Cutting Edge: TNF-Induced MicroRNAs Regulate
TNF-Induced Expression of E-Selectin and Intercellular
Adhesion Molecule-1 on Human Endothelial Cells:
Feedback Control of Inflammation
Yajaira Sua ´rez,1Chen Wang, Thomas D. Manes, and Jordan S. Pober
39 untranslated region of mRNAs to posttranscription-
TNF-mediated induction of endothelial adhesion mole-
cules can be regulated by miRNAs that are induced by
TNF. Specifically, E-selectin and ICAM-1 are targets of
TNF-induced miRNAs miR-31 and miR-17-3p, respec-
Conversely, transfections with mimics of these miRNAs
decreased neutrophil adhesion to endothelial cells. These
data suggest that miRNAs provide negative feedback con-
leukocytes (1–3). MicroRNAs (miRNAs) control gene ex-
pression by pairing with partially complementary target sites in
mRNA 39 untranslated regions (UTRs), resulting in trans-
lational repression and/or mRNA destabilization (4–6). Most
miRNAs are constitutively expressed in a lineage-specific
manner that peaks during embryological development, but
Vascular endothelial growth factor (VEGF) induces a subset of
EC miRNAs that regulate cell growth, survival, and angio-
by a variety of proinflammatory stimuli (10–15). A constitu-
tively expressed, EC-restricted miRNA (miR-126) modulates
TNF-induced VCAM-1 expression (16). In this study, we re-
port that TNF increases miR-155, -31, -17, and -191 without
changing miR-20a, -222, and -126 in human ECs and that
he proinflammatory cytokine TNF induces de novo
1, endothelial cell (EC) surface proteins that bind
SELE and ICAM-1 are targets of TNF-induced miR-31 and
-17-3p, respectively, regulating neutrophil binding to ECs.
These data reveal how inducible miRNAs make up a negative
feedback loop to control inflammation.
Materials and Methods
Human cells were obtained from discarded tissues or peripheral blood of
deidentified donors under protocols approved by the Yale Human In-
vestigation Committee. HUVECs and human dermal fibroblasts (HDFs) were
isolated and serially cultured, as described previously (8, 17, 18). Neutrophils
were isolated from whole blood of healthy adult donors (19).
miRNA array analyses
HUVEC miRNA expression was assessed following stimulation for 2 or 24 h
with 10 ng/ml TNF or buffer control using Exiqon miRCURY LNA Arrays as
described (8). The data have been deposited in MIAMExpress with Ar-
rayExpress accession number E-MTAB-150 and are accessible through www.
ebi.ac.uk/microarray-as/ae/. Array results were validated by Northern blot (8,
18) or by quantitative RT-PCR using a mirVanaTM qRT-PCR miRNA
Detection Kit (Ambion, Austin, TX) (8, 18).
Reporter gene assays
cDNAsencodingthe entire39UTRof ICAM-1(1.329kb)orSELE(1.883kb)
mRNAs were amplified by RT-PCR from total HUVEC RNA using XhoI and
NotI linker/primers and directionally cloned downstream of the Renilla lucif-
erase open reading frame in the psiCHECK2 vector (Promega, Madison, WI)
that also contains a constitutively expressed firefly luciferase gene. ICAM-1 or
SELE 39UTRs were cloned in reverse orientation as controls lacking the
miRNA target sequence (20). Additionally, the region complementary to the
miR-31 seed sequence in position 94–100 of the human SELE 39UTR,
TCTTGCC, was scrambled to CTGCCTT (mSELE 39UTR), and the region
complementary to the miR-17-3p seed sequence in positions 638–644 and
1148–1154 in human ICAM-1 39UTR, ACTGCAG, was scrambled to
GTAAGCC (mICAM-1 39UTR). All constructs were confirmed by sequenc-
ing. COS-7 cells (giftof Dr. WilliamSessa, YaleUniversity) werecotransfected
with 1 mg the indicated reporter construct and the indicated miRIDIAN
miRNA mimic or negative control mimic sequences (CM) (Dharmacon, La-
fayette, CO) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Renilla
luciferase activity was normalized to the corresponding firefly luciferase activity
Department of Immunobiology and Interdepartmental Program in Vascular Biology and
Therapeutics, Yale University School of Medicine, New Haven, CT 06520-8089
1Current address: Department of Medicine, Leon H. Charney Division of Cardiology
and the Marc and Ruti Bell Vascular Biology and Disease Program, New York Univer-
sity School of Medicine, New York, NY 10016.
Received for publication July 27, 2009. Accepted for publication October 21, 2009.
This work was supported by National Institutes of Health Grants R01-HL36003 and
HL51014 (to J.S.P.) and a Scientist Development Grant from the American Heart
Association (0835481N) (to Y.S.). C.W. was supported by a National Institutes of
Health Medical Scientist Training Program (T32-GM07205STP).
The sequences presented in this article have been submitted to MIAMExpress under
accession number E-MTAB-150.
Address correspondence and reprint requests to Jordan S. Pober, Yale University School
of Medicine, Amistad Research Building, 10 Amistad Street, New Haven, CT 06520 or
Yajaira Sua ´rez at the current address: Department of Medicine, Leon H. Charney Di-
vision of Cardiology and the Marc and Ruti Bell Vascular Biology and Disease Program,
New York University School of Medicine, Smilow 8, 522 First Avenue, New York, NY
10016. E-mail addresses: Jordan.firstname.lastname@example.org or email@example.com
The online version of this article contains supplemental material.
Abbreviations used in this paper: CI, control inhibitor sequence; CM, control mimic
sequence; EC, endothelial cell; HDF, human dermal fibroblast; miRNA, microRNA;
QRT-PCR, quantitative real-time PCR; SELE, E-selectin; UTR, untranslated region;
VE-cadherin, vascular endothelial cadherin; VEGF, vascular endothelial growth factor.
using a Dual-Glo Luciferase Assay System (Promega) and reported as the
percentage of control cells cotransfected with the same concentration of CM.
HUVECs were transfected with 40 nM miRIDIAN miRNA mimics or with
60 nM miRIDIAN miRNA inhibitors (Dharmacon) using Oligofectamine
(Invitrogen) (8, 18). Control samples were transfected with an equal con-
centration of CM or negative control inhibitor sequence (CI), as described
previously (8). The effects of transfections with miR-mimics/inhibitors was
assessed by quantitative real-time PCR (QRT-PCR). miR-31 and -17-3p
levels were efficiently increased after 18 h and persisted 36 h following
transfection, and transfection with miR-31 did not affect the endogenous
levels of miR-17-3p and vice versa (not shown). Transfection with miRNA
inhibitors for miR-31 or -17-3p efficiently reduced the TNF-stimulated levels
of miR-31 and -17-3p below levels in untreated cells, indicating an efficient
inhibition of indicated miRNAs (not shown).
Western blots were performed (8, 18) using mouse mAbs reactive with human
ICAM-1 and SELE (R&D Systems, Minneapolis, MN) under nonreducing
conditions or with Tie2 and vascular endothelial cadherin (VE-cadherin)
polyclonal Abs (Santa Cruz Biotechnology, Santa Cruz, CA) or Hsp-90 mAb
(BD Biosciences, San Jose, CA) under reducing conditions. Binding of flu-
orophore-conjugated secondary Abs (Rockland, Gilbertsville, PA) were visu-
alized using an Odyssey Infrared Imaging System (LI-COR, Lincoln, NE).
Cell surface immunostaining
HUVECs were transfected and/or treated with TNF as indicated, stained with
directly fluor-conjugated mAbs as described (18), and analyzed on a FACS-
calibur (BD Biosciences) using CellQuest analysis software, collecting 5000
gated cells per sample. Specific Abs used in these analyses were FITC or PE
directly conjugated and reactive with human SELE (R&D Systems) or
ICAM-1 (BD Pharmingen, San Diego, CA), respectively.
Neutrophil adhesion assays
Adhesion of neutrophils to the confluent EC monolayer was measured as de-
calcein-AM (Molecular Probes, Eugene, OR) for 10 min at 37˚C and sus-
pended in PBS containing 1 mM magnesium chloride, 0.5 mM calcium
chloride, and 0.1 g/l glucose (PBS plus). Transfected HUVECs were treated
1 ml labeled neutrophils (105) for 15 min at 37˚C under static conditions.
Samples were fixed, and bound neutrophils were counted in five randomly
selected 310 fields. Where indicated, HUVECs were incubated with anti-
SELE and/or anti–ICAM-1 blocking Abs (R&D Systems) at 50 or 10 mg/ml,
respectively, for 1 h before the addition of the neutrophils.
Statistical differences between groups were assessed by the two-tailed paired
to the control of each experiment, and 95% confidence intervals were calcu-
lated; differences were judged significant when the confidence interval did not
include the “1” or “100” value.
Results and Discussion
We characterized miRNA expression in HUVECs stimulated
for 2 or 24 h with 10 ng/ml TNF using microarrays. Several
miRNAs were induced after 2 h; some of these had increased
after 24 h, whereas others had decreased (Fig. 1, Supplemental
Table I). Levels of selected miRNA species identified in the
arrays (Fig. 1, highlighted in black) were confirmed by
Northern blotting (Supplemental Fig. 1A) and/or by QRT-
PCR (Supplemental Fig. 1B). Levels of miR-155, -31, -17-5p,
-191, and -125b were increased by TNF, but levels of miR-
222, -20a, and -126 were not (Supplemental Fig. 1A, 1B).
Several TNF-induced HUVEC miRNAs were not induced in
HDFs, suggesting at least partialspecificity of the EC response.
miR-155 is highly induced by TNF in HUVECs and HDFs
(Supplemental Fig. 1C). TNF also induces miR-155 in im-
munecells(12), insynovial fibroblasts(21), andinlymphomas
(9, 22). miR-155 is also upregulated in ECs by VEGF (8).
Other miRNAs induced by TNF also overlap with those
components of the c-myc/E2F–regulated oncogenic cluster
miR-17/92 (including miR-17-5p, -18a, and -20a) (8), but
only miR-17-5p is upregulated by TNF (Fig. 1, Supplemental
processing (23, 24) or selective blockade of pri-miRNA pro-
cessing (25) may result in differential expression of mature
and systemic lupus erythematosus (26, 27). In human pulmo-
STAT3 (28). Secretion ofIL-6is inducedin ECs by TNF (29),
raising the possibility of an indirect autocrine/paracrine effect.
In HUVECs, guide and passenger strands of miR-17 (5p and
3p, respectively) are induced by TNF (Supplemental Fig. 1A).
We used miRNA target prediction algorithms (5) to identify
a putative binding site in the 39UTR of SELE for miR-31
(Supplemental Fig. 3A), a highly TNF-induced miRNA
(Fig. 1), and an additional site for miR-221 and -222 (Sup-
plemental Fig. 3A), two highly homologous miRNAs, derived
from the same pri-miRNA transcript that contains identical
seed sequences and that are not regulated by TNF (Fig. 1).
The miR-31 predicted site is a canonical 7mer-m8 site sup-
ported by an additional 39 pairing optimally centered on
miRNA nt 13–16 (5). The human “seed” region of miR-31
complementary to the sequence in SELE 39UTR is conserved
across species (Supplemental Fig. 3A). The miR-221/222–
predicted site is a nonconserved 6mer in the center of the
SELE 39UTR (Supplemental Fig. 3A). We tested these pre-
dicted miRNA/mRNA interactions using a SELE 39UTR
luciferase reporter plasmid in COS cells. The relative luciferase
activity was significantly reduced (?25%) by cotransfection
with miR-31 but not with CM (Fig. 2A). miR-31 did not
inhibit reporter vectors lacking SELE 39UTR, with reverse
oriented 39UTR (control 39 UTR) or with a mutational
change in the sequence complementary to the miR-31 seed
sequence (mSELE 39UTR) (Fig. 2A). Luciferase activity of
SELE 39UTR was significantly reduced with as little as 10 nM
of miR-31 (Supplemental Fig. 4A). Luciferase activity of SELE
isolated from ECs that were treated or not for 2 or 24 h with TNF (10 ng/ml).
Data are presented as the log2ratio of miRNA expression of average treated (2
or 24 h) versus average untreated (0 h). miR-155, 31, 17-5p, -191, -126,
-222, -20a are indicated in black diamonds.
TNF regulation of miRNA levels in human ECs. RNA was
22 CUTTING EDGE: CONTROL OF INFLAMMATION BY TNF-INDUCED miRNAs
39UTR was not significantly reduced by miR-221 and -222
(Fig. 2B). Positioning in the center of a long 39UTR and lesser
efficacy of 6mer sites (5) may explain this lack of effect. SELE
39UTR luciferase activity also was not affected by TNF-in-
duced miR-17-3p, -17-5p, -155, or -125b (Fig. 2B), none of
which is predicted to bind to the SELE 39UTR.
To analyze the effect ofmiR-31 on SELE proteinexpression,
HUVECs were transfected with miR-31 mimic (M-miR-31)
TNF for 3 or 6 h, the peaks of SELE mRNA and protein ex-
pression, respectively (1, 17). By immunoblotting, M-miR-31
reduced TNF-induced SELE levels by ?35% at 3 h (Fig. 2C).
Furthermore, SELE levels increased by 25% and 20% at 3 and
6 h, respectively, when cells were transfected with an antisense
miR-31 inhibitor (I-miR-31) prior to TNF stimulation (Fig.
2D). Two other EC proteins, VE-cadherin and Tie2, were not
changed by these treatments (although a putative binding site
for miR-31 was predicted in Tie2). By FACS, TNF-induced
SELE cell surface expression increased when miR-31 was in-
hibited (Supplemental Fig. 5A).
Two canonical 7mer-m8 and one 7mer-A1 putative binding
TNF-induced miR-17-3p and for non-TNF-induced miR-
221/222, respectively (Supplemental Fig. 3B). None of these
sites is conserved across species but were independently pre-
dicted by three different target-prediction algorithms (Tar-
getScan, miRBase, and RegRNA), and a large fraction of
nonconserved sites can be functional (30). In reporter gene
assays, miR-17-3p efficiently repressed ICAM-1 39UTR lucif-
erase activity by ?40% (Fig. 3A). Luciferase activity was un-
reporter vectors lacking ICAM-1 39UTR, wtith reverse-ori-
ented 39UTR (control 39UTR) or with a mutation in the se-
quence complementary to the miR-17-3p seed sequence
(mICAM-1 39UTR). Luciferase activity of ICAM-1 39UTR
was significantly reduced at 40 nM (Supplemental Fig. 4B).
ICAM-1 39UTR luciferase activity was unaffected by miR-221
(5). It was recently reported that miR-222 can regulate the
expression of ICAM-1 in tumor cells by direct interaction
with its 39UTR (31). A key difference from our experiments
is that Ueda et al. (31) tested the isolated target sequence rather
than testing the sequence in the context of the entire 39UTR.
Contextual features of the 39UTR, such as secondary struc-
tures or local AU-rich regions, among others, can govern
the SELE 39UTR. COS-7 cells were cotransfected with the indicated con-
structs and with 40 nM of M-miR-31 or CM (A) or with SELE 39UTR
construct and 40 nM of the indicated miRNA mimics or CM (B). Data are
expressed as relative luciferase activity to control samples cotransfected with
an equal concentration of CM (mean 6 SEM of three experiments performed
in duplicate).*Significantly different from cells transfected with CM and with
control 39UTR and with the indicated miRNAs, p # 0.05. C and D, Western
blot analysis of SELE protein levels in ECs transfected for 12 h with M-miR-
31 or CM (C) or with I-miR-31 or CI (D); in both cases, the cells were
treated or not with TNF 24 h posttransfection. Hsp-90 was used as loading
control. VE-cadherin served as a control protein that is not targeted by miR-
31. Graphs on the right show the relative total SELE protein levels compared
with nontreated CM- or CI-transfected controls (mean 6 SEM of four ex-
periments). Significantly different from TNF-treated control, p # 0.05.
miR-31 regulates TNF-induced SELE expression by targeting
targeting the ICAM-1 39UTR. COS-7 cells were cotransfected with the in-
dicated constructs and with 40 nM of M-miR-17-3p or CM (A) or with
ICAM-1 39UTR construct and 40 nM of the indicated miRNA mimics or
CM (B). Data are expressed as relative luciferase activity to control samples
cotransfected with an equal concentration of CM (mean 6 SEM of three
experiments performed in duplicate). *Significantly different from cells
transfected with CM and with control 39UTR and with the indicated miR-
NAs, p # 0.05. C and D, Western blot analysis of ICAM-1 protein levels in
ECs transfected for 12 h with M-miR-17-3p or CM (C) or with I-miR-17-3p
or CI (D); in both cases, the cells were treated or not with TNF 24 h post-
transfection. Hsp-90 was used as loading control. Tie2 served as a control
protein that is not targeted by miR-17-3p. Graphs on the right show the
relative total ICAM-1 protein levels compared with nontreated CM- or CI-
transfected controls (mean 6 SEM of four experiments). *Significantly dif-
ferent from TNF-treated control, p # 0.05.
miR-17-3p regulates TNF-induced ICAM-1 expression by
The Journal of Immunology 23
miRNA–mRNA interactions (5). ICAM-1 39UTR luciferase
31,-17-5p,-155,and-125b) (Fig. 3B)notpredictedtobindto
the ICAM1 39UTR. In general, the miRNA strandthat has the
lowest thermodynamic stability at its 59-terminus acts as the
(miRNA*) is degraded. However, in some cases, both miRNA
strands may accumulate in tissues at significant levels (32), and
there are validated examples of trans-regulatory RNAs with
demonstrable activities (33). Although basal miR-17-3p levels
are very low compared with miR-17-5p levels, miR-17-3p is
clearly stimulated upon TNF treatment (Supplemental Fig. 6).
To analyze the effect of miR-17-3p on ICAM-1 protein
expression, HUVECs were transfected with miR-17-3p mimic
were stimulated with TNF for 12 h. M-miR-17-3p reduced
levels increased up to 35% when cells were transfected with an
antisense miR-17-3p inhibitor (I-miR-17-3p) prior to TNF
stimulation (Fig. 3D). Tie2 levels were not affected by miR-
17-3p (Fig. 3C, 3D). Although the total protein levels were
consistently induced by miR-17-3p antagonism, increases in
TNF-induced ICAM-1 cell surface expression observed by
FACS did not reach statistical significance (Supplemental Fig.
5B). The increase was significant in some HUVEC donors,
consistent with a polymorphic response.
We didnotfindpredictedsites forregulationofVCAM-1by
TNF-induced miRNAs. miR-126 is an EC-specific miRNA
any sites for miR-126 in the 39UTRs of SELE or ICAM-1.
Finally, we tested whether the miRNA effects on TNF-in-
TNF increased neutrophil adhesion to ECs. Exogenous over-
the two significantly reduced neutrophil binding (Fig. 4A,
Supplemental Fig. 7A), whereas the inhibition of miRNAs
miR-31 and/or miR-17-3p increased neutrophil adherence to
TNF-stimulated ECs (Fig. 4B, Supplemental Fig. 7B). It is
likely that TNF-induced miRNAs (miR-31 and -17-3p) regu-
late neutrophil adhesion through the regulation of TNF-
induced expression of SELE and ICAM-1, respectively, al-
though other actions of these miRNAs could affect neutrophil
binding. To test this possibility, we performed experiments
combining blocking Abs with miRNA mimic or inhibitor
transfections. As expected, the incubation with blocking Abs
alone profoundly reduced neutrophil binding (Supplemental
Fig. 7C, 7D). Transfection of mimics did not cause any evi-
(Supplemental Fig. 7C). Transfection of inhibitors produced
a small increase in neutrophil binding in the presence of
blocking Abs that did not reach statistical significance (Sup-
plemental Fig. 7D). These experiments do not rule out the
possibility that these TNF-induced miRNAs affect neutrophil
if such effects exist, they are too small to detect when inter-
actions with SELE or ICAM-1 are blocked.
and ICAM-1). This kind of regulation, in which miRNA-
directed target repression acts to oppose the overall outcome of
for fine-tuning of the process (34). Antisense oligos (antimiRs)
that target specific miRNAs were recently shown to be very
efficient in vivo (35). In the system we describe, miRNA de-
of endogenous mature miRNAs could be useful as an anti-
We thank Dr. Anjelica Gonzalez for helpful comments regarding neutrophil
adhesion assays; Louise Benson, Gwen Davis-Arrington, Lisa Gras, and Todd
versity of Pennsylvania and the Keck Facility at Yale University for miRNA
array analyses; Dr. William C. Sessa for providing COS-7 cells and access
to LI-COR; and Dr. Carlos Ferna ´ndez-Hernando for critical discussions
during the preparation of this manuscript.
The authors have no financial conflicts of interest.
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