Elevated microRNA-34a contributes to trophoblast cell apoptosis in preeclampsia by targeting BCL-2 Short running title: miR-34a in preeclampsia Man Guo 1 , Xinying Zhao 2 , Xiaolei Yuan 1 , Peiling Li 1* 1 Department of Obstetrics and Gynecology, the Second Affiliated Hospital of Harbin Medical University, Harbin, China 2 Blood 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 1 1 1 2 3 4 5 6 7 8 9 10 11 12 2
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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.
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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