Research A rticle
miR-155 Regulated Inflammation Response by
the SOCS1-STAT3-PDCD4 Axis in Atherogenesis
Jinshan Ye,
1,2
Ruiwei Guo,
2
Yankun Shi,
2
Feng Qi,
2
Chuanming Guo,
2
and Lixia Yang
1,2
1
DepartmentofPostgraduate,irdMilitaryMedicalUniversity,Chongqing400038,China
2
Department of Cardiology, K unming General Hospital of Chengdu Military Area, Yunnan 650032, China
Correspondence should be addressed to Lixia Yang; doctorylixia@aliyun.com
Received May ; Revised August ; Accepted August
Academic Editor: T
ˆ
ania Silvia Fr
¨
ode
Copyright © Jinshan Ye et al. is is an open access article distributed under the Crea tive Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Inammation response plays a critical role in all phases of atherosclerosis (AS). Increased evidence has demonstrated that miR-
mediates inammatory mediators in macrophages to promote plaque formation and rupture. However, the precise mechanism of
miR- remains unclear in AS. Here, we also found that miR- and PDCD were elevated in the aortic tissue of atherosclerotic
mice and ox-LDL treated RAW. cells. Further studies showed that miR- not only directly inhibited SOCS expression, but
also increased the expression of p-STAT and PDCD, as well as the production of proinammation mediators IL- and TNF-
𝛼. Downregulation of miR- and PDCD and upregulation of SOCS obviously decreased the IL- and TNF-𝛼 expression. In
addition, inhibition of miR- levels in atherosclerotic mice could notably reduce the IL- and TNF-𝛼 level in plasma and aortic
tissue, accompanied with increased p-STAT and PDCD and decreased SOCS. us, miR- mig ht mediate the inammation
in AS via the SOCS-STAT-PDCD axis. ese results provide a rationale for intervention of intracellular miR- as possible
antiatherosclerotic targets.
1. Introduction
A therosclerosis (AS) remains a major cause of mortality
worldwide, causing acute cardiovascular events and chronic
damage, including ischemic heart disease and ischemic stroke
[]. It is well known that inammation plays an important role
inallphasesofAS[,].Intheinitiationandprogression
ofAS,manyimmunecells,especiallyM-typemacrophages,
are recruited to the arterial wall where they produce and
secrete extensive amounts of inammation mediators and
chemokines and promote the formation of plaque [] and
plaque rupture []. is leads to plaque instability, thrombo-
sis, and, nally, cardiovascular events [].
MicroRNAs (miRNAs), which are a class of – nt
small noncoding RNAs, have served as negative regulators
of gene expression at a posttranscriptional level [] and play
an important role in cell development, metabolism, prolife-
ration, and apoptosis [, ]. miRNAs are involved in the path-
ogenesis of many diseases, from c ancer to cardiovascular
disease []. Studies have recently found that several miRNAs,
such as miR- [] and miR- [], are involved in the ini-
tiation and progress of AS. In particular, miR-, located
within a region known as the B-cell integration cluster
(BIC) in the genome, plays a key role in innate immunity
[, ]. A broad range of inammatory factors, including
ox-LDL, stimulate macrophages, and they in turn regulate
the expression of inammatory factors to enhance cellular
inammatory response []. In addition, other results showed
that miR- is mainly expressed in macrophages and SMCs
in late atherosclerotic lesions, and lesional macrophages are
an especially abundant source of miR- []. How the preci-
sion mechanism of miR- regulated the formation of
macrophage-derived foam cells during early atherogenesis is
still not clear.
Historically, programmed cell death (PDCD) was
always notably reduced or decient in various tumors [–
]. PDCD activation was involved in the apoptosis of cancer
cells [, ] and was therefore considered a tumor suppressor
[]. Interestingly, Billiard et al. showed that PDCD decient
mice were resistant to inammatory diseases []. Zhong
Hindawi Publishing Corporation
Mediators of Inflammation
Volume 2016, Article ID 8060182, 14 pages
http://dx.doi.org/10.1155/2016/8060182
Mediators of Inammation
et al. found that PDCD was involved in allergic pulmonary
inammation through regulated macrophage alternative acti-
vation []. Other studies showed that PDCD improved the
inammatory response via nuclear factor-𝜅B(NF-𝜅B) activa-
tion and inhibition in the production of interleukin- (IL-)
[, ]. Liang et al. demonstrated that PDCD was elevated
in atherosclerosis mice and in the foam cells, and inhibition
of PDCD could suppress the inammation mediator, such
as IL- and TNF-𝛼 []. Other studies showed that PDCD
deciency in mice increased the expression of IL- in
macrophagesandledtoadecreaseinatheroscleroticlesions
in ApoE
−/−
mice who were fed high fat diets (HFD) [].
ese results suggest the potential role of PDCD as a novel
therapeutic target in the clinical treatment of atherosclerosis.
Although several reports showed that PDCD was directly
regulated by miR- and miR- in macrophages, whether
miR- regulated the PDCD is unclear.
In this study, we explored elevated miR- and PDCD
in the aortic tissue of atherosclerotic mice and ox-LDL
treatedRAW.cells.WealsoshowedthatmiR-directly
inhibits SOCS expression and increase s the expression of
p-STAT and PDCD, thereby promoting the production
of proinammation mediators. Knocking down of miR -
, SOCS overexpression, and downregulation of PDCD
notably aected the production and release of the inamma-
tion mediator. Furthermore, inhibition of the miR- level in
atherosclerotic mice also elevated the SOCS expression and
decreased the expression of p-STAT, PDCD, and proin-
ammation cytokine levels. In summary, we determined that
miR- might promote an inammation response of AS
through the SOCS-STAT-PDCD a xis.
2. Methods
2.1. Cell Culture and Treatment. A macrophage Raw. cell
line was purchased from American Type Culture Collection
and maintained in DMEM medium with % FBS and %
antibiotics. ese cells were treated with 𝜇g/mL, 𝜇g/mL,
and 𝜇g/mL ox-LDL for h or 𝜇g/mL ox-LDL from –
h. STAT, PDCD siRNA, anti-miR-, and anti-NC were
transfected into macrophages for h, respectively, and these
cells were exposed with 𝜇g/mL ox-LDL for h. ere-
aer, the medium was changed to normal culture medium
to continue further study. ese siRNA target sites were
showed in Supplemental Tables S and S (in Supplementary
Materi al available online at http://dx.doi.org/.//
).
2.2. Total RNA Isolation and Real-Time PCR. Total RNA
from cells and aortic tissue was extracted by using the
TRIzol reagent according to the manufacturer’s protocol.
Reverse transcription polymerase chain reaction (RT-PCR)
was conducted in a two-step process using an RNA PCR
Kit according to the manufacturer’s instructions. e primers
of miR-, SOCS, STAT, and PDCD showed in Sup-
plemental Table S. Each PCR amplication was performed
under the following conditions:
∘
C for min,
∘
Cfors,
and
∘
C for s at the annealing temperature through
cycles.
2.3. Cytokine Assay by ELISA. e plasma of IL-, IL-
, and TNF-𝛼 levelwasdetectedbyELISAaccordingto
manufacturer’s instructions. e details had been shown in
the Lei and colleagues study [].
2.4. Western Blot. Macrophage cells and aortic tissue were
collected and homogenized with lysis buer (Pierce), and
then the concentrations of total protein were determined
using a BCA kit (Pierce). Immunoblotting was conducted fol-
lowing procedures outlined in a study []. Rabbit polyclonal
anti-mouse phospho-STAT (p-STAT), mouse monoclonal
anti-PDCD, and rabbit polyclonal SOCS were obtained
from Abcam, Inc.
2.5. Constructed SOCS1 Adenoviruses. SOCS wild-type
(WT) adenoviruses were constructed as outlined in the study
by Galam et al. []. Briey, T cells were transfected
with SOCS, cDNA vectors, and a packing system (Sys-
temViraPower Adenoviruses Expression Systems, Invitro-
gen). Aer T cells were transfected, all plasmids and
adenovirus-containing media were harvested at and h.
ereaer, t he virus was puried by CsCl gradients.
2.6. Luciferase Reporter Assay. e
-UTR of SOCS was
synthesized and annealed and then inserted into the SpeI
and HindIII sites of pMIR-reporter luciferase vector down-
stream of the stop codon of the gene for luciferase. For its
mutagenesis, the sequences complementary to the binding
site of miR- in the
-UTR (SOCS: AGCAUUA) were
replaced by AGCUAAU. ese constructs were validated
by sequencing. T cells were seeded into a -well plate
for a luciferase assay. Aer cultured overnight, cells were
cotransfected with the wild-type or mutated plasmid, pRL-
TK plasmid, and equal amounts of miR- or miR-NC. e
pRL-TK control vector was also transfected as a control.
Luciferase assays were performed h aer transfection using
the Dual Luciferase Reporter Assay System. Firey and
Renilla reniformis luciferase activities were measured h
later. Experiments were performed in three independent
replicates.
2.7. Animal Experiments. Male C wild-type (WT) mice
and A poE
−/−
mice were purchased from the Model Animal
Research Center of Nanjing University . ApoE
−/−
mice (
weeks, male) were fed HFDs (% fat, .% cholesterol)
for week and then randomized into two groups (𝑛=
10 mice, resp.): control antagomiR-injected and antagomiR-
-injected groups. e mice received two subcutaneous
injections of mg/kg antagomiR- or antagomiR for the
rst week, spaced days apart, and then weekly injections
of mg/kg antagomiR or antagomiR- thereaer for
weeks, at which point injections of antagomiR or antagomiR-
were stopped. ey were both still fed, however, with an
HFD for week. Aortic roots of mice were embedded in OCT
medium and frozen imme diately. All animal exp eriments
were carried out in accordance with the National Institutes of
Health Guide for the Care and Use of Laboratory Animals and
were approved by the Biological Research Ethics Committee
of the Institute of Health Sciences.
Mediators of Inammation
0
1
2
3
4
miR-155 relative expression
∗
∗∧
∗∧#
5𝜇g/mL 10 𝜇g/mL 20 𝜇g/mL
Control
ox-LDL for 24 h
(a)
0
1
2
3
4
miR-155 relative expression
∗
∗∧
∗∧#
6 h
12 h 24 h
Control
20 𝜇g/mL ox-LDL
(b)
0
2
4
6
miR-155 relative expression
∗∧
∗
c57 mice WT
ApoE
−/−
HFDApoE
−/−
ND
(c)
F : e relative expression of miR - in macrophages RAW. cells and the aortic tissue of ApoE
−/−
mice. (a) qPCR analyzed the
expression of miR- in RAW . cells when macrophages were treated by ox-LDL for h at the indicated dose:
∗
𝑝
< 0.05,relativeto
control,
∧
𝑝
< 0.05,relativeto𝜇g/mL ox-LDL group,
#
𝑝
< 0.05,relativeto𝜇g/mL ox-LDL group. (b) qPCR detected t he le vel of miR-
in macrophages, which were treated with 𝜇g/mL ox-LDL at the indicated time:
∗
𝑝
< 0.05,relativetocontrol,
∧
𝑝
< 0.05,relativetoh
group,
#
𝑝
< 0.05, relative to h group. (c) Expression of miR- in aortic tissue of ND-fed wild-type (WT) c mice, ND-fed ApoE
−/−
mice
(ApoE
−/−
ND), and HFD-fed ApoE
−/−
mice (ApoE
−/−
HFD) was determined by qPCR (𝑛=4):
∗
𝑝
< 0.05, relative to c mice WT;
∧
𝑝
< 0.05,
relative to ApoE
−/−
ND.
2.8. Atherosclerosis Analysis. oracoabdominal aortas were
xed by % formaldehyde (sigma) for h, and then the
plaques were stained with Oil Red O staining. e collagen
size of atherosclerotic plaques was analyzed by using Sirius
red and fast green collagen staining.
2.9. Statistical Analysis. Data were expressed as mean ±
standard error (SE) and were achieved via at least three
independent experiments. Two-tailed Student’s t-test and
one-way analyses of variance (ANOVA) were performed. e
signicant statistical dierence was dened according to 𝑝<
0.05.
3. Results
3.1. miR-155 Induced by ox-LDL in Macrophages RAW264.7
CellsandIncreasedintheAorticTissueofApoE
−/−
Mice. In
this study, we investigated the expression of miR- aer
RAW. cells were treated by ox-LDL at the indicated
dose and time (Figures (a) and (b)). e data showed that
ox-LDL gradually increased the expression of miR- with
increased concentrations from 𝜇g/mL to 𝜇g/mL treat-
ment with macrophages for h, compared to control (𝑝<
0.05), and the expression of miR- was peaked while
cells were treated with 𝜇g/mL ox-LDL. e result also
showed that miR- was signicantly higher aer the
RAW. cells (treated with 𝜇g/mL ox-LDL for h, h,
and h) than in control (𝑝 < 0.05). erefore, ox-LDL
induced miR- in a dose- and time-dependent manner.
Moreover, we evaluated the miR- expression in the aortic
tissue of ApoE
−/−
mice with normal food (ND) and HFD.
e results revealed that the expression of miR- elevated in
aortic tissue of ApoE
−/−
ND and ApoE
−/−
HFD, compared to
the c mice (𝑝 < 0.05),anditwasnotablyhigherinApoE
−/−
HFDthaninApoE
−/−
ND (𝑝 < 0.05). ese data suggest that
miR- was induced by ox-LDL, and it could be involved in
the pathogenesis of atherosclerosis [].
Mediators of Inammation
∗∧
∗∧
∗∧
∗∧#
∗∧#
∗∧#
∗
∗
TNF-𝛼 IL-10
IL-6
Control
5𝜇g/mL
10 𝜇g/mL
20 𝜇g/mL
0
2
4
6
8
Relative mRNA expression
(a)
∗∧
∗∧
∗∧
∗
∗
ApoE
−/−
HFD
c57 mice WT
ApoE
−/−
ND
TNF-𝛼 IL-10IL-6
(pg/mL)
0
20
40
60
80
Plasma levels of cytokines
(b)
F : e levels of cytokines IL-, TNF-𝛼, and IL- in RAW. cells aer ox-LDL treatment and atherosclerotic mice. (a) ox-LDL
induced the expression of IL-, TNF-𝛼, and IL- for h at indicated dose was determined by qPCR:
∗
𝑝
< 0.05,relativetocontrol,
∧
𝑝
< 0.05,
relative to 𝜇g/mL ox-LDL group;
#
𝑝
< 0.05,relativeto𝜇g/mL ox-LDL group. (b) e plasma levels of IL-, TNF-𝛼, and IL- of c mice
WT, ApoE
−/−
ND, and ApoE
−/−
HFD were detected by the ELISA kit (𝑛=6):
∗
𝑝
< 0.05,relativetocmiceWT;
∧
𝑝
< 0.05,relativeto
ApoE
−/−
ND.
3.2. ox-LDL M ediated the Production of Cytokines in
RAW264.7 Cells and Plasma Levels of ese Cytokines
Changed in Atherosclerotic Mice. e production and release
of inammation mediators by macrophages played a critical
role in AS [, , ]. In our study, we showed that the
expression of IL- and TNF-𝛼 mRNA was increased and
IL- mRNA expression was inhibited by increasing ox-LDL
concentration, compared to the control group in RAW.
cells (Figure (a)). In addition, the level of plasma IL- and
TNF-𝛼 was higher i n the ApoE
−/−
ND and ApoE
−/−
HFD
groups than in the c mice (𝑝 < 0.05). ese were notably
elevated in ApoE
−/−
HFD compared with ApoE
−/−
ND
(𝑝 < 0.05) (Figure (b)). On the contrary, the plasma IL-
level was signicantly reduced in ApoE
−/−
HFD, compared
to the ApoE
−/−
ND and c mice group (𝑝 < 0.05); however,
there was no dierence b etween the ApoE
−/−
ND and c
mice group (𝑝 > 0.05) (Figure (b)).
3.3. PDCD4 Regulated by miR-155 In Vitro. Previous studies
reveal that knocked down PDCD played an important
role in attenuating foam cell formation and atherosclerosis
in ApoE
−/−
mice []. Consistent with miR-, PDCD
expression was increased upon ox-LDL sti mulation in a
dose-dependent manner (Figures (a) and (b)). To furt her
investigate the relationship between miR- and PDCD,
RAW . cel ls were transfected with anti-miR- and miR-
NCandthenchallengedwithox-LDL.eseresultsshowed
that the inhibition of miR- could reduce the expression of
PDCD while RAW. cells are t reated by ox-LDL (Figures
(c) and (d)). Together, these results sug gest that miR-
could regulate PDCD expression in macrophages; however,
the mechanism was unclear.
3.4. Downregulation of PDCD4 and miR-155 Mediated Pro-
duction of Cytokines IL-6, TNF-𝛼, and IL-10 in RAW264.7
Cells. PD CD and miR- played important roles in t he
regulation of inammation response [, –]. ree
PDCD siRNA oligos were t ransfected into RAW. cells
to eva luate the suppression eective of PDCD expression.
is data showed that PD C D siRNA oligo () was more
eective in knocking down PDCD than other siRNA oligos
(Figure (a)). Moreover, the protein expression of PDCD
was signicantly decreased aer cells were treated with
PDCD siRNA oligo () (Figure (b)). In our study, the
inammation mediators were detected aer PDCD and
miR- were knocked down in RAW. cells. qPCR
detected demonstrated that downregulation of PDCD and
miR- could partly reverse the elevation of IL- and TNF-
𝛼 mRNA expression, which was induced by 𝜇g/mL ox-
LDL (Figures (c) and (d)). On the contrary, knocked down
PDCD and miR- obviously increased the expression of
IL- mRNA when cells were exposed to ox-LDL. ese
data suggested that miR- mediated the production of the
inammation mediator via PD CD.
3.5. miR-155 Regulated PDCD4 via SO CS1-STAT3 Signal
Pathway . S everal studies showed that miR- regulated the
production of inammation mediator via direct target [–
]. In our study, HEK cel ls were cotransfected with
the wild-typ e (WT) or mutated (Mut) SOCS luciferase
reporter vector, together with miR- mimic or miR-NC,
for h. Luciferase activity was sig nicantly inhibited in
cells transfected with WT SOCS and miR- mimic, but
not in cells transfected with mutation SOCS and miR-
mimic (Figures (a) and (b)). is data demonstrated
that SOCS was a direct target of miR [ ]. To further
Mediators of Inammation
PDCD4
0
1
2
3
4
5
PDCD4 protein relative expression
∗∧#
∗∧
𝛽-Actin
Control
10 𝜇g/mL 20 𝜇g/mL5𝜇g/mL
Control 5𝜇g/mL 10 𝜇g/mL 20 𝜇g/mL
(a)
0
2
4
6
PDCD4 mRNA relative expression
∗
∗∧#
∗∧
Control
10 𝜇g/mL 20 𝜇g/mL5𝜇g/mL
(b)
Anti-NC
Anti-miR-155
20 𝜇g/mL ox-LDL
PDCD4
𝛽-Actin
−−−+
−−+−
−
+++
Anti-NC
Anti-miR-155
0
1
2
3
4
PDCD4 protein relative expression
∗
∗
∗∧#
20 𝜇g/mL ox-LDL ++−+
−+−−
−−+−
(c)
Anti-NC
Anti-miR-155
0
1
2
3
4
5
PDCD4 mRNA relative expression
∗
∗
∗∧#
20 𝜇g/mL ox-LDL
++
−
+
−+−−
−−+−
(d)
F : e relative expression of PDCD in macrophages RAW. cells. (a and b) Western blot and qPCR analyzed the PDCD
protein and mRNA expression aer indicated ox-LDL treated macrophages RAW. cells for h. Representative bands show the
proteinexpressionofPDCD(theupperpanel),𝛽-actin (the middle panel), and the histograms showing quantication of PDCD bands
normalization relative to 𝛽-actin expression in (a):
∗
𝑝
< 0.05 versus control,
∧
𝑝
< 0.05 versus 𝜇g/mL group, and
#
𝑝
< 0.05 versus 𝜇g/mL
group. (c and d ) Western blot and qPCR analyzed the PDCD protein and mRNA expression while RAW. cells were treated by 𝜇g/mL
ox-LDL plus anti-miR- or anti-NC. pM anti-miR- or anti-NC had been transfected into RAW. cells for h, and then they were
treated by 𝜇g/mL ox-LDL for h. Representative bands show the protein expression of PDCD (the upper panel), 𝛽-actin (the middle
panel), and the histograms showing quantication of PDCD bands normalized relative to 𝛽-actin expression in (c):
∗
𝑝
< 0.05 versus control,
∧
𝑝
< 0.05 versus 𝜇g/mL ox-LDL group, and
#
𝑝
< 0.05 versus 𝜇g/mL ox-LDL plus anti-NC group.