Clinical features and placenta preparation

We enrolled patients according to the American College of Obstetricians and Gynecologists (ACOG) diagnostic criteria for PE [11]. Placenta samples were collected from women with PE (n = 43) and normal pregnancies (n = 40) during cesarean section at West China Second Hospital of Sichuan University between November 2019 and June 2021. Samples were taken within 30 minutes placenta isolation. Specifically, 1 cm3 of surface parent villous tissues containing a large number of trophoblasts were obtained, and calcification and blood vessels were avoided during collection. Samples were washed with ice phosphate buffered saline (PBS) to prevent the interference of blood cells, and immediately stored in a -150 °C refrigerator. This study was approved by the Ethics Committee of West China Second Hospital (No. MRER-2019-032), and all participants in the study signed informed consent forms.

Transcriptome sequencing

Five pairs of placentas in women with normal pregnancy or PE matched by gestational age were selected for transcriptome sequencing (Oebiotech, China). The clinical information of the five selected pairs of pregnant women is listed in Supplementary Table 1 (Additional file 1).

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

Total RNA was extracted with an RNA extraction kit (Bioteke, China), and the concentration of RNA was measured with an ultramicro spectrophotometer (Implen, Germany). Then, the PrimeScriptTM RT reagent kit (TaKaRa, Japan) was used for reverse transcription according to the manufacturer’s protocols. qRT–PCR was carried out using ChamQ Universal SYBR qPCR Master Mix (Vazyme, China). All experiments were conducted in triplicate. The relative expression levels were analyzed using the 2−ΔΔCt method, and β-actin was the internal control. The primers for the qRT–PCRs are shown in Supplementary Table 2 (Additional file 1).

Cell culture and transfection

The HTR8/SVneo cell line (National Collection of Authenticated Cell Cultures, China) and JAR cell line (Shanghai Zhong Qiao Xin Zhou Biotechnology, China) were cultured in RPMI 1640 medium (HyClone, USA) supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin–streptomycin (Gibco) in humidified air at 37 °C with 5% CO2.

An TARID overexpression pcDNA3.1(+) vector (pcDNA3.1-TARID) (BGI, China), TARID antisense oligonucleotide (ASO-TARID) (RiboBio, China), CXCL3 overexpression pcDNA3.1(+) vector (pcDNA3.1-CXCL3) (Tsingke, China), and small-interfering RNA against CXCL3 (si-CXCL3) (RiboBio, China) were transfected into cells using Lipofectamine™ 3000 transfection reagent (Invitrogen, USA) when cell confluency was 50–70% in six-well plates. Co-transfection of pcDNA3.1-TARID and si-CXCL3 or ASO-TARID or pcDNA3.1-CXCL3 was performed to study the mechanism of TARID effects downstream. The assays were carried out from 24 to 48 h after transfection.

Fluorescence in situ hybridization (FISH)

TARID, U6 or 18S probes (RiboBio, China) were analyzed to detect the localization and distribution of cells with a FIISH kit (RiboBio, China). First, placental paraffin sections or cell slides were prepared. Then, 2.5 μL of 20 μM probe was added and incubated overnight at 37 °C. The next day, the slides were rinsed and then incubated with Cytokeratin 7 rabbit monoclonal antibody (mAb) (1:200, ABclonal, China) overnight at 4 °C. On the third day, the cells were incubated with a fluorescent secondary antibody and then counterstained with 40′,6-diamidino-2-phenylindole (DAPI). Images were taken with a laser scanning confocal microscope (Olympus, Japan).

Subcellular fractionation

Nuclear and cytosolic fractions were prepared from HTR8/SVneo cells with a PARIS™ Kit (Life Technologies, USA). qPCR assays were performed to detect TARID, U6, and β-actin in the nucleus and cytoplasm of the cells. The subcellular expression levels of TARID, U6, and β-actin are expressed as relative percentages. U6 and β-actin were included as nuclear and cytosolic markers, respectively.

Transwell assays

A total of 5 × 104 HTR8/SVneo cells or 1 × 105 JAR-transfected cells were seeded in the upper compartment of a Transwell insert with an aperture of 8 μm for the cell migration assay. HTR8/SVneo cells (1 × 105) or JAR-transfected cells (2 × 105) were seeded on upper Matrigel-coated compartment for the cell invasion assay. After incubation for 24 h, 4% formaldehyde was used for fixation for 30 minutes, and crystal violet was added for 4 minutes. Then, the cells were rinsed with double distilled water, the cells on the top of the membrane were gently wiped with cotton swabs, and pictures were taken under the microscope. All experiments were conducted in triplicate.

Cell viability assay

The effect of TARID on trophoblast proliferation was measured by cell counting kit-8 (CCK-8) (Saint-Bio, China) assay. HTR8/SVneo- or JAR-transfected cells were seeded in 96-well plates at 4000 cells per well. The optical density (OD) value was measured at 490 nm with a microplate reader (TECAN, Switzerland) at a predetermined time. The results are representative of three individual experiments.

Tube formation assay

Angiogenesis slides (Ibidi, Germany) were coated with 10 μL of Matrigel (BD Biosciences, USA), which was allowed to condense at 37 °C for 2 h. A total of 2000 transfected HTR8/SVneo cells were plated on the top layer. After incubation for 4 h at 37 °C, images were acquired for tube formation analysis. All experiments were conducted in triplicate.

Flow cytometry for apoptosis

For an apoptosis analysis, the cells were stained with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) (KeyGen, China). Harvested cells were analyzed by flow cytometry (Millipore, USA) according to the manufacturer’s recommendations. Three individual experiments were performed.

UID mRNA-seq

Three groups of HTR8/SVneo cells transfected with pcDNA3.1-TARID or pcDNA3.1-NC were selected to extract RNA, and second-generation sequencing of UID mRNA was then performed (SEQHEALTH, China).

Western blot analysis

Proteins from placentas in women with normal pregnancy or PE (matched for gestational age, fetal sex and primiparity) and cell lines were extracted using a total protein extraction kit (Beyotime, China). The protein concentration was determined using a BCA protein quantification kit (Yeasen, China). All proteins were standardized to a concentration of 3 μg/μL and denatured at 100 °C for 5 minutes in SDS–PAGE protein loading buffer (5X) (Beyotime, China). The proteins were separated with a PAGE gel quick preparation kit (Yeasen, China) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). Subsequently, the membranes were blocked with 5% skim milk powder (Solarbio, China) for 1 h and incubated with primary antibodies against CXCL3 (1:500, Sigma, USA), ERK1/2 (1:1000, ABclonal, China), p-ERK1/2 (1:1000, ABclonal, China) or β-actin (1:5000, ABclonal, China) overnight at 4 °C. Next, the membranes were washed three times with TBST and then incubated with a secondary antibody (1:100000, ABclonal, China) for 1 h at room temperature. Finally, SuperKine™ West Femto Maximum Sensitivity Substrate (Abbkine, China) was used to detect the chemiluminescence intensity with a ChemiDoc™ MP Imaging System (Bio-Rad, USA). Image Lab 6.0 software (Bio-Rad, USA) was used for gray value calculation. All experiments were conducted in triplicate.

Immunofluorescence

Placental tissue from normal pregnancies and patients with preeclampsia were subjected to immunofluorescence staining with CXCL3 Rabbit mAb (1:200, Sigma, USA) and Cytokeratin 7 Rabbit mAb (1:200, ABclonal,China) (Cdllsw, China).

Statistical analysis

Statistical analyses were performed using SPSS version 26.0.0.0 (IBM, USA) and GraphPad Prism 9.0.1 (GraphPad Software, USA) software. The normality of the data was analyzed by Kolmogorov–Smirnov normality test. Differences between two groups were analyzed by Student’s t test for normally distributed continuous variables and by the Mann–Whitney U test for nonnormally distributed continuous variables. Pearson’s chi-squared test was used for analyzing categorical variables. The correlation between two variables was analyzed by Pearson correlation. Binary logistic regression was used to examine the association between placental TARID or CXCL3 mRNA levels, and the risk of developing PE was determined by calculating unadjusted and adjusted odds ratios (ORs). Body mass index (BMI) and gestational age were included in the analysis following the processes reported in a previous study [12, 13]. A P value less than 0.05 was regarded as statistically significant.