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 Table I. All corresponding blood samples were collected after delivery (within 1 h) and used according to the protocol approved by the Ethics Committee at Xuzhou Central Hospital. Pregnant women were excluded from the present study if they presented any of the following: <18 years of age without a legal guardian, was at >26 weeks of gestation, had a multifetal gestation, suffered from a cardiovascular disease (including chronic hypertension or valvular heart disease) or pregestational diabetes, or had a history of fenfluramine/phentermine use. All participants provided written informed consent.

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 (18).

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 (Fig. 2A and B). Next, a CCK-8 assay was performed to elucidate the function of ANGPTL8 in trophoblast cells, and the results revealed that reduced expression of ANGPTL8 increased the viability and proliferation of HTR8/SVneo cells (Fig. 2C). Western blotting was also performed to detect the levels of proteins involved in cell proliferation, including CDK2, p21 and PCNA. As shown in Fig. 2D, downregulation of ANGPTL8 decreased p21 expression, and increased the CDK2 and PCNA expression levels in HTR8/SVneo cells compared with the expression levels in the control and si-NC groups.

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 (Fig. 3A and B). Next, western blotting was used to evaluate the levels of proteins associated with migration and invasion, including MMP-2, MMP-9, TIMP-1 and TIMP-2. The data revealed that downregulation of ANGPTL8 markedly enhanced the levels of MMP-2 and MMP-9, while the protein expression levels of TIMP-1 and TIMP-2 were significantly decreased in the si-ANGPTL8-1 group as compared with those in the control and si-NC groups (Fig. 3C).