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Research Article
Control of Paneth cell function by HuR regulates gut
mucosal growth by altering stem cell activity
Lan Xiao
1
, Bridgette Warner
1
, Caroline G Mallard
1
, Hee K Chung
1
, Amol Shetty
2
, Christine A Brantner
3
, Jaladanki N Rao
1,4
,
Gregory S Yochum
5,6
, Walter A Koltun
5
, Kathleen B To
4
, Douglas J Turner
1,4
, Myriam Gorospe
7
, Jian-Ying Wang
1,4,8
Rapid self-renewal of the intestinal epithelium requires the ac-
tivity of intestinal stem cells (ISCs) that are intermingled with
Paneth cells (PCs) at the crypt base. PCs provide multiple secreted
and surface-bound niche signals and play an important role in the
regulation of ISC proliferation. Here, we show that control of PC
function by RNA-binding protein HuR via mitochondria affects
intestinal mucosal growth by altering ISC activity. Targeted de-
letion of HuR in mice disrupted PC gene expression profiles,
reduced PC-derived niche factors, and impaired ISC function,
leading to inhibited renewal of the intestinal epithelium. Human
intestinal mucosa from patients with critical surgical disorders
exhibited decreased levels of tissue HuR and PC/ISC niche dys-
function, along with disrupted mucosal growth. HuR deletion led
to mitochondrial impairment by decreasing the levels of several
mitochondrial-associated proteins including prohibitin 1 (PHB1)
in the intestinal epithelium, whereas HuR enhanced PHB1 ex-
pression by preventing microRNA-195 binding to the Phb1 mRNA.
These results indicate that HuR is essential for maintaining the
integrity of the PC/ISC niche and highlight a novel role for a
defective PC/ISC niche in the pathogenesis of intestinal mucosa
atrophy.
DOI 10.26508/lsa.202302152 | Received 12 May 2023 | Revised 29 August
2023 | Accepted 30 August 2023 | Published online 11 September 2023
Introduction
The maintenance of homeostasis in the gut epithelium is a complex
process that requires epithelial cells to rapidly alter gene ex-
pression patterns to regulate cell survival, proliferation, migration,
differentiation, and cell-to-cell interaction (Xian et al, 2017;Borrelli
et al, 2021;Yang et al, 2021). This homeostasis is disrupted in dif-
ferent situations, including surgical patients who undergo massive
gastrointestinal surgical resection and are then supported with
total parenteral nutrition. The disrupted renewal of the intestinal
epithelium in critically ill patients impairs mucosal adaptation and
causes gut barrier dysfunction, which can then lead to the trans-
location of luminal toxic substances and bacteria to the blood-
stream, sepsis, and, in some instances, multiple organ dysfunction
syndrome and death (Carter et al, 2013;Kumar et al, 2020). Effective
therapies to preserve the integrity of the intestinal epithelium in
patients with critical surgical illnesses are limited, as the mecha-
nisms that regulate gut mucosal renewal in stressful environments
are poorly understood.
The dynamic turnover rate of the human small intestinal epi-
thelium, which undergoes ~10
11
mitoses/day, is driven by intestinal
stem cells (ISCs) and is extensively regulated (Radtke & Clevers,
2005;Serra et al, 2019). ISCs divide daily and produce bipotent
progenitors, amplifying and differentiating into absorptive or se-
cretory lineages (Sailaja et al, 2016;Li et al, 2023). Paneth cells (PCs),
specialized intestinal epithelial cells (IECs) residing at the intestinal
crypt base, produce abundant antibacterial proteins and peptides
such as lysozyme and Reg3 lectins (Sato et al, 2011;Yu et al, 2020a).
PCs also create a niche for ISCs in the crypts and provide multiple
secreted and surface-bound niche signals that determine ISC fate
(Sato et al, 2011;Yilmaz et al, 2012;Chung et al, 2021). Emerging
evidence indicates that interactions between PCs and ISCs are
crucial for constant intestinal epithelial renewal under various
pathophysiological conditions (Rodriguez-Colman et al, 2017;Baulies
et al, 2020;Butto et al, 2020;Lueschow & McElroy, 2020). Coculturing of
sorted PCs with ISCs strongly enhances intestinal organoid formation
and growth (Sato et al, 2011;Yu et al, 2020a), whereas PC defects in the
crypts result in ISC dysfunction and inhibit intestinal mucosal growth
(Shroyer et al, 2007;Norona et al, 2020). When PCs are ablated ge-
netically in mice, intestinal enteroendocrine, tuft, and stromal cells can
also produce niche factors and support ISCs (Durand et al, 2012;van Es
et al, 2019).
HuR (encoded by the Elavl1 gene), one of the most prominent
RNA-binding proteins regulating mRNA translation and turnover, is
widely involved in many aspects of gut mucosal pathobiology (Li
et al, 2020;Palomo-Irigoyen et al, 2020). In our previous studies, we
1
Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD, USA
2
Institute for Genome Science, University of Maryland School
of Medicine, Baltimore, MD, USA
3
Electron Microscopy Core Imaging Facility, University of Maryland Baltimore, Baltimore, MD, USA
4
Baltimore Veterans Affairs Medical
Center, Baltimore, MD, USA
5
Department of Surgery, Pennsylvania State University College of Medicine, Hershey, PA, USA
6
Department of Biochemistry and Molecular
Biology, Pennsylvania State University College of Medicine, Hershey, PA, USA
7
Laboratory of Genetics and Genomics, National Institute on Aging-IRP, NIH, Baltimore, MD,
USA
8
Department of Pathology, University of Maryland School of Medicine, Baltimore, MD, USA
Correspondence: jywang@som.umaryland.edu
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found declines in the levels of tissue HuR - along with inhibition of
the growth of the intestinal mucosa - in patients with various ill-
nesses, including inflammatory bowel disease (IBD) (Xiao et al, 2019;
Li et al, 2020). Specific ablation of HuR in the intestinal epithelium of
mice (IE-HuR
−/−
) causes atrophy of the mucosa in the small in-
testine and compromises the regeneration of the gut mucosa and
adaptation after irradiation (Liu et al, 2014), septic stress (Zhang
et al, 2020), and mesenteric ischemia and reperfusion (Liu et al,
2017). Recently, HuR was shown to regulate PC function in the in-
testinal epithelium by altering the membrane localization of Toll-
like receptor 2 via posttranscriptional control of chaperone protein
CNPY3 (Xiao et al, 2019;Chung et al, 2021). However, little is known about
the role and mechanism of altered PCs by HuR, particularly their
interaction with ISCs, in the regulation of intestinal mucosal growth.
Mitochondria generate ATP to provide cells with energy and
function as factories that participate actively in the biosynthesis of
various macromolecules (Urbauer et al, 2020). As unveiled by recent
studies, by virtue of their ability to produce a plethora of metab-
olites, mitochondria in the gut epithelium appear to play an
emerging role as signaling organelles (Twig et al, 2008;Sato et al,
2021). Mitochondria sense the metabolic environment and integrate
host and microbial-derived signals (Berger et al, 2016;Ludikhuize et al,
2020). Mitochondrial metabolism, dynamics, and stress responses are
pivotal in regulating intestinal mucosal self-renewal and epithelial
differentiation (Berger et al, 2016;Jackson et al, 2020;Ludikhuize et al,
2020). Mitochondrial impairment delays gut mucosal repair and
weakens epithelium–host defenses. Targeted deletion of the genes
encoding mitochondrial proteins prohibitin 1 (PHB1) and heat shock
protein 60 (HSP60) in mice results in PC defects and causes a pre-
disposition to ileitis in mice (Jackson et al, 2020;Khaloian et al, 2020).
Mitochondrial activity is attenuated in human colonic mucosal tissues
from patients with ulcerative colitis (Ho & Theiss, 2022;Ozsoy et al, 2022).
In this study, we provide evidence that control of PC function by HuR
regulates the renewal of the small intestinal epithelium by affecting
ISC activity. We found that loss of HuR in mice altered cell type-specific
gene expression in PCs and led to dysfunction of the PC/ISC niche.
Humanintestinalmucosafrompatientswithcriticalsurgicaldisorders
exhibitedbothreducedHuRanddefectsinPC/ISCnichefunction,
which was associated with an inhibition of intestinal epithelial re-
newal. Our results further show that HuR enhances mitochondrial
function by increasing the levels of mitochondrial-associated proteins
including PHB1, and that it promotes PHB1 expression levels at least in
part by interfering with the function of microRNA-195 (miR-195). These
findings reveal that HuR is essential for sustaining integrity of the PC/
ISC niche in the intestinal epithelium and point to the HuR/miR-195/
PHB1 axis as novel therapeutic targets for interventions to enhance the
function of the PC/ISC niche and promote intestinal mucosal re-
generation and adaptation in patients with critical disorders.
Results
HuR deletion causes defects in the PC/ISC niche function in the
mucosa of the small intestine
To determine if HuR promotes intestinal epithelial renewal by
augmenting ISC proliferation via PCs, we used IE-HuR
−/−
mice that
were generated by crossing HuR
fl/fl
mice with villin-Cre–expressing
mice as described (Liu et al, 2014). HuR levels in the mucosa of the
small intestine and colon were undetectable in IE-HuR
−/−
mice, but
there were no changes in HuR expression in other tissues and
organs such as the gastric mucosa, lung, liver, kidney, and pancreas,
as reported previously (Liu et al, 2014,2017). Immunohistochemistry
analysis showed that HuR staining almost completely disappeared
in epithelial cells in the small intestinal mucosa of IE-HuR
−/−
mice,
although HuR expression levels were unaffected in submucosal
connective tissue (Fig 1A). Conditional HuR deletion in IECs inhibited
the renewal of the small intestinal epithelium, as indicated by a
decrease in the levels of BrdU-positive cells within the crypts and
subsequent shrinkages of crypt and villi, but it failed to affect the
growth of the colonic mucosa - similar to the observations reported
in our previous study (Liu et al, 2014,2017). Targeted HuR deletion in
mice did not alter the overall morphology or structure of the
mucosa of the small intestine or the colon. HuR deletion in the
intestinal epithelium led to abnormalities in PCs, as evidenced by
decreased lysozyme-positive cells in IE-HuR
−/−
mice relative to
control littermate mice (Fig 1B), as observed previously (Xiao et al,
2019). Interestingly, defects in PCs induced by HuR deletion were
associated with a loss of ISCs in the small intestinal mucosa.
Staining of whole mounts of the small intestine revealed that ISCs,
marked by OLFM4 and LGR5, were normally located at the base of
the crypts in littermate mice, but the numbers of OLFM4- and LGR5-
positive cells (Fig 1C and D) decreased markedly in IE-HuR
−/−
mice
when compared with control littermates. On the other hand, HuR
deletion did not affect the number of goblet cells and differentiated
enterocytes, as measured by Alcian blue staining and villin im-
munostaining analysis, respectively. To examine how HuR affected
the interaction of PCs with ISCs in the small intestinal mucosa after
HuR deletion, we examined changes in the levels of niche signals
derived from PCs in IE-HuR
−/−
and littermate mice. As shown in Fig
1E, immunostaining detection of WNT3 and NOTCH2 revealed
enriched signals at the base of small intestinal crypts in control
littermate mice, but their levels in intestinal crypts decreased
markedly in IE-HuR
−/−
mice. Semiquantitative analysis showed that
the intensities of WNT3 and NOTCH2 immunostaining decreased
by >90% in the HuR-deficient intestinal epithelium compared with
those observed in littermate mice (Fig 1F). Because WNT3 and
NOTCH2 are niche growth factors (Xian et al, 2017;Yu et al, 2018;
Bottcher et al, 2021), these results suggest that HuR deletion impairs
ISC proliferation at least partially by reducing PC-derived niche
signals.
Altered cell type-specific gene expression profiles in the
HuR-deficient intestinal epithelium
To obtain cell type-specific transcriptional regulation profiles in the
intestinal epithelium after HuR deletion, we performed single-cell
RNA-sequencing (scRNA-seq) analysis on the small intestinal
mucosa harvested from IE-HuR
−/−
and control littermate mice. In
total, 60,000 high-quality cells, as characterized (Fig S1A) and
validated by flow-cytometry analysis (Fig S1B–D), were grouped
into 18 clusters, which expressed classic cell type markers from
the absorptive or secretory lineages, and markers distinctive of
ISCs and daughter transient-amplifying cells (Fig S2), following
Regulation of PC/ISC niche by HuR Xiao et al. https://doi.org/10.26508/lsa.202302152 vol 6 | no 11 | e202302152 2of14
published single-cell transcriptomic signatures in the intestinal
epithelium (Islam et al, 2020;Elmentaite et al, 2021). Secretory cells
in the epithelial compartment of the small intestine consisted of
PCs, goblet cells, tuft cells, and enteroendocrine cells in both IE-
HuR
−/−
and littermate mice, whereas absorptive cells were marked
by various enterocytes (Fig 2A). Notably, PCs were marked by high
expression of Defe5 and Rg3a mRNAs, whereas ISCs were marked by
transcripts including Ascl2,Lgr5,Olfm4,Rgmb, and Smoc2 mRNAs,
as reported previously (Elmentaite et al, 2021;Luna Velez et al, 2023).
Cell cluster analysis revealed that the numbers of PCs and ISCs
decreased in IE-HuR
−/−
mice compared with control littermate mice,
which were consistent with the findings obtained from studies
using immunostaining assays as shown in Fig 1.
Profiling analysis of cell type-specific gene expression showed
the existence of transcriptionally distinct PCs and ISCs in the
small intestinal epithelium of IE-HuR
−/−
mice. A comparison of the
transcriptomic profiles in the mucosa from IE-HuR
−/−
mice relative
to control littermates demonstrated that ~2,116 RNAs were differ-
entially abundant in PCs; 1,144 were less abundant and 972 were more
abundant after HuR deletion (Fig 2B,top). In ISCs, ~400 RNAs were
differentially abundant - 289 RNAs were lower, and 120 RNAs were
higher in IE-HuR
−/−
mice compared with control littermates (Fig 2C,
top). The most highly increased and decreased RNAs in PCs and ISCs of
IE-HuR
−/−
mouse relative to control littermates are summarized in low
panels of Fig 2B and C. Amongst the top differentially abundant mRNAs
are many encoding niche growth factors and proteins that are nec-
essary for maintaining the PC structure and function. HuR deletion also
altered gene expression profiles of secretory progenitor cells (Atoh1+)
and goblet cells in the small intestinal epithelium, but the exact role
of HuR in the regulation of secretory progenitor and goblet cells
will be fully investigated in a separate study. Collectively, our single-
cell sequencing data analysis identified transcriptomic and cellular
Figure 1. Targeted deletion of HuR in mice
causes defects in the PC/intestinal stem
cell niche in the intestinal epithelium.
(A) Immunostaining of HuR in the small
intestinal mucosa of IE-HuR
−/−
and control
littermate mice. Red, HuR; and green, E-
cadherin (E-cad). Scale bars, 50 μm. Three
separate experiments showed similar
results. (B) Images of Paneth cells marked by
lysozymes in the small intestinal mucosa. Red,
lysozyme; and green, E-cad. Scale bars,
50 μm. (C) Immunostaining of OLFM4 in the
small intestinal crypts. Yellow, OLFM4; blue,
nucleus stained by DAPI. Scale bar, 25 μm.
(D) Immunostaining of LGR5 in the small
intestinal crypts, as shown in yellow–green.
Scale bar, 25 μm. (E) Immunostaining of
NOTCH2 (top) and WNT3 (bottom) in the small
intestinal crypts, as shown in red. Scale bar,
25 μm. (F) Quantitation of NOTCH2 (top) and
WNT3 (bottom) signals in the small intestinal
crypts treated as described in (E). Values are
means ± SEM (n= 5 biological replicates).
Unpaired, two-tailed ttest was used. *P< 0.05
compared with control littermates.
Regulation of PC/ISC niche by HuR Xiao et al. https://doi.org/10.26508/lsa.202302152 vol 6 | no 11 | e202302152 3of14
responses to HuR deletion in the small intestinal mucosa and strongly
suggested the importance of HuR in the PC/ISC niche to regulate the
renewal of the intestinal epithelium.
Reduced HuR levels associate with abnormal PC/ISC niche in
patients with critical illness
To explore the clinical relevance of HuR function in the human
PC/ISC niche, human ileal mucosal tissues were collected from
four Crohn’s disease (CD) patients who required urgent/emergent
intestinal resection because of severe complications (SC) such as
intestinal perforation, peritonitis, and necrotizing enteritis as well as
four healthy controls who had neither CD, nor emergency surgical
disorders. Consistent with findings observed in IBD patients without SC
(Xiao et al, 2019), the ileal mucosa from patients with CD/SC also
exhibited a significant decrease in the levels of HuR (Fig 3A), as
measured by immunostaining analysis. In the mucosa from the small
intestine,HuRwaslocatedinboththecytoplasmandnucleusin
control individuals, but the levels of total HuR and cytoplasmic HuR
were significantly lower in CD/SC patients relative to control patients.
Figure 2. Single-cell transcriptional profiles
of PCs and intestinal stem cells (ISCs) in
the small intestinal epithelium after HuR
deletion in mice.
(A) Uniform manifold approximation and
projection of key epithelial cell types of the
small intestinal mucosa from control
littermates (left) and IE-HuR
−/−
mice (right)
(n= 3). Annotated clusters of PCs are showed
in green; ISCs are shown in light-blue. (B) Top
panel, scatter plot depictions of genes
expressed differentially in PCs of control and
IE-HuR
−/−
mice, as measured by scRNA-seq
analysis. Low panel, differential expression
analysis of most altered genes in results
described in top panel. Values are the means
from three animals. The P-value cutoff used
for identifying differentially expressed genes
was 0.05. (C) Top panel, scatter plot depictions
of genes expressed differentially in ISCs.
Low panel, most altered genes in ISCs in
results described in the top panel. Values are
the means from three animals. The P-value
cutoff used for identifying differentially
expressed genes is 0.05.
Regulation of PC/ISC niche by HuR Xiao et al. https://doi.org/10.26508/lsa.202302152 vol 6 | no 11 | e202302152 4of14
Importantly, reduced levels of HuR in the intestinal mucosa in
patients with CD/SC were associated with significant defects in PC/
ISC niche function because the numbers of lysozyme-positive cells
and OLFM4- and LGR5-positive cells in the ileal mucosa from CD/SC
patients decreased markedly relative to those observed in control
patients (Fig 3B). In fact, PCs and ISCs were almost totally unde-
tectable in some ileal mucosal tissue samples from CD/SC patients.
Consistent with the observations in the small intestinal mucosa of
IE-HuR
−/−
mouse (Fig 1), decreased HuR levels in the mucosa of
patients with CD/SC were accompanied by reduced levels of PC-
derived niche signals, as evidenced by decreased intensities of
WNT3 and NOTCH2 immunostaining signals in the crypts of CD/SC
patients compared with control individuals (Fig 3C). Importantly,
the impaired function of the PC/ISC niche associated with HuR
inhibition in patients with CD/SC was linked to a significant inhi-
bition of the intestinal mucosal growth because the levels of the
proliferation marker Ki67 decreased markedly in CD/SC patients
relative to control individuals (Fig 3D). These results implicate PC/
ISC niche dysfunction in the impaired renewal of the intestinal
epithelium in critically ill patients after decreased tissue HuR levels.
HuR deletion impairs mitochondrial function in the
intestinal epithelium
Because mitochondria play an important role in maintaining in-
testinal epithelial homeostasis and its dysfunction is intimately
Figure 3. Reduced HuR associates with
defects in PC/intestinal stem cell niches in
patients with CD/SC.
(A) Immunostaining of HuR in the small
intestinal mucosa from control individuals
and Crohn’s disease patients who underwent
emergency surgery because of severe
complications (CD/SC). Purple, HuR; blue,
nucleus stained by DAPI. Scale bars, 50 μm. All
these experiments were repeated in four
controls and four patients with CD/SC and
showed similar results. (B) Images of PCs
(lysozyme-positive cells; top) and intestinal
stem cells marked by OLFM4 and LGR5
(bottom) in the human small intestinal
mucosa as examined by immunostaining
assays using anti-lysozyme (red), anti-
OLFM4, anti-LGR5 (yelloe-green), and anti-E-
cadherin (green). Blue, nucleus stained
by DAPI. Scale bars, 50 and 25 μm.
(C) Immunostaining of WNT3 (top)and
NOTCH2 (bottom) in the crypts of human small
intestine. Yellow–green, WNT3; red, NOTCH2;
and blue, nucleus stained by DAPI. Scale bar,
25 μm. (D) Small intestinal mucosal renewal in
patients with CD/SC, as assessed by
measuring Ki67 staining (pink). Scale bar,
50 μm.
Regulation of PC/ISC niche by HuR Xiao et al. https://doi.org/10.26508/lsa.202302152 vol 6 | no 11 | e202302152 5of14
involved in the pathogenesis of IBD and other mucosal pathologies
(Berger et al, 2016;Ludikhuize et al, 2020;Urbauer et al, 2020), we
tested the possibility that HuR regulates activity of the PC/ISC niche by
affecting mitochondrial metabolism. We further screened for expres-
sion of mitochondrial-associated genes in our single-cell sequencing
data from PCs and found that HuR deletion decreased the expression
levels of many mRNAs encoding proteins that control mitochondrial
catabolism and oxidation/reduction reactions (Fig 4A). To further
evaluate if IE-HuR
−/−
mice had mitochondrial abnormalities, we ex-
amined changes in expression of several mitochondrial-associated
proteins in the small intestinal mucosa. HuR ablation in mice de-
creased the levels of PHB1 and HSP60 (Fig 4B and C); these two proteins
are essential for mitochondrial integrity in the intestinal epithelium and
loss of PHB1 or HSP60 results in defective PCs and leads to ileitis in mice
(Jackson et al, 2020;Khaloian et al, 2020). PHB1 and HSP60 were highly
expressed in the small intestine mucosa of control littermate mice, but
their levels decreased markedly in IE-HuR
−/−
mice. In fact, PHB1 and
HSP60 proteins were undetectable in the small intestinal mucosa of IE-
HuR
−/−
mice by Western blot analysis (Fig 4B,top).
In addition, the HuR-deficient mucosa of the intestinal mucosa
also exhibited decreased levels of VDAC, cytochrome C (Cyto-C), and
PDHG without changes in the abundance of PHB2, SGK or HSC70 (Fig
4B,bottom;Fig S3A). Moreover, transmission electron microscopy
(TEM) at the base of crypts in the small intestinal epithelium
showed reduced numbers of mitochondria in PC-like cells from IE-
HuR
−/−
mice, and mitochondria with swollen morphology, disrup-
tion of cristae, decreased fused structures, and occasional dense
inclusion bodies, when compared with littermate mice (Fig 4D). On
the other hand, HuR deletion did not alter the levels or morphology
of mitochondria in enterocytes located at the villous area of the
small intestinal mucosa, where all mitochondrial features were
indistinguishable between IE-HuR
−/−
mice and littermate mice, as
examined by TEM (Fig S3B).
To analyze mitochondrial function in the HuR-deficient intestinal
epithelium, we analyzed primary cultured intestinal organoids and
cultured IECs. Consistent with the observations in the mouse ep-
ithelium, HuR deletion inhibited the growth of the intestinal
organoids ex vivo, with a marked decrease in the numbers of
Figure 4. HuR deletion in mice inhibits the
expression of mitochondrial proteins in
the intestinal epithelium.
(A) Causal analysis of metabolism/
mitochondria-associated pathways down-
or up-regulated in PCs after HuR deletion in
mice. Left panel, most altered pathways
involved in metabolism and mitochondrial
activity in HuR-deficient PCs. Right panel,
most altered genes in results described in left
panel. Values are the means from three
animals. The P-value cutoff used for
identifying differentially expressed genes was
0.05. (B) Immunoblots of HuR and various
mitochondrial proteins in the small intestinal
mucosa of littermate and IE-HuR
−/−
mice.
(C) Quantitative analysis derived from
densitometric scans of immunoblots of
mitochondrial proteins in results as described
in (B). Values are the means ± SEM (n=3).
Unpaired, two-tailed ttest was used. *P< 0.05
compared with control littermates.
(D) Transmission electron microscopy of
crypt bases of the small intestinal epithelium.
White arrows: mitochondria; black arrows:
dense inclusion body; yellow box:
unhealthy mitochondria. Scale bar = 2 and
0.5 μm. Three separate experiments showed
similar results.
Source data are available for this figure.
Regulation of PC/ISC niche by HuR Xiao et al. https://doi.org/10.26508/lsa.202302152 vol 6 | no 11 | e202302152 6of14
lysozyme-positive cells in intestinal organoids derived from IE-
HuR
−/−
mice and smaller organoids that contained fewer buds
when derived from HuR-deficient mice compared with those
generated from control littermate mice (Fig 5A). To examine the
mitochondrial respiratory capacity, we used a Cell Mito Stress test
and a Seahorse XF extracellular flux analyzer. We observed de-
creases in basal and maximal respiration levels and in ATP pro-
duction, and decreased spare respiratory capacity in the organoids
derived from IE-HuR
−/−
mice compared with the organoids gen-
erated from control littermate mice (Fig 5B). Using cultured human
colorectal adenocarcinoma Caco-2 cells, we found that HuR si-
lencing similarly disrupted mitochondrial homeostasis and re-
duced the levels of several mitochondrial proteins. Decreasing HuR
by transfection with a specific siRNA targeting HuR (siHuR) markedly
decreased the levels of PHB1 and COX-IV, although it only slightly
reduced PHB2 abundance and failed to alter the expression of
HSP60, SDHA or Cyto-C (Figs 5C and S4A). Basal levels of SGK1 and
OPA1 proteins in Caco-2 cells were undetectable. On the other
hand, ectopic overexpression of HuR by transfection with an HuR
expression vector significantly increased the levels of cellular PHB1
and modestly elevated COX-IV levels (Fig S4B). Seahorse analysis
showed similar inhibitory patterns of mitochondrial respiratory
capacity and ATP production in HuR-silenced Caco-2 cells (Fig 5D),
as observed in HuR-deficient intestinal organoids (Fig 5B).
The impairment of mitochondrial function induced by HuR si-
lencing was further confirmed by a decrease in the levels of
MitoTracker green (Fig 5E) and an increase in superoxide production
(Fig 5F), as examined by using MitoTracker Red and MitoSox kits
available commercially. Interestingly, mitochondrial dysfunction in
HuR-silenced cells was prevented by ectopically overexpressing PHB1,
Figure 5. Decreasing the levels of HuR
causes mitochondrial dysfunction.
(A) Immunostaining of lysozyme-positive
cells in the small intestinal organoids derived
from control littermate and IE-HuR
−/−
mice.
Red, lysozyme; green, E-cadherin. Scale bars,
80 μm. (B) Mitochondrial respiration in the
intestinal organoids treated as described in
(A), as measured by Seahorse analysis. Values
are the means ± SEM (n= 3). Two-way ANOVA
with Bonferroni post hos test was used. *P<
0.05 compared with control littermates.
(C) Immunoblots of HuR and mitochondrial
proteins in cultured intestinal epithelial
cells (IECs) transfected with siRNA directed at
silencing HuR (siHuR) or control siRNA (C-
siRNA). The levels of the proteins shown
were examined 48 h after the transfection.
(D) Mitochondrial respiration in cultured IECs
treated as described in (C). Values are the
means ± SEM (n= 3). Two-way ANOVA with
Bonferroni post hos test was used. *P< 0.05
compared with C-siRNA. (E, F) Levels of
MitoTracker Green and superoxide in cultured
IECs after transfection with siHuR alone or co-
transfection with siHuR and prohibitin 1
expression vector (n= 6). *
,+
P< 0.05 compared
with C-siRNA and siHuR, respectively.
Regulation of PC/ISC niche by HuR Xiao et al. https://doi.org/10.26508/lsa.202302152 vol 6 | no 11 | e202302152 7of14
as indicated by an increased level of MitoTracker Green and reduction
insuperoxidelevelwhenHuR-silencedcellsweretransfectedwitha
vector to overexpress PHB1 (Fig 5E and F,right). Together, these results
suggest that lower HuR levels trigger mitochondrial dysfunction at
least in part by inhibiting PHB1 expression. We propose that these
effects contribute to the impairment of the PC/ISC niche and sub-
sequent mucosal atrophy.
HuR regulates PHB1 expression by interfering with
miR-195 function
Given that PHB1 is required for maintaining mitochondrial ho-
meostasis and PC function as shown in the present study and
previous reports (Jackson et al, 2020;Liu et al, 2022), we investigated
the mechanism by which HuR regulates PHB1 expression in cul-
tured IECs. First, we examined if HuR associated with the Phb1 mRNA
by performing RNP immunoprecipitation (IP) assays using an anti-
HuR antibody under conditions that preserved RNP integrity (Yu
et al, 2011). The interaction of Phb1 mRNA with HuR was examined by
isolating RNA from the IP material and subjecting it RT, followed by
quantitative (Q)–PCR analysis. Although HuR overexpression in-
creased PHB1 levels (Fig S4B) and HuR silencing decreased Phb1
mRNA levels (Fig 6A), HuR did not specifically bind to Phb1 mRNA, as
the levels of Phb1 mRNA in HuR IP samples were similar to those
observed in control IgG (Fig 6B). As a positive control, the claudin 1
(Cldn1) mRNA was highly enriched in HuR samples compared with
control IgG. These results indicate that HuR may regulate PHB1
expression through mechanisms other than by directly interacting
with the Phb1 mRNA.
Second, we focused on the role of microRNA miR-195 (miR-
195) because miR-195 represses PHB1 production (Cirilo et al,
2017) and because HuR and miR-195 jointly regulate expression
of shared target transcripts antagonistically (Zhuang et al, 2013;
Kwon et al, 2021). As shown by pulldown analysis after trans-
fecting Caco-2 cells with a biotinylated miR-195 (Kwon et al,
2021), miR-195 directly interacted with the Phb1 mRNA in Caco-2
cells but not with Phb2 mRNA (Fig 6C and D); transfections with a
control biotinylated scramble RNA did not show enrichment in Phb1
mRNA. Ectopically expressing miR-195 by transfecting the miR-195
precursor (pre-miR-195) (Fig 6E) markedly decreased the levels of
cellular Phb1 mRNA (Fig 6F,top) and protein (Fig 6F,bottom;Fig S5A),
but it only slightly reduced PHB2 and failed to alter the abundance
of HSP60, COX-IV, and Cyto-C proteins. Interestingly, increasing the
levels of cellular HuR by transfection with the HuR expression
vector reduced the ability of biotinylated miR-195 to bind to the
Phb1 mRNA, relative to what was seen in the vector control group
(Fig 6G).
Furthermore, HuR-induced stimulation of PHB1 expression was
prevented by increasing miR-195 through transfection with pre-
miR-195 (Figs 6H and S5B). The levels of PHB1 protein in cells co-
transfectedwithHuRandpre-miR-195weresimilartothose
observed in cells transfected with control vector. In addition, there
were no significant differences in the levels of PHB2 between these
three groups. These results indicate that HuR promotes PHB1 ex-
pression primarily by inhibiting the association of Phb1 mRNA with
miR-195.
Discussion
Integrity and effectiveness of the PC/ISC niche are essential for
constant renewal of the intestinal epithelium (Sato et al, 2011;
Yilmaz et al, 2012;Butto et al, 2020), but the exact mechanism
underlying the activation of PC/ISC niche in response to stress
remains largely unknown. In the present study, we provide genetic
evidence that HuR plays an important role in the PC/ISC niche
function at least in part by controlling mitochondrial activity.
Targeted deletion of HuR in mice caused deregulation of cell type-
specific gene expression in PCs, resulted in defective PCs, and
impaired ISC proliferation, in turn inhibiting the growth of the
small intestine mucosa. Experiments aimed at characterizing HuR
targets in this process revealed that HuR deletion decreased mi-
tochondrial metabolism by reducing the levels of expression of
several mitochondrial-associated proteins including PHB1, and that
HuR enhanced PHB1 expression by preventing miR-195 binding to
the Phb1 mRNA. These findings link HuR-regulated functions in
the PC/ISC niche with intestinal epithelium renewal and highlight
the connections between a dysfunctional PC/ISC niche, HuR de-
ficiency, and intestinal epithelium pathology in patients with
critical disorders.
The small intestinal epithelium is the fastest self-renewing
tissue in mammals and this continuous growth is carried out by
active ISCs, which reside at the base of the crypts and are inter-
mingled with postmitotic and differentiated PCs (Sato et al, 2011;
Sailaja et al, 2016). PCs constitute the ISC niche, secrete stem cell
growth signals, and are thus essential for maintaining ISC function
(Sato et al, 2011;Tian et al, 2015;Lueschow & McElroy, 2020). This
dependence of ISCs on PC-mediated paracrine signaling can be
easily recapitulated in an ex vivo system. For example, single ISCs
can be expanded ex vivo into epithelial organoids or “mini-gut,”but
the outgrowth efficiency of single ISCs is quite low; when ISCs are
plated together with PCs, however, their outgrowth efficiency in-
creased by 10fold (Sato et al, 2011;Dayton & Clevers, 2017). In
addition, PCs also metabolically support ISCs by providing them
with a metabolic fuel source (Yu et al, 2020a). In the present study,
we observed that HuR regulates ISC proliferation in the intestinal
epithelium at least partially by altering PC function. Conditional
deletion of HuR in mice altered the transcriptomic profiles of PCs
and decreased the levels of PC-derived growth factors WNT3 and
NOTCH2 in the crypt bases. Because ISCs are located at a niche
growth factor-rich environment that relies on constant secretion of
PCs, decreased levels of WNT3 and NOTCH2 in the HuR-deficient
epithelium definitely contribute to ISC dysfunction and subsequent
mucosal growth inhibition observed in IE-HuR
−/−
mice. In support of
these results, HuR deletion in mice also decreases the expression
of WNT co-receptor LRP6 at the posttranscription level in the in-
testinal epithelium (Liu et al, 2014).
The results presented here also show that HuR regulates PC
function by controlling mitochondrial metabolism. Targeted HuR
deletion resulted in mitochondrial dysfunction, as evidenced by
decreased levels of mitochondrial-associated proteins in the in-
testinal epithelium and by an inhibition of mitochondrial respi-
ratory capacity ex vivo and in vitro. PCs are highly susceptible to
mitochondrial dysfunction driven by HuR deletion because cell
Regulation of PC/ISC niche by HuR Xiao et al. https://doi.org/10.26508/lsa.202302152 vol 6 | no 11 | e202302152 8of14
Figure 6. HuR regulates prohibitin 1 (PHB1) expression via interaction with miR-195.
(A) Levels of Phb1 mRNA in Caco-2 cells 48 h after transfection with C-siRNA or siHuR. Values are the means ± SEM (n= 3 biological replicates). Unpaired, two-tailed ttest
was used. *P< 0.05 compared with C-siRNA. (B) Left panel, association of endogenous HuR with endogenous mRNAs in Caco-2 cells as measured by RIP using either anti-
HuR antibody (Ab) or control IgG, followed by RT–PCR analysis. Right panel, levels of input mRNAs. Values are the means ± SEM (n= 3 biological replicates). Unpaired, two-
tailed ttest was used. *P< 0.05 compared with control IgG. (C) Levels of miR-195 in Caco-2 cells 24 h after transfection with biotinylated miR-195. Values are the means ±
SEM (n= 3 biological replicates). Unpaired, two-tailed ttest was used. *P< 0.05 compared with contro l scramble oligomer. (D) Levels of Phb1 mRNA in the materials pulled
down by biotin-miR-195 in cells treated as described in (C). Values are the means ± SEM (n= 3). Unpaired, two-tailed ttest was used. *P< 0.05 compared with control
scramble oligomer. (E) Levels of miR-195 (left) and U6 RNA (right) in Caco-2 cells 48 h after transfection with pre-miR-195. Values are the means ± SEM (n= 3 biological
replicates). Unpaired, two-tailed ttest was used. *P< 0.05 compared with scramble. (F) Levels of Phb1 mRNA (top) and protein (bottom) in cells treated as described in (E).
Values are the means ± SEM (n= 3 biological replicates). Unpaired, two-tailed ttest was used. *P< 0.05 compared with scramble. (G) Levels of Phb1 mRNA in the materials
pulled down by miR-195 (top panel) and total input mRNA (bottom) after co-transfection with HuR expression vector and bio-miR-195. Va lues are the means ± SEM (n=3
biological replicates). *
,+
P< 0.05 compared with cells transfected with scramble oligomer or cells transfected with bio-miR-195 with control vector, respectively.
(H) Immunoblots of HuR and PHB1 proteins in Caco-2 cells 48 h after transfection with HuR expression vector alone or co-transfection with HuR vector and pre-miR-195.
Experiments were repeated three times and showed similar results.
Source data are available for this figure.
Regulation of PC/ISC niche by HuR Xiao et al. https://doi.org/10.26508/lsa.202302152 vol 6 | no 11 | e202302152 9of14
type-specific gene expression analysis revealed the existence of
transcriptionally distinct PCs in IE-HuR
−/−
mice and because HuR
deletion caused ultrastructural abnormalities in the mitochondria
of PC-like cells located at the bases of crypts. It has been reported
that targeted deletion of the Phb1 or Hsp60 gene in IECs causes
defects in PCs primarily by disrupting mitochondrial function, which
compromises the epithelium–host defense and is the central to the
pathogenesis of ileitis (Jackson et al, 2020;Khaloian et al, 2020).
Interestingly, HuR deletion almost completely inhibited the ex-
pression of PHB1 and HSP60 in the intestinal epithelium, whereas
ectopicallyexpressedPHB1restoredmitochondrialfunctioninHuR-
deficient cells. Like other secretory cells such as goblet and tuft cells
in the intestinal epithelium, PCs are mitochondrial-rich to sustain
energy-expending secretion and other function; therefore, maintaining
the mitochondrial health and effectiveness in PCs are especially
crucial for their activity (Rath et al, 2018). In addition, mitochondrial
impairment is deleterious in terminally differentiated long-lived cells
such as PCs, because damaged mitochondria are not diluted or
repaired by cell replication (Clevers & Bevins, 2013).
Another important finding of this study is that HuR regulates
PHB1 expression by interacting with miR-195. Although HuR deletion
decreased the levels of PHB1 in mouse intestinal mucosa and
ectopic overexpression of HuR increased PHB1 levels in cultured
cells, HuR failed to directly bind to the Phb1 mRNA. In contrast, miR-
195 was found to associate extensively with the Phb1 transcript and
inhibited PHB1 expression. Our study further shows that increasing
the levels of cellular HuR blocked miR-195 association with the Phb1
mRNA and that miR-195 overexpression abolished HuR-induced
stimulation of PHB1 expression. As reported previously (Xiao &
Wang, 2014;Wang et al, 2017;Ma et al, 2023), HuR can perform its
regulatory function by antagonizing miRNAs and small ncRNAs
besides its RNA binding affinity. For example, HuR antagonizes miR-
548c-3p to regulate the expression of TOP2A (Srikantan et al, 2011),
prevents miR-122-mediated repression of CAT-1 expression
(Bhattacharyya et al, 2006), blocks miR-494 to regulate the ex-
pression of nucleolin (Tominaga et al, 2011), and co-operates with
let-7 in repressing c-Myc expression (Kim et al, 2009). Although the
exact process by which HuR competes with miR-195 for association
with the Phb1 mRNA remains unknown at present, HuR formed a
complex with miR-195, thus abolishing miR-195 binding affinity for
Phb1 mRNA and preventing miR-195-induced inhibition of PHB1
expression. In this regard, HuR regulates stability of the Stim1,Dclk1,
and Cldn2 mRNAs by competing with miR-195 to bind to these
transcripts, thus contributing to HuR-enhanced homeostasis of the
intestinal epithelium (Zhuang et al, 2013;Kwon et al, 2021). Another
possibility is that HuR may affect the processing of pre-miR-195, but
our previous studies revealed that ectopically expressed HuR did
not alter the levels of mature miR-195 (Kwon et al, 2021).
Our results are of particular importance from a clinical point of
view, because human intestinal mucosa from patients with CD/SC
exhibited both decreased levels of HuR and PC/ISC niche dys-
function. Although defective PCs and mitochondrial impairment are
commonly observed in patients with IBD (Xiao et al, 2019;Ozsoy
et al, 2022), the present study demonstrates for the first time that
there were significant defects in PC/ISC niche in the intestinal
mucosa of patients with critical surgical disorders after HuR in-
hibition. Notably, abnormalities in PC/ISC niche function in the
intestinal epithelium of IE-HuR
−/−
mouse are similar to those ob-
served in the mucosa of patients with CD/SC. Our study also es-
tablishes a cause–effect relationship between HuR and control of
PC/ISC niche function via mitochondria in in vivo, ex vivo, and cell
culture models. Although most mechanistic studies were con-
ducted in cultured Caco-2 cells and these in vitro observations
should be verified in ex vivo and in vivo systems, our findings
strongly support a model whereby HuR functionally interacts with
miR-195 to regulate PC/ISC niche function by altering PHB1 ex-
pression and mitochondrial metabolism. These findings provide
better understanding of the mechanisms underlying the mainte-
nance of intestinal homeostasis in stressful environments and
point to potential therapeutic targets to enhance regeneration and
adaptation of the intestinal mucosa in surgical patients with critical
disorders.
Materials and Methods
Studies in murine and human tissues
Age- and gender-matched 6- to 8-wk-old mice of C57BL/6 back-
ground were used. Intestinal epithelial tissue-specific HuR deletion
(IE-HuR
−/−
) mice were generated by crossing the HuR
flox/flox
(HuR
fl/fl
) and villin-Cre mice purchased from the Jackson Labora-
tory, as described in our previous studies (Liu et al, 2014;Xiao et al,
2019). HuR
fl/fl
-Cre
−
mice served as control littermates. Both IE-
HuR
−/−
mice and control littermates were housed and handled in a
pathogen-free breeding barrier and were cared for by trained
technicians and veterinarians. Animals were deprived of food but
were allowed free access to tap water for 24 h before experiments.
Two portions of the middle small intestine were taken, one for
histological examination and the other for extraction of protein and
RNA. The tissues were fixed in formalin and paraffin for immu-
nohistochemical staining, whereas the mucosa was scraped with a
glass slide for various measurements, as described previously (Yu
et al, 2011;Xiao et al, 2018). All animal experiments were performed
in accordance with NIH guidelines and were approved by the In-
stitutional Animal Care and Use Committee of University of Mary-
land School of Medicine and Baltimore VA Hospital.
Human tissue samples were obtained from surplus discarded
tissue from the University of Maryland Health Science Center and
Penn State Hershey Carlino Family Inflammatory Bowel and Co-
lorectal Disease Biobank. All patients gave informed consent to
have surgically resected tissue collected for this study. The dis-
eased intestinal tissues, as determined by a pathologist and sur-
geon, were stored in liquid nitrogen until they were assayed.
Dissected and opened intestines were mounted onto a solid
surface and fixed in formalin and paraffin. The study was approved
by the Institutional Review Boards of University Maryland and the
Pennsylvania State College of Medicine.
Cell and intestinal organoid culture
Human colorectal carcinoma Caco-2 cells were purchased from
the American Type Culture Collection and were maintained under
Regulation of PC/ISC niche by HuR Xiao et al. https://doi.org/10.26508/lsa.202302152 vol 6 | no 11 | e202302152 10 of 14
standard culture conditions (Liu et al, 2017;Xiao et al, 2018). The
culture medium and fetal bovine serum were purchased from
Invitrogen and biochemical reagents were from Sigma-Aldrich. Iso-
lation and culture of primary enterocytes were conducted following
the method described previously (Yu et al, 2020b;Yu et al, 2022). Briefly,
primary crypts were released from the small intestinal mucosa of mice;
isolated crypts were mixed with Matrigel (Corning) and cultured in
mouse IntestiCult organoid growth medium (Stemcell technology). The
levels of DNA synthesis were measured by assaying BrdU incorpo-
ration, and the growth of organoids was examined by measuring
organoid cross-sections using NIS-Elements AR4.30.02 program.
Plasmid construction and RNA interference
An expression vector containing the human HuR cDNA under the
control of pCMV promoter was purchased from Origene and used to
increase cellular HuR levels as described previously (Liu et al, 2009).
Transient transfections were performed using the Lipofectamine
reagent following the manufacturer’s recommendations (Invi-
trogen). 48 h after transfection using LipofectAMINE, cells were
harvested for analysis. Expression of HuR was silenced by trans-
fection with siHuR as described (Liu et al, 2017). The siHuR and
C-siRNA were purchased from Santa Cruz Biotechnologies. For each
60-mm cell culture dish, 15 μl of the 20 μM stock duplex siHuR or
C-siRNA was used. 48 h after transfection using LipofectAMINE
(116668019; Invitrogen), the cells were harvested for analysis.
Q-PCR and immunoblotting analyses
Total RNA was isolated by using the RNeasy mini kit (QIAGEN) and
used in RT and quantitative (Q)-PCR amplification reactions as
described (Zhuang et al, 2013;Yu et al, 2022). Q-PCR analysis was
performed using Step-one-plus Systems with specific primers,
probes, and software (Applied Biosystems). All primers used for
Q-PCR analysis were purchased from Thermo Fisher Scientific. The
levels of Gapdh mRNA were assessed to monitor the evenness in
RNA input in Q-PCR analysis.
To examine protein levels, whole-cell lysates were prepared
using 2% SDS, sonicated, and centrifuged (Xiao et al, 2016). The
supernatants were boiled and size-fractionated by SDS–PAGE. After
transferring proteins onto nitrocellulose filters, the blots were
incubated with primary antibody, after incubations with secondary
antibody. Antibodies recognizing HuR, PHB1, PHB2, HSP60, SGK,
VDAC, Cyto-C, PDHG, and GAPDH were obtained from Santa Cruz
Biotechnology and BD Biosciences and Invitrogen. Secondary an-
tibodies conjugated to horseradish peroxidase were purchased
from Sigma-Aldrich. All antibodies used in this study were validated
for species specificity. Antibody dilutions used for Western blots to
detect HuR, PHB1, PHB2, HSP60, SGK, VDAC, Cytochrome C, Lgr5,
PDHG, and GAPDH were 1:800 or 1,000 (first Ab) and 1:2,000 (second
Ab), respectively, whereas antibody dilutions for immunostaining
were 1:200 (first Ab) and 1:2,000 (second Ab). Relative protein levels
were analyzed by using Bio-Rad Chemidoc and XRS system
equipped with Image Lab Software (version 4.1). We also used
“Quantity tool”to determine the band intensity volume; the values
were normalized with internal loading control GAPDH.
Immunofluorescence staining and TEM
The procedures of immunofluorescence staining were carried out
according to the method described (Xiao et al, 2019;Yu et al, 2020b).
Slides were fixed in 3.7% formaldehyde in PBS and rehydrated. All
slides were incubated with a primary antibody against different
proteins in the blocking buffer at concentration of 1:200 or 1:300
dilution at 4°C overnight and then incubated with a secondary
antibody conjugated with Alexa Fluor-594 (Invitrogen) or Alexa
Fluor-488 (Invitrogen) for 2 h at RT. After rinsing three times, the slides
were incubated with 1 μM DAPI (Invitrogen) for 10 min to stain cell
nuclei. Finally, the slides were mounted and viewed through a Zeiss
confocal microscope (LSM710; model). Slides were examined in a
blinded fashion by coding, and decoded only after examination was
completed. Images were processed using Photoshop software (Adobe).
For TEM, the small intestines from the animals were rapidly
removed, cut into small pieces, and placed into 2% paraformal-
dehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer
for 1 h. Samples were incubated in 1% osmium tetroxide for 1 h and
then dehydrated through an ethanol series. They were embedded
in EPON resin and ultrathin sections of 80–100 nm were collected
on grids. The samples were imaged in a FEI Tecnai T12 TEM (Thermo
Fisher Scientific) at 80 kV with an XR60B AMT CCD camera (2 × 2 k).
Isolation of IECs and scRNA-seq analysis
The small intestinal mucosal tissues harvested from littermate and
IE-HuR
−/−
mice were weighed before being washed in cold D-PBS
and diced with scalpel, as described previously (Xiao et al, 2016).
Briefly, cleaned mucosal tissues were incubated in chelation me-
dium (20 mM; EDTA-DPBS) at 37°C for 90 min with agitation. Villus-
rich supernatant was collected and later combined with a pellet
that was further dissociated with TrypLE for 10 min at 37°C (crypt-
rich cells). The epithelial single-cell suspension was then washed
and passed through 40-μmfilters. Before proceeding to scRNA-
seq, the purity of epithelial populations was confirmed by flow
cytometry analysis. Once satisfactory viability and EPCAM purity
were demonstrated, these high-quality IECs were directly loaded
for droplet-based scRNA-seq according to the manufacturer’s
protocol for the Chromium Single Cell Platfor (39V2; 10X Genomics)
to obtain 10,000 cells per reaction. Library preparation was carried
out according to the manufacturer’s protocol. 10× Genomics scRNA-
seq gene expression raw sequencing data were processed using the
CellRanger software v.3.0.2 and the 10X human transcriptome
GRCh38-3.0.0 as the reference. The 10X Genomics V (D) Ig heavy and
light chains were processed using Cellranger vdj v.3.1.0 and the
reference Cellranger-vdj-GRCh38-alts-ensembl-3.1.0 with default
settings. The dimensionality reduction, Leiden clustering, differ-
ential abundance analysis, cell-type composition analysis, gene
enrichment analysis, mitochondrion Gene Ontology Term (GO:
0005739) were performed as reported previously (Islam et al, 2020;
Elmentaite et al, 2021).
Seahorse metabolic analyzer assays
Seahorse Bioscience XFe24 Analyzer was used to measure mito-
chondrial respiratory capacity in intestinal organoids and cultured
Regulation of PC/ISC niche by HuR Xiao et al. https://doi.org/10.26508/lsa.202302152 vol 6 | no 11 | e202302152 11 of 14
IECs (Thompson et al, 2019;Kleele et al, 2021). 2-D intestinal
organoids and Caco-2 cells were grown on a 96-well XFe96 plate at a
cell density of 20,000 cells/well. Cartridge plates for metabolic
stress injections were hydrated for at least 12 h at 37°C before the
assay with Calibrant Solution. 1 h before running the Seahorse
assay, the cell culture medium was removed and replaced with
Seahorse Assay Medium. The following compounds (final con-
centrations) were sequentially injected into each well: oligomycin
(1.5 μM), FCCP (0.25 μM), and rotenone/antimycin (0.5 μM). Oxygen
consumption rate was measured under basal conditions and after
each injection using an XFe96 extracellular flux analyzer (Seahorse
Bioscience). Key parameters of mitochondrial function, including
basal respiration, ATP-linked respiration, proton leak, maximal
respiration and spare capacity, were calculated and analyzed on
Wave (Agilent). Mitochondrial activity and intracellular superoxide
production were examined by using MitoTracker Red and MitoSox
kits (Invitrogen) and performed, according to the manufacturer’s
instruction.
RNP-IP and biotin-labeled miR-195 pull-down assays
Immunoprecipitation (IP) of RNP complexes was carried out to
assess the association of endogenous HuR with endogenous
mRNAs encoding PHB1, PHB2, HSP60, and claudin-1 as described
(Liu et al, 2009). Twenty million cells were collected per sample, and
lysates were used for IP for 4 h at RT in the presence of excess
(30 μg) IP antibody (IgG, or anti-HuR). RNA in IP materials was used
in RT reactions followed by Q-PCR analysis. The amplification of
Gapdh mRNA, found in all samples as low-level contaminating
housekeeping transcripts (not HuR target), served to monitor the
evenness of sample input, as reported previously (Zhuang et al,
2013).
Biotinylated RNA pull-down assays were conducted as described
previously (Kwon et al, 2021). After biotin-labeled miR-195 was
incubated with cytoplasmic proteins in RT for 1 h, the mixture was
mixed with Streptavidin–Dynabeads (Invitrogen) and incubated at
4°C on a rotator overnight. The beads were washed thoroughly, and
the beads-bound RNA was isolated and subjected to RT followed by
Q-PCR analysis.
Statistical analysis
All values were expressed as the means ± SEM. Unpaired, two-tailed
ttest was used when indicated with P< 0.05 considered significant.
When assessing multiple groups, one-way ANOVA was used with
Tukey’s post hoc test (Harter, 1960). The statistical software used
was GraphPad InStat Prism 9.0. For nonparametric analysis rank
comparison, the Kruskal–Wallis test was conducted.
Data Availability
Primary datasets of single-cell transcriptional profiles of PCs and
ISCs in IE-HuR
−/−
and littermate mice have been generated and
deposited on NCBI with GEO Accession number GSE242410.
Supplementary Information
Supplementary Information is available at https://doi.org/10.26508/lsa.
202302152.
Acknowledgements
This work was supported by Merit Review Awards (to J-Y Wang and JN Rao)
from US Department of Veterans Affairs; grants from National Institutes of
Health (NIH) (DK57819, DK61972, DK68491 to J-Y Wang); and funding from the
National Institute on Aging-Intramural Research Program, NIH (to M Gor-
ospe) and from the Peter and Marshia Carlino Fund for IBD Research (to GS
Yochum and WA Koltun).
Author Contributions
L Xiao: data curation, formal analysis, validation, and methodology.
B Warner: data curation.
CG Mallard: data curation.
HK Chung: data curation.
A Shetty: software, validation, and methodology.
CA Brantner: data curation and methodology.
JN Rao: data curation and supervision.
GS Yochum: data curation.
WA Koltun: data curation.
KB To: investigation.
DJ Turner: data curation and methodology.
M Gorospe: investigation.
J-Y Wang: conceptualization, supervision, funding acquisition, inves-
tigation, methodology, project administration, and writing—original
draft, review, and editing.
Conflict of Interest Statement
The author discloses the following: J-Y Wang is a Senior Research Career
Scientist at the Biomedical Laboratory Research and Development Service
(US Department of Veterans Affairs). The remaining authors disclose no
conflicts of interest.
References
Baulies A, Angelis N, Foglizzo V, Danielsen ET, Patel H, Novellasdemunt L,
Kucharska A, Carvalho J, Nye E, De Coppi P, et al (2020) The
transcription co-repressors MTG8 and MTG16 regulate exit of
intestinal stem cells from their niche and differentiation into
enterocyte vs secretory lineages. Gastroenterology 159: 1328–1341.e3.
doi:10.1053/j.gastro.2020.06.012
Berger E, Rath E, Yuan D, Waldschmitt N, Khaloian S, Allgauer M, Staszewski O,
Lobner EM, Schottl T, Giesbertz P, et al (2016) Mitochondrial function
controls intestinal epithelial stemness and proliferation. Nat
Commun 7: 13171. doi:10.1038/ncomms13171
Bhattacharyya SN, Habermacher R, Martine U, Closs EI, Filipowicz W (2006)
Relief of microRNA-mediated translational repression in human cells
subjected to stress. Cell 125: 1111–1124. doi:10.1016/j.cell.2006.04.031
Borrelli C, Valenta T, Handler K, Velez K, Gurtner A, Moro G, Lafzi A, Roditi LdV,
Hausmann G, Arnold IC, et al (2021) Differential regulation of
β-catenin-mediated transcription via N- and C-terminal co-factors
Regulation of PC/ISC niche by HuR Xiao et al. https://doi.org/10.26508/lsa.202302152 vol 6 | no 11 | e202302152 12 of 14
governs identity of murine intestinal epithelial stem cells. Nat
Commun 12: 1368. doi:10.1038/s41467-021-21591-9
Bottcher A, Buttner M, Tritschler S, Sterr M, Aliluev A, Oppenlander L,
Burtscher I, Sass S, Irmler M, Beckers J, et al (2021) Non-canonical Wnt/
PCP signalling regulates intestinal stem cell lineage priming towards
enteroendocrine and Paneth cell fates. Nat Cell Biol 23: 23–31.
doi:10.1038/s41556-020-00617-2
Butto LF, Pelletier A, More SK, Zhao N, Osme A, Hager CL, Ghannoum MA,
Sekaly RP, Cominelli F, Dave M (2020) Intestinal stem cell niche defects
result in impaired 3D organoid formation in mouse models of Crohn’s
disease-like ileitis. Stem Cell Rep 15: 389–407. doi:10.1016/
j.stemcr.2020.06.017
Carter SR, Zahs A, Palmer JL, Wang L, Ramirez L, Gamelli RL, Kovacs EJ (2013)
Intestinal barrier disruption as a cause of mortality in combined
radiation and burn injury. Shock 40: 281–289. doi:10.1097/
SHK.0b013e3182a2c5b5
Chung HK, Xiao L, Jaladanki KC, Wang JY (2021) Regulation of Paneth cell
function by RNA-binding proteins and noncoding RNAs. Cells 10: 2107.
doi:10.3390/cells10082107
Cirilo PDR, de Sousa Andrade LN, Correa BRS, Qiao M, Furuya TK, Chammas R,
Penalva LOF (2017) MicroRNA-195 acts as an anti-proliferative miRNA
in human melanoma cells by targeting Prohibitin 1. BMC Cancer 17:
750. doi:10.1186/s12885-017-3721-7
Clevers HC, Bevins CL (2013) Paneth cells: Maestros of the small intestinal
crypts. Annu Rev Physiol 75: 289–311. doi:10.1146/annurev-physiol-
030212-183744
Dayton TL, Clevers H (2017) Beyond growth signaling: Paneth cells
metabolically support ISCs. Cell Res 27: 851–852. doi:10.1038/cr.2017.59
Durand A, Donahue B, Peignon G, Letourneur F, Cagnard N, Slomianny C,
Perret C, Shroyer NF, Romagnolo B (2012) Functional intestinal stem
cells after Paneth cell ablation induced by the loss of transcription
factor Math1 (Atoh1). Proc Natl Acad Sci U S A 109: 8965–8970.
doi:10.1073/pnas.1201652109
Elmentaite R, Kumasaka N, Roberts K, Fleming A, Dann E, King HW,
Kleshchevnikov V, Dabrowska M, Pritchard S, Bolt L, et al (2021) Cells of
the human intestinal tract mapped across space and time. Nature 597:
250–255. doi:10.1038/s41586-021-03852-1
Harter HL (1960) Critical values for Duncan’s new multiple range test.
Biometrics 16: 671–685. doi:10.2307/2527770
Ho GT, Theiss AL (2022) Mitochondria and inflammatory bowel diseases:
Toward a stratified therapeutic intervention. Annu Rev Physiol 84:
435–459. doi:10.1146/annurev-physiol-060821-083306
Islam M, Chen B, Spraggins JM, Kelly RT, Lau KS (2020) Use of single-cell-omic
technologies to study the gastrointestinal tract and diseases, from
single cell identities to patient features. Gastroenterology 159:
453–466.e1. doi:10.1053/j.gastro.2020.04.073
Jackson DN, Panopoulos M, Neumann WL, Turner K, Cantarel BL, Thompson-
Snipes L, Dassopoulos T, Feagins LA, Souza RF, Mills JC, et al (2020)
Mitochondrial dysfunction during loss of prohibitin 1 triggers Paneth
cell defects and ileitis. Gut 69: 1928–1938. doi:10.1136/gutjnl-2019-
319523
Khaloian S, Rath E, Hammoudi N, Gleisinger E, Blutke A, Giesbertz P, Berger E,
Metwaly A, Waldschmitt N, Allez M, et al (2020) Mitochondrial
impairment drives intestinal stem cell transition into dysfunctional
Paneth cells predicting Crohn’s disease recurrence. Gut 69: 1939–1951.
doi:10.1136/gutjnl-2019-319514
Kim HH, Kuwano Y, Srikantan S, Lee EK, Martindale JL, Gorospe M (2009) HuR
recruits let-7/RISC to repress c-Myc expression. Genes Dev 23:
1743–1748. doi:10.1101/gad.1812509
Kleele T, Rey T, Winter J, Zaganelli S, Mahecic D, Perreten Lambert H, Ruberto
FP, Nemir M, Wai T, Pedrazzini T, et al (2021) Distinct fission signatures
predict mitochondrial degradation or biogenesis. Nature 593: 435–439.
doi:10.1038/s41586-021-03510-6
Kumar M, Leon Coria A, Cornick S, Petri B, Mayengbam S, Jijon HB, Moreau F,
Shearer J, Chadee K (2020) Increased intestinal permeability
exacerbates sepsis through reduced hepatic SCD-1 activity and
dysregulated iron recycling. Nat Commun 11: 483. doi:10.1038/s41467-
019-14182-2
Kwon MS, Chung HK, Xiao L, Yu TX, Wang SR, Piao JJ, Rao JN, Gorospe M, Wang JY
(2021) MicroRNA-195 regulates Tuft cell function in the intestinal
epithelium by altering translation of DCLK1. Am J Physiol Cell Physiol
320: C1042–C1054. doi:10.1152/ajpcell.00597.2020
Li XX, Xiao L, Chung HK, Ma XX, Liu X, Song JL, Jin CZ, Rao JN, Gorospe M, Wang JY
(2020) Interaction between HuR and circPABPN1 modulates
autophagy in the intestinal epithelium by altering atg16l1 translation.
Mol Cell Biol 40: e00492-19. doi:10.1128/MCB.00492-19
Li C, Zhou Y, Wei R, Napier DL, Sengoku T, Alstott MC, Liu J, Wang C, Zaytseva YY,
Weiss HL, et al (2023) Glycolytic regulation of intestinal stem cell self-
renewal and differentiation. Cell Mol Gastroenterol Hepatol 15:
931–947. doi:10.1016/j.jcmgh.2022.12.012
Liu L, Rao JN, Zou T, Xiao L, Wang PY, Turner DJ, Gorospe M, Wang JY (2009)
Polyamines regulate c-Myc translation through Chk2-dependent HuR
phosphorylation. Mol Biol Cell 20: 4885–4898. doi:10.1091/mbc.e09-07-
0550
Liu L, Christodoulou-Vafeiadou E, Rao JN, Zou T, Xiao L, Chung HK, Yang H,
Gorospe M, Kontoyiannis D, Wang JY (2014) RNA-binding protein HuR
promotes growth of small intestinal mucosa by activating the Wnt
signaling pathway. Mol Biol Cell 25: 3308–3318. doi:10.1091/mbc.E14-
03-0853
Liu L, Zhuang R, Xiao L, Chung HK, Luo J, Turner DJ, Rao JN, Gorospe M, Wang JY
(2017) HuR enhances early restitution of the intestinal epithelium by
increasing Cdc42 translation. Mol Cell Biol 37: e00574-16. doi:10.1128/
MCB.00574-16
Liu H, Fan H, He P, Zhuang H, Liu X, Chen M, Zhong W, Zhang Y, Zhen C, Li Y, et al
(2022) Prohibitin 1 regulates mtDNA release and downstream
inflammatory responses. EMBO J 41: e111173. doi:10.15252/
embj.2022111173
Ludikhuize MC, Meerlo M, Gallego MP, Xanthakis D, Burgaya Julia M, Nguyen
NTB, Brombacher EC, Liv N, Maurice MM, Paik JH, et al (2020)
Mitochondria define intestinal stem cell differentiation downstream
of a FOXO/Notch axis. Cell Metab 32: 889–900.e7. doi:10.1016/
j.cmet.2020.10.005
Lueschow SR, McElroy SJ (2020) The Paneth cell: The curator and defender of
the immature small intestine. Front Immunol 11: 587. doi:10.3389/
fimmu.2020.00587
Luna Velez MV, Neikes HK, Snabel RR, Quint Y, Qian C, Martens A, Veenstra GJC,
Freeman MR, van Heeringen SJ, Vermeulen M (2023) ONECUT2
regulates RANKL-dependent enterocyte and microfold cell
differentiation in the small intestine; a multi-omics study. Nucleic
Acids Res 51: 1277–1296. doi:10.1093/nar/gkac1236
Ma XX, Xiao L, Wen SJ, Yu TX, Sharma S, Chung HK, Warner B, Mallard CG, Rao JN,
Gorospe M, et al (2023) Small noncoding vault RNA2-1 disrupts gut
epithelial barrier function via interaction with HuR. EMBO Rep 24:
e54925. doi:10.15252/embr.202254925
Norona J, Apostolova P, Schmidt D, Ihlemann R, Reischmann N, Taylor G,
Kohler N, de Heer J, Heeg S, Andrieux G, et al (2020) Glucagon-like
peptide 2 for intestinal stem cell and Paneth cell repair during graft-
versus-host disease in mice and humans. Blood 136: 1442–1455.
doi:10.1182/blood.2020005957
Ozsoy M, Stummer N, Zimmermann FA, Feichtinger RG, Sperl W, Weghuber D,
Schneider AM (2022) Role of energy metabolism and mitochondrial
function in inflammatory bowel disease. Inflamm Bowel Dis 28:
1443–1450. doi:10.1093/ibd/izac024
Palomo-Irigoyen M, Perez-Andres E, Iruarrizaga-Lejarreta M, Barreira-
Manrique A, Tamayo-Caro M, Vila-Vecilla L, Moreno-Cugnon L, Beitia N,
Medrano D, Fernandez-Ramos D, et al (2020) HuR/ELAVL1 drives
Regulation of PC/ISC niche by HuR Xiao et al. https://doi.org/10.26508/lsa.202302152 vol 6 | no 11 | e202302152 13 of 14
malignant peripheral nerve sheath tumor growth and metastasis. J
Clin Invest 130: 3848–3864. doi:10.1172/JCI130379
Radtke F, Clevers H (2005) Self-renewal and cancer of the gut: Two sides of a
coin. Science 307: 1904–1909. doi:10.1126/science.1104815
Rath E, Moschetta A, Haller D (2018) Mitochondrial function - gatekeeper of
intestinal epithelial cell homeostasis. Nat Rev Gastroenterol Hepatol
15: 497–516. doi:10.1038/s41575-018-0021-x
Rodriguez-Colman MJ, Schewe M, Meerlo M, Stigter E, Gerrits J, Pras-Raves M,
Sacchetti A, Hornsveld M, Oost KC, Snippert HJ, et al (2017) Interplay
between metabolic identities in the intestinal crypt supports stem
cell function. Nature 543: 424–427. doi:10.1038/nature21673
Sailaja BS, He XC, Li L (2016) The regulatory niche of intestinal stem cells. J
Physiol 594: 4827–4836. doi:10.1113/JP271931
Sato T, van Es JH, Snippert HJ, Stange DE, Vries RG, van den Born M, Barker N,
Shroyer NF, van de Wetering M, Clevers H (2011) Paneth cells
constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 469:
415–418. doi:10.1038/nature09637
Sato M, Kadomatsu T, Miyata K, Warren JS, Tian Z, Zhu S, Horiguchi H, Makaju A,
Bakhtina A, Morinaga J, et al (2021) The lncRNA Caren antagonizes
heart failure by inactivating DNA damage response and activating
mitochondrial biogenesis. Nat Commun 12: 2529. doi:10.1038/s41467-
021-22735-7
Serra D, Mayr U, Boni A, Lukonin I, Rempfler M, Challet Meylan L, Stadler MB,
Strnad P, Papasaikas P, Vischi D, et al (2019) Self-organization and
symmetry breaking in intestinal organoid development. Nature 569:
66–72. doi:10.1038/s41586-019-1146-y
Shroyer NF, Helmrath MA, Wang VY, Antalffy B, Henning SJ, Zoghbi HY (2007)
Intestine-specific ablation of mouse atonal homolog 1 (Math1) reveals
a role in cellular homeostasis. Gastroenterology 132: 2478–2488.
doi:10.1053/j.gastro.2007.03.047
Srikantan S, Abdelmohsen K, Lee EK, Tominaga K, Subaran SS, Kuwano Y,
Kulshrestha R, Panchakshari R, Kim HH, Yang X, et al (2011)
Translational control of TOP2A influences doxorubicin efficacy. Mol
Cell Biol 31: 3790–3801. doi:10.1128/MCB.05639-11
Thompson LP, Song H, Polster BM (2019) Fetal programming and sexual
dimorphism of mitochondrial protein expression and activity of
hearts of prenatally hypoxic Guinea pig offspring. Oxid Med Cell
Longev 2019: 7210249. doi:10.1155/2019/7210249
Tian H, Biehs B, Chiu C, Siebel CW, Wu Y, Costa M, de Sauvage FJ, Klein OD
(2015) Opposing activities of Notch and Wnt signaling regulate
intestinal stem cells and gut homeostasis. Cell Rep 11: 33–42.
doi:10.1016/j.celrep.2015.03.007
Tominaga K, Srikantan S, Lee EK, Subaran SS, Martindale JL, Abdelmohsen K,
Gorospe M (2011) Competitive regulation of nucleolin expression by
HuR and miR-494. Mol Cell Biol 31: 4219–4231. doi:10.1128/MCB.05955-11
Twig G, Elorza A, Molina AJ, Mohamed H, Wikstrom JD, Walzer G, Stiles L, Haigh
SE, Katz S, Las G, et al (2008) Fission and selective fusion govern
mitochondrial segregation and elimination by autophagy. EMBO J 27:
433–446. doi:10.1038/sj.emboj.7601963
Urbauer E, Rath E, Haller D (2020) Mitochondrial metabolism in the intestinal
stem cell niche-sensing and signaling in health and disease. Front
Cell Dev Biol 8: 602814. doi:10.3389/fcell.2020.602814
van Es JH, Wiebrands K, Lopez-Iglesias C, van de Wetering M, Zeinstra L, van
den Born M, Korving J, Sasaki N, Peters PJ, van Oudenaarden A, et al
(2019) Enteroendocrine and tuft cells support Lgr5 stem cells on
Paneth cell depletion. Proc Natl Acad Sci U S A 116: 26599–26605.
doi:10.1073/pnas.1801888117
Wang JY, Xiao L, Wang JY (2017) Posttranscriptional regulation of intestinal
epithelial integrity by noncoding RNAs. Wiley Interdiscip Rev RNA 8:
e1399. doi:10.1002/wrna.1399
Xian L, Georgess D, Huso T, Cope L, Belton A, Chang YT, Kuang W, Gu Q, Zhang X,
Senger S, et al (2017) HMGA1 amplifies Wnt signalling and expands the
intestinal stem cell compartment and Paneth cell niche. Nat Commun
8: 15008. doi:10.1038/ncomms15008
Xiao L, Wang JY (2014) RNA-binding proteins and microRNAs in
gastrointestinal epithelial homeostasis and diseases. Curr Opin
Pharmacol 19: 46–53. doi:10.1016/j.coph.2014.07.006
Xiao L, Rao JN, Cao S, Liu L, Chung HK, Zhang Y, Zhang J, Liu Y, Gorospe M, Wang
JY (2016) Long noncoding RNA SPRY4-IT1 regulates intestinal
epithelial barrier function by modulating the expression levels of
tight junction proteins. Mol Biol Cell 27: 617–626. doi:10.1091/mbc.E15-
10-0703
Xiao L, Wu J, Wang JY, Chung HK, Kalakonda S, Rao JN, Gorospe M, Wang JY
(2018) Long noncoding RNA uc.173 promotes renewal of the intestinal
mucosa by inducing degradation of microRNA 195. Gastroenterology
154: 599–611. doi:10.1053/j.gastro.2017.10.009
Xiao L, Li XX, Chung HK, Kalakonda S, Cai JZ, Cao S, Chen N, Liu Y, Rao JN, Wang
HY, et al (2019) RNA-binding protein HuR regulates Paneth cell
function by altering membrane localization of TLR2 via post-
transcriptional control of CNPY3. Gastroenterology 157: 731–743.
doi:10.1053/j.gastro.2019.05.010
Yang L, Yang H, Chu Y, Song Y, Ding L, Zhu B, Zhai W, Wang X, Kuang Y, Ren F,
et al (2021) CREPT is required for murine stem cell maintenance
during intestinal regeneration. Nat Commun 12: 270. doi:10.1038/
s41467-020-20636-9
Yilmaz OH, Katajisto P, Lamming DW, Gultekin Y, Bauer-Rowe KE, Sengupta S,
Birsoy K, Dursun A, Yilmaz VO, Selig M, et al (2012) mTORC1 in the
Paneth cell niche couples intestinal stem-cell function to calorie
intake. Nature 486: 490–495. doi:10.1038/nature11163
Yu TX, Wang PY, Rao JN, Zou T, Liu L, Xiao L, Gorospe M, Wang JY (2011) Chk2-
dependent HuR phosphorylation regulates occludin mRNA
translation and epithelial barrier function. Nucleic Acids Res 39:
8472–8487. doi:10.1093/nar/gkr567
Yu S, Tong K, Zhao Y, Balasubramanian I, Yap GS, Ferraris RP, Bonder EM, Verzi
MP, Gao N (2018) Paneth cell multipotency induced by notch
activation following injury. Cell Stem Cell 23: 46–59.e5. doi:10.1016/
j.stem.2018.05.002
Yu S, Balasubramanian I, Laubitz D, Tong K, Bandyopadhyay S, Lin X, Flores J,
Singh R, Liu Y, Macazana C, et al (2020a) Paneth cell-derived lysozyme
defines the composition of mucolytic microbiota and the
inflammatory tone of the intestine. Immunity 53: 398–416.e8.
doi:10.1016/j.immuni.2020.07.010
Yu TX, Chung HK, Xiao L, Piao JJ, Lan S, Jaladanki SK, Turner DJ, Raufman JP,
Gorospe M, Wang JY (2020b) Long noncoding RNA H19 impairs the
intestinal barrier by suppressing autophagy and lowering paneth and
goblet cell function. Cell Mol Gastroenterol Hepatol 9: 611–625.
doi:10.1016/j.jcmgh.2019.12.002
Yu TX, Kalakonda S, Liu X, Han N, Chung HK, Xiao L, Rao JN, He TC, Raufman JP,
Wang JY (2022) Long noncoding RNA uc.230/CUG-binding protein 1
axis sustains intestinal epithelial homeostasis and response to tissue
injury. JCI Insight 7: e156612. doi:10.1172/jci.insight.156612
Zhang Y, Cai JZ, Xiao L, Chung HK, Ma XX, Chen LL, Rao JN, Wang JY (2020) RNA-
binding protein HuR regulates translation of vitamin D receptor
modulating rapid epithelial restitution after wounding. Am J Physiol
Cell Physiol 319: C208–C217. doi:10.1152/ajpcell.00009.2020
Zhuang R, Rao JN, Zou T, Liu L, Xiao L, Cao S, Hansraj NZ, Gorospe M, Wang JY
(2013) miR-195 competes with HuR to modulate stim1 mRNA stability
and regulate cell migration. Nucleic Acids Res 41: 7905–7919.
doi:10.1093/nar/gkt565
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