miR-185/ P2Y6 Axis Inhibits Angiotensin II-Induced Human Aortic Vascular Smooth Muscle Cell Proliferation

Abstract

The abnormal proliferation and apoptosis of human aortic vascular smooth muscle cells (HAVSMCs) play an important role in the pathogenesis of hypertension. Recent study revealed that angiotensin II (Ang II) could elicit HAVSMC dysfunction, to induce or aggravate hypertension. Purinergic receptor P2Y6, an inflammation-inducible G protein-coupled receptor, promoted Ang II-induced hypertension. In the present study, we revealed that Ang II induced HAVSMC proliferation and upregulated P2Y6 protein levels. After knockdown of P2Y6, the promotive effect of Ang II on HAVSMC proliferation was restored. microRNAs (miRNAs) involve in most biological processes. In this study, we scanned out seven candidate miRNAs, which were predicted to contain binding site of P2Y6's 3'-UTR by online tools. Among them, miR-185 was significantly downregulated by Ang II treatment. miR-185 reduced P2Y6 protein levels by direct binding to the 3'UTR of P2Y6. miR-185 overexpression suppressed HAVSMC proliferation; P2Y6 overexpression or Ang II treatment promoted HAVSMC proliferation, and restored the suppressive effect of miR-185 on HAVSMC proliferation. Besides, miR-185/P2Y6 axis also affected pERK1/2 protein levels. Taken together, the present study indicated that miR-185/P2Y6 axis might inhibit Ang II-induced HAVSMC proliferation through miR-185 negatively regulating P2Y6 expression and the downstream ERK pathway; rescuing miR-185 expression to inhibit P2Y6 may represent a therapeutic strategy against HAVSMC dysfunction and hypertension.

Full text

ORIGINAL RESEARCH ARTICLE
miR-185/P2Y6 Axis Inhibits Angiotensin II-Induced Human
Aortic Vascular Smooth Muscle Cell Proliferation
Shunmin Wang,
1,2
Lujun Tang,
1
Qian Zhou,
1
Duomei Lu,
1
Wulei Duan,
1
Cheng Chen,
1
Lu Huang,
2
and Yuansheng Tan
3,4
The abnormal proliferation and apoptosis of human aortic vascular smooth muscle cells (HAVSMCs) play an
important role in the pathogenesis of hypertension. Recent study revealed that angiotensin II (Ang II) could
elicit HAVSMC dysfunction, to induce or aggravate hypertension. Purinergic receptor P2Y6, an inflammation-
inducible G protein-coupled receptor, promoted Ang II-induced hypertension. In the present study, we revealed
that Ang II induced HAVSMC proliferation and upregulated P2Y6 protein levels. After knockdown of P2Y6,
the promotive effect of Ang II on HAVSMC proliferation was restored. microRNAs (miRNAs) involve in most
biological processes. In this study, we scanned out seven candidate miRNAs, which were predicted to contain
binding site of P2Y6’s 3¢-UTR by online tools. Among them, miR-185 was significantly downregulated by Ang
II treatment. miR-185 reduced P2Y6 protein levels by direct binding to the 3¢UTR of P2Y6. miR-185 over-
expression suppressed HAVSMC proliferation; P2Y6 overexpression or Ang II treatment promoted HAVSMC
proliferation, and restored the suppressive effect of miR-185 on HAVSMC proliferation. Besides, miR-185/
P2Y6 axis also affected pERK1/2 protein levels. Taken together, the present study indicated that miR-185/P2Y6
axis might inhibit Ang II-induced HAVSMC proliferation through miR-185 negatively regulating P2Y6 ex-
pression and the downstream ERK pathway; rescuing miR-185 expression to inhibit P2Y6 may represent a
therapeutic strategy against HAVSMC dysfunction and hypertension.
Keywords: miR-185, human aortic vascular smooth muscle cells, angiotensin II, P2Y6, proliferation, hypertension
Introduction
Human aortic vascular smooth muscle cells
(HAVSMCs), which are present under the vascular
endothelial cells, are the main cellular components that form
the structure of the vascular wall and maintain the blood
vessel. In normal adults, vascular smooth muscle cells
maintain the tension of the blood vessel wall by slow and
mild contraction. Blood pressure is largely maintained by
vascular tension. Abnormal proliferation of vascular smooth
muscle cells plays an important role in the pathogenesis of
hypertension (Voelkel and Tuder, 1997). Overproliferation of
vascular smooth muscle cells lead to smaller vascular lumen,
thickening of the vascular wall, and plays an important role in
pathogenesis of hypertension (Voelkel and Tuder, 1997).
The rennin–angiotensin system (RAS) plays an important
role in the pathogenesis of hypertension. The system in-
cludes renin, angiotensin (Ang II), angiotensin-converting
enzyme and so on. Ang II has strong capability of vaso-
constriction, and can promote vascular smooth muscle cell
proliferation, hardens the vessel wall, and causes the lumen
to narrow (Touyz, 2005). Ang II could increase blood
pressure and promote arterial reconstruction through the
strong contraction of blood vessels; on the other hand, Ang
II can directly promote vascular smooth muscle cell hy-
pertrophy and collagen fiber proliferation, leading to vas-
cular remodeling (Pueyo and Michel, 1998; Takemoto and
Egashira, 1999). Ang II can also promote many processes
of cardiovascular tissue, including cell growth, migration,
differentiation, and apoptosis. Therefore, Ang II may be
one of the important factors affecting the cardiovascu-
lar remodeling (Komuro and Kudoh, 1999). However, how
HAVSMCs determine the responsiveness to Ang II under
different developmental and region-specific conditions is
mostly unclear.
Purinergic P2Y receptors are a family of inflammation-
inducible G protein-coupled receptors that respond to extra-
cellular mononucleotides (Abbracchio et al.,2006).Multiple
1
The Graduate Institute, Hunan University of Chinese Medicine, Changsha City, China.
2
Department of Cardiovascular, The First Hospital of Hunan University of Chinese Medicine, Changsha, China.
3
Department of Cardiovascular, First College of Traditional Chinese Medicine, Hunan University of Chinese Medicine, Changsha, China.
4
Department of Cardiovascular, College of Integrated Traditional Chinese and Western Medicine, Hunan Academy of Chinese Medicine,
Changsha, China.
DNA AND CELL BIOLOGY
Volume 36, Number 5, 2017
ªMary Ann Liebert, Inc.
Pp. 1–9
DOI: 10.1089/dna.2016.3605
1

P2Y receptor subtypes, including P2Y6 (Pediani et al., 1999;
Wang et al., 2002), are present in blood vessels. P2Y6 acti-
vation induces a Ca
2+
response in vascular smooth muscle
cells that results in contraction of isolated blood vessels
(Abbracchio et al., 2006). These pharmacological studies
suggest that P2Y6 plays an important role in cardiovascular
function. In addition, P2Y6 ablation attenuates vascular in-
flammation in vivo under pathological conditions (Riegel
et al., 2011; Stachon et al., 2014). These results suggest that
P2Y6 participates in the development of pathological vas-
cular remodeling. However, the functional role of P2Y6 on
HAVSMCs’ proliferation still remains to be validated.
The discovery of microRNAs (miRNAs) has opened new
avenues for studying and understanding hypertension and
hypertension-associated endothelial dysfunction, featuring a
postgenomic era of biomedical research (Nemecz et al.,
2016). These noncoding regulatory RNA molecules of *22
nucleotides have emerged as potential biomarkers, effectors,
and targets for diagnosis, prognosis, and therapy in hyper-
tension. Lately, many studies have been done on under-
standing miRNA biology and function. It seems that
miRNAs play an important role in the regulation of almost
every cellular process. Usually, a single miRNA can interact
with hundreds of mRNA molecules and a specific mRNA
molecule may be the target of multiple miRNAs. Thus,
miRNA–mRNA interactions may delineate the complex
regulatory networks with consequence on the target gene
expression and hence on some biological processes. Ad-
ditionally, the disruptions of miRNA regulation are fre-
quently associated with some pathological states, including
hypertension-associated endothelial dysfunction (Nemecz
et al., 2016).
In the present study, we validated the functional role
of Ang II in HAVSMCs proliferation and P2Y6 expres-
sion, and the effect of Ang II-induced P2Y6 expression
on HAVSMC proliferation. By using the online tools, we
scanned out some candidate miRNAs that contain the binding
site in 3¢UTR of P2Y6 and were associated with cell pro-
liferation. Among them, miR-185 could be downregulated
by Ang II treatment. As verified by performing a series of
functional and mechanistic assays, we revealed that miR-
185/P2Y6 axis could suppress HAVSMCs proliferation
through ERK signaling.
Materials and Methods
Cell lines and cell transfection
HAVSMCs were obtained from ATCC, cultured in
RPMI-1640 medium (Invitrogen, CA) adding 10% fetal calf
serum (FBS) (Gibco, CA) as the supplement, and incubated
at 37C in a humidified atmosphere with 5% CO
2
.
P2Y6-siRNA1 and P2Y6-siRNA2 were used to achieve
P2Y6 silence (GeneCopoecia, Guangzhou, China). pCMV-
P2Y6 vector was used to achieve exogenous P2Y6 expres-
sion (GeneCopoecia, Guangzhou, China). miR-185 mimics
or miR-185 inhibitor (GeneCopoecia, Guangzhou, China)
was used to achieve miR-185 overexpression or inhibition.
After being plated in 96- or 6-well plates, HAVSMCs were
then transfected with indicated siRNAs, mimics or inhibitor
with lipo2000 (Invitrogen), cultured for 24 h, and conse-
quently used for the next experiments.
RNA extraction and real-time PCR assays
We extracted total RNA from target cells by using TRIzol
reagent (Invitrogen). Mature miR-185, miR-483, miR-20a,
miR-1182, miR-3151, miR-149, and miR-877 expression in
cells was detected using the Hairpin-itmiRNAs qPCR Kit
(GenePharma, Shanghai, China). P2Y6 mRNA expression
was monitored by using SYBR Green qPCR assay. RNU6B
expression was used as an endogenous normalization. Data
were analyzed using 2
-DDCT
method.
Cell Counting Kit-8 cell proliferation assay
Cell Counting Kit-8 (CCK-8) (Beyotime, Hangzhou, China)
was used to measure HAVSMCs proliferation rates. We see-
ded 0.5 ·10
4
cells in each 96-well plate for 24 h, transfected
them with the indicated siRNA, miRNA mimics or inhibitor,
and further incubated cells for 24, 48, 72, 96, and 120 h, re-
spectively. At 1 h before the endpoint of incubation, we added
10 mL CCK-8 reagents to each well. A microplate reader was
used to determine OD
570nm
value in each well.
5-Bromo-2-deoxyUridine cell proliferation assay
By measuring 5-Bromo-2-deoxyUridine (BrdU) incorpo-
ration, the DNA synthesis in proliferating cells was deter-
mined. BrdU assays were conducted at 24 and 48 h after
HAVSMCs were transfected with the indicated vectors. Cells
were seeded in 96-well culture plates at a density of 2·10
3
cells/well, cultured for 48 h, then incubated with a final con-
centration of 10mM BrdU (BD Pharmingen, San Diego, CA)
for 2 h. When the incubation period ended, the medium was
removed, the cells were fixed for 30 min at RT, incubated with
peroxidase-coupled anti-BrdU-antibody (Sigma-Aldrich) for
60 min at RT, washed three times with PBS, incubated with
peroxidase substrate (tetramethylbenzidine) for 30 min, and
the 450 nm absorbance values were measured for each well.
Background BrdU immunofluorescence was determined in
cells not exposed to BrdU, but stained with the BrdU antibody.
Western blotting
RIPA buffer (Cell-Signaling Tech.) was used to homo-
genize the cells. The expression of P2Y6, ERK1/2, and
pERK1/2 in HAVSMC was detected by performing immu-
noblotting. After treatments, HAVSMCs were lysed in 1%
PMSF supplemented RIPA buffer. Protein were loaded onto
SDS-PAGE minigel, and then transferred onto PVDF
membrane. The blots were probed with 1:1000 diluted rabbit
polyclonal P2Y6, ERK1/2, and pERK1/2 antibody (Cat no.
ab92504, ab17942, ab50011; Abcam) at 4C overnight, and
incubated with Horseradish Peroxidase (HRP)-conjugated
secondary antibody (1:5000) (Cat nos. A0208 and A0216;
Beyotime, China). Signals were visualized using enhance
chemiluminescence (ECL) Substrates (Millipore). The pro-
tein expression was normalized to endogenous GAPDH
(AF0006; Beyotime, China).
Luciferase reporter assay
UTR luciferase reporter assays were performed in 293T
cells (ATCC). After being cultured overnight, cells were
cotransfected with the wild-type (wt-P2Y6)/mutated-type
(mut-P2Y6 3¢UTR1 and mut-P2Y6 3¢UTR2) reporter plas-
mids (purchased from Yrbio Tech, China) and miR-185
2 WANG ET AL.

mimics or miR-185 inhibitor. Luciferase assays were per-
formed 48 h after transfection using the Dual Luciferase
Reporter Assay System (Promega, WI).
Statistical analysis
Data from at least three independent experiments were
exhibited as mean –standard deviation, analyzed by SPSS
17.0 statistical software (SPSS, Chicago). Paired Student’s
t-test was used to compare the differences between two
groups. One-way ANOVA was used to analyze the differ-
ences between groups in the proliferation assays. A pvalue
of <0.05 was considered statistically significant.
Results
Ang II induced HAVSMCs proliferation
and upregulated P2Y6 protein level
Ang II has been reported to induce vascular smooth muscle
cells’ proliferation. In this study, we treated HAVSMC with
different concentrations of Ang II (0, 10nM, 100 nM, 1 mM),
or for different durations (0, 12, 24, 48h), and determined the
cell proliferation by using CCK-8 assays, determined the
protein levels of P2Y6 by using western blot assays. Results
showed that the proliferation of HAVSMC was promoted by
Ang II in a concentration- or time-dependent manner (Fig. 1A,
B). P2Y6 protein levels were also upregulated by Ang II in a
concentration- or time-dependent manner (Fig. 1C, D). These
data indicated that Ang II promoted HAVSMC proliferation
and P2Y6 protein levels in HAVSMC in a concentration- and
time-dependent manner.
P2Y6 silence could partially restore the promotive
effect of Ang II on HAVSMCs proliferation
To validate the role of P2Y6 in Ang II-induced HAVSMC
proliferation, two siRNAs were used to achieve P2Y6 si-
lence. Inhibitory efficiency of two siRNAs was verified by
using real-time PCR assays, and P2Y6-siRNA1 reduced
P2Y6 expression more effectively (Fig. 2A). Protein levels
of P2Y6 were determined by using western blot assays, and
similar results were observed: P2Y6-siRNA1 reduced P2Y6
protein level more effectively (Fig. 2B). Then P2Y6-
siRNA1 was transfected into HAVSMC with the presence of
Ang II, and the cell proliferation was determined by using
CCK-8 and BrdU assays. Results showed that Ang II sig-
nificantly promoted HAVSMCs proliferation, whereas P2Y6
silence suppressed HAVSMCs proliferation, compared with
the control group (Fig. 2C, D); after being transfected with
P2Y6-siRNA1, the promotive effect of Ang II on HAVSMCs
proliferation was partially restored (Fig. 2C, D). These data
indicated that P2Y6 is involved in Ang II-induced HAVSMC
proliferation; P2Y6 silence by P2Y6-siRNA could par-
tially restore the promotive effect of Ang II on HAVSMC
proliferation.
Prediction and screening of upstream miRNAs of P2Y6
The disruptions of miRNA regulation are frequently asso-
ciated with some pathological states including hypertension-
associated endothelial dysfunction. Ang II has previously
been reported to regulate the expression of miR-29b, miR-
129-3p, and miR-212 through mechanisms depending on
Gaq/11 and ERK1/2 activation (Jeppesen et al.,2011).
FIG. 1. Ang II induced HAVSMCs pro-
liferation and upregulated P2Y6 protein le-
vel (A) The cell proliferation of HAVSMCs
in response to different concentrations of
Ang II treatment for 24 h (0, 10 nM, 100 nM,
1mM) determined by using CCK-8 assays.
(B) The cell proliferation of HAVSMC in
response to 100 nM Ang II treatment for 0,
12, 24, and 48 h determined by using CCK-8
assays. (C) P2Y6 protein levels in response
to different concentrations of Ang II treat-
ment for 24 h (0, 10 nM, 100 nM, 1 mM)
determined by using western blot assays. (D)
P2Y6 protein levels in response to100 nM
Ang II treatment for 0, 12, 24, and 48 h
determined by using western blot assays.
Data are presented as mean –SD, n=8.
*p<0.05, **p<0.01. Ang II, angiotensin II;
CCK-8, Cell Counting Kit-8; HAVSMC,
human aortic vascular smooth muscle cell;
SD, standard deviation.
MIR-185/P2Y6 AXIS REGULATES HAVSMCSGROWTH 3

FIG. 2. P2Y6 silence could partially
restore the promotive effect of Ang II on
HAVSMC proliferation (A) P2Y6-siRNA1
and P2Y6-siRNA2 were constructed to
achieve P2Y6 silence. The mRNA levels
of P2Y6 were determined by using real-time
PCR assays. (B) The protein levels of P2Y6
after P2Y6-siRNA1 or P2Y6-siRNA2 trans-
fection were determined by using western
blot assays. (C) The cell viability
of HAVSMC in response to P2Y6-siRNA1
transfection with the presence of Ang II
treatment (100 nM) was determined by using
CCK-8 assays. (D) The cell proliferation
of HAVSMC in response to P2Y6-siRNA1
transfection with the presence of Ang II
treatment was determined by using BrdU
assays. Data are presented as mean –SD,
n=6. *p<0.05 and **p<0.01 versus
si-NC group,
##
p<0.01 versus P2Y6-
siRNA1 group,
DD
p<0.01 versus
AngII +si-NC group.
FIG. 3. Prediction and
screening of upstream miR-
NAs of P2Y6 (A) Six candi-
date miRNAs (miR-185,
miR-483, miR-20a, miR-
1182, miR-149, and miR-
877), which had the potential
of targeting P2Y6 were
screened out by using online
tools, including miRwalk,
miRanda, RNA22, TargetS-
can, and microT-CDS, and
referring to the previous
studies. (B) The expression
levels of the indicated can-
didate miRNAs in response
to Ang II treatment (100 nM,
24 h) were determined by
using real-time PCR assays.
Data are presented as
mean –SD, n=6. *p<0.05,
**p<0.01 versus DMSO
group. miRNAs, micro-
RNAs.
4 WANG ET AL.

Likewise, recent data suggested that miR-132 and miR-212
are involved in Ang II-induced hypertension (Eskildsen et al.,
2013). In this study, we used online tools, including miRwalk,
miRanda, RNA22, TargetScan, and microT-CDS to scan out
several candidate miRNAs that could potentially target P2Y6
(Fig. 3A). According to the previous literature, we chose six
miRNAs (miR-185, miR-483, miR-20a, miR-1182, miR-149,
and miR-877) to determine their expression levels in response
to Ang II treatment (100 nM, 24 h). By using real-time PCR,
we observed that the expression of miR-185, miR-483, miR-
20a, miR-1182, and miR-877 was modulated by Ang II
treatment, except miR149, among which miR-185 expression
was significantly downregulated (Fig. 3B). These data sug-
gested that miR-185 might be involved in Ang II-induced
HAVSMC proliferation and P2Y6 expression.
miR-185 inhibited P2Y6 expression by direct
binding to the 3¢UTR of P2Y6
To verify the association between miR-185 and P2Y6,
miR-185 mimics or inhibitor was transfected into HAVSMCs
to achieve miR-185 overexpression or inhibition (Fig. 4A).
The protein levels of P2Y6 were determined by using western
blot assays after transfection with miR-185 mimics or in-
hibitor. Results showed that P2Y6 protein was downregulated
by miR-185 overexpression, whereas upregulated by miR-
FIG. 4. miR-185 inhibited P2Y6 expres-
sion by direct binding to the 3¢UTR of P2Y6
(A) miR-185 mimics or inhibitor was used
to achieve miR-185 overexpression or inhi-
bition. The expression efficiency was veri-
fied by using real-time PCR. (B) The protein
levels of P2Y6 in response to miR-185
overexpression or inhibition were deter-
mined by using western blot assays. (C) A
wt-P2Y6 3¢UTR luciferase reporter gene
vector, as well as a mut-P2Y6 3¢UTR vector
was constructed by mutating the two pre-
dicted binding sites of miR-185 in the
3¢UTR of P2Y6.(D) The indicated luciferase
vectors were cotransfected into 293T cells
with miR-185 mimics or inhibitor. The lu-
ciferase activity was then determined by
using dual luciferase assays. Data are pre-
sented as mean –SD, n=6. *p<0.05,
**p<0.01.
MIR-185/P2Y6 AXIS REGULATES HAVSMCSGROWTH 5

185 inhibition (Fig. 4B). We constructed a wt-P2Y6 3¢UTR
luciferase reporter gene vector, as well as a mut-P2Y6 3¢UTR
vector by mutating the two predicted binding sites of miR-
185 in the 3¢UTR of P2Y6 (Fig. 4C). The indicated vectors
were cotransfected into 293T cells with miR-185 mimics or
inhibitor, and the luciferase activity was determined by using
dual luciferase assays. Results showed that the luciferase
activity of wt-P2Y6 3¢UTR vectors was reduced by miR-185
mimics, whereas promoted by miR-185 inhibitor, compared
with the NC (negative control) group; the effect of miR-185
mimics or inhibitor on luciferase activity was offset by either
mutation in the 3¢UTR of P2Y6 (Fig. 4D). These data indi-
cated that miR-185 could reduce P2Y6 protein level by direct
binding to the 3¢UTR of P2Y6.
miR-185 regulated HAVSMC proliferation
and downstream ERK pathway
through targeting P2Y6
We revealed that miR-185 inhibits P2Y6 protein level through
direct binding to its 3¢UTR; next we validated the functional role
of miR-185 in regulating HAVSMCs proliferation. As deter-
mined by CCK-8 and BrdU assays, miR-185 overexpression
significantly inhibited HAVSMCs proliferation, P2Y6 over-
expression promoted HAVSMCs proliferation; P2Y6 could
partially restore the inhibitory effect of miR-185 on HAVSMCs
proliferation (Fig. 5A, B). According to previous studies,
ERK pathway is involved in the pathophysiology of hyper-
tension (Roberts, 2012), Moreover, P2Y receptors, including
P2Y6, could mediate ERK activation to prevent cell apo-
ptosis (Lenz et al., 2000; Kim et al., 2003). In this study, we
further monitored the protein levels of total ERK1/2 and p-
ERK1/2, as well as P2Y6 protein level in response to co-
transfection of miR-185 mimics and pCMV-P2Y6 by using
western blot assays. Results showed that miR-185 over-
expression significantly downregulated P2Y6 and pERK1/2
protein levels without affecting total ERK1/2 protein.
pCMV-P2Y6 transfection exerted an opposite function: up-
regulated both P2Y6 and pERK1/2 protein levels without
affecting total ERK1/2 protein. In addition, P2Y6 partially
restored the effect of miR-185 on the indicated factors
(Fig. 5C). These data indicated that miR-185 inhibits
FIG. 5. miR-185 regulated HAVSMCs
proliferation and downstream ERK pathway
through targeting P2Y6. (A) The cell via-
bility of HAVSMCs in response to co-
transfection of miR-185 mimics and pCMV-
P2Y6 was determined by using CCK-8 as-
says. (B) The cell proliferation of HAVSMC
in response to cotransfection of miR-185
mimics and pCMV-P2Y6 was determined
by using BrdU assays. (C) The protein levels
of ERK1/2 and pERK1/2 in HAVSMC in
response to cotransfection of miR-185
mimics and pCMV-P2Y6 was determined
by using western blot assays. Data are pre-
sented as mean –SD, n=6. *p<0.05,
**p<0.01 versus NC mimics +Vector
group;
##
p<0.01 versus miR-185 mimics +
Vector group;
DD
p<0.01 versus NC mimics
+pCMV-P2Y6 group. BrdU, 5-Bromo-2-
deoxyUridine.
6 WANG ET AL.

HAVSMC proliferation through inhibiting P2Y6 expression
and regulating the downstream ERK pathway.
miR-185 restored the promotive effect of Ang II
on P2Y6 expression and HAVSMCs proliferation
We have revealed that miR-185 inhibits HAVSMC pro-
liferation through inhibiting P2Y6 expression; we further
investigated whether ectopic miR-185 expression could re-
store the effect of Ang II on P2Y6 and HAVSMC prolifer-
ation. In HAVSMCs, miR-185 expression was significantly
upregulated by miR-185 mimics; when Ang II treatment was
conducted, miR-185 mimics-induced miR-185 expression
was partially reduced (Fig. 6A). Then the protein levels of
P2Y6 were monitored. As shown by western blot assays,
P2Y6 protein was reduced by ectopic miR-185 expression,
increased by Ang II treatment; the promotive effect of Ang II
treatment on P2Y6 protein could be partially reversed by
ectopic miR-185 expression (Fig. 6B). Furthermore, the cell
proliferation of HAVSMC was monitored by using CCK-8
and BrdU assays. Similarly, the promotive effect of Ang II
treatment on HAVSMC proliferation could be partially re-
versed by miR-185 (Fig. 6C, D). These data further indicated
that miR-185 restores the promotive effect of Ang II on P2Y6
expression; miR-185/P2Y6 axis regulates Ang II-induced
HAVSMC proliferation.
Discussion
Excessive proliferation of vascular smooth muscle cells is
a crucial event in the pathogenesis of several cardiovascular
diseases, including hypertension. In the present study, we
found that Ang II induced HAVSMC proliferation and P2Y6
protein levels in a concentration- or time-dependent manner.
After silence of P2Y6 by P2Y6-siRNA, HAVSMC prolif-
eration was downregulated; in addition, P2Y6 silence could
also partially restore the effect of Ang II on HAVSMC
proliferation. Through online tools and previous literatures,
we chose several candidate miRNAs that might potentially
target P2Y6 and were associated with cell proliferation.
Among them, miR-185 expression could be significantly
downregulated by Ang II treatment. By direct binding to the
FIG. 6. miR-185 restored the promotive effect of Ang II on P2Y6 expression and HAVSMCs proliferation (A) miR-185
mimics was transfected into HAVSMC under Ang II treatment. The expression levels of miR-185 were determined by using
real-time PCR assays. (B) The protein levels of P2Y6 in response to ectopic miR-185 expression under Ang II treatment
were determined by using western blot assays. (C) and (D) The cell proliferation of HAVSMC in response to ectopic miR-
185 expression under Ang II treatment was determined by using CCK-8 and BrdU assays. Data are presented as mean –SD,
n=6. *p<0.05, **p<0.01 versus NC mimics group;
#
p<0.05,
##
p<0.01, versus miR-185 mimics group,
DD
p<0.01, versus
Ang II +NC mimics group.
MIR-185/P2Y6 AXIS REGULATES HAVSMCSGROWTH 7

3¢UTR of P2Y6, miR-185 inhibited P2Y6 expression and
HAVSMCs proliferation. Moreover, downstream ERK
pathway might be involved in this process.
The excessive proliferation of vascular smooth muscle cell
can be induced by various cytokines and growth factors, such as
Ang II, insulin, and platelet-derived growth factors (Touyz and
Schiffrin, 1996). Ang II has been frequently reported to be
associated with cardiovascular smooth muscle cell proliferation
and apoptosis (Pueyo and Michel, 1998; Takemoto and Ega-
shira, 1999). Ang II could increase human brain vascular
smooth muscle cell migration, proliferation, and apoptosis, and
increased the blood pressure, neurological deficit score, middle
cerebral artery (MCA) remodeling, and hemorrhage volume in
intracerebral hemorrhage (ICH) mice (Bihl et al., 2015). One
micromole of Ang II significantly promoted the proliferation of
A7r5 cell, an aortic smooth muscle cell line, from 100% to
131% (Li et al., 2015). In the present study, we treated
HAVSMCs with different concentrations of Ang II, for differ-
ent durations. HAVSMCs proliferation was promoted by Ang II
in a concentration- or time-dependent manner. Besides, Ang II
treatment could also upregulate P2Y6 protein levels in a con-
centration- or time-dependent manner.
P2Y receptors are G-protein-coupled receptors activated
by extracellular nucleotides. The P2Y6 receptor is selec-
tively activated by UDP, and its transcript has been detected
in numerous organs, including the spleen, thymus, intestine,
blood leukocytes, and aorta. Nishimura et al. reported that
through heterodimerization with the angiotensin type 1 re-
ceptor, P2Y6 could promote Ang II-induced hypertension
(Nishimura et al., 2016). By generating P2Y6
-/-
mice, Bar
et al. (2008) revealed that the P2Y6 receptor is indeed re-
sponsible for the contractile action of both UDP and UTP in
aorta, and P2Y6 is expressed and functional in vascular
smooth muscle cells. In the present study, we investigated
the detailed role of P2Y6 in HAVSMCs proliferation. After
P2Y6-siRNA transfection, HAVSMCs proliferation was
suppressed. Moreover, Ang II-induced HAVSMC prolifer-
ation could be partially restored by P2Y6 silence. However,
the mechanism by which P2Y6 affects Ang II-induced
HAVSMC proliferation and Ang II-upregulated P2Y6 ex-
pression still remains to be validated.
The RAS has an essential role in blood pressure regula-
tion by affecting cardiac contractility, vascular resistance,
and blood volume. RAS overactivation is a major patho-
genetic factor in hypertension. Several miRNAs have been
shown to interact with the RAS. miR-155 has been found to
regulate AGTR1 expression (Martin et al., 2006). miR-124
and miR-135a independently repressed the translation of
NR3C2, a ligand-dependent transcription factor that regu-
lates water and ion transporter expression, without affecting
mRNA levels. The authors proposed that miR-124 and miR-
135a may contribute to the modulation of the RAAS and
thereby to blood pressure regulation (Sober et al., 2010). In
this study, we used online tools to screen out several can-
didate miRNAs, which were predicted to have the potential
of binding to P2Y6. These candidate miRNAs were also
reported to be associated with cell proliferation: miR-185,
miR-483, miR-20a, miR-1182, miR-149, and miR-877
(Bertero et al., 2011; Fan et al., 2015; Zhang et al., 2015;
Chen et al., 2016; Li et al., 2016; Zhu et al., 2016). After
Ang II treatment, the expression of all candidate miRNAs was
modulated by Ang II, except miR-149, among which miR-185
expression was more strongly downregulated. We then veri-
fied that miR-185 could bind to the 3¢UTR of P2Y6 on the two
predicted binding sites, and inhibit P2Y6 expression through
direct targeting. These data suggested the involvement of miR-
185 in regulation of HAVSMCs proliferation; so we per-
formed a series of functional assays to investigate whether
miR-185 and P2Y6 constitute a regulatory axis to affect
HAVSMC proliferation together.
We observed a significant inhibitory effect of miR-185 on
HAVSMC proliferation by CCK-8 and BrdU assays; in ad-
dition, exogenous P2Y6 expression could partially restore the
inhibitory effect of miR-185 on HAVSMCs proliferation,
indicating that miR-185/P2Y6 axis affected HAVSMC pro-
liferation. A similar result has been reported by Shan et al.
that miR-185-5p inhibitor transfection increased the viability
and proliferation of HUVECs; lncRNA-RNCR3, miR-185-
5p, and KLF2 constitute a regulatory network to regulate the
viability and proliferation of HUVECs (Shan et al., 2016).
Recent studies reported that miRNA–mRNA interactions
may delineate the complex regulatory networks with conse-
quence on the target gene expression and hence on some
biological processes. Due to its major role in pathophysiology
of hypertension (Guo et al., 2014; Li et al.,2017),wethen
validated whether ERK pathway was involved in regulation
of HAVSMC proliferation by miR-185. As shown by western
blot, miR-185 reduced pERK1/2 protein level; P2Y6 in-
creased pERK1/2 protein level, and partially restored the
effect of miR-185 on pERK protein. These data suggested
that ERK pathway was involved in the process of miR-185/
P2Y6 axis inhibiting Ang II-induced HAVSMC proliferation.
Taken together, we indicated that miR-185/P2Y6 axis
inhibits Ang II-induced HAVSMC proliferation through the
downstream ERK pathway; rescuing miR-185 expression to
inhibit P2Y6 expression may thus represent a therapeutic
strategy against HAVSMCs dysfunction and hypertension.
Acknowledgments
This study was supported by the National Natural Science
Foundation of China (program no.: 81473616), Science and
Technology Program Major Project of China Hunan Pro-
vincial Science & Technology Department (2016DK2002),
and Hunan Traditional Chinese Medicine Scientific Re-
search Key Project (201753).
Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Yuansheng Tan, MD
Hunan University of Chinese Medicine
Hanpu science and Technology Park
Changsha 410208
China
E-mail: tanyuansheng_hn@163.com
Received for publication December 14, 2016; received in
revised form February 6, 2017; accepted February 6, 2017.
MIR-185/P2Y6 AXIS REGULATES HAVSMCSGROWTH 9