Preeclampsia (PE) is a pregnancy-specific systemic disorder characterized by various manifestations of organ dysfunction. Inadequate trophoblastic invasion of the uterine wall is involved in the pathogenesis of PE. Angiopoietin-like protein 8 (ANGPTL8) serves an important role in cardiovascular disease development and may have a potential effect on cell proliferation. In the present study, downregulation of ANGPTL8 promoted cell proliferation, decreased p21 expression, and increased the expression levels of cyclin-dependent kinase 2 and proliferating cell nuclear antigen in HTR8/SVneo cells. Silencing of ANGPTL8 led to significant acceleration in cell migration and invasion, and markedly enhanced the matrix metalloproteinase (MMP)-2 and MMP-9 expression levels. In addition, the protein expression levels of tissue inhibitor of matrix metalloproteinase (TIMP)-1 and TIMP-2 were decreased in the group transfected with small interfering RNA (si)-ANGPTL8-1 as compared with those in the control and si-negative control groups. Taken together, these results indicated that ANGPTL8 downregulation promoted the proliferation, migration and invasion of trophoblast cells. Thus, ANGPTL8 suppresses the viability, proliferation, migration and invasion of trophoblast cells, and may be a potential therapeutic target for the clinical treatment of PE.
Preeclampsia (PE) is a pregnancy-specific disorder that causes considerable maternal and perinatal morbidity and mortality worldwide (
Angiopoietin-like proteins (ANGPTLs) comprise a class of secreted factors that are characterized by an N-terminal coiled-coil domain and a C-terminal fibrinogen-like domain, and are structurally similar to angiopoietin. However, unlike other ANGPTLs, ANGPTL8 lacks the C-terminal fibrinogen-like domain (
The present study aimed to investigate the effect of ANGPTL8 and the molecular mechanisms underlying the pathogenesis of PE in HTR8/SVneo cells. Furthermore, the serum expression of ANGPTL8 and its potential correlation in patients with PE and healthy subjects were explored. The current study results provided evidence that ANGPTL8 affects the proliferation, invasion and migration of trophoblast cells to promote PE.
A total of 30 patients with PE and 30 healthy pregnant women who underwent cesarean deliveries and hospitalized (July 2016 to April 2018) in Xuzhou Central Hospital (Xuzhou, China) were recruited into the present study. The main clinicopathological characteristics of the participants are summarized in
The immortalized human trophoblast cell line HTR8/SVneo was purchased from the Type Culture Collection of the Chinese Academy of Sciences. HTR/SVneo cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.). The cells were incubated in a humidified atmosphere containing 95% air and 5% CO2 at 37̊C.
siRNAs were synthesized by Santa Cruz Biotechnology, Inc., and transfected into the HTR8/SVneo cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. The cells were divided into three groups, as follows: Control group (without any treatment), si-negative control (NC) group (transfected with an unrelated sequence), and si-ANGPTL8 group (transfected with ANGPTL8-siRNA). At 48 h post-transfection, the cells were harvested and subjected to western blotting or reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay to detect the efficiency of transfection. The sequences of si-ANGPTL8 used are as follows: si-ANGPTL8-1: 5'-AAGCCCACCAAGAATTTGAGA-3'; si-ANGPTL8-2: 5'-TATGACAGAGCACTGGAATTC-3'.
Serum was separated by centrifugation (900 x g; 5 min) of the blood samples at 4˚C and stored at -80̊C until further assay. Subsequently, the serum concentration of ANGPTL8 was quantified using an ELISA kit (cat. no. NBP2-68217; R&D Systems) according to the manufacturer's protocol.
Cells were washed with ice-cold phosphate buffer, and total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and measured using a Qubit machine (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Next, RNA (1 µg) was reversed transcribed using RT reagents (Takara Bio, Inc.) to synthesize complementary DNA. qPCR was then conducted using a 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) and the following PCR cycling protocol was used: 95˚C for 2 min and then 40 cycles of 94˚C for 15 sec, 60˚C for 20 sec, 72˚C for 20 sec, followed by 72˚C for 7 min. The primer sequences for qPCR were as follows: ANGPTL8 forward, 5'-ATTCCTGGGGACAGAAGTCA-3', and reverse, 5'-GCTTTACACCTTCGAGCTGA-3'; and GAPDH forward, 5'-CTCACCGGATGCACCAATGTT-3', and reverse, 5'-CGCGTTGCTCACAATGTTCAT-3'. GAPDH was used as an internal control for mRNA expression, and the relative mRNA expression was calculated by the 2-ΔΔCq method (
After transfection for 24 h, cells were harvested, and total protein was extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology). Protein concentration was then measured with a BCA kit (Thermo Fisher Scientific, Inc.). A total of 25 µg protein was separated by SDS-PAGE (10%), transferred to PVDF membranes (EMD Millipore) and blocked in 5% non-fat milk for 2 h at room temperature. The membranes were incubated with the primary antibodies overnight at 4˚C, followed by incubation with HRP-conjugated goat anti-rabbit IgG (cat. no. R2004; 1:5,000; Sigma-Aldrich; Merck KGaA) or goat anti-mouse IgG (cat. no. A6715; 1:5,000; Sigma-Aldrich; Merck KGaA) for 1 h at room temperature. The antibodies used in the present study were as follows: Anti-ANGPTL8 (1:1,000; cat. no. CSB-PA757793LA01HU), purchased from Cusabio; anti-cyclin-dependent kinase 2 (CDK2; 1:1,000; cat. no. 2546), anti-p21 (1:1,000; cat. no. 2947), anti-proliferating cell nuclear antigen (PCNA; 1:1,000; cat. no. 13110), anti-matrix metalloproteinase (MMP)-2 (1:1,000; cat. no. 40994), anti-MMP-9 (1:1,000; cat. no. 13667), anti-tissue inhibitor of matrix metalloproteinase (TIMP)-1 (1:1,000; cat. no. 8946) and anti-TIMP-2 (1:1,000; cat. no. 5738) antibodies, obtained from Cell Signaling Technology, Inc. The proteins were visualized using a Tanon-5200 Chemiluminescence Imager (Tanon Science and Technology Co., Ltd.) and an enhanced chemiluminescence western blotting substrate (EMD Millipore).
A Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc.) was performed to detect cell viability. Briefly, HTR8/SVneo cells were seeded into 96-well plates (5x103 cells/well) and incubated at 37̊C with 5% CO2. At the indicated time points (24, 48 and 72 h) after transfection, 10 µl CCK-8 solution was added into each well and incubated for 2 h at 37̊C in the dark. The absorbance of each well was then measured with a microplate reader (ELx808; BioTek Instruments, Inc.) at 450 nm.
A wound healing assay was performed to detect the migration ability of HTR8/SVneo cells. Briefly, cells (4x104) were cultured in six-well plates for 24 h. The cells were transfected with si-NC or si-ANGPTL8-1 for 72 h. HTR8/SVneo cells without treatment served as a control. The monolayers were then scratched vertically using a 200-µl sterile pipette tip, and any floating cells were washed off with PBS. Cells were photographed at the indicated time points (0 and 24 h) using a Leica DM IL LED microscope equipped with an Integrated 5.0 Mega-Pixel MC170 HD camera (Leica Microsystems GmbH).
A Transwell assay was performed to evaluate the invasive capability of HTR8/SVneo cells with an 8-µm pore polycarbonate membrane chamber insert in a 24-well plate (Corning, Inc.). For the invasion assays, the chamber inserts were coated with 50 µl Matrigel (200 mg/ml; BD Biosciences). Briefly, at 24 h post-transfection, cells were resuspended in 200 µl serum-free medium and seeded in the upper chamber of the transwell inserts. A total of 600 µl RPMI 1640 medium containing 10% FBS was added to the lower chambers. After 24 h of incubation, the non-invading cells were gently removed with a cotton swab, and the invasive cells attached to the lower surface of the chamber membranes were fixed with polyoxymethylene and stained with 0.1% crystal violet solution. Finally, the number of cells in five random fields was counted, and images were captured under an inverted microscope (Olympus Corporation).
The data are presented as the mean ± standard error of the mean. Differences among the data were analyzed using Student's t-test (unpaired) or analysis of variance followed by Bonferroni correction, as appropriate. Statistical data were analyzed using SPSS software, version 13.0 (SPSS, Inc.). P<0.05 was considered to indicate a statistically significant difference.
Successful interference on ANGPTL8 expression in trophoblast cells by siRNA transfection was confirmed by western blotting and RT-qPCR. The results demonstrated that the silencing effect of si-ANGPTL8-1 was better than that of si-ANGPTL8-2; thus, si-ANGPTL8-1 was selected for use in subsequent experiments (
Wound healing and transwell assays were performed to investigate the effect of ANGPTL8 on trophoblast cell migration and invasion, respectively. The results demonstrated that silencing of ANGPTL8 led to a significant acceleration in the migratory and invasive capacities of trophoblast cells (
PE is a systemic disorder that occurs during pregnancy and is characterized by various manifestations of organ dysfunction. PE causes gestational hypertension and proteinuria, which is a sign of renal dysfunction (
ANGPTL8 is a newly identified hormone with the ability to regulate the glucose and lipid metabolic pathways. Abnormal expression of ANGPTL8 has been reported in nonalcoholic fatty liver disease, insulin resistance and diabetes mellitus (
The current study further employed HTR8/SVneo cells to investigate the aberrant trophoblastic invasion in the pathogenesis of PE
In conclusion, the present study demonstrated that ANGPTL8 serves an important role in the pathogenesis of PE, and that the downregulation of ANGPTL8 significantly promoted trophoblast cell proliferation, invasion and migration. Collectively, these data support the potential application of ANGPTL8 as a target for clinical diagnosis and treatment of PE.
Not applicable.
No funding was received.
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
WW and XL designed the experiments and drafted the manuscript. DJ mainly performed the experiments and analyzed the data. WW performed the western blot and RT-qPCR experiments. XL reviewed the manuscript. All authors read and approved the final manuscript.
The present study was approved by the Ethics Committee of Xuzhou Central Hospital (Xuzhou, China). All participants provided written informed consent.
All participants provided written informed consent for publication.
The authors declare that they have no competing interests.
ANGPTL8 expression levels in preeclampsia patients and correlation analysis with baseline characteristics. (A) Levels of ANGPTL8 in the serum of the study population. (B) Correlation analysis of body mass index (BMI), systolic pressure, diastolic pressure and proteinuria with ANGPTL8 levels. ***P<0.001 vs. control subjects. ANGPTL8, angiopoietin-like protein 8.
Effect of ANGPTL8 on the viability and proliferation of trophoblast cells. HTR8/SVneo cells were transfected with si-NC or si-ANGPTL8. (A) Western blotting and (B) reverse transcription-quantitative polymerase chain reaction were performed to evaluate the protein and mRNA expression of ANGPTL8, respectively. (C) Cell Counting Kit-8 assay was used to detect the viability of HTR8/SVneo cells. (D) The expression levels of CDK2, p21 and PCNA were analyzed by western blotting. The data represent the mean ± standard error of the mean of three independent experiments. *P<0.05, **P<0.01 and ***P<0.001, vs. control or si-NC group. ANGPTL8, angiopoietin-like protein 8; si-, small interfering RNA; NC, negative control; CDK2, cyclin-dependent kinase 2; PCNA, proliferating cell nuclear antigen.
Effect of ANGPTL8 on the migration and invasion of trophoblast cells. (A) Wound healing assay and (B) Transwell assay were performed to investigate the effect of ANGPTL8 on cell migration and invasion, respectively (magnification, x100). (C) The effect of ANGPTL8 on the protein expression levels of MMP-2, MMP-9, TIMP-1 and TIMP-2 was analyzed by western blotting. The data represent the mean ± standard error of the mean of three independent experiments. ***P<0.001 vs. control or si-NC group. ANGPTL8, angiopoietin-like protein 8; si-, small interfering RNA; NC, negative control; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of matrix metalloproteinase.
Baseline characteristics of subjects in the study population (n=30 per group).
Characteristics | Preeclampsia (n=30) | Healthy controls (n=30) | P-value |
---|---|---|---|
Maternal age (years) |
|||
≤35 | 25 (83.3%) | 27 (90.0%) | 0.54 |
>35 | 5 (16.7%) | 3 (10.0%) | |
Body mass index | 28.3±1.86 (22-32) | 25±1.12 (21-29) | <0.001 |
Blood pressure | |||
Systolic | 139±10 (118-160) | 115±10 (103-127) | <0.001 |
Diastolic | 95±5 (84-100) | 78.7±12.3 (65-93) | <0.001 |
Proteinuria | 2.5±0.65 (0.5-4.5) | 0.13±0.05 (0-0.3) | <0.001 |
Gestational age at delivery (years) | 33.3±1.84 (31-35) | 33.2±2.53 (31-36) | 0.71 |
Previous pregnancy |
14 (46.7%) | 15 (50%) | 0.32 |
aPresented as n (%). Data are expressed as the mean ± standard error (range), unless otherwise specified.