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Elevated microRNA-34a contributes to trophoblast cell apoptosis in
preeclampsia by targeting BCL-2
Short running title: miR-34a in preeclampsiaMan Guo1, Xinying Zhao2, Xiaolei Yuan1, Peiling Li1*
1Department of Obstetrics and Gynecology, the Second Affiliated Hospital of Harbin
Medical University, Harbin, China
2Blood Dialysis Center, General Hospital of Heilongjiang Agricultural Reclamation
Bureau, Harbin, China
Address: Department of Obstetrics and Gynecology, the Second Affiliated Hospital of
Harbin Medical University, 246 Xuefu Road, Nangang District, Harbin, Hei
Longjiang Province, 150081, People’s Republic of China.
Fax: +86-451-86297003; Tel: +86-451-86296140
*Corresponding author: Peiling Li, [email protected]
Other authors: Man Guo, [email protected]; Xinying Zhao, [email protected];
Xiaolei Yuan, [email protected]
Abstract word count: 182
Word count: 2623
Number of figures: 4
Number of tables: 2
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Summary Table
What is known about this topic
1. Preeclampsia (PE) is one of the most common pregnancy-specific pathologic
complications, and is characterized by onset of hypertension and proteinuria.
2. Some studies have reported that trophoblast cell apoptosis occurs in PE and may
play a crucial role in the disease process.
3. miRNA (miR)-34a has been widely studied cell apoptosis.
What this study adds
1. This study showed that upregulation of miR-34a induced trophoblast cell apoptosis
in PE.
2. miR-34a inhibition reversed miR-34a-induced apoptosis in the HTR-8/SVneo
human trophoblast cell line.
3. miR-34a may be linked to the occurrence of PE via effects on BCL2 in the human
placenta, and may therefore provide a potential therapeutic target for PE.
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Abbreviations
AMO-34a Anti-miRNA-34a oligonucleotide
BCL-2 B cell CLL/lymphoma 2
Ctrl Control
miRNA/miR MicroRNA
MTT 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide
NC Negative control
qPCR Quantitative PCR
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Abstract
Preeclampsia (PE) is one of the most common pregnancy-specific pathologic
complications, and is characterized by onset of hypertension and proteinuria.
Placental trophoblast cell apoptosis is generally accepted as a major cause of PE.
However, the details of the mechanism underlying the condition remain unclear. Here,
we aimed to investigate a possible association between microRNA (miR)-34a and
human trophoblast cell apoptosis during PE. We evaluated miR-34a expression in
placentas from patients with PE compared with those from healthy pregnant
individuals. Furthermore, we measured apoptosis rate after miR-34a mimic and/or
inhibitor transfection in vitro, and identified B cell CLL/lymphoma 2 (BCL2) as a
target of miR-34a. We found that miR-34a levels were significantly higher in
placental tissues from patients with PE than in normal placentas. Upregulation of
miR-34a induced trophoblast cell apoptosis in PE by inhibiting expression of BCL-2
protein. miR-34a inhibition reversed miR-34a-induced apoptosis in the HTR-8/SVneo
human trophoblast cell line. Our findings indicate that miR-34a may be linked to the
occurrence of PE via effects on BCL2 in the human placenta, and may therefore
provide a potential therapeutic target for PE.
Keywords: Preeclampsia; miR-34a; Apoptosis; Placenta
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1. Introduction
Preeclampsia (PE) is a major hypertensive disorder specific to pregnant women
and the leading cause of maternal morbidity worldwide [1]. It is characterized by
new-onset hypertension, along with proteinuria after 20 weeks of gestation [1]. The
causes of the disease may relate to the mother, the placenta and/or the fetus, and can
include many factors such as abnormal immune regulation, endothelial cell damage,
genetic factors, and nutritional factors [2,3]. However, there is no single factor that
can satisfactorily explain the cause and mechanism of PE. Some studies have reported
that trophoblast cell apoptosis occurs in PE and may play a crucial role in the disease
process [4,5]. However, the details of the mechanisms underlying trophoblast cell
apoptosis in PE remain to be further studied.
microRNAs (miRNAs) are small noncoding RNA molecules (19–22 nt), which
are involved in post-transcriptional regulation of target mRNAs [6]. Previous studies
have indicated that miRNAs are closely linked to many biological and pathological
processes, including cell proliferation [7], apoptosis [8], oncogenesis [9], type 2
diabetes [10] and cardiovascular disease [11]. Growing evidence supports that there
have been many miRNAs which contribute to PE [12,13]. miRNA (miR)-34a, a
classic regulator closely associated with apoptosis, has been widely studied in cancer
cells [14], tubular cells [15]. However, limited studies have reported on the role of
miR-34a in PE.
In this study, we found that miR-34a is upregulated in placental tissues from
patients with PE. In vitro inhibition of miR-34a reversed cell apoptosis in
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HTR-8/SVneo cells. miR-34a may contribute to trophoblast cell apoptosis in PE by
targeting BCL-2, an anti-apoptosis member of Bcl-2 family. The Bcl-2 family includes
both pro-apoptosis members (Bax, Bak, and Bad) and anti-apoptosis members (Bcl-2,
Bcl-XL, and Bcl-w) [16,17]. These findings highlight the important role of miR-34a
in the pathogenesis of PE, and provide new insight into the development of the
disease.
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2. Materials and Methods
2.1. Patients and sample collection
All experimental procedures were approved by the Ethical Committee for the use of
Human Samples of Harbin Medical University, and written informed consent was
provided by all participants. Placental tissues were obtained from 26 healthy and 29
severe preeclamptic pregnant women aged 28–36 years who were hospitalized in the
Department of Gynecology and Obstetrics of the Second Affiliated Hospital of Harbin
Medical University, China. The grade of PE was diagnosed according to the definition
in 12th Five-Year ordinary higher education undergraduate national planning textbook
of Obstetrics and Gynecology (eighth edition), People's Medical Publishing House,
page 64-72. Briefly, patients with PE were defined as those who exhibited systolic
blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg on two or more
occasions, accompanied by proteinuria, after gestational week 20. Women with
chronic hypertension, renal disease or other complications were excluded from the
study. Severe PE was defined by the presence of more than one of the following
points listed in supplementary material. All placental tissues were collected
immediately after placental caesarean delivery.
2.2. HE staining and immunohistochemistry
Tissues were fixed in 4% paraformaldehyde and embedded in paraffin. BCL-2 was
then immunostained with rabbit anti-BCL-2 monoclonal antibody (Cell Signaling
Technology, Cat. No. 15071, 1:400)
2.3. Cell culture
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HTR-8/SVneo cells were obtained from ATCC and cultured in RPMI-1640
supplemented with 10% fetal bovine serum, 50 µg/ml streptomycin and 50 IU/ml
penicillin. Experimenters were blind to group assignment and outcome assessment.
2.4. Cell viability measurements
Cell death was evaluated by assessment of lactate dehydrogenase (LDH) release and
MTT assay. For LDH measurement, the culture media were collected and assessed
using an LDH assay kit (Thermo Fisher Scientific Inc., Cat. No. 88953). For the MTT
assay, HTR-8/SVneo cells were seeded in 96-well plates. The supernatant media were
discarded and 100 µl of MTT solution (5.0 mg/ml) was added per well. After
incubation for 4 h at 37 °C, the crystals that had formed were dissolved in dimethyl
sulfoxide, and the formazan salt extracted was quantified by measuring absorbance at
570 nm using a SpectraMax M2 microplate reader (Molecular Devices, USA).
2.5. Apoptosis assay
Cells were harvested and stained with anti-annexin V antibody and propidium iodide
solution, and fluorescence was detected by flow cytometry (BD LSRFortessa,
Franklin Lakes, NJ, USA).
2.6. Luciferase activity assay
We obtained a pmiR-RB-REPORT™ dual luciferase reporter vector carrying
fragments of the BCL2 3′-UTRs that contain target sites for miR-34a, from
Guangzhou RiboBio Co., Ltd, Guangzhou, China. The plasmid construct (200 ng)
was transfected into HEK293 cells (1 × 105 cells/well, 24-well plate) using
Lipofectamine 2000 (Invitrogen, Cat. No. 11668019). The HEK293 cells were
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obtained from SGST.CN. A dual luciferase reporter assay kit (Promega, Madison, WI)
and a GloMax 20/20 Luminometer (Promega) were used to measure firefly and renilla
luciferase activities 24 h after transfection.
2.7. Western blot analysis
Protein samples were extracted from placental tissues and HTR-8/SVneo cells as
described previously [18]. Protein concentration was detected using a BCA protein
assay kit (Beyotime, Cat. No. P0010). Protein samples (approximately 80 µg) were
separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and
transferred to polyvinylidene fluoride membranes. Membranes were probed with
primary antibodies against PARP-1 (Cell Signaling Technology, Cat. No. 9532, rabbit
monoclonal, 1:1000), Caspase-3 (Cell Signaling Technology, Cat. No. 9662, rabbit
polyclonal, 1:1000), BCL-2 (Cell Signaling Technology, Cat. No. 2876, rabbit
polyclonal, 1:1000) and α-tubulin (Santa Cruz, Cat. No. sc-32293, mouse monoclonal,
1:1000). Alkaline phosphatase-conjugated secondary antibody was used (Promega,
s3728, s3738).
Quantitative real-time polymerase chain reaction (PCR)
miRNA was isolated from placental tissues and HTR-8/SVneo cells using a mirVana
miRNA isolation kit (Ambion, Austin, TX). Quantitative real-time PCR (qPCR) was
performed using SYBR Green PCR Master Mix (Applied Biosystems, Foster City,
CA), with U6 used as a control. The sequences of the primers used in this study are
listed in Table 1.
2.9. Transfection
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HTR-8/SVneo cells were transfected with miRNA and/or anti-miRNA
oligonucleotides (AMOs) or negative control (Guangzhou RiboBio Co., Ltd.,
Guangzhou, China) using Lipofectamine 2000 (Invitrogen), as described previously
[19]. Cells were collected for RNA or protein detection 48 h after transfection.
2.10. Statistical Analysis
Values are expressed as mean ± SD. Multiple groups were analyzed with one-way
ANOVA followed by a Student–Newman–Keuls test. The ANOVA results were
displayed in Suppl. Table 1, 2 and 3. Two-group-only comparisons were carried out
by t test. P < 0.05 was considered statistically significant.
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3. Results
3.1. Clinical characteristics
Clinical data were obtained from 29 pregnant women with PE and 26 healthy
pregnant control participants. Blood pressure, 24-h urine protein and body mass index
were significantly higher, and gestational day at delivery and infant birth weight were
significantly lower, in preeclamptic women than in control participants (p < 0.01).
There were no significant differences in maternal age and maternal smoking number
between the two groups. The clinical characteristics are summarized in Table 2.
3.2. Apoptosis occurs in placentas from patients with PE
Immunohistochemical analysis showed that BCL-2 expression levels were
significantly lower in placentas from patients with PE than that in those from control
participants (normal pregnancy), which revealed that apoptosis occurred in PE-
affected placental tissues (Figure 1A). Furthermore, western blot analysis revealed
that expression levels of cleaved Caspase-3 and cleaved PARP-1 were significantly
higher in placentas from patients with PE than in those from control participants
(Figure 1B).
3.3. miR-34a is upregulated in placentas from patients with PE
We examined miR-34a expression levels in control and preeclamptic placental tissues
using qPCR analysis. miR-34a levels were four times higher in preeclamptic tissue
than in control tissue (p < 0.01; Figure 2).
3.4. miR-34a induces apoptosis in HTR-8/SVneo cells
To explore the role of miR-34a in preeclamptic trophoblast cells, we transfected a
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miR-34a mimic into HTR-8/SVneo cells. An MTT assay (Figure 3A) and LDH
analysis (Figure 3B) 24 h after transfection indicated that the rate of cell death was
significantly increased by expressing the miR-34a mimic. This effect was reversed by
AMO-34a administration. Moreover, flow cytometry analysis revealed that miR-34a
significantly increased apoptosis in HTR-8/SVneo cells (Figure 3C). Furthermore,
expression of cleaved Caspase-3 and cleaved PARP-1 were significantly increased
after transfection of miR-34a; again, this effect was reversed by AMO-34a (Figure
3D). Transfection of AMO-34a into HTR-8/SVneo cells efficiently reduced
intracellular expression of miR-34a (up to 90%) (Suppl. Figure 1).
3.5. Validation of BCL2 as a direct target of miR-34a
We next sought to identify specific target genes of miR-34a. Expression of BCL-2
protein and the mRNA BCL2 and BCL2L2 level, which contributes to anti-apoptotic
pathway regulation [20], was significantly lower in placentas from patients with PE
than in those from control participants by western blot (Figure 4A, Suppl. Figure 2).
In HTR-8/SVneo cells, overexpressing miR-34a decreased BCL-2 levels; this effect
was efficiently reversed by addition of AMO-34a (Figure 4B). Furthermore, a
luciferase assay verified that miR-34a overexpression inhibited luciferase activity in
HEK293 cells transfected with a plasmid carrying the 3′-UTR of BCL2 gene (Figure
4C).
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4. Discussion
Accumulating evidence indicates that miRNAs are irregularly expressed in
preeclamptic placentas and closely associated with PE [12,13]. However, the
molecular mechanisms of the involvement of miRNAs in the modulation of the
trophoblast cell function are still unclear; especially the role of miRNAs in the
trophoblast cell apoptosis in PE remains largely unknown. In this study, we provide
evidence that miR-34a is upregulated in placental tissues of patients with PE. Our
mechanistic studies revealed that miR-34a upregulation modulates trophoblast cell
apoptosis in PE by inhibiting expression of BCL-2 protein, implying that miR-34a
plays a fundamental role in PE development.
The mechanisms underlying the development and progression of PE are very
complex. The pathogenic process begins in the first three month of pregnancy, long
before clinical signs emerge. Hence, it is difficult to identify early biomarkers. It is
critically important to find new methods to predict PE occurrence, and to develop
effective approaches to stop the process. Although extensive research on the
mechanism of PE has been conducted recently, the pathogenic mechanisms remain
unclear. PE is a vascular disease induced by multiple factors; growing evidence
indicates that endoplasmic reticulum stress, inflammatory response, apoptosis and
miRNAs play important roles in the disease process [4,5,12,13,21,22].
Apoptosis occurs in normal placental tissue, in a dynamic balance with
proliferation during different stages of pregnancy [23]. Recently, interest has been
raised by the observation of increased levels of villous trophoblast apoptosis in
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placental pathologies, including early pregnancy loss [24] and PE. In PE, there is a
reduction in trophoblast cell number within the spiral arteries; this is related to
reduced luminal size and increased apoptosis in severe PE [25]. Growing body of
evidence indicates that apoptosis plays an important role in the development of PE
[4,5]. In this study, we used immunohistochemistry and western blotting to detect
apoptosis in placentas from patients with PE, and obtained strong positive results. Our
results further support the involvement of apoptosis in PE.
The mechanistic pathways responsible for trophoblast cell apoptosis in PE are
not fully understood. Recent evidence has provided new clues that miRNAs are
involved in the regulation of PE and human placental diseases [12,13,26]. Although
alteration of the miRNA profile of PE individuals has been widely investigated
[12,13], the miRNA that is most directly associated with PE remains unclear. In our
study, we selected miR-34a, a well-established regulator of apoptosis [14], that are
differentially expressed in placental tissues of PE patients relative to normal
pregnancy. miR-34a displayed the significant fold-change increase, which was in line
with previous observations that high level of miR-34a was present in the placentas of
20 preeclamptic patients [27]. However, Doridot et al. reported that pri-miR-34a was
overexpressed in the preeclamptic placentas but the mature miR-34a level was
decreased [28], of which the contradictory results may be attributed to the technical
flaw in miRNA extension [28]. To demonstrate the potential role of miR-34a-
mediated apoptosis in trophoblast cells, endogenous miR-34a was abrogated by
AMO-34a in HTR-8/SVneo cells. Over-expression of miR-34a significantly enhanced
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apoptosis. AMO-34a relieved this cytotoxic effect, and reversed the upregulation of
apoptosis-related protein expressions on HTR-8/SVneo cells. These results suggest
miR-34a play a crucial role in trophoblast cell apoptosis in PE, which may accelerate
the progress of PE.
The mechanism of action of miR-34a in PE, and whether it regulates apoptosis,
are questioned that remain to be answered. Other studies have revealed BCL-2 is a
target of miR-34a in apoptosis in cancer cells [29]. Axt-Fliedner found that the BCL-2
gene is widely expressed in many embryonic organizations [30]. The balance of
expression of BCL-2 family proteins in placental tissues plays an important role in
fetal development. Aban and Ishihara further revealed that BCL-2 is downregulated in
the placentas from patients with PE compared with in normal healthy pregnant
women [31,32]. Here, we too observed this phenomenon. Bioinformatics target
prediction identified BCL-2 as a target of miR-34a. In HTR-8/SVneo cells,
overexpressing miR-34a decreased BCL-2 levels; this effect was efficiently reversed
by addition of AMO-34a. Moreover, we used a luciferase assay to verify this target in
HEK293 cells as previous studies [33,34]. Our results indicate that miR-34a
overexpression can inhibit BCL-2 expression in vitro. On the basis of these results, we
suggest that miR-34a may be involved in trophoblast cell apoptosis in PE by targeting
BCL-2.
Some issues remain unsolved by this study. First, the sample size is limited and a
pregnant cohort would be needed for further validation of miR-34a or other results.
Second, although it would be ethically challenging clinically, the verification of the
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action of miR-34a in vivo model was necessary to be clarified in the future studies.
Meanwhile, it will be essential to identify the factors that induce miR-34a
upregulation. Additionally, further research will be performed in the future study for
analyzing the function of other miRNAs in this processing.
In conclusion, we provide evidence that miR-34a is elevated in placental tissues
from patients with PE. It appears that miR-34a upregulation and the associated
inhibition on BCL-2 plays a critical role in mediating trophoblast cell apoptosis in PE.
Collectively, these results allow us to propose a novel signaling pathway linking
trophoblast cell apoptosis to PE. Our findings provide novel insight into trophoblast
cell apoptosis-induced PE progression, whereby lowering miR-34a might be an
effective strategy for improving apoptosis in trophoblast cells.
Conflict of interest
The authors have no conflicts of interest to declare.
Acknowledgments
This study was supported by the Science and Technology Grant from Education
Department of Heilongjiang Province, China (12521347).
M.G. and P.L. conceived and designed the experiments. M.G., X.Z., and X.Y.
performed the experiments. M.G. and X.Y. analyzed data and wrote the manuscript.
P.L. reviewed and edited the manuscript.
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Figure legends
Figure 1
Apoptosis exists in PE placental tissues.
(A) Immunohistochemical assay shows the localization of BCL-2 in human placenta.
HE staining was performed in paraffin sections of human placenta. Direct
magnification ×200. Scale bar, 10 μm. (B) Western blot analysis of cleaved-PARP-1/-
Caspase 3 in placental tissues from normal and PE patients. *p < 0.05 vs Ctrl group. n
= 6 for each group.
Figure 2
Differential expression of miR-34a in placental tissues from PE patients.
qPCR verifies upregulation of miR-34a in placental tissues with PE. **p < 0.01 vs
Ctrl; n = 29 placentas derived from PE patients and n = 26 placentas from normal
pregnant women.
Figure 3
miR-34a induced cell death in HTR-8/SVneo cells.
(A) Effect of miR-34a on HTR-8/SVneo cells survival rate. (B) Cells death was
measured with LDH release. (C) Cell apoptosis was assessed by FCM with FITC and
PI staining after transfection of miR-34a. (D) Western blots verifying the effect of
miR-34a on expression of cleaved PARP-1 and cleaved Caspase 3 proteins. *p < 0.05
vs Ctrl group; ^p < 0.05 vs miR-34a group; each in vitro test was performed 5 times.
Ctrl: transfection of miR-NC (negative control) only in HTR-8/SVneo cells; miR-34a:
transfection of miR-34a alone in HTR-8/SVneo cells; miR-34a+AMO-34a: cells
transfected with AMO-34a after miR-34a treatment.
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Figure 4
Repression of BCL-2 genes by miR-34a.
(A) Downregulation of BCL-2 in placental tissues from PE patients. (B) Verification
of the specificity of miR-34a mimics to block expressions of BCL-2. (C) Effect of
miR-34a on 3′UTR of BCL2 determined by luciferase activity assay. *p < 0.05 vs Ctrl,
^p < 0.05 for the indicated comparison. n = 6 placentas per group; each in vitro test
was performed 5 times. NC, negative control.
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