Dataset collection and preprocessing

Datasets GSE75010 and GSE73374 were selected and downloaded from the GEO database. Both consist of human placental transcriptome data. GSE75010 (GPL6244), extracted solely from BioBank, includes 77 control samples and 80 PE samples [20, 21]. GSE73374 (GPL16686) included 17 control samples and 19 PE samples [22] (Supplementary Table 1). Probe annotation information for the respective GPL platform was used to annotate probes into the corresponding gene symbols, and unannotated probes were removed. Additionally, a list of 326 GPCRs was extracted from a previous article [23].

Analysis of weighted gene co-expression network

The gene expression matrix, annotated and normalized, was subjected to outlier removal and Pearson correlation analysis to compute the inter-gene relationships, leading to the construction of a correlation matrix. An appropriate soft threshold was calculated based on this matrix to define network connection weights, transforming the correlation matrix into a Topological Overlap Matrix (TOM). Gene connectivity in the network was assessed using the TOM, and hierarchical clustering was used to establish gene modules, each representing a set of genes with similar expression patterns. Subsequently, the average expression profile of genes in each module was correlated with the external phenotype to identify modules most closely associated with PE. All analyses were performed using the R package “WGCNA”.

Hub PE-related GPCRs screening

The PE-related gene interaction was obtained from WGCNA analysis. Cytoscape was used for visualizing the interaction network. The top 20 key genes were extracted using Cytohubba with the following four algorithms: maximum neighborhood component (MNC), maximal clique centrality (MCC), Degree (degree of a node in the network) of Network-based Method with Clustering Analysis (DNMC), and Degree. LASSO regression with 7-fold cross-validation was used for the selection of the PE-related feature, performed using the “glmnet” R package. The genes obtained from both methods were intersected to identify hub genes.

Hub PE-related GPCRs validation

The R packages “ggplot2,” “stats,” and “car” were used for validating the differential expression of hub GPCRs. The “ggplot2” package was used for graphical representation, while the “stats” package facilitated statistical analyses. Additionally, the “car” package was used to enhance the robustness of statistical testing. ROC curves were generated using the “pROC” package to assess the diagnostic performance of hub GPCRs.

Cell culture and transfection

Immortalized human trophoblast cell line HTR-8/SVneo was obtained from Saibainuo Limited Company Biotechnology Corporation (Shanghai). Cells were cultured in 1640 medium (Meilunbio, Dalian, China) containing 1% penicillin-streptomycin (Meilunbio, Dalian, China) and 10% fetal bovine serum (Meilunbio, Dalian, China) at 37 °C with 5% CO2. CCBP2 siRNA and GPR87 siRNA (Tsingke, Wuhan, China) were separately transfected into HTR8/SVneo cells using Lipo8000TM (Beyotime). Each siRNA was diluted and transfected according to the recommended dilution ratios provided in the product instructions. Cells were collected 48 h after transfection for subsequent experiments.

Western blot analysis

Cell total proteins were extracted using RIPA lysis buffer (Solarbio) containing PMSF (Solarbio, Beijing, China) and a proteinase inhibitor (Meilunbio). The extracted proteins (20 µg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a PVDF membrane (Millipore). The membrane was treated with the primary antibodies CCBP2 (1:1000, A05491-2, Boster, Wuhan, China) GPR87 (1:1000, DF4798, Affinity, Jiangsu, China), and β-actin(1:5000, BM0627, Boster), the latter used as the loading control and incubated overnight at 4 °C. Next, the membrane was treated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000, BA1050/1054, Boster) and incubated at room temperature for 1 h. ImageJ software was used for the quantitative analysis based on grayscale values.

Cell proliferation assay

Cells transfected with siCCBP2, siGPR87, and their corresponding negative controls were seeded in a 96-well plate (1 × 10^3 cells/well). Cell Counting Kit-8 (CCK8) solution (10 µl, Meilunbio) was added at 24, 48, 72, and 96 h post-culture and incubated for 2 h at 37 °C under 5% CO2. Cell proliferation was assessed by measuring the absorbance at 450 nm.

Wound healing assay

Transfected cells were seeded in a 6-well plate and incubated for at least 24 h until reaching the confluence. A sterile yellow micropipette tip (200 µl) was used to create a wound in the monolayer of cells. Detached cell fragments were removed by washing with PBS and the cells were further incubated in culture medium without FBS. Cell images were captured at 0 h and 24 h post-wounding. ImageJ software was used to process the images and calculate the wound closure rate.

Migration and invasion assays

Transwell chambers (8.0 μm, CORNING, Shanghai, China) were placed into the lower part of a 12-well plate. The transfected cells were resuspended in serum-free RPMI 1640 medium, and 200 µL cell suspension (1 × 10^5 cells/mL) was added to the upper chamber, which was either coated with Matrigel (1 mg/ml, BD Biosciences) (for invasion assays) or left uncoated (for migration assays). Then, 500 µL RPMI 1640 medium containing 10% FBS was added to the lower chamber. After 24 h of incubation, the upper chambers were removed, and the non-migrated/invaded cells were gently wiped off. The cells that had migrated or invaded to the lower surface of the membrane were fixed with 4% paraformaldehyde (Solarbio Science) for 15 min, followed by staining with 0.1% crystal violet. Images were captured under a microscope, and the number of invasive cells was quantified using ImageJ software.

Statistical analysis

Statistical analysis was performed using R (version 4.1.2) and GraphPad Prism (version 9.4.1). Student t-test was used for the comparison of two groups. Results are expressed as mean ± SD/SEM. A value of p < 0.05 was considered statistically significant.