/reperfusion injury via a mechanism involving the inhibition of intestinal mucosal cell apoptosis
Citation: Cell Death and Disease
MicroRNA-378 protects against intestinal ischemia/ reperfusion injury via a mechanism involving the inhibition of intestinal mucosal cell apoptosis
Intestinal ischemia/reperfusion (I/R) injury remains a major clinical event and contributes to high morbidity and mortality rates, but the underlying mechanisms remain elusive. Recent studies have demonstrated that microRNAs (miRNAs) have important roles in organ I/R injury, but the changes and potential roles of miRNAs in intestinal I/R-induced intestinal injury are unclear. This study was designed to analyze the miRNA expression profiles in intestinal mucosa after I/R injury and to explore the role of target miRNA during this process. Using miRNA microarray analysis, we found changes of 19 miRNAs from the expression profile of miRNAs in a mouse model of intestinal I/R and further verified them by RT-qPCR. Here, we report that miR-378 is one of the markedly decreased miRNAs and found the putative target mRNA that is linked to cell death after applying the TargetScan, miRanda, CLIP-Seq and miRDB prediction algorithms. Our results show that the overexpression of miR-378 significantly ameliorated intestinal tissue damage in wild-type and transgenic mice and oxygen glucose deprivation/reperfusion-challenged IEC-6 cell injury. Moreover, miR-378 overexpression reduced intestinal epithelial cell apoptosis in both in vivo and in vitro ischemic models and attenuated cleaved caspase-3 expression. Collectively, our results revealed that the suppression of caspase-3 activation by miRNA-378 overexpression may be involved in the protective effects of intestinal ischemic damage. MiRNA-378 may serve as a key regulator and therapeutic target in intestinal I/R injury. Cell Death and Disease (2017) 8, e3127; doi:10.1038/cddis.2017.508; published online 12 October 2017
Intestinal ischemia/reperfusion (I/R) injury is a potentially
serious consequence of acute mesenteric ischemia,
hemorrhagic, traumatic or septic shock, severe burns or some
surgical procedures, including small bowel transplantation
and aortic aneurysm repair.1 Intestinal I/R not only leads to
injury of the intestine itself, but also involves severe
destruction of distant tissues because of disruption of the intestinal
mucosal barrier, which is associated with local and systemic
injuries that ultimately progress to multiple organ dysfunction
and often death.2 Therefore, the development of effective
treatment strategies against intestinal I/R is important for
improving the prognosis of critically ill patients.
The factors contributing to intestinal I/R injury were
complex, including microvascular dysfunction,3 reactive
oxygen species over-production,4,5 inflammation6,7 and even
intestinal epithelial cell death. Many evidence reveals that
apoptosis is a major mode of cell death caused by intestinal I/
R,8,9 which is a complex biological process that can be
triggered by the death receptor and mitochondrial death
signaling pathways.10 The small intestine is prone to be
subjected to ischemic-induced apoptosis because of the
priority of blood flow supplied to brain and heart when
hypovolemic shock occurs. Various studies demonstrated that
prophylactic anti-apoptotic treatment could be an effective
therapeutic strategy for the prevention of intestinal I/R
injury,11?13 including pharmacological blockade of protein
kinase C ?2,14 PI3K/AKT pathway activation15 and so on.
However, the underlying molecular mechanism of I/R-induced
intestinal epithelium apoptosis is remained poorly understood
and effective pharmacological or genetic agents would be
rational to develop.
MicroRNAs (miRNAs) are a novel class of endogenous,
small non-coding single-stranded RNAs, with highly
conserved sequences among species. Through imperfect
sequence-specific binding to the 3?-untranslated region
(UTR) of target messenger RNAs (mRNAs), miRNAs
downregulate gene expression by degrading target mRNAs and/or
inhibiting protein synthesis.16,17 Meanwhile, it has been found
to be not only crucial for the development and maintenance of
physiological homeostasis, but have also been causally
implicated in tissue injury and repair.18?20 Recent studies
showed that characteristic changes of miRNAs have important
roles in cardiac,21 cerebral,22 renal23 I/R injury, which are
associated with cell apoptosis, oxidative stress and
inflammation. Meanwhile, emerging evidence suggests that miRNAs
are of paramount importance in gastrointestinal development
and physiology.24,25 However, the differential miRNAs
expression of intestinal mucosa in response to intestinal I/R are still
unclear, and the contributions of miRNAs to post-ischemic
intestine remain to be elucidated.
Thus, studies are needed to better understand the
characterization of miRNAs expression and their function
involved in intestinal I/R-induced injury. In this study, we aimed
to analyze miRNAs expression profiles in intestinal mucosa
after I/R injury and explore the role and potential mechanisms
of target miRNAs on intestinal epithelial cell apoptosis induced
by intestinal I/R injury.
Evaluation of intestinal I/R injury. Representative intestine
sections (n = 8 per group) revealed that 60 min of ischemia
and 120 min of reperfusion caused significant intestinal
mucosal damage that was primarily manifested as severe
edema of the mucosal villi, infiltration of inflammatory cells
and increased gaps between epithelial cells. By contrast,
normal mucosal architecture was observed in the
shamoperated mice (Figure 1a). Consistent with the histological
changes in the intestinal mucosa, Chiu?s score, which reflects
the severity of histological damage, was significantly higher in
the injury group than in the sham group (Figure 1b).
Moreover, the plasma diamine oxidase (DAO) levels in the injury
group were markedly higher than those in the sham group
(Figure 1c). This well-established model was used in the
Identification of downregulated miR-378 in the intestines
of mice following intestinal I/R. MiRNA microarray analysis
revealed that 19 differentially expressed miRNAs were
identified in response to intestinal I/R, including 1 upregulated
and 18 downregulated miRNA candidates, all of which
exhibited a P-value o0.05 calculated by t-test when
compared with the sham group (n = 5 per group, Table 1).
The above differential miRNA expression levels in the
ischemic intestine were further validated by RT-qPCR. To
characterize intestinal I/R-responsive miRNA candidates, the
following parameters were defined: a fold change of at least
1.5, and putative target mRNAs that were linked to cell death
as identified using the prediction algorithms TargetScan,
miRanda, CLIP-Seq and miRDB. Consequently, miR-378
was found to exhibit a significant fold change (3.38-fold
downregulation, validated by RT-qPCR) among the
I/Rinduced miRNAs (Table 2). Moreover, among the 40
predicted putative targets of miR-378, three are common to
the above four algorithms, including MTSS1L, NEUROD1
and CASPASE-3 (Table 3).
MiR-378 agomir protects against ischemic intestinal
injury and cell apoptosis in the wild-type (WT) mouse
intestinal I/R model. Three days after injection of
agomir378, antagomir-378 or their negative control (NC), miR-378
expression was significantly preserved in the agomir group
compared with the other ischemic insult groups. Moreover,
miR-378 expression in the antagomir group was significantly
lower than that in the injury group (Figure 2a). In addition, the
WT mice that received agomiR-378 exhibited slight edema
and mucosal architecture destruction, and the damage was
further ameliorated compared with injury group. Conversely,
the intestinal injury induced by I/R was further aggravated
after receiving antagomiR-378, as evidenced by increased
Chiu?s scores and DAO levels (Figures 2b-d).
As shown in Figure 2e, the ileal tissues demonstrated the
marked appearance of dark brown (TUNEL positive) apoptotic
cells from the detached epithelium at the tips to the lower part
&: only experimental evidence. Not found in mouse genome. Confidence:undefined.
Microarray analysis identified 19 differentially expressed miRNAs in response to intestinal I/R, all of which exhibited a P-value o0.05 calculated by t-test when
compared with the sham group (n = 5 per group)
of the villi in the injury mice. However, fewer positive-staining
to the lower part of the villi in the WT injury mice. However, fewer
cells were observed in the ileum sections taken from the
positive-staining cells were observed in the ileal sections taken
agomir mice. The intestinal mucosa apoptotic index was
from the TG Injury mice. The apoptotic index of WT injury group
significantly reduced after pretreatment with agomir-378. By
was markedly higher than that of the TG injury group (Figure 3f).
contrast, the antagomiR-378 treatment increased the number
Moreover, the significant reduction in epithelial apoptosis
of apoptotic cells (Figure 2f). Consistently, pretreatment with
correlated with lower cleaved caspase-3 expression in the TG
agomir-378 resulted in a significant reduction in cleaved
injury mice (Figures 3g and h).
caspase-3 expression (Figures 2g and h).
In vivo overexpression of miR-378 ameliorates the
intestinal mucosal injury and cell apoptosis induced by
intestinal I/R. The miR-378 level was detected by RT-qPCR to
confirm the construction of the miR-378 transgenic (TG) mouse
In vitro overexpression of miR-378 improves IEC-6 cell
survival after OGD/R challenge. As illustrated in Figure 4a,
the transfection of the pre-miR-378 (mimic) or miR-378
inhibitor, but not their NCs, significantly increased or
decreased the miR-378 levels in the normoxic cells. Cell
model. As illustrated, the expression of miR-378 in the TG
viability analysis using the MTT assay revealed that 4 h of
mouse was 3.39 times that in the WT mice (Figure 3a).
OGD followed by 4 h of reoxygenation resulted in obvious cell
Compared with the WT mice, the intestinal injury induced by
death, whereas pre-miR-378 treatment markedly augmented
intestinal I/R in the TG mice was mitigated as evidenced by the
cell survival after OGD/R challenge. Moreover, pretreatment
improved histological injury and decreased DAO levels (Figures
with the miR-378 inhibitor further exacerbated cell death
3b-d). Moreover, as illustrated in Figure 3e, the ileal tissues
when compared with the OGD/R group (Figure 4b). Flow
demonstrated the marked appearance of dark brown (TUNEL
cytometric analysis was used to further confirm whether
positive) apoptotic cells from the detached epithelium at the tips
miR-378 could affect 4 h OGD/4 h reoxygenation-induced
IEC-6 cell apoptosis. As shown in the dual staining
(Figure 4c), Annexin V+/PI+ and Annexin V+/PI- (quadrants
2 and 4 represent apoptosis and necrosis) cells were rarely
seen in the sham group, whereas OGD/R increased the
numbers of apoptotic and necrotic cells. Transfection with
pre-miR-378 could significantly reduce, whereas the miR-378
inhibitor aggravated, the numbers of apoptotic and necrotic
OGD/R cells (Figure 4d). Consistent with the flow cytometric
analysis, miR-378 mimic transfection resulted in a significant
downregulation of cleaved caspase-3 when compared with
the OGD/R group. Furthermore, pretreatment with the
miR-378 inhibitor further increased the cleaved caspase-3
expression (Figures 4e and f).
MiR-378 reduces caspase-3 expression in the intestine
by targeting the 3?-UTR of caspase-3. Caspase-3, a critical
executor of apoptosis, is one of the predicted putative targets
of miR-378 based on the prediction algorithms. One
miR-378binding site was identified within the 3?-UTR of the caspase-3
mRNA (Figure 5a). A dual-luciferase reporter assay was
performed to validate caspase-3 as a putative target of
miR-378. The co-transfection of 293 T cells with
caspase-3luc and a miR-378 mimic reduced the expression of luciferase
by 46% compared with the results following NC
caspase-3luc/miR-378 mimic co-transfection (Figure 5b). These results
indicated that miR-378 decreased the expression of
caspase3 through its direct binding with the caspase-3 3?-UTR.
Intestinal I/R is a serious clinical event that is associated with
high morbidity and mortality. This study presented two
important findings. First, with a miRNA microarray approach,
we searched for miRNAs that were differentially expressed in
the intestinal mucosa in response to intestinal I/R. We
identified 18 downregulated miRNAs with expression that
was decreased at least 1.5-fold. MiR-378 is one of these
markedly decreased miRNAs and was found to be the putative
target mRNA linked to cell death based on the application of
the TargetScan, miRanda, CLIP-Seq and miRDB prediction
algorithms. Furthermore, we demonstrated that the
overexpression of miR-378 significantly ameliorated intestinal
tissue damage in WT/TG mice and OGD-challenged IEC-6
cell injury. Second, overexpression of miR-378 reduced
intestinal epithelial cell apoptosis in both in vivo and in vitro
ischemic models and attenuated cleaved caspase-3
expression. These results imply that the protective effects of miR-378
following intestinal ischemia are likely mediated by the
inhibition of cell apoptosis via the translational repression of
MiRNAs are known to be important mediators of gene
regulation in response to cell-to-cell signaling and to act in the
negative feedback of gene regulation,16,17 which affects
several biological processes, such as development,26
differentiation,27 apoptosis28?30 and oncogenesis.31 MiRNA
analyses in organ I/R injury have been reported in recent
studies. Xu et al.32 found 78 miRNAs that exhibit more than
twofold differences in the liver upon I/R injury. Among these
miRNAs, four miRNAs, including miR-23a, miR-326,
miR-346_MM 1 and miR-370, were further significantly
downregulated by ischemic preconditioning compared with
the levels in non-preconditioned controls. Furthermore, Song
et al.33 reviewed the differential expression of miRNAs in
ischemic heart disease. To the best of our knowledge, there
are no miRNA microarray analyses that have identified global
changes in miRNA expression in the intestines of mice
subjected to I/R injury that can be used to characterize the
potential roles of miRNAs. In this study, we identified 19
miRNAs in the intestinal mucosa that changed by 41.5-fold
relative to the sham group (Table 1). Similar to previous
studies,34 we selected some miRNAs that were closely related
to organs I/R injury. Among these differentially expressed
miRNAs, let-7b, miR-26b, miR-182, miR-192, let-7d, miR-15b,
miR-16, let-7a and miR-378, were confirmed by RT-qPCR to
exhibit marked decreases in expression in the intestinal
mucosa following intestinal I/R (Table 2). Previous studies
have demonstrated the role of miR-378 in the regulation of cell
apoptosis. For example, a recent study demonstrated that the
downregulation of miR-378 supports cell survival by targeting
the insulin-like growth factor receptor in cardiomyocytes and
acts as a negative regulator.35 Paradoxically, some
researchers have found that miR-378 may suppress luteal cell
apoptosis by targeting the interferon gamma receptor 1
gene,36 and the overexpression of miR-378 attenuates
I/Rinduced cell apoptosis by inhibiting caspase-3 expression in
cardiomyocytes.37 Therefore, the role of miR-378 in the
regulation of intestinal epithelial cell death remains an enigma.
Thus, we selected miR-378 for further functional studies.
Chemically modified agomir and antagomir have been used
to increase or decrease miRNA expression in vivo.38
Therefore, we first used agomiR-378 and antagomiR-378 and found
that they significantly increased and decreased miR-378
expression in the intestinal mucosa, respectively (Figure 2a).
Furthermore, when pretreated with agomiR-378, intestinal I/R
injury was attenuated, as evidenced by the significantly
decreased Chiu?s scores, DAO activities and
TUNELpositive epithelial cells. By contrast, miR-378 antagomir
pretreatment aggravated the intestinal tissue injury (Figures
2b-f). Obviously, the current results indicated that miR-378
may have a protective role in intestinal I/R-induced intestinal
MiR-378 is predicted to have many potential targets.39,40
More importantly, these assumed targets include both
wellcharacterized pro-apoptotic and anti-apoptotic targets.
Therefore, it is necessary to evaluate the long-term and global
consequences of miR-378 overexpression in adult mice. To
generate miRNA-378 overexpression TG mice, the specific
promoter and pronuclear injection were used to drive the
expression of miR-378 in mice. RT-qPCR analysis revealed
that miR-378 was successfully overexpressed in TG mice to a
level of 3.39-fold that observed in WT mice (Figure 3a).
Thereafter, we performed further biochemical and
physiological studies. As demonstrated, miR-378 overexpression in TG
mice alleviated intestinal I/R injury (Figures 3b-f). OGD/R
served to create an in vitro model of intestinal I/R injury that
has previously been proven to be more amenable to the
molecular dissection of cell death mechanisms.41 Consistent
with evidence from the intestinal protection in vivo, our in vitro
data revealed that IEC-6 cell viability was significantly
preserved after transfection with the miR-378 mimic, whereas
OGD/R challenge led to obvious cell death. By contrast, the
IEC-6 cell viability was further decreased compared with the
OGD/R group following pretreatment with the miR-378
inhibitor (Figure 4b). Taken together, our results highlight the
crucial role of miR-378 in reducing the intestinal injury induced
Previous studies have documented that apoptosis is the
main mode of intestinal mucosal cell death after intestinal I/
R,8,9 and the exploration of the mechanisms of apoptosis
might lead to effective therapy for organ I/R injury.5,42,43
Therefore, we investigated the role of miR-378 in the
regulation of intestinal epithelial cell apoptosis. In this study,
we found that the intestinal mucosal cell apoptotic index was
reduced following pretreatment with miR-378 agomir, whereas
miR-378 antagomir administration increased the apoptotic
index. Consistent with the evidence of intestinal protection
in vivo, our in vitro data demonstrated that the intestinal
apoptotic/necrotic cells were also markedly reduced when
pretransfected with miR-378 mimic in the OGD/R-challenged
IEC-6 cells, whereas miR-378 inhibitor transfection
aggravated the apoptotic and necrotic cells (Figure 4d). These data
indicate that increased expression of miR-378 exerts
antiapoptotic properties. As intestinal epithelial cell apoptosis has
been known to contribute to intestinal I/R injury, and a
reduction in intestinal mucosa apoptosis could reduce the
intestinal injury induced by I/R, it can be concluded that the
overexpression of miR-378 attenuated intestinal I/R injury by
inhibiting intestinal epithelial cell apoptosis.
The regulation of miRNAs and their interactions is complex.
It is well accepted that an miRNA can bind to a large number of
mRNAs in a manner dependent on a signature sequence in
the 3?-UTR region of the target mRNAs. A specific mRNA can
be controlled by many miRNAs if the miRNAs contain
complementary binding sites. Structural studies have
estimated that most miRNAs can inhibit approximately 200
mRNAs.44 Although our study has identified a relationship
between miR-378 and its target caspase-3 in the ischemic
intestine using prediction algorithms (i.e., TargetScan,
miRanda, CLIP-Seq and miRDB) and the dual-luciferase reporter
assay, the protection afforded by the miR-378 mimetic is only
partial, and therefore, other miRNAs and mRNAs might also
have a role in the damage-sparing mechanism.
Our observation is consistent with recent reports that have
demonstrated that miR-378 targets cleaved caspase-3. Wang
et al.45 reported that miR-378 inhibits cell growth and
enhances L-OHP-induced apoptosis in human colorectal
cancer. Lee et al.46 found that miR-378 transfection could
enhance cell survival and could reduce caspase-3 activity by
inhibiting the expression of suppressor of fused and Fus-1.
Caspase-3 is a well-established executor of apoptosis that
acts by cleaving various substrate proteins and also amplifies
the death signal from the plasma membrane by activating
additional caspases. Our previous studies demonstrated that
inhibiting the activation of caspase-3 could reduce ischemic
intestinal injury. In this study, we found that intestinal I/R
caused severe intestinal injury accompanied by a significant
magnitude of miRNA expression, and overexpression
of miR-378 decreased caspase-3 activation and reduced
intestinal mucosa cell apoptosis/necrosis both in vivo and
There are issues that remain to be resolved in this study.
First, apoptosis is an ATP-dependent form of cell death
program that is related to mitochondrial dysfunction and
caspase activation. Our results showed that, in miRNA-378
overexpressing TG mice, the addition of agomiR-378 and
miR-378 mimic decreased caspase-3 cleavage. However,
whether miR-378 changes the mitochondrial function or ATP
biogenesis remains to be determined in the future. Second,
because several miRNAs responded rapidly to intestinal I/R,
this study only investigated the function of miR-378 in ischemic
intestine; whether other differentially expressed miRNAs
(miR-182, miR-192, let-7a, etc.) have roles during the process
was not explored. In addition, the parallel existence of
necroptosis,41 apoptosis and autophagy of epithelial cells
following ischemic stimulus demonstrates the complexity of
the pathophysiology of intestinal I/R, and the regulatory
process by which miRNAs are differentially expressed
requires further exploration.
In summary, the modulation of miRNA expression offers a
potential therapeutic option for intestinal I/R injury. This study
was the first to analyze miRNA expression profiles in the
intestinal mucosa after intestinal I/R and to find that miR-378 is
significantly downregulated during the process. Further
studies revealed that increased expression of miR-378
attenuated intestinal I/R injury by inhibiting intestinal mucosal
cell apoptosis, which is associated with the regulatory effects
Figure 5 The luciferase reporter assay of miR-378. MiR-378 mimic, miR-378
inhibitor and NC mimic were co-transfected with a modified control vector containing the
caspase-3 3?-UTR. (a) A schematic representation of the interaction between miR-378
and the 3?-UTR of caspase-3. (b) The luciferase assay showed that miR-378
downregulated 46% of the expression of caspase-3 in the Reporter+mimic miR-378
group, whereas the miR-378 inhibitor reversed the effect of miR-378. The results indicated
that miR-378 acts on the 3?-UTR. The data are expressed as the mean ? S.D. (n = 5
independent experiments). **Po0.01 compared with the Reporter+mimic NC group
of miR-378 on caspase-3 signaling. The protection rendered
by miR-378 may represent a potential novel therapeutic target
for the treatment of diseases related to intestinal I/R injury.
Materials and Methods
In vivo experiments
Animal model and preparation of specimens: All studies were approved
by the Animal Care Committee of Sun Yat-sen University (China) and were
performed in accordance with National Institutes of Health guidelines for the use of
experimental animals. Ninety-six adult pathogen-free male mice (weighing 25?30 g)
were housed in individual cages in a temperature-controlled room under a fixed
circadian rhythm with free access to food and water.
Male FVB/N mice (8?10 weeks old) were anesthetized with pentobarbital (30 mg/
kg, intraperitoneal injection). The small intestine was exteriorized by midline
laparotomy. The intestinal I/R injury was established by occluding the superior
mesenteric artery (SMA) with a microvessel clip for 60 min followed by 120 min of
reperfusion according to our previous study.9 Ischemia was recognized by the
existence of pulselessness or a pale color of the small intestine. The return of pulses
and the re-establishment of the pink color were assumed to indicate valid reperfusion
of the intestine. At the end of the reperfusion, a segment of 10 cm of the intestine was
cut 5 cm away from the ileocecal valve and was divided into two segments. The
segments were fixed in 10% neutral formaldehyde, paraffin embedded for
morphological analysis and washed with cold saline after being scraped off. The
intestinal mucosa was dried with filter paper and preserved at ?80 ?C for detection.
Histological assessments of intestinal injury: The segment of small
intestine was stained with hematoxylin and eosin. Damage of intestinal mucosa was
evaluated independently by two pathologists who were blinded to the study groups.
The degree of injury was evaluated using the criteria of Chiu's score as previously
described.47 A minimum of five randomly chosen fields from each mouse were
evaluated and averaged to determine mucosal damage.
Detection of DAO activity in the plasma: To further confirm intestinal injury,
serum DAO, a sensitive marker that reflects small intestinal mucosal injuries, was
detected using a chemical assay kit (Nanjing Jiancheng Biochemicals Ltd, Nanjing,
China) with an ultraviolet spectrophotometer at the wavelength of 436 nm according
to the manufacturer?s protocol.
MiRNA microarray analysis: Mice were randomly assigned to the sham or the
injury group (n = 5 per group). Total miRNAs were isolated from the intestinal mucosa and
processed for miRNA microarray analysis using the miRCURY LNA Array (version 11.0
Exiqon A/S, Vedbaek, Denmark) system. RNA samples were labeled with an
ExiqonmiRCURY Hy3/Hy5 power labeling kit and hybridized on a miRCURY LNA Array
station. Scanning was performed with an Axon GenePix 4000B microarray scanner.
GenePix pro version 6.0 was used to read the raw image intensities. The intensity of the
green signal was calculated after background subtraction, and replicated spots on the
same slide were averaged to obtain median intensity. The median normalization method
was used to acquire normalized data (foreground minus background divided by median).
The median was the 50th percentile of miRNA intensity and was 450 in all samples after
background correction. The threshold value for significance used to define the upregulation
or downregulation of miRNAs was a P-value o0.05 calculated by t-test. The miRNAs that
were selected for investigation in our study were further filtered on the basis of expression
levels and previously published data.
Prediction of miRNA-mRNA targets: MiRNA targets are difficult to identify
because of the lack of strict base pairing between miRNA and mRNA target
sequences. Several computation algorithms, including TargetScan,48 miRanda,49
CLIP-Seq50 and miRDB,51 aid this task by examining base-pairing rules between
miRNAs and the locations of mRNA target sites with binding sequences within the
target?s 3?-UTR and the conservation of target binding sequences within related
genomes. After all the predicted targets were identified, we further calculated the
intersection of the above four algorithms and drew a Venn diagram to analyze the
specific novel candidate predicted targets (Supplementary Figure 1). Genes that
were predicted by three of the TargetScan, miRanda, CLIP-Seq and miRDB
algorithms were regarded as potential targets of a certain miRNA.52
In vivo administration of agomiR-378 and antagomiR-378: To
investigate the effects of miRNA-378 following intestinal I/R injury, the chemically
modified agomir and antagomir were used to increase or decrease miRNA
expression in vivo.38 The miRNA agomir is a chemically modified, cholesterylated,
stable miRNA mimic, and its in vivo delivery resulted in target silencing similar to the
effects induced by the overexpression of endogenous miRNA. The miRNA
antagomir is a chemically modified, cholesterol-conjugated, single-stranded RNA
analog that complements the miRNAs and could efficiently and specifically silence
the endogenous miRNA. AgomiR-378 and antagomiR-378 were synthesized by
RiboBio (Guangzhou, China). Scrambled mimics that did not target any miRNA
were injected as a NC. Their sequences are listed as follows: agomir-378:
agomir-378 NC: 5?-UCACAACCUCCUAGAAAGAGUAGA-3?, 3?-AGUGUUGGAGG
AUCUUUCUCAUCU-5?, antagomir-378: 5?-CCUUCUGACUCCAAGUCCAGU-3?;
and antagomir-378 NC: 5?-CAGUACUUUUGUGUAGUACAAA-3?.
The mice received either agomir, antagomir or their NCs (100 ?l) via tail vein
injection (40 mg/kg body weight, n = 8 per group) for three consecutive days.
Expression of miR-378 was detected on the fourth day by RT-qPCR.
Generation of a miR-378 TG mouse model: MiRNA-378-overexpressing TG
(Cyagen Biosciences, Guangzhou, China) mice were produced by pronuclear injection.
Briefly, we first acquired the genomic sequence of miR-378 from the Origene company
(OriGene Technologies, Rockville, MD, USA) website, then intercepted Villin genes
upstream of the transcription start 7-kb site and the first intron 5.5 kb of sequence based
on previous studies. We next added the linearization restriction site MluI between the
two homology arms of the inserted vector pStar-K using Gap-repair ways to obtain a
final vector. To generate miRNA-378-overexpressing TG mice, the specific promoter and
pronuclear injection were used to drive the expression of miR-378 in founder mice. The
offspring of FVB/N mice propagation was based on founder FVB/N mice using full-sib
mating. The level of miR-378 was detected in the third-generation mice by RT-qPCR,
normalized to U6, and expressed as the fold change relative to the WT control. The
primer designs and sequential methods are listed as follows: Primer design:
homologous arm before PCR amplification: BamHI-upstream-F: 5?-ACTCGCGGAT
CCTTTAATCCCATCACTTGGGAGG-3?, MluI-upstream-R: 5?-GCGTAGTCCCATCTG
GGAAATACGCGTGGCAATGGCAGAGTGAAGAG-3?. Homologous arm after PCR
amplification: MluI-upstream-F: 5?-ACGCGTATTTCCCAGATGGGACTACGC-3?,
ACTGTAG-3?. DsRed2 amplification: Downstream-DsRed2-F: 5?-GCCACCATGGA
TAGCACTGAGAACGTC-3?, DsRed2-mir-378-R: 5?-CACTGCTTCTGCTGACAACTG
CTACTGGAACAGGTGGTGG-3?. Mir-378 amplification: DsRed2-mir-378-F: 5?-GT
TCCAGTAGCAGTTGTCAGCAGAAGCAGTG-3?, NotI-mir-378-R: 5?-AAGGAAAAAAGC
GGCCGCCTGGGTTAGCCACCAAAGAC-3?, Vector of pStar-K for subsequent
restructuring: pStar-K-F: 5?-ATTTCCCAGATGGGACTACGC-3?, pStar-K-R:
Real-time quantitative polymerase chain reaction (RT-qPCR): Total
RNA from fresh tissues and cell lines was extracted using Trizol Reagent (Invitrogen,
Grand Island, NY, USA) following the manufacturer's instructions. The quality of RNA
was examined using a UV-Vis spectrophotometer UV-1800 (Shimadzu, Japan). RNA
integrity was verified using 1.5% agarose gel electrophoresis with OD260/280 between
1.8 and 2.0, and RNA 28 s/18 s41. RT-qPCR analysis of the miRNA was performed
using RT primer and TaqMan probe for the miRNAs (Ribobio) on an ABI 7500 (Applied
Biosystems, Foster City, CA, USA) according to the manufacturer's protocol. Their
primers are listed in Supplementary Table 1. The expression of RNA U6 small nuclear 2
(RNU6B) was used as an endogenous reference control. Each RT-qPCR analysis was
repeated twice using three independent specimens. The relative abundance of miRNA
in the tissues and cell lines was calculated using the equation RQ = 2 ? ??CT.
In situ TUNEL for intestinal mucosa apoptosis assay: The ileal
fragments were fixed in 10% neutral formaldehyde and embedded in paraffin.
The apoptosis of the intestinal mucosal epithelial cells was performed with the
terminal deoxynucleotidyltransferase (TdT)-mediated dUDP-biotin nick end labeling
(TUNEL) method as previously described. Cell death was assessed using an In Situ
Detection assay kit (Roche, Indianapolis, IN, USA). TUNEL-positive cells were
characterized by dark brown staining of the nucleus and nuclear membrane.
Quantitation was performed by counting the numbers of positive cells in five
randomly chosen fields within each slide at 400 ? independently by two pathologists
who were blinded to the study groups. The rate of cell apoptosis (apoptotic index) is
expressed as a percentage of the TUNEL-positive cells using the following formula:
the number of TUNEL-positive cell nuclei/the number of total cell nuclei ? 100.
In vitro experiments
IEC-6 cell culture and oxygen and glucose deprivation/reperfusion
(OGD/R) model: Intestinal epithelial cells (IEC-6, catalog no. RL-1592) were
obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and
were cultured in Dulbecco?s modified Eagle?s medium containing 4.5 g/l D-glucose, 10%
v/v fetal bovine serum (FBS) and 1% penicillin/streptomycin antibiotics (Gibco, Invitrogen
Ltd, Shanghai, China). The cells were maintained under standard cell culture conditions
at 37 ?C under 5% CO2 and 21% O2. To simulate the situation of intestinal ischemia
in vivo, oxygen and glucose deprivation (OGD) was used in vitro. Briefly, after the IEC-6
cells were grown under normoxic conditions up to a confluence of 80%, OGD was
induced by exchanging the normoxic medium with D-Hanks buffer (OGD medium), and
the cells were then switched to a modular incubator chamber filled with a 95% N2 and
5% CO2 gas mixture for 4 h at 37 ?C. Following the OGD, the medium was changed
back to the normoxic medium, and the cells were incubated under the normal conditions
for an additional 4 h of reoxygenation.53,54 The cells that were not subjected to OGD/R
were incubated at 37 ?C with 5% CO2 for the same time periods and served as control
(sham). Six independent in vitro experiments were performed in the present work.
miRNA transfection: To assess the function of elevated levels of miR-378 in IEC-6
cells, pre-miR-378 (mimic) and miR-378 inhibitor were transfected for miR-378
overexpression and inhibition, respectively. Before large-scale transfection, the conditions
were optimized with cells cultured in 96-well plates in Opti-MEM-I serum-reduced
medium, NC oligonucleotide with FAM moiety at the 5? end, and Lipofectamine 2000
Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer?s procedure. The
transfection efficiency was determined by the use of the NC oligonucleotide with 6-FAM
at the 5? end. After optimization, transfection complexes were added to the cells at a final
oligonucleotide concentration of 50 nM.55 MiR-378 mimic and inhibitor NCs were also
involved in this study. All oligonucleotides were purchased synthesized (RiboBio). All
experiments were performed in triplicate. After 48 h transfection, the cells were
harvested and used for molecular and cellular studies.
Dual-luciferase reporter analysis: 293 T cells (5 ? 104cells/well) were
cultured in 48-well plates for 24 h and then transfected with
PMIR-RB-REPORTcaspase-3-3?-UTR (1 ?g/well) (RiboBio) and miR-378 mimic (100 nm per well) or a
NC, separately. The plasmid (PMIR-RB-REPORT-caspase-3-3?-UTR) contained a
synthetic Firefly luciferase gene that served as the reference gene and caspase-3
3?-UTR downstream from Renilla luciferase served as a reporter gene. The cells
were harvested 24 h after transfection, and the ratio of Renilla and Firefly luciferase
activities was measured with a dual-luciferase reporter assay kit (Promega,
Madison, WI, USA) according to the manufacturer?s instructions.
Cell viability assessment: The cells (1 ? 105) were plated into each of the wells
of the 96-well plates. After OGD/R, the MTT assay was used to detect the IEC-6
cells viabilities. Briefly, MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2-H-tetrazolium bromide, 5 mg/ml in phosphate-buffered saline, Sigma, Shanghai, China) was
added to each well and incubated for 4 h at 37 ?C. Then, the medium was replaced
with 150 ?l buffered DMSO. The optical density (OD) was recorded using a
microplate reader at the wavelength of 490 nm. Cell viability is expressed as a
percentage of the sham value.
Flow cytometric analysis: IEC-6 cell apoptosis was assayed by flow cytometry
according to the protocol provided by the manufacturer after OGD/R. Briefly, the cells
were washed twice with cold PBS before staining with FITC Annexin V and propidium
iodide (PI) using the Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences, Franklin
Lakes, NJ, USA) for 15 min at room temperature in the dark. The stained cells were
analyzed using flow cytometry within 1 h. The FITC Annexin V+/PI-and FITC Annexin
V+/PI+ cell populations were considered to represent necrotic and apoptotic cells.
Western blot analysis: Protein was extracted from intestinal mucosal samples
and IEC-6 cells. The levels of cleaved caspase-3 expression were determined using
a specific antibody against the large fragment (17 kDa) of activated caspase-3 that
resulted from cleavage. Twenty micrograms of total protein was electrophoresed on
a 12% (w/v) SDS-PAGE gel, transferred onto a nitrocellulose membrane and
blocked with 5% (w/v) nonfat dried milk. The membranes were probed with the
caspase-3 primary antibody (Cell Signaling Technology, Danvers, MA, USA, 9665,
1 : 200) followed by the peroxidase-conjugated secondary antibody. The protein
signal was visualized with chemiluminescence reagents under a GeneGnome Bio
Imaging System (Syngene, MD, USA). The amount of cleaved caspase-3 was
quantified by densitometry and normalized to the internal control (GAPDH).
Statistics analysis. All data are expressed as the mean ? S.D. Differences
between two groups were analyzed by Student?s t-test and between multiple groups
by a one-way ANOVA post hoc procedure. Po0.05 (two-sided tests) was
considered statistically significant.
Conflict of Interest
The authors declare no conflict of interest.
Acknowledgements. We thank Professor Weikang Wu (Department of
Pathophysiology, Sun Yat-sen University, Guangdong, China) for technical
assistance. We thank the KangChen Bio-Tech Inc. for microarray data and analysis.
We thank Dr. Yihong Ling and Yuanzhong Yang (Department of Pathology, Sun
Yatsen University Cancer Center, Guangdong, China) for histopathological help. This
work was supported by grants from National Natural Science Foundation, Beijing,
China (81671955 to Kexuan Liu, 81401630 to Cai Li, 81101407 to Yunsheng
Li and 81772116 to Shihong Wen), and grant from Key Program of Natural
Science Foundation of Guangdong Province, China
Springer Nature remains neutral with regard to jurisdictional claims in published maps
and institutional affiliations.
1. Mallick IH YW , Winslet MC , Seifalian AM . Ischemia-reperfusion injury of the intestine and protective strategies against injury . Dig Dis Sci 2004 ; 49 : 1359 - 1377 .
2. Martin B . Prevention of gastrointestinal complications in the critically ill patient . AACN Adv Crit Care 2007 ; 18 : 158 - 166 .
3. Yang J , Zong CH , Zhao ZH , Hu XD , Shi QD , Xiao XL et al. Vasoactive intestinal peptide in rats with focal cerebral ischemia enhances angiogenesis . Neuroscience 2009 ; 161 : 413 - 421 .
4. Zhou J , Huang WQ , Li C , Wu GY , Li YS , Wen SH et al. Intestinal ischemia/reperfusion enhances microglial activation and induces cerebral injury and memory dysfunction in rats . Crit Care Med 2012 ; 40 : 2438 - 2448 .
5. Liu KX , Li C , Li YS , Yuan BL , Xu M , Xia Z et al. Proteomic analysis of intestinal ischemia/ reperfusion injury and ischemic preconditioning in rats reveals the protective role of aldose reductase . Proteomics 2010 ; 10 : 4463 - 4475 .
6. Wen SH , Ling YH , Liu WF , Qiu YX , Li YS , Wu Y et al. Role of 15-F2t-isoprostane in intestinal injury induced by intestinal ischemia/reperfusion in rats . Free Radic Res 2014 ; 48 : 907 - 918 .
7. Li C , Xu M , Wen SH , Yao X , Wu Y , Huang CY et al. Limb remote ischemic preconditioning for intestinal and pulmonary protection during elective open infrarenal abdominal aortic aneurysm repair . Anesthesiology 2013 ; 118 : 842 - 852 .
8. Ikeda HSY , Suzuki M , Koike M , Tamura J , Tong J , Nomura M et al. Apoptosis is a major mode of cell death caused by ischaemia and ischaemia reperfusion injury to the rat intestinal epithelium . Gut 1998 ; 42 : 530 - 537 .
9. Wen SH , Li Y , Li C , Xia ZQ , Liu WF , Zhang XY et al. Ischemic postconditioning during reperfusion attenuates intestinal injury and mucosal cell apoptosis by inhibiting JAK/STAT signaling activation . Shock 2012 ; 38 : 411 - 419 .
10. Gillies LA , Kuwana T. Apoptosis regulation at the mitochondrial outer membrane . J Cell Biochem 2014 ; 115 : 632 - 640 .
11. Liu KX , He W , Rinne T , Liu Y , Zhao MQ , Wu WK . The effect of ginkgo biloba extract (EGb 761)pretreatment on intestinal epithelial apoptosis induced by intestinal ischemia reperfusion in rats role of ceramide . Am J Chin Med 2007 ; 35 : 805 - 819 .
12. Chen G , Zhang Z, Cheng Y , Xiao W , Qiu Y , Yu M et al. The canonical Notch signaling was involved in the regulation of intestinal epithelial cells apoptosis after intestinal ischemia/ reperfusion injury . Int J Mol Sci 2014 ; 15 : 7883 - 7896 .
13. Yucel AF , Kanter M , Pergel A , Erboga M , Guzel A . The role of curcumin on intestinal oxidative stress, cell proliferation and apoptosis after ischemia/reperfusion injury in rats . J Mol Histol 2011 ; 42 : 579 - 587 .
14. Chen Z , Wang G , Zhai X , Hu Y , Gao D , Ma L et al. Selective inhibition of protein kinase C beta2 attenuates the adaptor P66 Shc-mediated intestinal ischemia-reperfusion injury . Cell Death Dis 2014 ; 5 : e1164 .
15. Liu C , Shen Z , Liu Y , Peng J , Miao L , Zeng W et al. Sevoflurane protects against intestinal ischemia-reperfusion injury partly by phosphatidylinositol 3 kinases/Akt pathway in rats . Surgery 2015 ; 157 : 924 - 933 .
16. Ambros V. microRNAs: tiny regulators with great potential . Cell 2001 ; 107 : 823 - 826 .
17. Ke X-S , Liu C-M , Liu D-P , Liang C-C. MicroRNAs: key participants in gene regulatory networks . Curr Opin Chem Biol 2003 ; 7 : 516 - 523 .
18. Baehrecke EH . miRNAs: micro managers of programmed cell death . Curr Biol 2003 ; 13 : R473 - R475 .
19. Hezova R , Slaby O , Faltejskova P , Mikulkova Z , Buresova I , Raja KR et al. MicroRNA- 342 , microRNA -191 and microRNA-510 are differentially expressed in T regulatory cells of type 1 diabetic patients . Cell Immunol 2010 ; 260 : 70 - 74 .
20. Mendell JT , Olson EN. MicroRNAs in stress signaling and human disease . Cell 2012 ; 148 : 1172 - 1187 .
21. Zhang B , Zhou M , Li C , Zhou J , Li H , Zhu D et al. MicroRNA -92a inhibition attenuates hypoxia/reoxygenation-induced myocardiocyte apoptosis by targeting Smad7 . PLoS ONE 2014 ; 9 : e100298 .
22. Zhu F , Liu JL , Li JP , Xiao F , Zhang ZX , Zhang L. MicroRNA- 124 (miR-124) regulates Ku70 expression and is correlated with neuronal death induced by ischemia/reperfusion . J Mol Neurosci 2014 ; 52 : 148 - 155 .
23. Hu H , Jiang W , Xi X , Zou C , Ye Z. MicroRNA -21 attenuates renal ischemia reperfusion injury via targeting caspase signaling in mice . Am J Nephrol 2014 ; 40 : 215 - 223 .
24. McKenna LB , Schug J , Vourekas A , McKenna JB , Bramswig NC , Friedman JR et al. MicroRNAs control intestinal epithelial differentiation, architecture, and barrier function . Gastroenterology 2010 ; 139 : 1654 - 1664 1664 e1651 .
25. Zhang L, Cheng J , Fan XM . MicroRNAs: new therapeutic targets for intestinal barrier dysfunction . World J Gastroenterol 2014 ; 20 : 5818 - 5825 .
26. Iwasaki YW , Siomi H. miRNA regulatory ecosystem in early development . Mol Cell 2014 ; 56 : 615 - 616 .
27. Navarro C , Cruz-Oro E , Prat S. Conserved function of FLOWERING LOCUS T (FT) homologues as signals for storage organ differentiation . Curr Opin Plant Biol 2015 ; 23 : 45 - 53 .
28. He JF LY , Wan XH , Jiang D. Biogenesis of MiRNA-195 and its role in biogenesis, the cell cycle, and apoptosis . J Biochem Mol Toxicol 2011 ; 25 : 404 - 408 .
29. Wojtas B , Ferraz C , Stokowy T , Hauptmann S , Lange D , Dralle H et al. Differential miRNA expression defines migration and reduced apoptosis in follicular thyroid carcinomas . Mol Cell Endocrinol 2014 ; 388 : 1 - 9 .
30. Zhang Y , Chen N , Zhang J , Tong Y . Hsa-let-7g miRNA targets caspase-3 and inhibits the apoptosis induced by ox-LDL in endothelial cells . Int J Mol Sci 2013 ; 14 : 22708 - 22720 .
31. Hatziapostolou M , Polytarchou C , Aggelidou E , Drakaki A , Poultsides GA , Jaeger SA et al. An HNF4alpha-miRNA inflammatory feedback circuit regulates hepatocellular oncogenesis . Cell 2011 ; 147 : 1233 - 1247 .
32. Xu CF YC , Li YM . Regulation of hepatic microRNA expression in response to ischemic preconditioning following ischemia reperfusion injury in mice . OMICS 2009 ; 13 : 513 - 520 .
33. Song MA , Paradis AN , Gay MS , Shin J , Zhang L . Differential expression of microRNAs in ischemic heart disease . Drug Discov Today 2015 ; 20 : 223 - 235 .
34. Lenaerts K , Ceulemans LJ , Hundscheid IH , Grootjans J , Dejong CH , Olde Damink SW . New insights in intestinal ischemia-reperfusion injury: implications for intestinal transplantation . Curr Opin Organ Transplant 2013 ; 18 : 298 - 303 .
35. Knezevic I , Patel A , Sundaresan NR , Gupta MP , Solaro RJ , Nagalingam RS et al. A novel cardiomyocyte-enriched microRNA, miR-378, targets insulin-like growth factor 1 receptor: implications in postnatal cardiac remodeling and cell survival . J Biol Chem 2012 ; 287 : 12913 - 12926 .
36. Ma T , Jiang H , Gao Y , Zhao Y , Dai L , Xiong Q et al. Microarray analysis of differentially expressed microRNAs in non-regressed and regressed bovine corpus luteum tissue; microRNA-378 may suppress luteal cell apoptosis by targeting the interferon gamma receptor 1 gene . J Appl Genet 2011 ; 52 : 481 - 486 .
37. Fang J , Song XW , Tian J , Chen HY , Li DF , Wang JF et al. Overexpression of microRNA-378 attenuates ischemia-induced apoptosis by inhibiting caspase-3 expression in cardiac myocytes . Apoptosis 2012 ; 17 : 410 - 423 .
38. Wang X , Zhang X , Ren XP , Chen J , Liu H , Yang J et al. MicroRNA -494 targeting both proapoptotic and antiapoptotic proteins protects against ischemia/reperfusion-induced cardiac injury . Circulation 2010 ; 122 : 1308 - 1318 .
39. Feng M , Li Z , Aau M , Wong CH , Yang X , Yu Q . Myc/miR-378/ TOB2/cyclin D1 functional module regulates oncogenic transformation . Oncogene 2011 ; 30 : 2242 - 2251 .
40. Fei B , Wu H. MiR -378 inhibits progression of human gastric cancer MGC-803 cells by targeting MAPK1 in vitro . Oncol Res 2012 ; 20 : 557 - 564 .
41. Wen SH , Ling YH , Yang WJ , Shen JT , Li C , Deng WT et al. Necroptosis is a key mediator of enterocytes loss in intestinal ischaemia/reperfusion injury . J Cell Mol Med 2017 ; 21 : 432 - 443 .
42. Yang BYD , Wang Y . Caspase-3 as a therapeutic target for heart failure . Exp Opin Ther Targets 2013 ; 17 : 255 - 263 .
43. Wen SH , Ling YH , Li Y , Li C , Liu JX , Li YS et al. Ischemic postconditioning during reperfusion attenuates oxidative stress and intestinal mucosal apoptosis induced by intestinal ischemia/ reperfusion via aldose reductase . Surgery 2013 ; 153 : 555 - 564 .
44. Lewis BP SI , Jones-Rhoades MW , Bartel DP , Burge CB . Prediction of mammalian microRNA targets . Cell 2003 ; 115 : 787 - 798 .
45. Wang KY , Ma J , Zhang FX , Yu MJ , Xue JS , Zhao JS . MicroRNA-378 inhibits cell growth and enhances L-OHP-induced apoptosis in human colorectal cancer . IUBMB Life 2014 ; 66 : 645 - 654 .
46. Lee DY , Deng Z , Wang CH , Yang BB . MicroRNA-378 promotes cell survival, tumor growth, and angiogenesis by targeting SuFu and Fus-1 expression . Proc Natl Acad Sci USA 2007 ; 104 : 20350 - 20355 .
47. Chiu CJ MA , Brown R , Scott HJ , Gurd FN . Intestinal mucosal lesion in low-flow states . Arch Surg 1970 ; 101 : 478 - 483 .
48. Agarwal V , Bell GW , Nam JW , Bartel DP . Predicting effective microRNA target sites in mammalian mRNAs . Elife 2015 ; 4 : e05005 .
49. John B , Enright AJ , Aravin A , Tuschl T , Sander C , Marks DS . Human microRNA targets . PLoS Biol 2004 ; 2 : e363 .
50. Li JH , Liu S , Zhou H , Qu LH , Yang JH . starBase v2 . 0: decoding miRNA-ceRNA, miRNAncRNA and protein-RNA interaction networks from large-scale CLIP-Seq data . Nucleic Acids Res 2014 ; 42 (Database issue): D92 - D97 .
51. Wong N , Wang X. miRDB: an online resource for microRNA target prediction and functional annotations . Nucleic Acids Res 2015 ; 43 (Database issue): D146 - D152 .
52. Yu S , Kim J , Min H , Yoon S . Ensemble learning can significantly improve human microRNA target prediction . Methods (San Diego, CA) 2014 ; 69 : 220 - 229 .
53. Shen JT , Li YS , Xia ZQ , Wen SH , Yao X , Yang WJ et al. Remifentanil preconditioning protects the small intestine against ischemia/reperfusion injury via intestinal delta- and mu-opioid receptors . Surgery 2016 ; 159 : 548 - 559 .
54. Ma YL QP , Li Y , Shen L , Wang SQ , Dong HL , Hou WG et al. The effects of different doses of estradiol (E2) on cerebral ischemia in an in vitro model of oxygen and glucose deprivation and reperfusion and in a rat model of middle carotid artery occlusion . BMC Neurosci 2013 ; 14 : 118 .
55. Zhang GJ ZH , Xiao HX , Li Y , Zhou T . MiR -378 is an independent prognostic factor and inhibits cell growth and invasion in colorectal cancer . BMC Cancer 2014 ; 14 : 109 .