International Journal of
Molecular Sciences
Article
miR-30e-5p and miR-15a Synergistically Regulate
Fatty Acid Metabolism in Goat Mammary Epithelial
Cells via LRP6 and YAP1
Zhi Chen 1,† , Huiling Qiu 2,† , Liuan Ma 2,† , Jun Luo 1, *, Shuang Sun 1 , Kang Kang 2 ,
Deming Gou 2, * and Juan J. Loor 3
1
2
3
*
†
Shanxi Key Laboratory of Molecular Biology for Agriculture, College of Animal Science and Technology,
Northwest A&F University, Yangling 712100, Shaanxi, China; [email protected] (Z.C.);
[email protected] (S.S.)
College of Life Sciences, Shenzhen University, Shenzhen 518060, China; [email protected] (H.Q.);
[email protected] (L.M.); [email protected] (K.K.)
Mammalian NutriPhysioGenomics, Department of Animal Sciences, Division of Nutritional Sciences,
University of Illinois, Urbana, IL 61801, USA; [email protected]
Correspondence: [email protected] (J.L.); [email protected] (D.G.);
Tel.: +86-29-8708-2891 (J.L.); Fax: +86-29-8708-2892 (J.L.)
These authors contributed equally to this work.
Academic Editor: Kumiko UI-TEI
Received: 31 August 2016; Accepted: 9 November 2016; Published: 16 November 2016
Abstract: MicroRNA (miRNA) regulates the expression of genes and influences a series of biological
processes, including fatty acid metabolism. We screened the expression of miRNA in goat mammary
glands during peak-lactation and non-lactating (“dry”) periods, and performed an in vitro study
with goat mammary epithelial cells (GMEC) prior to sequencing analysis. Results illustrated that
miR-30e-5p and miR-15a were highly expressed. Utilizing a luciferase reporter assay and Western
blot, low-density lipoprotein receptor-related protein 6 (LRP6) and Yes associated protein 1 (YAP1) genes
were demonstrated to be a target of miR-30e-5p and miR-15a in GMEC. Moreover, we demonstrated
that the overexpression of miR-30e-5p and miR-15a in GMEC promoted fat metabolism while their
knockdown impaired fat metabolism. These findings extend the discovery of a key role of miR-30e-5p
and miR-15a in mediating adipocyte differentiation by suggesting a role in promoting milk fat
synthesis. In conclusion, our findings indicate that miR-30e-5p, together with miR-15a, represses
expression of LRP6 and promotes fat metabolism in GMEC. The data expanded our knowledge on
the function of miRNAs in milk fat metabolism and synthesis in ruminant mammary cells.
Keywords: miR-30e-5p; miR-15a; fat metabolism; LRP6; YAP1
1. Introduction
Goat milk contains high concentrations short-chain fatty acids and unsaturated fatty acids,
which can elicit positive effects on human health [1–3]. Thus, investigating the control of goat milk
fat metabolism is of great significance [4,5]. Although the focus in the last few decades has been on
the analysis of a single gene or function, more comprehensive studies of molecular regulation of milk
fat metabolism are needed [6–8]. MicroRNA are members of the non-coding RNA family, and are
composed of 21–23 nucleotides [9–11]. They can negatively regulate target genes via mRNA splicing
and inhibiting protein translation [12–15]. Currently, hundreds of miRNA have been uncovered in
non-ruminants, and bioinformatics predictions indicate that they could regulate the expression of
approximately thirty percent of the genes in the genome [16–19]. Previous research indicates that
over 60% of protein-coding genes have more than one conserved site for miRNA [1,20]. Various
Int. J. Mol. Sci. 2016, 17, 1909; doi:10.3390/ijms17111909
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reports illustrated that miRNA were expressed in a spatio-temporal specific manner, and influence a
variety of procedures, such as immune regulation, fat metabolism, cell apoptosis, cell proliferation,
and cell differentiation, among others. Despite the vast amount of work on miRNA in non-ruminant
species [21], there are few studies focused on the function and mechanisms whereby miRNA
synergistically regulates the process of milk fat metabolism [22–24]. In addition, mechanisms of
miRNA working in concert with other miRNA are likely to be uncovered.
By working in concert with transcription factors, and promoting the expression of certain genes,
Yes-associated protein (YAP) and its downstream proteins in the Hippo signaling pathway, play a
crucial role in promoting cell growth and inhibiting apoptosis. The protein encoded by LRP6 is a
component of the cell-surface receptors for Wnt proteins, and Wnt is known to promote recruitment
of Axin via LRP6 all of which leads to inhibiting degradation of β-catenin [25]. Increasing evidence
demonstrates that the Hippo/YAP and Wnt/-catenin pathways (crosstalk) control the growth and
development of cells, tissues, and organs through various types of interactions [26,27]. Studying the
mechanism of the two pathways and the interaction with miRNA will provide a new strategy for
regulating milk fat metabolism. Our results revealed that miR-30e-5p and miR-15a are highly-expressed
not only in the mammary gland of goats, but also in goat mammary epithelial cells (GMEC) cultured
with prolactin. Furthermore, miR-30e-5p cooperates with miR-15a promoting fat metabolism by
repressing the expression of YAP1 in GMEC. Our research illustrates that miR-30e-5p and miR-15a
play a significant role in the accumulation of milk fat in ruminants.
2. Results
2.1. Screening of miRNA at Peak-Lactation and the Non-Lactating Period and Prolactin Effects on miRNA
in GMEC
Some reports have showed that miRNA participates in the regulation of milk fat metabolism [28–30].
We profiled the differentially-expressed miRNA between peak-lactation and non-lactating periods in
goat mammary glands to investigate the connection between miRNA regulation and this physiological
process in a more comprehensive way. We applied the S-Poly (T) real method, including 267 Capra
hircus primary miRNA and 793 Bos taurus primary miRNA from miRBase. We have underscored the
overlapping data and more details can be found in Supplementary Data 1. Firstly, all of the miRNA
with four-fold change and p < 0.05 were chosen as candidate miRNA. (Figure 1A and Supplementary
Data 1). Overall, we identified 55 differentially-expressed miRNA between peak and non-lactating
mammary tissue, with 31 up-regulated and 24 down-regulated. To better understand the link between
these miRNA and mammary cell growth and milk fat metabolism, we screened miRNA in GMEC
cultured with increasing concentrations of prolactin (an essential lactogenic hormone).
In order to detect the expression of miRNAs in GMECs, we used optimal concentration of
prolactin. We found that 2.5 µg/mL was the most significant one (Figure 1B–D), so 2.5 µg/mL was our
experimental concentration. Then we took a second of screening with the same criterion (Figure 1E,
Supplementary Data 2). The analysis indicated that miR-30e-5p and miR-15a were highly expressed
(Figure 1A,E).
We also performed bioinformatics analysis to screen the differentially-expressed miRNA. Based
on the 30 -UTR complementary prediction with Target Scan 6.2 (Whitehead Institute for Biomedical
Research, Boston, MA, USA) and miRNA functional analysis by DAVID [31,32], it was evident that
many genes are potential targets of the highly-expressed miRNA. Previous studies have revealed that
miR-30e-5p and miR-15a are not only associated with milk fat metabolism (Figures S1 and S2; Tables S1
and S2), but their potential target genes appear to have overlapping functions. Therefore, we selected
miR-30e-5p and miR-15a for further detailed studies. Next, we measured the expression level of
miR-30e-5p and miR-15a in different tissues of dairy goats (Figure 2A,B), and observed that miR-30e-5p
and miR-15a were primarily expressed in mammary tissue. Furthermore, we investigated the miRNA
expression in mammary glands at different stages of lactation (Figure 2C,D). miR-30e-5p and miR-15a
Int. J. Mol. Sci. 2016, 17, 1909
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3 of 17
(Figure
2A,B),
and
observed that miR-30e-5p and miR-15a were primarily expressed in mammary
Int.
J. Mol.2A,B),
Sci. 2016,
17, observed
1909
3 of 17
(Figure
and
that miR-30e-5p and miR-15a were primarily expressed in mammary
tissue. Furthermore, we investigated the miRNA expression in mammary glands at different stages
tissue. Furthermore, we investigated the miRNA expression in mammary glands at different stages
of lactation (Figure 2C,D). miR-30e-5p and miR-15a were up-regulated at early lactation and
of lactation (Figure 2C,D). miR-30e-5p and miR-15a were up-regulated at early lactation and
were
up-regulated
at early lactation
and down-regulated
results provided
strong
down-regulated
thereafter.
These results
provided strongthereafter.
evidence These
that miR-30e-5p
and miR-15a
down-regulated thereafter. These results provided strong evidence that miR-30e-5p and miR-15a
evidence
that
miR-30e-5p
and
miR-15a
may
play
an
important
role
in
lactation.
may play an important role in lactation.
may play an important role in lactation.
Figure 1. (A) Screening of microRNA (miRNA) expressed at peak-lactation and non-lactating stages.
Figure1.1.(A)
(A)Screening
ScreeningofofmicroRNA
microRNA(miRNA)
(miRNA)expressed
expressedatatpeak-lactation
peak-lactationand
andnon-lactating
non-lactatingstages.
stages.
Figure
Three mammary samples from goats at each stage of lactation were used; (B) Triglyceride levels in
Three mammary
mammary samples from
lactation
were
used;
(B)(B)
Triglyceride
levels
in cells
Three
fromgoats
goatsatateach
eachstage
stageofof
lactation
were
used;
Triglyceride
levels
in
cells treated
with increasing
concentrations
of prolactin;
triglyceride
levels were compared
with
treated
with with
increasing
concentrations
of prolactin;
triglyceride
levels were
with controls
cells
treated
increasing
concentrations
of prolactin;
triglyceride
levelscompared
were compared
with
controls
6), * p < <0.05;
** p <Goat
0.01; (C) Goat mammary
epithelial
cells
(GMEC)
were
cultured
with
(n
= 6), *(n
p(n<==0.05;
0.01;
epithelial cells
(GMEC)
were
cultured
with
prolactin
for
controls
6), * p**<p0.05;
** (C)
p < 0.01; mammary
(C) Goat mammary
epithelial
cells
(GMEC)
were
cultured
with
prolactin
for
48
h,
and
the
expression
of
β-casein
was
quantified
by
RT-qPCR
(n
=
6),
*
p
<
0.05.
All
48
h,
and
the
expression
of
β-casein
was
quantified
by
RT-qPCR
(n
=
6),
*
p
<
0.05.
All
experiments
were
prolactin for 48 h, and the expression of β-casein was quantified by RT-qPCR (n = 6), * p < 0.05. All
experimentsand
were
duplicated
and
repeated
three
times. Values
are presented
as means(D)
± standard
duplicated
repeated
threeand
times.
Valuesthree
are presented
as means
± standard
Western
experiments
were
duplicated
repeated
times. Values
are presented
as errors;
means ± standard
errors;
(D)
Western
blot
analyses
of
the
expression
of
β-casein
in
the
prolactin
treatment
experiments.
blot analyses
of the expression
of β-casein
in the prolactin
treatment
The effect
of prolactin
errors;
(D) Western
blot analyses
of the expression
of β-casein
in theexperiments.
prolactin treatment
experiments.
Theβ-casein
effect of prolactin
on β-casein
protein
expression
was blot
evaluated
by Western
blot in GMEC;
(E)
on
expression
was
evaluated
by Western
in GMEC;
(E) Screening
miRNAs
The
effect ofprotein
prolactin
on β-casein
protein
expression
was evaluated
by Western
blot in for
GMEC;
(E)
Screening
for
miRNAs
involves
in
the
0ug/ml
prolactin
and
2.5ug/ml
prolactin,
respectively.
The
involves infor
themiRNAs
0 µg/mLinvolves
prolactininand
µg/mL
prolactin,
respectively.
The expression
of 18s rRNA
Screening
the2.5
0ug/ml
prolactin
and
2.5ug/ml prolactin,
respectively.
The
expression
of
18s rRNA is control.
used as a normalization control.
is
used
as
a
normalization
expression of 18s rRNA is used as a normalization control.
Figure 2. (A) miR-30e-5p expression in various tissues of dairy goats; (B) miR-15a expression in
Figure
Figure 2.2. (A)
(A)miR-30e-5p
miR-30e-5pexpression
expressionininvarious
varioustissues
tissuesofofdairy
dairygoats;
goats;(B)
(B)miR-15a
miR-15aexpression
expressionin
in
various tissues of dairy goats; (C) miR-30e-5p expression in various tissues of dairy goats; (D)
various
tissues
of
dairy
goats;
(C)
miR-30e-5p
expression
in
various
tissues
of
dairy
goats;
(D)
various
of dairy goats; (C) miR-30e-5p expression in various tissues of dairy goats; (D)
miR-15a
miR-15a expression in various tissues of dairy goats. All experiments were duplicated and repeated
miR-15a
expression
intissues
variousoftissues
of dairy
All experiments
were duplicated
andthree
repeated
expression
in various
dairy goats.
Allgoats.
experiments
were duplicated
and repeated
times.
three times. Values are presented as means ± standard errors
three
times.
Values areaspresented
means errors
± standard errors
Values
are presented
means ± as
standard
Int. J. Mol. Sci. 2016, 17, 1909
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TAG via
via LRP6
LRP6 and
and YAP1
YAP1
2.2. miR-30e-5p, miR-15 Regulate TAG
Based on target prediction analysis (Target Scan 6.2) and miRNA functional analysis
analysis (DAVID),
(DAVID),
evident that
that many
many genes
genes are
are potential
potential targets
targets of
of miR-30e-5p,
miR-30e-5p, including
including LRP6,
LRP6, which
which encodes
encodes
it was evident
a transmembrane
transmembrane cell surface protein involved in receptor-mediated endocytosis [29], and a Wnt
co-receptor ininthe
thecanonical
canonical
signaling
pathway
We chose
LRP6
for functional
validation
co-receptor
signaling
pathway
[30].[30].
We chose
LRP6 for
functional
validation
because
because
it isto known
to beregulator
a crucialofregulator
of multiple
metabolic
processes,
including
genetic
it
is known
be a crucial
multiple metabolic
processes,
including
genetic
variations
in
variations
in
LRP6
being
associated
with
high
serum
LDL
(low
density
lipoprotein)
cholesterol
LRP6 being associated with high serum LDL (low density lipoprotein) cholesterol levels in humans.
0 -UTR.
levels in humans.
Furthermore,
3C illustrates
potential
bindinginsites
miR-30e-5p
in the
Furthermore,
Figure
3C illustratesFigure
the potential
bindingthe
sites
of miR-30e-5p
the 3of
We observed
3′-UTR.
We observed
overexpression
anddown-regulated
inhibition of miR-30e-5p
down-regulated
and
that
overexpression
and that
inhibition
of miR-30e-5p
and up-regulated
the mRNA level
up-regulated
the mRNA
level3A).
of LRP6, respectively (Figure 3A).
of
LRP6, respectively
(Figure
RT-qPCR quantification
quantification of
of the
the LRP6
LRP6 expression
expression (n
(n == 6). Red bars represent the negative
Figure 3. (A) RT-qPCR
miR-30e-5p in
control; black bars represent miR-30e-5p mimic or inhibitor; (B,C) Target site of miR-30e-5p
0
0 -UTR.
LRP63
-UTR and
construction
of theofluciferase
(Luc) expression
vector fused
with fused
the LRP63
LRP63′-UTR
andthethe
construction
the luciferase
(Luc) expression
vector
with
the
0
WT
representsWT
the represents
Luc reporterthe
vector
the WT
LRP6
3 -UTR
(2155–2163);
MU represents
the Luc
LRP63′-UTR.
Luc with
reporter
vector
with
the WT
LRP6 3′-UTR
(2155–2163);
MU
reporter
vector
withreporter
the mutation
the miR-30e-5p
site
30 -UTR; site
(D) in
The
effect
of miR-30e-5p
represents
the Luc
vectoratwith
the mutation
at in
theLRP6
miR-30e-5p
LRP6
3′-UTR;
(D) The
mimicking
and inhibiting
LRP6 protein
expression
wasprotein
evaluated
by Western
analysis
GMEC;
effect of miR-30e-5p
mimicking
and inhibiting
LRP6
expression
was blot
evaluated
byinWestern
(E)
The
YAP1
expression
level
was
quantified
by
RT-qPCR
(n
=
6).
Red
bars
represent
the
negative
blot analysis in GMEC; (E) The YAP1 expression level was quantified by RT-qPCR (n = 6). Red bars
0 -UTR
control;
black
represent
miR-15a
or inhibitor;
(F,G) Target
miR-15a(F,G)
in theTarget
YAP13site
represent
the bars
negative
control;
blackmimic
bars represent
miR-15a
mimicsite
or of
inhibitor;
of
0
and
the construction
of the luciferase
(Luc)
expression vector
with the
YAP13
-UTR. WT
represents
miR-15a
in the YAP13′-UTR
and the
construction
of the fused
luciferase
(Luc)
expression
vector
fused
0
the
Luc
vector with
WT YAP13
(167–173);
MU represents
the Luc
reporter vector
with
with
thereporter
YAP13′-UTR.
WT the
represents
the -UTR
Luc reporter
vector
with the WT
YAP13′-UTR
(167–173);
0 -UTR; (H) The effect of miR-15a mimicking and inhibiting
the
mutation
at
the
miR-15a
site
in
YAP13
MU represents
Luc reporter vector with the mutation at the miR-15a site in YAP13′-UTR; (H)
YAP1
protein
expression
was evaluated
by Western
analysis
in GMEC.
experiments
were
The effect
of miR-15a
mimicking
and inhibiting
YAP1blot
protein
expression
was All
evaluated
by Western
duplicated
and
times. Values
are duplicated
presented as
means
± standard
errors,Values
* p < 0.05;
blot analysis
inrepeated
GMEC. three
All experiments
were
and
repeated
three times.
are
**
p < 0.01. as means ± standard errors, * p < 0.05; ** p < 0.01.
presented
0 -UTR segment of LRP6,
To verify
verify that
that miR-30e-5p
miR-30e-5p directly targeted
targeted this site, we synthesized a 33′-UTR
To
themiR-30e-5p
miR-30e-5ptarget
target
site,
cloned
it the
intopsi-CHECK2
the psi-CHECK2
to construct
including the
site,
andand
cloned
it into
vectorvector
to construct
a 30 -UTRa
3′-UTR
reporter
plasmid.
The
luciferase
reporter
assay
indicated
that
over-expression
of
miR-30e-5p
reporter plasmid. The luciferase reporter assay indicated that over-expression of miR-30e-5p decreased
decreased the relative luciferase activity of the reporter with a wild-type 3′-UTR rather than the one
with mutations in the seed sequences (Figure 3B,C). Furthermore, the expression level of the LRP6
Int. J. Mol. Sci. 2016, 17, 1909
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the relative luciferase activity of the reporter with a wild-type 30 -UTR rather than the one with
mutations
in the
seed sequences
(Figure
3B,C).
Furthermore,
expression
level of the
LRP6 protein
protein was
consistent
with the
mRNA
expression
data the
after
the miR-30e-5p
over-expression
was
consistent
with
the
mRNA
expression
the miR-30e-5p
over-expression
treatment
treatment
(Figure
3D).
The
findings
illustratedata
that after
miR-30e-5p
directly interacts
with the target
site
(Figure
3D).
The
findings
illustrate
that
miR-30e-5p
directly
interacts
with
the
target
site
of
the
LRP6
of the LRP6 mRNA and negatively regulates its expression, which partly explained the function of
mRNA
and during
negatively
regulates its expression, which partly explained the function of miR-30e-5p
miR-30e-5p
lactation.
during
lactation.
Our
results also revealed that YAP1 was down-regulated by overexpression of miR-15a, and
Our
results also
that
was (Figure
down-regulated
by overexpression
and goat
was
was up-regulated
byrevealed
inhibition
ofYAP1
miR-15a
3E). Furthermore,
as shownofinmiR-15a,
Figure 3G,
up-regulated
inhibition
of miR-15a
(Figure
3E). Furthermore,
as shown
in Figure
goat YAP1
has
YAP1 has a by
potential
binding
site for
miR-15a
in the 3′-UTR.
To verify
that 3G,
miR-15a
directly
0 -UTR. To verify that miR-15a directly targeted this site,
atargeted
potential
binding
site
for
miR-15a
in
the
3
this site, we synthesized a 3′-UTR segment of YAP1 including the miR-15a target site, and
we
synthesized
a 30 -UTR
segmentvector
of YAP1
miR-15areporter
target site,
and cloned
into the
cloned
it into the
psi-CHECK2
toincluding
constructthe
a 3′-UTR
plasmid.
The itluciferase
0
psi-CHECK2
vector
to construct
a 3 -UTR reporter
plasmid.decreased
The luciferase
reporterluciferase
assay indicated
that
reporter assay
indicated
that over-expression
of miR-15a
the relative
activity
of
over-expression
of
miR-15a
decreased
the
relative
luciferase
activity
of
the
reporter
with
a
wild-type
the reporter with a wild-type 3′-UTR rather than the one with mutations in the seed sequences
0 -UTR rather than the one with mutations in the seed sequences (Figure 3F,G). Furthermore, the
3(Figure
3F,G). Furthermore, the expression level of YAP1 protein was consistent with the expression
expression
level after
of YAP1
was
consistent withtreatment
the expression
of mRNA
data
after the
miR-15a
of mRNA data
theprotein
miR-15a
over-expression
(Figure
3H). The
findings
illustrated
over-expression
treatment
(Figure
3H).
The
findings
illustrated
that
miR-15a
directly
interacts
with
that miR-15a directly interacts with the target site of the YAP1 mRNA and negatively regulates
its
the
target site
of thepartly
YAP1explained
mRNA and
regulates
expression,
which partly explained the
expression,
which
thenegatively
function of
miR-15aits
during
lactation.
function of miR-15a during lactation.
2.3. miR-30e-5p Is Associated with β-Catenin and Cooperates with miR-15a in Repressing YAP1 in GMEC
2.3. miR-30e-5p Is Associated with β-Catenin and Cooperates with miR-15a in Repressing YAP1 in GMEC
Some studies have shown that LRP6 is an important factor in the Wnt phosphorylation
Some and
studies
have shown
LRP6 issignal
an important
factor
in the
Wnt phosphorylation
pathway,
pathway,
β-catenin
is an that
important
mediating
Wnt
signaling
from molecules
to the
and
β-catenin
is
an
important
signal
mediating
Wnt
signaling
from
molecules
to
the
nucleus
[25].
nucleus [25]. Our initial experiment revealed that LRP6 is a miR-30e-5p’s target gene. To confirm
Our
initial
experiment
revealed
that
LRP6
is
a
miR-30e-5p’s
target
gene.
To
confirm
the
functional
the functional relationship between miR-30e and β-catenin, we examined the mRNA level of
relationship
between
miR-30e
β-catenin,
examined
mRNA
level of
β-catenin
using RT-qPCR
β-catenin using
RT-qPCR
andand
Western
blot.we
Figure
4A,B the
show
that there
was
a significant
decrease
and
Western
blot.
Figure
4A,B
show
that
there
was
a
significant
decrease
in
β-catenin
expression
in
in β-catenin expression in GMEC transfected with a miR-30e-5p mimic in comparison
with the
GMEC
with athat
miR-30e-5p
mimic
comparison
with the
control group, indicating that
control transfected
group, indicating
miR-30e-5p
did in
impact
the β-catenin
expression.
miR-30e-5p
did
impact
the
β-catenin
expression.
Data indicated that miR-30-5p and miR-15a promote fat metabolism. To address the
Data indicated
that
miR-30-5pand
andmiR-15a
miR-15awe
promote
fat metabolism.
To address
the relationship
relationship
between
miR-30-5p
measured
the expression
of miR-15a
in GMEC
between
miR-30-5p
and
miR-15a
we
measured
the
expression
of
miR-15a
in
GMEC
with
miR-30e-5p
with miR-30e-5p as being overexpressed or inhibited, respectively. As shown in Figure 4C,
as
being overexpressed
or inhibited,
respectively.
As shown
Figure 4C,
compared
the negative
compared
with the negative
control,
the expression
of in
miR-15a
increased
by with
1.5 times
in the
control,
the
expression
of
miR-15a
increased
by
1.5
times
in
the
miR-30e-5p
mimic
transfected
cells
miR-30e-5p mimic transfected cells (p < 0.05). On the other hand, the expression of miR-15a
(p
< 0.05). On
the other hand,
expression
of miR-15a
whenthe
miR-30e-5p
decreased
significantly
when the
miR-30e-5p
was
inhibited.decreased
However,significantly
compared with
negative
was
inhibited.
However,
compared
with
the
negative
control,
the
expression
of
miR-30e-5p
did not
control, the expression of miR-30e-5p did not change in the miR-30e-5p mimic or inhibited
change
in the
miR-30e-5p
or4D).
inhibited
transfectedstudy
cells (panalyzed
< 0.05) (Figure
4D). The relationship
subsequent
transfected
cells
(p < 0.05)mimic
(Figure
The subsequent
the expression
study
analyzed
the
expression
relationship
between
target
genes
and
miRNA.
To
resolve
this
between target genes and miRNA. To resolve this question, we examined the mRNA question,
level of
we
examined
theRT-qPCR
mRNA level
β-catenin
using
Western
As shown
Figure
4E,F,
β-catenin
using
and of
Western
blot.
As RT-qPCR
shown inand
Figure
4E,F,blot.
compared
withinthe
negative
compared
with
the negative
control,
the expression
of YAP1 decreased
in miR-30e-5p
transfected
control, the
expression
of YAP1
decreased
in miR-30e-5p
mimic transfected
cells, mimic
(p < 0.05).
On the
cells,
(p
<
0.05).
On
the
other
hand,
the
expression
of
YAP1
increased
significantly
when
miR-30e-5p
was
other hand, the expression of YAP1 increased significantly when miR-30e-5p was inhibited.
inhibited.
Interestingly,
the
expression
of
YAP1
was
significantly
decreased
by
Si-β-catenin,
suggesting
Interestingly, the expression of YAP1 was significantly decreased by Si-β-catenin, suggesting a
amechanistic
mechanisticrelationship
relationshipbetween
betweenthese
theseproteins
proteins(Figure
(Figure4G,H).
4G,H).
Figure 4. Cont.
Int. J. Mol. Sci. 2016, 17, 1909
Int. J. Mol. Sci. 2016, 17, 1909
6 of 17
6 of 17
Figure
Figure4.4.(A)
(A)RT-qPCR
RT-qPCRquantification
quantificationof
of the
the β-catenin
β-cateninexpression
expressionlevel
level(n
(n==6).
6).Red
Redbars
barsrepresent
representthe
the
negative
negativecontrol;
control;black
blackbars
barsrepresent
representmiR-30e-5p
miR-30e-5pmimic
mimicor
orinhibitor;
inhibitor;(B)
(B)The
Theeffect
effectof
ofmiR-30e-5p
miR-30e-5p
mimicking
inhibitingβ-catenin
β-cateninprotein
protein
expression
evaluated
by Western
blot analysis
in
mimicking and inhibiting
expression
waswas
evaluated
by Western
blot analysis
in GMEC;
GMEC;
(C)were
GMEC
were transfected
withmimic
miR-30e-5p
mimic
and the level
miR-15a
(C) GMEC
transfected
with miR-30e-5p
or inhibitor,
andor
theinhibitor,
miR-15a expression
was
expression
level
was quantified
by bars
RT-qPCR
(n =the
6). negative
Red barscontrol;
represent
thebars
negative
control;
black
quantified by
RT-qPCR
(n = 6). Red
represent
black
represent
miR-30e-5p
bars
represent
miR-30e-5p
mimic
or transfected
inhibitor; (D)
GMEC
were
transfected
with and
miR-15a
mimic or
mimic
or inhibitor;
(D) GMEC
were
with
miR-15a
mimic
or inhibitor,
the miR-30e-5p
expressionand
level
quantifiedexpression
by RT-qPCR
(n =was
6). Red
bars represent
the negative
control;
inhibitor,
thewas
miR-30e-5p
level
quantified
by RT-qPCR
(n = 6).
Red black
bars
bars represent
miR-15a mimic
inhibitor;
the YAP1miR-15a
expression
levelor
was
quantified
RT-qPCR
represent
the negative
control;orblack
bars(E)
represent
mimic
inhibitor;
(E)bythe
YAP1
(n = 6). Redlevel
bars was
represent
the negative
control;(nblack
the miR-30e-5p
mimic
or inhibitor;
expression
quantified
by RT-qPCR
= 6). bars
Red represent
bars represent
the negative
control;
black
(F) The
effect ofthe
miR-30e-5p
mimicking
inhibiting
protein
evaluated
by
bars
represent
miR-30e-5p
mimic orand
inhibitor;
(F) YAP1
The effect
of expression
miR-30e-5pwas
mimicking
and
Western blot
in GMEC;
GMEC were
Si-NC (60
nM)
SiRNA-β-catenin
nM)
inhibiting
YAP1
protein(G)
expression
wastransfected
evaluated with
by Western
blot
inorGMEC;
(G) GMEC(60
were
for 48 h, the
mRNA
expression
of SiRNA-β-catenin
YAP1 was quantified
by RT-qPCR
(n =
6); (H)expression
The effect of
of YAP1
Si-NC
transfected
with
Si-NC
(60 nM) or
(60 nM)
for 48 h, the
mRNA
(60 nM)
or SiRNA-β-catenin
(60=nM)
forThe
48 heffect
on YAP1
protein
was evaluated(60
by nM)
Western
was
quantified
by RT-qPCR (n
6); (H)
of Si-NC
(60expression
nM) or SiRNA-β-catenin
for
blot
in GMEC.
All experiments
andWestern
repeatedblot
three
times. Values
are presented
as
48
h on
YAP1 protein
expressionwere
was duplicated
evaluated by
in GMEC.
All experiments
were
means ± standard
errors,three
* p < 0.05;
p < 0.01.are presented as means ± standard errors, * p < 0.05;
duplicated
and repeated
times.**Values
** p < 0.01.
2.4. Functional Evaluation of miR-30e-5p, miR-15a, LRP6, and YAP1 in GMEC
2.4. Functional Evaluation of miR-30e-5p, miR-15a, LRP6, and YAP1 in GMEC
The expression of miR-30e-5p was 66 times higher in the miR-30e-5p mimic-transfected GMEC
Thethan
expression
miR-30e-5p
was 66
higher decreased
in the miR-30e-5p
mimic-transfected
GMEC
group
the NC of
(negative
control),
buttimes
expression
more than
99% in the miR-30e-5p
group
thangroup
the NC
(negative
but expression
decreased
than 99%
in the
miR-30e-5p
inhibited
(Figure
S3).control),
The expression
of miR-15a
wasmore
95 times
higher
in the
miR-15a
inhibited
group (Figure
S3).
Thetheexpression
of miR-15a
was 95 more
times than
higher
mimic-transfected
GMECs
than
NC, but expression
decreased
99%ininthe
the miR-15a
miR-15a
mimic-transfected
GMECs
inhibited group (Figure
S4).than the NC, but expression decreased more than 99% in the miR-15a
inhibited
groupmilk
(Figure
S4). as lipid droplets composed of TAG (Triglyceride) [32,33]. Compared with
In GMEC,
fat exists
In GMEC,
milk fat
as lipid decreased
droplets composed
of 1.8
TAG
(Triglyceride)
[32,33].
the negative
control,
theexists
TAG content
(p < 0.05) by
times
in GMEC with
the Compared
miR-30e-5p
with
the(Figure
negative
TAGthe
content
decreased
(p <was
0.05)
by 1.8 times
in GMEC with the
mimic
5A).control,
Similarthe
to TAG,
cholesterol
content
increased
in miR-30e-5p-transfected
miR-30e-5p
(Figure 5A).
Similar
TAG, in
thethecholesterol
was increased
in
cells (Figure mimic
5B). Furthermore,
β-casein
was to
increased
miR-30e-5pcontent
(Figure 5C,D).
Our findings
miR-30e-5p-transfected
cells
(FigureTAG
5B).and
Furthermore,
increased
in the
miR-30e-5p
revealed that miR-30e-5p
enhanced
cholesterol β-casein
synthesiswas
in goat
mammary
cells
(Figure 4).
(Figure 5C,D). Our findings revealed that miR-30e-5p enhanced TAG and cholesterol synthesis in
goat mammary cells (Figure 4).
Int. J. Mol. Sci. 2016, 17, 1909
Int. J. Mol. Sci. 2016, 17, 1909
7 of 17
7 of 17
Figure 5. (A) Triglyceride levels in cells transfected with miR-30e-5p mimic or inhibitor; Triglyceride
Figure 5. (A) Triglyceride levels in cells transfected with miR-30e-5p mimic or inhibitor; Triglyceride
levels in transfected cells compared with miR-30e-5p controls (n = 6). Red bars represent the
levels in transfected cells compared with miR-30e-5p controls (n = 6). Red bars represent the negative
negative control; black bars represent miR-30e-5p mimic or inhibitor; (B) Cholesterol levels in
control; black bars represent miR-30e-5p mimic or inhibitor; (B) Cholesterol levels in transfected cells
transfected cells compared with controls (n = 6). Red bars represent the negative control; black bars
compared with controls (n = 6). Red bars represent the negative control; black bars represent miR-30e-5p
represent miR-30e-5p mimic or inhibitor; (C) The expression of β-casein was quantified by RT-qPCR
mimic or inhibitor; (C) The expression of β-casein was quantified by RT-qPCR (n = 6); (D): The effect of
(n
= 6); (D): The
effect
of miR-30e-5p
mimic
or inhibitor
on β-casein
protein expression
miR-30e-5p
mimic
or inhibitor
on β-casein
protein
expression
was evaluated
by Westernwas
blotevaluated
in GMEC.
by
Western
blot
in
GMEC.
All
experiments
were
duplicated
and
repeated
three
times.
Values
are
All experiments were duplicated and repeated three times. Values are presented as means ±
standard
presented
as0.05;
means
errors, * p < 0.05; ** p < 0.01.
errors, * p <
** p±<standard
0.01.
Compared with the negative control, the TAG content increased (p < 0.05) by 2.0 times in
Compared with the negative control, the TAG content increased (p < 0.05) by 2.0 times in miR-15a
miR-15a mimic-transfected cells (Figure 6A). On the other hand, the TAG content decreased
mimic-transfected cells (Figure 6A). On the other hand, the TAG content decreased significantly when
significantly when miR-15a was inhibited (Figure 6A). Compared with the negative control, the
miR-15a was inhibited (Figure 6A). Compared with the negative control, the cholesterol content
cholesterol content increased (p < 0.05) by 1.2 times in miR-15a mimic-transfected cells, but
increased (p < 0.05) by 1.2 times in miR-15a mimic-transfected cells, but decreased significantly when
decreased significantly when miR-15a was inhibited (Figure 6B). MiR-15a also enhanced the
miR-15a was inhibited (Figure 6B). MiR-15a also enhanced the expression and protein level of milk fat
expression and protein level of milk fat metabolic marker genes (Figure 6C,D). Our findings
metabolic marker genes (Figure 6C,D). Our findings revealed that miR-15a seems to plays a crucial
revealed that miR-15a seems to plays a crucial role in milk TAG synthesis and promotes milk fat
role in milk TAG synthesis and promotes milk fat metabolism in goat mammary gland (Figure 6).
metabolism in goat mammary gland (Figure 6).
Several genes work in a coordinated fashion to control ruminant mammary lipid and protein
Several genes work in a coordinated fashion to control ruminant mammary lipid and protein
metabolism [32–34]. Ectopic overexpression of miR-30e-5p strongly up-regulated the mRNA expression
metabolism [32–34]. Ectopic overexpression of miR-30e-5p strongly up-regulated the mRNA
of PPARγ, LPL, DGAT1, and CD36 (Figure 7A and Figure S3). In contrast, miR-30e-5p inhibition
expression of PPARγ, LPL, DGAT1, and CD36 (Figure 7A and Figure S3). In contrast, miR-30e-5p
led to a remarkable down-regulation of HSL (Figure 7A and Figure S3). These data indicate that
inhibition led to a remarkable down-regulation of HSL (Figure 7A and Figure S3). These data
miR-30e-5p plays an important role in the regulation of genes associated with different aspects of fatty
indicate that miR-30e-5p plays an important role in the regulation of genes associated with different
acid metabolism.
aspects of fatty acid metabolism.
The results also indicated that ectopic over expression of miR-15a strongly up-regulated the
The results also indicated that ectopic over expression of miR-15a strongly up-regulated the
mRNA expression of PPARγ, LPL, SCD1, and FASN (Figure 7B and Figure S3). In contrast, cells
mRNA expression of PPARγ, LPL, SCD1, and FASN (Figure 7B and Figure S3). In contrast, cells
transfected with the miR-15a inhibitor led to a remarkable down-regulation of a number of genes
transfected with the miR-15a inhibitor led to a remarkable down-regulation of a number of genes
related to fat metabolism, including HSL (Figure 7B and Figure S3). Thus, similar to miR-30e-5p,
related to fat metabolism, including HSL (Figure 7B and Figure S3). Thus, similar to miR-30e-5p,
miR-15a plays an important role in the regulation of genes associated with different aspects of fatty
miR-15a plays an important role in the regulation of genes associated with different aspects of fatty
acid metabolism.
metabolism.
acid
Int.Int.
J. Mol.
Sci.
2016,
17,
J. Mol.
Sci.
2016,
17,1909
1909
Int. J. Mol. Sci. 2016, 17, 1909
of 17
8 of817
8 of 17
Figure 6. (A) Triglyceride levels in cells transfect with miR-15a mimic or inhibitor; triglyceride levels
Figure
(A)
Triglyceride
levels
cellstransfect
transfectwith
withmiR-15a
miR-15amimic
mimicor
or inhibitor; triglyceride
triglyceride levels
levels in
Figure
6. 6.
(A)
Triglyceride
inincells
in transfected
cells werelevels
compared
with that of
miR-15a controls
(n =inhibitor;
6). Red bars represent
the
in
transfected
cells
were
compared
with
that
of
miR-15a
controls
(n
=
6).
Red
bars
represent
the
transfected
cells
were
compared
with
that
of
miR-15a
controls
(n
=
6).
Red
bars
represent
the
negative
negative control; black bars represent miR-15a mimic or inhibitor; (B) Cholesterol levels in
negative
control;
black
bars
represent
miR-15a
mimic
or
inhibitor;
(B)
Cholesterol
levels
in
control;
black cells
bars were
represent
miR-15a
mimic
orcontrol
inhibitor;
levels inthe
transfected
cells were
transfected
compared
with
that of
(n =(B)
6).Cholesterol
Red bars represent
negative control;
transfected
cells
were
compared
with
that
of
control
(n
=
6).
Red
bars
represent
the
negative
control;
compared
with
that ofmiR-15a
control mimic
(n = 6).orRed
bars represent
the negative
control; black
bars represent
black bars
represent
inhibitor;
(C) The expression
of β-casein
was quantified
by
black bars
represent
miR-15a
mimic
or inhibitor;
(C) The was
expression
of β-casein
was quantified
by
miR-15a
mimic
or
inhibitor;
(C)
The
expression
of
β-casein
quantified
by
RT-qPCR
(n
= 6); (D)
The
RT-qPCR (n = 6); (D) The effect of miR-15a mimic or inhibitor on β-casein protein expression
was
RT-qPCR
(n = 6);
(D) The
effect of miR-15a
mimic
or inhibitor
on β-casein
proteinby
expression
was in
effect
of
miR-15a
mimic
or
inhibitor
on
β-casein
protein
expression
was
evaluated
Western
blot
evaluated by Western blot in GMEC. Total protein was harvested 48 h post-treatment, respectively.
evaluated
Western
in GMEC.
protein was harvested 48 All
h post-treatment,
respectively.
GMEC.
Totalby
protein
wasblot
harvested
48and
hTotal
post-treatment,
experiments
duplicated
All experiments
were
duplicated
repeated threerespectively.
times. Values are
presentedwere
as means
±
All
experiments
were
duplicated
and
repeated
three
times.
Values
are
presented
asp means
and
repeated
three
times.
Values
are
presented
as
means
±
standard
errors,
*
p
<
0.05;
**
<
0.01. ±
standard errors, * p < 0.05; ** p < 0.01.
standard errors, * p < 0.05; ** p < 0.01.
Figure 7. (A) Expression of fatty acid metabolism-related genes. The mRNA expression of PPARγ,
Figure
7. (A)
Expression
of of
fatty
acid
metabolism-related
genes.
The
mRNA
expression
of of
PPARγ,
LPL,
Figure
7. (A)
Expression
fatty
acid
metabolism-related
genes.
The
mRNA
expression
PPARγ,
LPL, CD36, DGAT1, and HSL was quantified by RT-qPCR (n = 6). Red bars represent the miR-30e-5p
CD36,
and HSL
was
quantified
by RT-qPCR
(n = 6).(nRed
the miR-30e-5p
mimic;
LPL,DGAT1,
CD36, DGAT1,
and
HSL
was quantified
by RT-qPCR
= 6).bars
Redrepresent
bars represent
the miR-30e-5p
mimic; black bars represent the miR-30e-5p inhibitor; (B) Expression of fatty acid
black
bars represent
the miR-30e-5p
inhibitor;
(B) Expression
of fatty
metabolism-related
genes.
mimic;
black bars
represent the
miR-30e-5p
inhibitor;
(B) acid
Expression
of fatty acid
metabolism-related genes. The mRNA expression of PPARγ, LPL, SCD1, FASN, and HSL was
The
mRNA expression
of PPARγ,
LPL, SCD1,
FASN,ofand
HSL was
by RT-qPCR
= 6).
metabolism-related
genes.
The mRNA
expression
PPARγ,
LPL, quantified
SCD1, FASN,
and HSL (n
was
quantified by RT-qPCR (n = 6). Red bars represent the miR-15a mimic; black bars represent the
Red
bars represent
the miR-15a
mimic;
black
bars represent
the miR-15a
experiments
quantified
by RT-qPCR
(n = 6).
Red bars
represent
the miR-15a
mimic; inhibitor.
black barsAll
represent
the
miR-15a inhibitor. All experiments were duplicated and repeated three times. Values are presented
miR-15a
inhibitor.
experiments
were duplicated
repeated
three times.
Valueserrors,
are presented
were
duplicated
and All
repeated
three times.
Values are and
presented
as means
± standard
* p < 0.05;
as means ± standard errors, * p < 0.05; ** p < 0.01.
** pas<means
0.01. ± standard errors, * p < 0.05; ** p < 0.01.
Int. J. Mol. Sci. 2016, 17, 1909
9 of 17
Int. J. Mol. Sci. 2016, 17, 1909
9 of 17
Both LRP6
LRP6 siRNA
siRNA and
and YAP1
YAP1 siRNA
siRNA were
were used
used to
to explore
explore their
their functional
functional role
role in
in GMEC
GMEC from
from
Both
individual
lactating
goats.
Compared
with
the
negative
control,
the
level
of
LRP6
decreased
by
75%
individual lactating goats. Compared with the negative control, the level of LRP6 decreased by 75%
in GMEC
GMEC transfected
transfected with
with the
the LRP6
LRP6 siRNA
siRNA (Figures
(Figures S5
S5 and
and S6,
S6, Supplementary
Supplementary Data
Data 4).
4). Similarly,
Similarly,
in
compared
with
the
negative
control,
the
YAP1
level
decreased
by
72%
in
the
GMEC
transfected
with
compared with the negative control, the YAP1 level decreased by 72% in the GMEC transfected
YAP1
siRNA
(Figures
S5
and
S6).
As
depicted
in
Figure
8A,
compared
with
the
negative
control,
the
with YAP1 siRNA (Figures S5 and S6). As depicted in Figure 8A, compared with the negative
TAG content
increased
< 0.05) by(p1.25
times
GMEC
with LRP6 siRNA.
Compared
control,
the TAG
content(pincreased
< 0.05)
byin
1.25
timestransfected
in GMEC transfected
with LRP6
siRNA.
with
the
negative
control,
the
cholesterol
content
increased
(p
<
0.05)
by
1.2
times
in
GMEC
transfected
Compared with the negative control, the cholesterol content increased (p < 0.05) by 1.2 times in
with LRP6
siRNA (Figure
8B). In
addition,
we uncovered
that LRP6
promoted the
expression
of milk
GMEC
transfected
with LRP6
siRNA
(Figure
8B). In addition,
we uncovered
that
LRP6 promoted
fat
metabolic
marker
genes,
including
miRNAs
and
proteins
(Figure
8C,D).
Our
findings
revealed
the expression of milk fat metabolic marker genes, including miRNAs and proteins (Figure 8C,D).
that findings
LRP6 plays
a crucial
milk aTAG
synthesis
milk fat
in goat
Our
revealed
that role
LRP6inplays
crucial
role in and
milkpromotes
TAG synthesis
andmetabolism
promotes milk
fat
mammary
cells.
metabolism in goat mammary cells.
Compared with
thethe
TAG
content
increased
(p < 0.05)
by 1.35
GMEC
Compared
with the
thenegative
negativecontrol,
control,
TAG
content
increased
(p < 0.05)
bytimes
1.35 in
times
in
transfected
with
YAP1
siRNA
(Figure
9A).
Compared
with
the
negative
control,
the
cholesterol
content
GMEC transfected with YAP1 siRNA (Figure 9A). Compared with the negative control, the
increased (p content
< 0.05) by
1.6 times (p
in GMEC
withinYAP1
siRNA
(Figure 9B).
We YAP1
also observed
cholesterol
increased
< 0.05) transfected
by 1.6 times
GMEC
transfected
with
siRNA
that
YAP1
promoted
the
expression
of
milk
fat
metabolic
marker
genes,
including
miRNAs
and
(Figure 9B). We also observed that YAP1 promoted the expression of milk fat metabolic marker
proteinsincluding
(Figure 9C,D).
Our and
findings
revealed
that YAP1
crucial role
in milkthat
TAGYAP1
synthesis
and
genes,
miRNAs
proteins
(Figure
9C,D).plays
Ourafindings
revealed
plays
a
promotes
milk
TAG
synthesis
in
goat
mammary
cells.
crucial role in milk TAG synthesis and promotes milk TAG synthesis in goat mammary cells.
Figure
Triglyceride levels
levelsin
incells
cellstransfected
transfectedwith
withSi-NC
Si-NCororSiRNA-LRP6;
SiRNA-LRP6;
triglyceride
levels
Figure 8. (A) Triglyceride
triglyceride
levels
in
in
transfected
cells
were
compared
with
Si-NC
6). Red
represent
the negative
control;
transfected
cells
were
compared
with
Si-NC
(n =(n6).= Red
barsbars
represent
the negative
control;
blackblack
bars
bars
represent
SiRNA-LRP6;
(B) Cholesterol
in transfected
cellscompared
were compared
with controls
represent
SiRNA-LRP6;
(B) Cholesterol
levelslevels
in transfected
cells were
with controls
(n = 6).
(n
=
6).
Red
bars
represent
the
negative
control;
black
bars
represent
the
siRNA-LRP6;
(C)
The
Red bars represent the negative control; black bars represent the siRNA-LRP6; (C) The expression
of
β-casein wasofquantified
RT-qPCR
(n =by
6); RT-qPCR
(D) The effect
(60 nM)
or of
siRNA-LRP6
expression
β-casein by
was
quantified
(n = of
6);si-NC
(D) The
effect
si-NC (60 (60
nM)nM)
or
on β-casein protein
expression
was evaluated
by Westernwas
blot in
GMEC; (E)
levels
in cells
siRNA-LRP6
(60 nM)
on β-casein
protein expression
evaluated
by Triglyceride
Western blot
in GMEC;
transfected
with levels
controlininhibitor
(50 nM) +with
control
siRNA(50
nM),(50
inhibitor
nM) + control
(E)
Triglyceride
cells transfected
control
inhibitor
nM) + 30e-5p
control(50
siRNA(50
nM),
siRNA
(50
nM)
and
inhibitor
30e-5p
(50
nM)
+
siRNA-LRP6
(50
nM);
triglyceride
levels
were
compared
inhibitor 30e-5p (50 nM) + control siRNA (50 nM) and inhibitor 30e-5p (50 nM) + siRNA-LRP6
withnM);
that triglyceride
of controls (nlevels
= 6). Values
are presented
means
± standard
* p < 0.05.
(50
were compared
withasthat
of controls
(n =errors,
6). Values
are presented as
means ± standard errors, * p < 0.05.
We applied a “rescue” experiment to demonstrate that miR-30e-5p and miR 15a exert their
We applied
a “rescue”
experiment
to demonstrate
miR-30e-5p
miR 15a
exert8E)
their
functions
via LRP6
and YAP1.
The siRNA-LRP6
rescuethat
increased
TAG and
in GMEC
(Figure
in
functions
via
LRP6
and
YAP1.
The
siRNA-LRP6
rescue
increased
TAG
in
GMEC
(Figure
8E)
in
response to the ectopic expression of inhibitor miR-30e-5p. The decrease of TAG was partly alleviated
response
to the ectopic
expression
of pinhibitor
miR-30e-5p.
The decrease
of TAG of
was
partly
by the siRNA-LRP6
rescue
(TAG assay,
< 0.05, Figure
7E). Interestingly,
the increase
TAG
was
alleviated
by the by
siRNA-LRP6
rescuerescue
(TAG (TAG
assay,assay,
p < 0.05,
7E). Interestingly,
the increase of
partly alleviated
the siRNA-YAP1
p < Figure
0.05, Figure
9E).
TAG was partly alleviated by the siRNA-YAP1 rescue (TAG assay, p < 0.05, Figure 9E).
Int. J. Mol. Sci. 2016, 17, 1909
Int. J. Mol. Sci. 2016, 17, 1909
10 of 17
10 of 17
Figure
levelsinintransfected
transfectedcells
cellswere
were
compared
with
of control
6). Red
Figure 9.
9. (A)
(A) Triglyceride
Triglyceride levels
compared
with
thatthat
of control
(n =(n
6).=Red
bars
bars
represent
the
negative
control;
black
bars
represent
miR-15a
mimic
or
inhibitor;
(B)
Cholesterol
represent the negative control; black bars represent miR-15a mimic or inhibitor; (B) Cholesterol levels
levels
in transfected
cellscompared
were compared
with
of the (n
control
= bars
6). Red
bars represent
the
in transfected
cells were
with that
of that
the control
= 6). (n
Red
represent
the negative
wasby
quantified
negative
control;
bars represent
siRNA-YAP1;
(C) The expression
ofβ-casein
control; black
barsblack
represent
siRNA-YAP1;
(C) The expression
of β-casein was
quantified
RT-qPCR
(n =
6); (D) The
of si-NC
(60 nM)
or siRNA-YAP1
nM) on β-casein
protein
expression
was
by
RT-qPCR
(n =effect
6); (D)
The effect
of si-NC
(60 nM) or (60
siRNA-YAP1
(60 nM)
on β-casein
protein
evaluated by
Western
blotby
in Western
GMEC; (E)
Triglyceride
levels
in cells transfected
with transfected
control inhibitor
expression
was
evaluated
blot
in GMEC; (E)
Triglyceride
levels in cells
with
(50 nM)inhibitor
+ control(50
siRNA
nM), inhibitor
15a
(50 nM)
+ Control
siRNA
nM), and
inhibitor
15a
control
nM) +(50
control
siRNA (50
nM),
inhibitor
15a (50
nM) +(50
Control
siRNA
(50 nM),
(50 nM)
+ siRNA-YAP1
(50 +nM).
All experiments
were
duplicated
and were
repeated
three times.
are
and
inhibitor
15a (50 nM)
siRNA-YAP1
(50 nM).
All
experiments
duplicated
and Values
repeated
presented
meansare
± standard
* p <±0.05;
** p <errors,
0.01. * p < 0.05; ** p < 0.01.
three
times.asValues
presentederrors,
as means
standard
3.
3. Discussion
Discussion
3.1. miRNA
miRNAExpression
ExpressionScreening
Screening at
at Peak-Lactation,
Peak-Lactation, Non-Lactating
Non-Lactating Stage,
Stage, and
and in
in Response
Response to Prolactin
3.1.
in GMEC
GMEC
in
Previous research
research illustrated
illustrated that
that miRNA
miRNA plays
plays an
an important
important role
role in
in mammary
mammary development
development
Previous
and lactation
lactation[35–37].
[35–37].For
Forinstance,
instance,
Avril-Sassen
screened
compared
the expression
of
and
Avril-Sassen
et et
al. al.
screened
andand
compared
the expression
of 102
102
miRNA
in
different
stages
of
lactation
in
mice
and
speculated
that
some
differentially-expressed
miRNA in different stages of lactation in mice and speculated that some differentially-expressed
miRNA were
were related
related to
to fatty
fatty acid
acid metabolism
metabolism [38].
[38]. AA more
more recent
recent study
study using
using Wendeng
Wendeng goats
goats
miRNA
compared
miRNA
expression
profiles
between
pregnancy
and
early
lactation
and
uncovered
several
compared miRNA expression profiles between pregnancy and early lactation and uncovered
differentially-expressed
miRNA associated
with development
and lactation
[39].
Lin et al.
[40]Lin
detected
several
differentially-expressed
miRNA associated
with development
and
lactation
[39].
et al.
that
miR-27
suppresses
TAG
accumulation
in
GMEC.
However,
most
of
these
studies
were
based
[40] detected that miR-27 suppresses TAG accumulation in GMEC. However, most of these studies
on sequencing
and array and
chiparray
technology.
The mainThe
objective
of our study
to was
better
were
based on sequencing
chip technology.
main objective
of ourwas
study
to define
better
the
relationship
of
known
miRNA
in
regulating
mammary
cell
fatty
acid
metabolism.
To this
this
define the relationship of known miRNA in regulating mammary cell fatty acid metabolism. To
end, we
wescreened
screenedpotential
potentialmiRNA
miRNAusing
usingdifferential
differentialexpression
expression
profiling.
Overall,
detected
end,
profiling.
Overall,
we we
detected
54
54
differentially-expressed
miRNA
in
peak-lactation
and
late-lactation
stages
with
30
up-regulated
differentially-expressed miRNA in peak-lactation and late-lactation stages with 30 up-regulated
and 24
24 down-regulated.
down-regulated. In
Inthis
thisstudy,
study, we
we established
established the
the miRNA
miRNA library
library including
including 267
267 Capra
Caprahircus
hircus
and
primary
miRNAs
and
793
Bostaurus
primary
miRNAs
from
miRBase,
as
well
as
the
sequencing
results
primary miRNAs and 793 Bostaurus primary miRNAs from miRBase, as well as the sequencing
via Solexa
throughthrough
which we
screened
a series ofa potential
miRNA involving
in regulation
results
via sequencing,
Solexa sequencing,
which
we screened
series of potential
miRNA involving
in
of
mammary
metabolism.
Lin
et
al.
detected
that
miR-27
suppresses
TAG
accumulation
in
GMEC.
regulation of mammary metabolism. Lin et al. detected that miR-27 suppresses TAG accumulation
However,
most of these
were
basedwere
on sequencing
and array and
chiparray
technology.
The main
in
GMEC. However,
moststudies
of these
studies
based on sequencing
chip technology.
purpose
of
the
present
study
was
to
better
define
the
relationship
of
known
miRNA
regulating
The main purpose of the present study was to better define the relationship of known miRNA
mammary mammary
cell fatty acid
Overall, we
detected
differentially-expressed
miRNA in
regulating
cell metabolism.
fatty acid metabolism.
Overall,
we54detected
54 differentially-expressed
peak-lactation
and
non-lactation
stages
with
30
up-regulated
and
24
down-regulated.
We
established
miRNA in peak-lactation and non-lactation stages with 30 up-regulated and 24 down-regulated.a
miRNA
library that
includes
793 Bos
taurus
primary
andprimary
267 Capra
hircus primary
We
established
a miRNA
library
that
includes
793 miRNAs
Bos taurus
miRNAs
and 267miRNA
Capra
hircus primary miRNA from miRBase, with which we screened several potential miRNA involved
in regulation of mammary fatty acid metabolism.
Int. J. Mol. Sci. 2016, 17, 1909
11 of 17
from miRBase, with which we screened several potential miRNA involved in regulation of mammary
fatty acid metabolism.
To establish which miRNA are associated with the process of growth and development of
mammary cells, we cultured GMEC with increasing concentrations of prolactin (a key lactogenic
hormone) for subsequent miRNA profiling. We used concentrations of 0 µg/mL, 0.5 µg/mL,
1 µg/mL, 2.5 µg/mL and 5 µg/mL prolactin, and determined that 2.5 µg/mL was the optimal for our
specific objective.
3.2. miR-30e-5p and miR-15a Target LRP6 and YAP1, Respectively
The protein encoded by LRP6 is a member of the LDL receptor-related family, a type of
transmembrane cell surface protein involved in receptor-mediated endocytosis. LRP6, however,
is widely known as a Wnt co-receptor in the canonical signaling pathway during embryonic
development [30]. Wenzhong Liu suggested that the impairment of Wnt signaling in LRP6+ mice
induced a reduction in body fat mass, diminished hepatic gluconeogenesis, and enhanced BAT and
hepatic insulin sensitivity [41]. To confirm that LRP6 is a direct target of miR-30e-5p, we first cloned
the 30 -UTR of LRP6 for miR-30e-5p into a luciferase reporter plasmid to detect whether miR-30e-5p
had a suppressing effect on this gene. Our findings indicated that miR-30e-5p significantly inhibited
the luciferase activity, suggesting that miR-30e-5p functions through the 30 -UTR of LRP6 to inhibit
the reporter gene expression. Furthermore, we made a mutation of the potential binding site for
miR-30e-5p in the 30 -UTR, and this mutation abrogated the suppressive effect of miR-30e-5p on the
30 -UTR of LRP6.
QiuYue Liu reported a time-shifted relationship between the expression Lats2 and two adipocyte
markers C/EBP α and PPARγ, and an inverse relationship between Lats2 and transcriptional
co-activator YAP1, which positively regulated cell growth and proliferation during differentiation in
3T3-L1 pre-adipocytes [42]. Eunjeong Seo demonstrated a Hippo-independent regulation of YAP1 by
SOX2 that cooperatively antagonized Wnt/β-catenin signaling and regulated PPARγ to determine
osteogenic or adipocytic fates [25]. To confirm that YAP1 is a direct target of miR-15a, we first cloned
the 30 -UTR of YAP1 for miR-15a into a luciferase reporter plasmid to detect whether miR-15a had
a suppressing effect on this gene. Our findings indicated that miR-15a significantly inhibited the
luciferase activity, suggesting that miR-15a functioned through the 30 -UTR of YAP1 to inhibit the
reporter gene expression. Furthermore, we made a mutation of the potential binding site for miR-15a
in the 30 -UTR, and this mutation abrogated the suppressive effect of miR-15a a on the 30 -UTR of YAP1.
The rescue experiments dealing with siRNA-LRP6 and siRNA-YAP1 partly abolished the decrease
of the TAG level induced by inhibitor miR-30e-5p and inhibitor miR-15a. Thus, the rescue experiments
illustrated that miR-30e-5p and miR-15a work via LRP6 and YAP1, respectively. These data provided
the basis for additional research on LRP6 and YAP1 to determine the synergistic mechanism regulating
fat oxidation in mammary epithelial cells.
3.3. Relationship between miR-30e-5p, β-Catenin, and YAP1
Several reports demonstrated the function of the miR-30 family in adipogenesis and inhibition
of osteogenesis. Fang Hu et al. found that miR-30b/c is a key regulator of thermogenesis and
uncovered a new mechanism underlying the regulation of brown adipose tissue function and the
development of beige fat [43]. Wang showed miR-30e regulated adipocyte by directly targeting the
Wnt/LRP6/β-catenin/TCF signaling. [44]. However, all of these studies did not explore the functional
and mechanistic role of miR-30e-5p in lactation.
β-catenin and LRP6 play an important role in the Wnt pathway. We verified the LRP6 is one of
miR-30e-5p’s target genes, and RT-qPCR and Western blot data revealed that miR-30e-5p could regulate
β-catenin. Interestingly, when we knocked out β-catenin by siRNA and measured YAP1 expression,
it was surprising to find that β-catenin inhibited the expression of YAP1. In conclusion, our findings
indicated that β-catenin could regulate YAP1 during fat metabolism in GMEC.
Int. J. Mol. Sci. 2016, 17, 1909
Int. J. Mol. Sci. 2016, 17, 1909
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3.4. miR-30e-5p
miR-30e-5p Cooperates
Cooperates with
with miR-15a
miR-15a to Repress YAP1
Although the
the specific
specificbiological
biologicalroles
roles
of some
miRNA
been reported,
that
of some
miRNA
have have
been reported,
the rolethe
thatrole
miRNA
miRNA
playsmammary
in goat mammary
glandcells
epithelial
cellsmiR-15a/miR-16
is limited. miR-15a/miR-16
control
cell cycle
plays in goat
gland epithelial
is limited.
control cell cycle
in non-small
in
non-small
lung
cancer
cells
[45].
Since
different
tissues
expressing
miR-15a
have
lung cancer cells [45]. Since different tissues expressing miR-15a have different functionsdifferent
[46], we
functions
speculated
that miR-15a
can regulate
the by
role
mammary
gland
by controlling
speculated[46],
that we
miR-15a
can regulate
the role mammary
gland
controlling
lipid
metabolism.
When
lipid
metabolism.
When of
themiR-15a
mimic and
were
in GMEC
at the
the mimic
and inhibitor
wereinhibitor
culturedofinmiR-15a
GMEC at
the cultured
same time,
we found
thatsame
they
time,
we
found
that
they
elicited
stronger
responses
together
than
they
did
individually.
We
also
elicited stronger responses together than they did individually. We also found that miR-30e-5p could
found
that
miR-30e-5p
could regulate
miR-15a,
butregulate
miR-15amiR-30e-5p.
could not, inInterestingly,
turn, regulateusing
miR-30e-5p.
regulate
miR-15a,
but miR-15a
could not,
in turn,
siRNA,
Interestingly,
using
siRNA, we
observed
thatYAP1,
miR-30e-5p
regulate
YAP1,
which
is reduced
a target
we observed that
miR-30e-5p
could
regulate
which iscould
a target
gene of
miR-15a,
and
gene
of miR-15a,
and reduced
the (Figure
expression
of β-catenin
in GMECthat
(Figure
the expression
of β-catenin
in GMEC
9). These
results suggested
YAP19).
is aThese
factor results
whose
suggested
YAP1by
is aWnt/β-catenin
factor whose expression
driven To
by conclude,
Wnt/β-catenin
signaling
inmiR-30e-5p
GMEC. To
expressionthat
is driven
signaling inisGMEC.
we proved
that
conclude,
we
proved
miR-30e-5p
could target
and regress
LRP6, which
coordinated
could target
and
regressthat
LRP6,
which coordinated
with miR-15a
in regulating
YAP1 and
mammarywith
cell
miR-15a
in
regulating
YAP1
and
mammary
cell
fatty
acid
metabolism
(Figure
10).
fatty acid metabolism (Figure 10).
summarizing our findings: miR-30e-5p
miR-30e-5p and
and miR-15a
miR-15a synergistically
synergistically regulate
regulate fat
fat
Figure 10. Diagram summarizing
metabolism via LRP6 and YAP1 in goat mammary epithelial cells.
4. Material
Materialand
andMethods
Methods
4.1. Ethics
Ethics Statement
Statement
The animal
animal use
use and
and care protocol was approved by the Animal Use and Care Committee at the
Northwest Agricultural and Forestry University,
University, Yang
YangLing,
Ling,China.
China.
4.2. Animals
Animals and
and RNA
RNA Extraction
Extraction
4.2.
The elite
goats
used
in this
research
waswas
fromfrom
the experimental
farm
The
elite herd
herdof
ofXinong
XinongSaanen
Saanendairy
dairy
goats
used
in this
research
the experimental
of Northwest
Agricultural
and Forestry
University
of China.
Three
healthy
goatsgoats
(three
yearsyears
old)
farm
of Northwest
Agricultural
and Forestry
University
of China.
Three
healthy
(three
with
similar
body
weight
were
selected
at
different
stages
of
lactation:
non-pregnant,
early-lactation
old) with similar body weight were selected at different stages of lactation: non-pregnant,
(15 days after parturition),
peak-lactation
days after parturition),
late-lactation
days after
early-lactation
(15 days after
parturition), (60
peak-lactation
(60 days after
parturition),(150
late-lactation
parturition),
andparturition),
non-lactating
(“dry”
period). (“dry”
All goats
gave birth
to kids
in the
second
lactation.
(150
days after
and
non-lactating
period).
All goats
gave
birth
to kids
in the
Spleen,
stomach,
heart,
liver,
sebum,
mammary
gland
tissue,
muscle,
lung,
and
kidney
were
collected
second lactation. Spleen, stomach, heart, liver, sebum, mammary gland tissue, muscle, lung,
and
after slaughter.
Three biological
samples
per biological
tissue weresamples
snap-frozen
in liquid
as soon
kidney
were collected
after slaughter.
Three
per tissue
werenitrogen
snap-frozen
in
as
possible.
Using
Trizol
reagent
(Invitrogen,
Carlsbad,
CA,
USA),
total
RNA
was
extracted
in
liquid nitrogen as soon as possible. Using Trizol reagent (Invitrogen, Carlsbad, CA, USA), total
accordance
with
the
instructions.
The
quality
and
quantity
of
RNA
were
detected
by
a
ND-1000
RNA was extracted in accordance with the instructions. The quality and quantity of RNA were
◦ C before
spectrophotometer
(NanoDrop,
Waltham, MA,
USA) and
the RNAMA,
wasUSA)
storedand
in −
80 RNA
detected
by a ND-1000
spectrophotometer
(NanoDrop,
Waltham,
the
was
stored in −80 °C before experimentation. The samples are a mixture of three goats (at the same
period) in non-pregnant, early-lactation (15 days after parturition), peak-lactation (60 days after
Int. J. Mol. Sci. 2016, 17, 1909
13 of 17
experimentation. The samples are a mixture of three goats (at the same period) in non-pregnant,
early-lactation (15 days after parturition), peak-lactation (60 days after parturition), late-lactation
(150 days after parturition), and non-lactating (“dry” period) stages, respectively.
4.3. Cell Culture and Transfection
The GMEC were cultured in DMEM/ F12 medium (Invitrogen, Carlsbad, CA, USA) containing
10% FBS, 10 ng/mL EGF-1 (epidermal growth factor 1, Gibco, Carlsbad, CA, USA), 5 mg/mL insulin,
50 U/mL penicillin/mL streptomycin, and 0.25 mmol/L hydrocortisone in 37 ◦ C in a humidified
atmosphere with 5% CO2 . The GMECs were cultured and fractionated in accordance with a previous
study [47]. To induce lactogenesis, GMEC were cultured in a lactogenic medium for 48 h prior to
initial experiments [48,49]. On the other hand, GMEC were also processed with 0 µg/mL, 0.5 µg/mL,
1 µg/mL, 2.5 µg/mL, and 5 µg/mL prolactin. Cells were cultured and transfected with either mimic of
miR-30e-5p and miR-15a (60 nM) or inhibitor (60 nM) (Invitrogen) using Lipofectamine™ RNAiMAX
(Invitrogen) on the basis of the manufacturer’s instructions. Cells were transfected with si-NC (60 nM),
siRNA-LRP6 (60 nM), siRNA-YAP1, or siRNA-β catenin. Cells were harvested 48 h after transfection.
4.4. Assay of Cellular TAG Content
The GMECs were transfected with either miR-30e-5p and miR-15a mimic or inhibitor. Cells
were stained with lysis buffer (1% Triton X-100, pH 7.4, 150 mmol/NaCl, 50 mmol/L Tris-HCL)
after 48 h of incubation. TAG was measured using a commercial kit on the basis of manufacturer’s
instructions (Loogen, Beijing, China) on an XD 811G Biochemistry Analyzer (Odin Science and
Technology Company, Shanghai, China). The values acquired were normalized to the content of total
protein with the BCA protein assay kit (Thermo Corp., Waltham, MA, USA, Prod#23227).
4.5. Assay of The Cellular Cholesterol Content
The GMECs were transfected with either miR-30e-5p/miR-15a mimic or inhibitor and siRNA
(20 ng). Cells were stained with lysis buffer (1% Triton X-100, pH 7.4, 150 mmol/NaCl, 50 mmol/L
Tris-HCL) after 48 h of incubation. Cholesterol was measured using a serum cholesterol kit on the
basis of the manufacturer’s instructions (Loogen) on an XD 811G Biochemistry Analyzer (Odin Science
and Technology Company, Shanghai, China). The values obtained were normalized to the content of
total protein.
4.6. RT-qPCR and Western Blot
Both the miRNA detection sensitivity and specificity are improved utilizing the S-Poly (T)
assay. In this study, we used the S-Poly (T) real method for the profile expression of 789 miRNA.
The expression of 18s rRNA was used as a normalization control. The S1 and S2 were used as
specific reverse primers. In brief, reverse transcription was performed as follows: 2.5 µL of 4×
reaction buffer, a 10 µL reaction including 0.2 µg total RNA, 1 µL of 0.5 µM RT primer, and 1 µL
of poly A/RT enzyme mix. The reaction was performed in 37 ◦ C for 25 min, followed by 42 ◦ C
for 25 min, and 72 ◦ C for 10 min. The products of RT were amplified and detected by 20 µL PCR
reaction containing 0.3 µL of RT products, a universal Taqman® probe, 0.5 units of Go Taq® Start
Polymerase (Promega, Fitchburg, WI, USA), universal reverse primer, 4 µL of 5× qPCR probe Mix,
0.5 µM forward primer and 0.2 mM universal Taqman probe. The PCR reaction was performed at
95 ◦ C for 3 min, followed by 42 cycles at 93 ◦ C for 10 s and 62 ◦ C for 30 s. For the mRNA expression
level, 0.3 µg of total RNA were synthesized into cDNA using the PrimeScript® RT Reagent Kit (Perfect
Real-Time, Takara, Japan) [49–51]. The expression was normalized to UXT. The sequences of primers
are listed in Supplementary data 3. All of the real-time reactions, including controls with no templates,
were carried out in a Bio-Rad CFX96 real-time PCR detection system (Bio-Rad, Foster City, CA, USA)
in triplicate. Relative expression was measured by the 2−∆∆Ct method [40,47,52,53].
Int. J. Mol. Sci. 2016, 17, 1909
14 of 17
As to Western blot analyses, cells were obtained and lysed in RIPA buffer (Solarbio, Beijing,
China). Proteins extracted from cells were separated by SDS-PAGE, transferred to nitrocellulose
membrane (Millipore, Billerica, MA, USA) and probed with the primary antibodies polyclonal rabbit
anti-LRP6 (Santa Cruz Biotechnology, Santa Cruz, CA, sc-15399, USA), monoclonal rabbit anti-YAP1
(Cell Signaling Technology Kit#8579), polyclonal rabbit anti-β-catenin, rabbit anti-β-casein (Biorbyt,
orb2053, Wuhan, China), and monoclonal mouse anti-β-actin (ProteintechGronup, 66009-1-IG, Wuhan,
China), respectively. Polyclonal anti-rabbit HRP-conjugated IgG goat (Tiangen, Beijing, China) was
used as a secondary antibody. Signals were detected by the chemiluminescent ECL Western blot
system (Pierce, Holmdel, NJ, USA).
4.7. Assay of Luciferase Reporter
To generate reporter constructs for the assay of luciferase, a segment containing a miRNA target
site in the 30 -UTR of LRP6 and YAP1 was inserted into the psi CHECK-2 vector (Promega) between the
NotI and Xho sites immediately downstream of the Renilla luciferase gene. The wild-type segment and
mutant-type sequence were established by PCR overlap technology. The sequences of primers were
listed in Supplementary Data 5. All constructs were verified using sequencing.
The GMECs were seeded in 384-well plates at a density of 50,000 cells per well one day before
transfection. A total of 0.33 g of each reporter construct was transiently transfected using the X-treme
GENE HP DNA Transfection Reagent (Roche, Basel, Switzerland) on the basis of the protocol. Cells
were then transfected either with mimic or inhibitor using LipofectamineTM RNAi MAX on the basis
of the manufacturer’s protocol after a 6 h recovery period in medium. After 48 h post-transfection,
Firefly and Renilla luciferase activities were measured with the Dual-Glo luciferase assay system on the
basis of the manufacturer’s instructions (Promega, Beijing, China).
4.8. Statistical Analysis
Analysis of statistics was calculated by the SPSS software package, SPSS 19.0 (Beijing, China).
Data are presented as means ± SE (standard error) of three independent experiments. Significant
differences were detected when * p < 0.05 and ** p < 0.01.
5. Conclusions
Our results revealed that miR-30e-5p and miR-15 play an important role in fatty acid metabolism
in GMEC. In addition, miR-30e-5p not only affected Wnt signaling pathways by regulating LRP6 and
β-catenin, but also affects the Hippo signaling pathway by regulating miR-15a and YAP1 (miR-15 target
genes) in GMEC. MiR-30e-5p and miR-15a synergistically regulate fat metabolism via LRP6 and YAP1
in goat mammary epithelial cells. In the long-term, these findings might be helpful in developing
practical means to improve the quality of ruminant milk.
Supplementary Materials: Supplementary materials can be found at www.mdpi.com/1422-0067/17/11/1909/s1.
Acknowledgments: This research was jointly supported by the National Natural Science Foundation of China
(No. 31372281), the Special Fund for Agro-scientific Research in the Public Interest (201103038), and the Transgenic
New Species Breeding Program of China (2014ZX08009-051B).
Author Contributions: Jun Luo and Zhi Chen conceived and designed the experiments; Zhi Chen, Huiling Qiu
and Liuan Ma performed the experiments; Zhi Chen, Shuang Sun, Kang Kang, Deming Gou and Juan J. Loor
analyzed the data; Zhi Chen, Huiling Qiu and Liuan Ma contributed reagents/materials/analysis tools; Zhi Chen
wrote the paper. All the authors have read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
Int. J. Mol. Sci. 2016, 17, 1909
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