Placenta 51 (2017) 76e81

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Placenta
journal homepage: www.elsevier.com/locate/placenta

Increased NDRG1 expression attenuate trophoblast invasion through

ERK/MMP-9 pathway in preeclampsia
Yufen Fu a, b, Jufeng Wei a, c, Xueli Dai b, Yuanhua Ye a, d, *
a
Department of Obstetrics and Gynecology, Qingdao University, Qingdao 266000, China
b
Department of Obstetrics, Zibo Maternity and Child Health Hospital, Zibo 255000, China
c
Department of Obstetrics, Qingdao Central Hospital, Qingdao 266000, China
d
Department of Obstetrics, Afliated Hospital of Qingdao University, Qingdao 266000, China

a r t i c l e i n f o a b s t r a c t

Article history: Objective: The aim of this study was to investigate the expression of N-myc downstream-regulated
Received 31 October 2016 gene1(NDRG1)in the placentas of pregnancies complicated with early-onset and late-onset preeclamp-
Received in revised form sia (PE) and its underlying mechanism on the pathophysiology of PE.
6 January 2017
Methods: The expressions of NDRG-1 in placentas of pregnancies complicated with early-onset PE and
Accepted 25 January 2017
late-onset PE were detected using immunohistochemistry, western blot assays and uorescence quan-
titative PCR. The expressions of MMP-2, MMP-9 and ERK1/2 protein were detected by western blot
Keywords:
analysis and cell invasion assay was performed using transwell chambers in NDRG1 silenced JEG-3 cells.
NDRG1
Preeclampsia
Results: Compared with the normal term pregnancies, the expression of both NDRG1 mRNA and protein
Placenta were signicantly high in placentas from PE, and the expression of NDRG1 in early-onset PE was higher
Invasion than that in late-onset PE. In NDRG1-silenced JEG-3 cells, MMP-2, MMP-9 and phosphorylation of ERK1/2
ERK1/2 protein increased obviously and the number of cells that penetrated the membrane increased.
Conclusion: Upregulation of NDRG1 is associated with impaired trophoblast invasion in PE by inhibition
ERK/MMP-2 and MMP-9 Pathway.
2017 Elsevier Ltd. All rights reserved.

1. Introduction genes and N-myc downstream-regulated gene is involved [6].

Preeclampsia (PE) is a pregnancy-specic systemic complica-
tion, characterized by the new onset hypertension, edema and
proteinuria after 20 weeks of gestation. According to the gesta-
tional age by which it develops, PE can be classied as early-onset
(before 34 weeks of gestation) or late-onset (at or after 34 weeks of
gestation) [1]. Even though unremitting efforts have been taken for
many years, there is no consensus about the exact etiology and
pathogenesis of PE. Therefore, PE remains high incidence and a
major cause of maternal and neonatal mortality and morbidity
worldwide [2e4]. The pathophysiology of PE is characterized by
impaired trophoblast invasion of decidua spiral arteries, which
subsequently leads to inadequate uteroplacental blood ow, injury
to trophoblast tissue, and maternal multisystem dysfunction [5].
Trophoblast differentiation and apoptosis are regulated by several
* Corresponding author. Department of Obstetrics, Afliated Hospital of Qingdao
University, Qingdao University, Qingdao 266000, China.
E-mail address: yeyuanhua8067@126.com (Y. Ye).
N-myc downstream-regulated gene belongs to a family of pro-
teins (NDRG1-4) implicate in many cellular processes, including
differentiation, proliferation and invasion [7]. Diverse physiologic
or pathologic conditions, such as hypoxia, cellular differentiation,
exposure to heavy metal and neoplasia, modulate NDRG1 expres-
sion [8e10]. NDRG1 expression in tumor cells is directly involved in
the regulation of invasion and downregulation of NDRG1 can be
enhanced in vitro invasion [7]. NDRG1 has been identied to ex-
press in the placenta. In the second- and third-trimester placenta,
NDRG1 mRNA is expressed predominantly in syncytiotrophoblast.
Recent studies [11,12] have shown the expression of NDRG1 in
trophoblasts is upregulated under hypoxic conditions, such as in-
trauterine growth restriction. Nevertheless, the expression of
NDRG1 in placenta tissue of early-onset or late-onset PE and the
associated regulatory mechanisms remain unknown. Therefore, the
aim of the present study was to investigate the expression and
mechanism of NDRG1 in the placentas of early-onset and late
-onset PE.

http://dx.doi.org/10.1016/j.placenta.2017.01.126
0143-4004/ 2017 Elsevier Ltd. All rights reserved.
 Y. Fu et al. / Placenta 51 (2017) 76e81
2. Materials and methods
2.1. Patient information and placenta collection
The ethics committee of Afliated Hospital of Qingdao Univer-
sity approved the present protocol of the study. The experimental
groups were shown as follows. Control group (n 20) included
normal singleton term pregnancies. Early gestation group (n 20)
included healthy women undergoing legal abortion for nonmedical
reasons. Premature delivery group (n 20) included 24e37 years
old women with gestational age less than 37 weeks without other
complications. PE group included 40 individuals with PE between
the ages of 25e44 years who were recruited from September 2014
to March 2015. The diagnosis of the PE was based on clinical evi-
dence. According to onset time, they were divided into two sub-
groups, early-onset PE (early PE, n 20), and late-onset PE (late PE,
n 20). Women were excluded from the study who were smokers,
drinkers, chronic hypertension, gestational diabetes mellitus,
eclampsia, or women carrying multiple pregnancies. Each patient
signed the informed consent.
Placentas were collected immediately after a vaginal or
abdominal delivery under sterile conditions. After removing the
decidual layer, three pieces of tissue (each about
1.0  1.0  1.0 cm3) were obtained from each placenta, and rinsed
several times with ice-cold phosphate-buffered saline (PBS) to
remove excess of blood cells. Two samples were immediately
placed into cryotubes and stored at 80  C refrigerator for later
analysis, and the other piece of tissue was xed in 4% para-
formaldehyde for immunohistochemistry. Villous tissues were
collected after articial abortion, with the same treatment method
as the placentas.
2.2. Cell line
The behavior of trophoblast invasion is similar to tumor cell, so
JEG-3 was used in the study. JEG-3 was obtained from the cell bank
at the Chinese Academy of Sciences (Shanghai, China) and cultured
in minimum essential medium (MEM, Gibco, USA), containing 10%
(v/v) fetal bovine serum (FBS, HyClone, USA), and 1% penicillin/
streptomycin (Gibco, USA), maintained at 37  C in a humidied
atmosphere of 5% CO2.
JEG-3 cells were seeded in 6-well plates and allowed to attach
overnight to reach 30%e50% conuence at the time of transfection.
To suppress native NDRG1 expression, transfections were per-
formed with 50 nM NDRG1 siRNA for 72 h according to the man-
ufacturer's instructions (Ribobio Life Technologies, Guangzhou,
China), same volumes sterile PBS were added as the control cells.
2.3. Immunohistochemistry (IHC) analysis
Immunohistochemical staining for all parafn-embedded
placental tissues were performed by the streptavidin ebiotin-
peroxidase complex method. Briey, 5-mm thick sections were
prepared and incubated with primary antibody (NDRG1,1:150,
Rabbit anti human monoclonal antibody, Sigma) at 4  C overnight,
then incubated with the secondary antibody (Gene Tech, Shanghai,
China) for 30 min at 37  C. After rinsing in PBS, the sections were
incubated with 3,3'-diaminobenzidine liquid (DAB, ZSGB-BIO, Bei-
jing, China), counterstained with Mayer's hematoxylin, then
dehydrated. Immunoreactivity was evaluated independently by
two investigators who were blinded to experimental protocol ac-
cording to the intensity and extent of staining. The Image-Pro Plus
software (version 6.0; Media Cybernetics) was used to for densi-
tometry analysis.
77
2.4. Fluorescence quantitative PCR analysis
The total RNA was extracted from the tissue of the placenta
using Trizol regent (Invitrogen, Carlsbad, CA, USA) according to the
manufacturer's instructions. cDNA was synthesized using Super-
script II reverse transcriptase (Invitrogen) from 2 mg of total RNA.
Fluorescence quantitative PCR was performed using the TaKaRa
SYBR Premix Ex Taq II kit by ABI Prism 7500 Real-Time PCR System
(Applied Biosystems, Foster City, CA, USA). The PCR primers were as
follows: NDRG1, 50 -GAGGCTAGAGGCATTTGGAA-30 (Forward),50 -
CTTTTGCTGCACATTAAGAGGA-3' (Reverse), b-actin,50 -ATAGTTGCG
TTACACCCTTTCTTG-3' (Forward), 50 eTCACCTTCACCGTTCCAGTTT-
3(Reverse). A total of 2 ml of cDNA were mixed with 1.6 ml primers,
10 ml SYBR Premix Ex Taq II and 0.4 ml ROX Reference Dye II. Initial
denaturation was performed at 95  C for10min, followed by 40
cycles of denaturation at 95  C for 30s, then annealing at 60  C for
30s and extension at 68  C for 30s. A melting curve for primer
validation and a template standard curve were performed to show
template independent amplication results. Relative mRNA levels
were calculated using the 2-Ct method and normalized against
b-actin.
2.5. Western blot analysis
Frozen placental tissues were homogenized in lysis buffer con-
taining: 20 mM Tris (pH 7.4), 137 mM NaCl, 2 mM EDTA, 1 mM
PMSF, 1% Triton-X100, 20 mM leupeptin, 10% glycerol, 0.1% SDS,
0.5% deoxycholate and 5 mg/ml aprotinin. To prepare whole-cell
extracts, JEG-3 cells were washed in PBS before incubation with
RIPA lysis buffer. The insoluble material was removed by centrifu-
gation at 15 000 g at 4  C for 15 min and the protein concentration
of the supernatant was measured using the Bradford method of
protein quantitation by spectrophotometry at 595 nm (Beckman
DU530). Equivalent amounts of protein from each lysate sample
were mixed with protein loading buffer, heated at 95  C for 5 min.
The protein samples (20 mg) were separated by 10% sodium dodecyl
sulfate (SDS)-polyacrylamide gel electrophoresis, and then elec-
trotransferred to a nitrocellulose membrane. After being blocked
with 5% nonfat milk for 1 h at room temperature, the membranes
were incubated with primary antibodies (1: 2000, Abcam) against
NDRG1, MMP2, MMP9, ERK1/2 and PERK, GAPDH (1:2500 Abcam)
at 4  C overnight. The membranes were washed and incubated with
monoclonal HRP-conjugated antibody for 1 h at room temperature.
The immunoreactive bands were detected by using enhanced
chemiluminescence (Pierce Chemical Co., Rockford, IL, USA).
2.6. Cell invasion assay
Cell invasion assay was performed using transwell chambers
precoated with Matrigel (BD, USA). MEM with 10% FBS was added
into the lower chamber. First JEG-3 cells were pre-cultured with or
without NDRG1 siRNA (50 nM) for 72 h. Then the cells were washed
with PBS and suspended in MEM. 200 ml cell suspension
(1  105 cells/ml) was added into the upper chamber. After
culturing at 37  C for 24 h, the upper cells that did not invade
through the membrane were wiped out by a cotton tipped swab.
The lters were xed in methanol and stained with DAPI (40 ,6
-diamidino-2-phenylindole). The number of invasive cells whose
nuclei were stained in blue were counted under uorescence
microscopic. The experiment was performed three times.
2.7. Statistical analysis
The data were analyzed by statistical package SPSS22.0. Data are
presented as mean SD. Statistical analysis was performed using
78 Y. Fu et al. / Placenta 51 (2017) 76e81

Table 1
The demographic and clinical characteristics of the participants.

Characteristics Control Early gestation Premature delivery Early PE Late PE

Cases 20 20 20 20 20
Times of gravidity 3.01 1.62 2.32 1.08 2.17 0.98 2.85 1.57 2.69 1.32
Gestational age (week) 39.02 3.16 8.5 1.32 32.58 3.36 33.49 2.83 37.95 2.66
Proteinuria (cases) 0 0 0 20 20
Systolic blood pressure (mmHg) 113.69 10.70 110.46 11.34 120.65 17.47 153.39 10.72b 158.74 18.84b
Diastolic blood pressure (mmHg) 74.52 10.56 76.49 8.55 71.95 6.72 113.58 9.8b 103.74 13.79b
Birth weight (g) 3773 935.8 2526 226.7 1728 190.1bc 2230 272.1a

a. p < 0.05, versus the control, b. p < 0.01, versus the control, c. p < 0.01, versus late PE.

one-way analysis of variance (ANOVA) followed by Student-
Newmann-Keuls multiple comparison tests and independent
samples t-test. P < 0.05 was considered statistically signicant.
3. Results
3.1. Clinical characteristics
The clinical characteristics of these subjects were presented in
Table 1. There were no signicant differences in times of gravidity
among all groups. All the PE patients suffered from proteinuria.
Obviously, in either early PE or late PE group, systolic blood pres-
sure or diastolic blood pressure was higher than in other groups
(P < 0.01). However, birth weight in PE group was particularly lower
than that in control group. Neonates from early-onset PE had lower
birth weight than those from late-onset PE (P < 0.05).
3.2. The expression of NDRG1 was upregulated in placentas of PE
Frist we evaluated the expression of NDRG1protein in placental
villi at different stages of pregnancy by immunohistochemistry
staining. No obvious immunostaining was observed during early
gestation, while NDRG1 protein was specically stained in the term
placenta (Fig. 1). NDRG-1 was located in the cytotrophoblast, syn-
cytiotrophoblast and intercellular substance. The expression of
NDRG1 in placentas from pregnancies complicated with early-
onset PE was the highest in all groups, and the expression in late
onset PE group was still higher than others groups. Furthermore,
the expression of NDRG1 in early-onset PE was higher than that in
late onset PE.
To compare the expression of NDRG1 mRNA in all groups, we
also performed quantitative PCR analysis. Compared with term
pregnancies, the expression of NDRG1mRNA in villous tissues from
early gestation group was lower, while it was much higher in pla-
centas from early-onset PE. It was noteworthy that the expression
of NDRG1mRNA from early-onset PE was higher than that from
premature delivery group, and the expression of NDRG1mRNA
from late-onset PE was higher than that from control. Compared
with control group, the relative expression of NDRG1mRNA in late-
onset PE increased by 53.3%. Compared with premature delivery
group, the relative expression in early-onset PE increased by 53.41%
(Fig. 2A). Moreover, the expression of NDRG1 in placentas from

Fig. 1. Expression of NDRG1 in placental villi by immunohistochemistry (400).

A. The representative images of NDRG-1 expression in human placentas tissue were detected by immunohistochemistry, the black arrows showed positive staining. a. control group,
b. early gestation group, c. premature delivery group, d. early PE group, e. late PE group. B. The average optical density assay of immunohistochemistry staining (n 16).*P < 0.05,
**P < 0.01.
 Y. Fu et al. / Placenta 51 (2017) 76e81
Fig. 2. The level of NDRG1 mRNA and protein increased signicantly in placentas from
the early PE or late PE group.
A. Fluorescence quantitative PCR analysis of NDRG1 in placentas tissue. Relative mRNA
levels were calculated using the 2Ct method, normalized against b-actin (n 20).
*P < 0.05, **P < 0.01. B. Western blot analysis of NDRG1 in placentas tissue. The level of
NDRG1 protein in placentas from the early PE or late PE group was signicantly higher
than that from control (n 10). *P < 0.05, **P < 0.01.
early PE was higher than that from late-onset PE. The similar results
were obtained by using western blot analysis of NDRG1 protein
(Fig. 2B). These results support the ndings obtained with immu-
nohistochemistry and indicate that NDRG1 expression is enhanced
in PE.
3.3. Silencing of NDRG1 increased the invasion ability and elevated
the levels of MMP-2, MMP-9 and phosphorylated ERK of JEG-3 cells
To evaluate the biological roles of NDRG1 in trophoblast inva-
sion of placentas in PE, JEG-3 cells were transfected with a specic
siRNA to silence NDRG1 and used for the subsequent studies. The
expression of NDRG1 was markedly decreased by 91.14% in NDRG1-
silenced cells compared with control cells, as conrmed by western
blot analysis (Fig. 3A).
To investigate the effects of NDRG1 on the invasion of JEG-
3 cells, transwell chambers with Matrigel-coated lter were used.
Following 24 h incubation, the number of NDRG1 silenced JEG-
3 cells that penetrated the membrane (50.3 5.9) was even higher
79
compared with that of control cells (21 5) (P 0.0027; Fig. 4). So
the data suggested that NDRG1 would lead to a signicantly
decreased invasion of JEG-3 cells.
Next, MMP-2 and MMP-9 protein in NDRG1 silenced JEG-3 cells
were detected by western blot. As shown in Fig. 3AeB, compared
with control group, the expression of MMP-2 protein signicantly
increased by 53.7% and the expression of MMP-9 obviously
increased by 48.83% in NDRG1 silenced cells. These results sug-
gested that NDRG1 was involved in the regulation of both expres-
sion and the secretion of MMP-2 and MMP-9 in JEG-3 cells.
Mitogen-activated protein kinase (MAPK) cascade is an impor-
tant pathway that responds to extracellular stimuli and regulates
MMP-2 or MMP-9 expression, and the extracellular signal-
regulated kinase-1/2 (ERK1/2) signaling pathway is preferentially
activated, so ERK1/2 was also examined by immunoblotting. There
was a signicant increase in phosphorylated of ERK1 at 44 kDa
(Thr202/Tyr204) and ERK2 at 42 kDa (Thr185/Tyr187) in NDRG1
silenced JEG-3 cells. However, there was no obvious alteration in
total ERK (Fig. 3AeB). These results demonstrated that the
increased invasion capability in vitro of NDRG1-decient cells was
likely mediated by MAPK/ERK cascade-regulated MMP-2 and
MMP-9 expression.
4. Discussion
Preeclampsia is one of the most common complications of
pregnancy and associated with high risks of preterm delivery, in-
trauterine growth restriction, placental abruption and perinatal
mortality. Despite extensive research, the cause of preeclampsia
remains elusive. The extravillous trophoblast invasion is an essen-
tial process for embryo implantation and placental formation. If
this invasion is inhibited, consequent placental dysfunction will
cause various obstetrical disorders such as preeclampsia [5].
NDRG1 is known to have a role in regulation of trophoblast dif-
ferentiation and response to injury [8e10]. To date, the determined
function of NDRG1 in trophoblast invasion is still unclear.
NDRG1 is one of the proteins that were markedly upregulated in
hypoxic trophoblasts [9,11]. The expression of NDRG1 was upre-
gulated in placentas from pregnancies complicated by IUGR
compared with placental samples from appropriately grown fe-
tuses [9]. In the present work, we found that the fetuses in PE group
developed lower weight than those in control group, and the fe-
tuses in early-onset PE group developed lower weight than those in
late-onset PE group. We also demonstrated that expression of both
NDRG1 mRNA and protein was upregulated in placentas derived
from early-onset PE compared with late-onset PE, and the
expression of NDRG1 in early-onset PE and late-onset PE were
higher than that in control, early gestation or premature delivery. In
order to further clarify the precise role of NDRG1 in PE, in vitro
approaches were also used in human choriocarcinoma cell line -
JEG-3 cells, a suitable cell line to study cell motility and migration
[13,14].
NDRG1 expression in tumor cells is directly involved in the
regulation of invasion and migration, and downregulation of
NDRG1 can strengthen invasion in vitro [15]. To investigate whether
NDRG1 can indeed inuence the trophoblast invasion progress, we
downregulated NDRG1expression in JEG-3 cells, which demon-
strated that exceptionally low expression of NDRG1 resulted in a
signicant increase in the invasion of JEG3 cells.
The trophoblast invasion process includes complex mechanisms
which can be inuenced by many molecules that relate to cell
growth, adhesion, differentiation, and degradation of extracellular
matrix (ECM), etc, among which, the proteolytic degradation of
ECM has been considered to help trophoblast to invade into uterine
endometrium [16]. The MMPs are the key family of proteolytic
80 Y. Fu et al. / Placenta 51 (2017) 76e81

Fig. 3. NDRG1, MMP-2, MMP-9 and ERK1/2 protein expression in NDRG1 silenced JEG-3 cells.
A. JEG-3 cells were transfected with 50 nM NDRG1 siRNA for 72 h. The protein expression of NDRG1, MMP-2, MMP-9, ERK1/2 and pERK1/2 were evaluated by western blot in
transfected cells. B. Statistical analysis of the western blot results (n 3). *P < 0.05, **P < 0.01 versus control.
 Y. Fu et al. / Placenta 51 (2017) 76e81
Fig. 4. Effects of NDRG1 on invasion of JEG-3 cell.
JEG-3 cells were pre cultured with or without NDRG1 siRNA (50 nM) for 72 h. Then
200 ml cell suspension (1  105 cells/ml) was added into the upper chamber. MEM with
10% FBS was added into the lower chamber. Following incubation for 24 h, the invasive
cells were stained with DAPI and recorded under uorescence microscope. The upper
pictures showed the nuclei of the invasive cells and the lower picture showed statis-
tical analysis of the number of invasive cells (n 3). *P < 0.01 versus control.
enzymes involved in the trophoblast invasion, and MMP-2 has been
shown to be one of the important enzymes for degrading type IV
collagen in the invasion, MMP-9 is identied as trophoblast derived
MMP [17]. To investigate how NDRG1 inuence the invasion pro-
cess, we detected the expression of MMP-2 and MMP-9 in NDRG1
silenced cells. The results showed the expression of MMP-2 and
MMP-9 was upregulated in NDRG1 silenced cells. It suggests
NDRG1 expression may inhibit the trophoblast invasion via
decreasing MMP-2 and MMP-9 expression. Previous studies have
indicated that ERK1/2 phosphorylation is responsible for the MMP
activation [18]. Hence, we examined the response of phosphory-
lated ERK1 and ERK2 (p-ERK1/2) and total ERK1/2. NDRG1 silenced
cells displayed increased levels of phosphorylated ERK1/2, how-
ever, the levels of ERK1/2 protein were not different between
NDRG1silenced cells and control. This indicated NDRG1 was at least
partly responsible for the decrease in p-ERK1/2. Moreover, the
expression of MMP-2 and MMP-9 was increased in NDRG1 silenced
cells. Thus, we infer NDRG1 may suppress p-ERK1/2 signaling and
its downstream effects on MMP-2 and MMP-9. (Fig. 3AeB).
In summary, our results suggest that upregulation of NDRG1 is
associated with impaired trophoblast invasion in PE by inhibition
MMP-2 and MMP-9 expression. However, the in vitro model can't
fully mimic the normal human endometrium in vivo. Thus, the
exploration of these ndings requires further investigation. Future
studies will be performed to demonstrate the regulating factors and
signaling pathways involved in the NDRG1 of PE. We believe that
future studies addressing the mechanisms by which NDRG1
81
inhibits trophoblast invasion may bring more knowledge regarding
its roles in PE therapy.
Disclosure of conict of interest
All authors declare they do not have any conicts of interest.
Acknowledgments
We thank the teachers in Central Laboratory of Afliated Hos-
pital of Qingdao University for their help and support.
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