Patient samples

Patients, aged 18–32 years, were recruited at the Department of Obstetrics, Shandong Provincial Hospital, affiliated with Shandong First Medical University (Jinan, China). Written informed consent was obtained from all participants. PE was defined according to the guidelines of the American College of Obstetricians and Gynecologists. Serum samples were prepared from blood collected from patients with PE (n = 10) and normal pregnancy (controls; n = 10).

Inclusion criteria were as follows: new-onset hypertension systolic blood pressure at least twice ≥ 140 mmHg and/or diastolic blood pressure ≥ 90 mmHg and proteinuria (≥ 300 mg), after 20 weeks of gestation. The exclusion criteria were as follows: (i) multiple pregnancies, (ii) smoking, and (iii) history of other clinical disorders, such as chronic hypertension or renal disease. More clinical information about the sample is found in Table 1. Peripheral blood (PB) samples were collected from patients with PE in EDTA-anticoagulant tubes (5 mL per sample) and centrifuged at 2000 × g for 15 min at 4 °C within 1 h to separate the plasma and blood cells. The plasma samples were immediately stored at − 80 °C for further use. This study complied with the World Medical Association Declaration of Helsinki guidelines and was approved by the Ethics Committee of the Shandong Provincial Hospital Affiliated to Shandong First Medical University (SWYX:NO.2023–279).

Table 1 The clinical characteristics of the study populationPrimary human trophoblast cell isolation

Primary human trophoblast cells were isolated as previously described [18, 19]. The villus tissue of maternal surface was taken, and blood clots and fetal 16 membranes were removed. After washing three times with sterile D-Hanks, the placentas were cut into pieces of about 1 mm3. A mixture of 0.25% trypsin, 0.1% collagenase, and deionized water was added to the tube and incubated for digestion in a 37 °C water bath for 25 min. When the single cell formation was observed under a microscope, the supernatant was collected, filtered, and centrifuged at 250 × g for 10 min. The resultant cell pellet was resuspended in Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12 (1:1) suspension supplemented with 10% fetal bovine plasma (FBS). Cells were maintained in DMEM (Gibco, NY, USA), supplemented with 10% FBS, 25 mM 17 HEPES, 2 mM glutamine, 100 g/mL streptomycin, and 100 UI/mL penicillin, and incubated in humidified air with 5% CO2 at 37 °C.

Cell culture and transfection

HTR-8/SVneo cells were maintained in RPMI-1640 (Gibco, NY, USA), supplemented with 10% fetal bovine serum (FBS), at 37 °C in a humidified incubator with 5% CO2. HEK293T cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% exosome-free FBS at 37 °C in a humidified incubator with 5% CO2.

HTR-8/SVneo cells in the logarithmic phase of culture were seeded in a six-well plate at 3 × 105 cells/well and cultured to 60% confluence. The cells were transfected with 50 nM miR-3198 mimic or miR-NC (Ribobio Co., Ltd., Shanghai, China) using Lipofectamine 2000 reagent (11668019, Invitrogen, CA, USA) per the manufacturer’s instructions. After transfection for 48 h, the HTR-8/SVneo cells were harvested and used for functional assays.

Exosome isolation

Exosomes were separated from PB plasma following a previously described protocol with modifications [20, 21]. Briefly, PB plasma was mixed with an equal volume of cold phosphate-buffered saline (PBS), and the mixture was sequentially centrifuged at 300 × g for 10 min, at 2000 × g for 30 min, and at 12,000 × g for 45 min. After centrifugation at 2000 × g, the supernatant was gently filtered through a 0.22-µm sterile filter (Steritop, Millipore, USA) and centrifuged twice at 120,000 × g for 70 min (Beckman Coulter). The supernatant was removed, and the pellets were resuspended in 200 µL sterile PBS. All the centrifugation steps were performed at 4 °C. The protein concentration of exosomes was measured using the bicinchoninic acid method. The resuspended exosomes were immediately frozen at − 80 °C or used in subsequent experiments.

Electroporation of exosomes

The electroporation method has higher purity and stability, and it can directly load biologically active substances such as drugs and siRNAs into exosomes without needing other media [22]. Electroporation uses a pulsed electric field to apply voltage to exosomes, resulting in the opening of the exosome membrane and allowing drugs to enter the exosomes. Electroporation requires the collection of exosomes from the culture medium, followed by purification of the obtained exosomes through ultracentrifugation or other methods. The transfection complex is prepared by packaging the exosomes with DNA or RNA. Finally, electroporation parameters are adjusted to ensure optimal transfection efficiency. Parameters such as voltage, pulse duration, and electrode spacing need to be optimized according to cell type and exosome type [23,24,25]. Exosomes and the miR-3198 mimic were placed on ice for 10 min. The exosomes were diluted to 0.5 mg/mL in electroporation buffer. The miR-3198 mimic was added at a final concentration of 100 nM. The mixture was transferred to a precooled 2 mm cuvette and subjected to three 400 V, 125 µF capacitance pulses for 5 ms. After electroporation, the exosomes were placed on ice for 10 min.

Coculture of exosomes with HTR-8/SVneo cells

HTR-8/SVneo cells were divided into a blank group (HTR-8/SVneo cells cultured under normal conditions), an NC-EXO group (exosomes electroporated with miR-NC and cocultured with HTR-8/SVneo cells), and an miR-3198-EXO group (exosomes electroporated with the miR-3198 mimic and cocultured with HTR-8/SVneo cells). HTR-8/SVneo cells were cocultured with electroporated exosomes in accordance with the grouping in 100 µg/mL for 48 h and used for further studies.

Western blot

The Western blotting was carried out as our previous protocol [21]. All proteins were extracted using RIPA-PMSF methods and quantified through BCA kit (Solarbio, Beijing, China). Briefly, 20 μg of loaded protein was separated with 7.5%, 10%, or 12.5% SDS‒PAGE (Epizyme, Shanghai, China) and then transferred onto PVDF membranes (Millipore, MA, USA). The membranes were blocked with 5% nonfat milk at room temperature for 1 h and incubated at 4 °C overnight with the primary antibodies. The next day, the membranes were incubated with the corresponding secondary antibody (Proteintech) for 2 h. Finally, the blots were visualized by Amersham Imager 600 system (GE, Boston, MA, USA). Primary antibodies were listed as follows: CD63 (1:1000, ab134045, Abcam), TSG101 (1:1000, ab125011, Abcam), CD81 (1:1000, ab79559, Abcam), WNT3 (1:1000, ab116222, Abcam), and GAPDH (1:3000, ab9485, Abcam).

RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)

Total exosomal RNA of PB plasma from patients with PE or controls was extracted using the AG RNAex Pro reagent (Accurate Biotechnology, Jinan, China) according to the manufacturer’s instructions. The concentration of total RNA was determined using a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific). To avoid RNA degradation, it was reverse transcribed into cDNA as soon as possible. For reverse transcription into cDNA, RNA was treated with the Evo M-MLV RT Kit with gDNA Clean for qPCR (Accurate Biotechnology). The cDNA was used for qRT-PCR with SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology), and the level of expression of genes relative to GAPDH was determined using the 2−△△CT method [14]. All the primers were synthesized by JEKAIYER (Shandong, China). The primer sequences are shown in Table 2.

Table 2 Sequences of primers used for qRT-PCRCell counting kit-8 (CCK-8) assay

HTR-8/SVneo cells subjected to pretreatment (transfection or coculture with exosomes) were incubated along with 10% CCK-8 (Bioss, Beijing, China) solution in 96-well plates for 2 h. Thereafter, the absorbance at 450 nm was measured using a microplate reader, according to the manufacturer’s instructions.

Migration assay

HTR-8/SVneo cells were resuspended in a plasma-free medium and plated in the upper chamber of a transwell plate (Corning, NY, USA). Completed medium containing 10% FBS was added to the lower chamber. After incubation for 24 h, non-migrating cells were removed using a cotton swab, and migrating cells were stained with 0.1% crystal violet. In each well, three microscopic fields were photographed randomly, and the number of cells was counted manually.

Scratch assay

To determine the migration ability of HTR-8/SVneo cells, pretreated cells were seeded in six-well plates and incubated until they reached a confluent state. The cell monolayer was scratched with a 200-μL pipette tip, and the wells were rinsed with PBS to remove detached cells. After culturing in plasma-free medium for 24 h, images were captured at 0 and 24 h. The width of the scratches was measured using the ImageJ software (NIH, USA).

Apoptosis assay

HTR-8/SVneo cells were cultured in the presence of exosomes for 24 h. The apoptosis assay was performed using an apoptosis kit (BD Biosciences) according to the manufacturer’s instructions. Briefly, cells were resuspended at a concentration of 1 × 106 cells/mL and 500 μL binding buffer was mixed with 100 μL cell suspension. The staining solution was added and gently blended on ice for 15 min in the dark. Flow cytometry (BD Accuri™ C6 Plus) was used to detect the apoptotic cells within 1 h.

Cell cycle assay

Cell cycle assay was performed using a cell cycle detection kit (Key GEN BioTECH, Jiangsu, China) according to the manufacturer’s instructions. Briefly, harvested HTR-8/SVneo cells were rinsed with PBS and then fixed with precooled 70% ethanol at − 20 °C. After overnight incubation, the cells were rinsed with PBS twice. RNase A and propidium iodide (PI) were mixed at a 1:9 ratio. After removing the supernatant, 500 μL PI/RNase A staining solution was added to the cells, and the cells were incubated at room temperature for 40 min. Flow cytometry (BD Accuri™ C6 Plus) was performed immediately to analyze the cell cycle.

Dual-luciferase reporter assay

The seed sequence in the 3-UTR of human WNT3 mRNA binding by miR-3198 was analyzed by TargetScan. 3-UTR of WNT3 and a sequence with mutant nucleotides in the miR-3198 binding site were inserted into pGL3 vector for luciferase. HEK393t cells were co-transfected with luciferase vector and miR-3198 mimics or control oligonucleotides. Luciferase activity was examined using the Dual-luciferase Reporter Assay System in accordance with the manufacturer’s protocol (Promega, USA) at 48 h after transfection.

Statistical analysis

Data were analyzed using the GraphPad Prism software version 9.0. Data were compared using analysis of variance (ANOVA) or t-test. A value of p < 0.05 was considered to indicate statistical significance.