GSE147776 is a gene expression profile obtained from placental samples by microarray analysis (Medina-Bastidas et al., 2020). GEO2R (https://www.ncbi.nlm.nih.gov/geo/geo2r/), as a convenient online tool, was used to screen the significantly-downregulated genes (log2FC < −1, p < 0.05) in PE placental tissues comparing to normal samples. Gene Ontology (GO) enrichment analysis of these genes was performed using the database for annotation, visualization and, integrated discovery (DAVID, https://david.ncifcrf.gov/home.jsp) (Sherman et al., 2022).

Healthy female mice (10–15 weeks old) were prepared for the experiment. The mice were kept in a stable environment of free food and water, temperature (22 ± 1°C), and lighting for 12 h light/dark cycle. When the female mice were mated with male mice, the day that a vaginal plug appeared was regarded as day 0 of pregnancy. Pregnant mice were randomly divided into the control group and the PE group. The mice in PE group were injected with 50 mg/kg N(ω)-nitro-L-arginine methyl ester (L-NAME) subcutaneously from day 7 to day 17, and the mice in the control group were injected with the same amount of solvent daily. Mean arterial pressure (MAP) was monitored every 2 days from day 2 to day 18. The 24 h-urine was collected on day 17 to detect total protein content using a urine protein test kit (Nanjing Jiancheng, Nanjing, China). Placental tissues were collected on day 18 for follow-up experiments. Before being sacrificed, the mice were injected with 200 mg/kg of pentobarbital sodium. All efforts were made to minimize the suffering of animals. The procedures used in this research were in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Ethical Committee of Nankai University (No. 2022-SYDWLL-000273).

Human chorionic trophoblast HTR-8/SVneo cells were purchased from Zhong Qiao Xin Zhou Biotechnology (Shanghai, China). The cells were cultured in RPMI-1640 (Solarbio, Beijing, China) with 10% fetal bovine serum (FBS) in an incubator with 5% CO₂ at 37°C. We purchased a commercialized plasmid named pDONR223-UCK2 cDNA clone (Cat#G163428) from YouBio Inc. (Changsha, China) to upregulate UCK2 expression. UCK2 was knocked down using specific siRNAs synthesized by General Biol. (Chuzhou, China). The target mRNA sequences of UCK2 were as follows: siUCK2-1: 5′-AGAAAUCACUGAAGGGAAA-3′, siUCK2-2: 5′-CCCUAGAGGUGCAGAUAAU-3′. Vector and siRNAs were transfected into cells using lipofectamine 3000 (Cat#L3000008, Invitrogen, Carlsbad, CA, USA). After transfection, cells were treated with 1 μM stattic (Shanghai yuanye Bio-Technology, Shanghai, China) to block the expression of STAT3.

Total RNA was isolated from cells or tissues by TRIpure (BioTeke, Beijing, China). The concentration of RNA was detected by ultraviolet spectrophotometer NANO 2000. RNA was reverse-transcribed using M-MLV reverse transcriptase (Beyotime, Shanghai, China) and RNase inhibitor (BioTeke, Beijing, China). 2 × Taq PCR MasterMix (Solarbio, Beijing, China), SYBR Green (Solarbio, Beijing, China), and primers were mixed with cDNA to examine the relative mRNA expression, which was normalized to GAPDH (glyceraldehyde 3-phosphate dehydrogenase) expression. Primer information is listed in Table 1.

Total proteins were extracted from trophoblast cells or the placenta tissues by RIPA and PMSF (Solarbio, Beijing, China). The protein concentrations were determined using Solarbio BCA Protein Assay Kit. Proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were transferred onto a polyvinylidene fluoride membrane (Millipore, Bedford, USA). After blocking by skim milk, the proteins were incubated with specific primary antibodies at 4°C overnight. Then the secondary antibodies were incubated for 1 h at 37°C. The details of the antibodies are listed in Table 2.

The cells were seeded in the 96-well plates with 3 × 10³ cells in each well, and five repeated wells were set in each group. When the cells were cultured for a specific time, the medium was removed, 20 μL of MTT staining solution (KeyGEN, Nanjing, China) was added, and the cells were incubated in the incubator for 4 h. After carefully absorbing the supernatant, 150 μL DMSO was added to cells for 10 min, then the optical density (OD) value was measured by an enzyme-labeled instrument (BIOTEK, VT, USA).

After 48 h transfection, the cells were washed with phosphate buffer-saline (PBS), fixed in 70% cold ethanol for 12 h, and then treated with 25 μL propidium iodide (PI) and 10 μL RNase A at 37°C for 30 min. According to the instructions of the cell cycle assay kit (Beyotime, Shanghai, China), cell cycle distribution was examined using NovoCyte flow cytometry (ACEA Biosciences, CA, USA). Forty-eight hours after transfection, the cells were washed with PBS and treated with Annexin V-FITC Apoptosis Detection Kit (KeyGEN, Nanjing, China). The cell apoptosis was detected by NovoCyte flow cytometry.

Cells were infiltrated with 0.1% Triton X-100 (Beyotime, Shanghai, China) and incubated with TUNEL reaction liquid of In Situ Cell Death Detection Kit (Roche, Basel, Switzerland) at 37°C for 60 min. After washing with PBS 3 times, cells were stained with DAPI for 5 min. BX53 fluorescence microscope (Olympus, Tokyo, Japan) was used to take pictures.

To estimate the effects of UCK2 on the migration ability of trophoblast cells, a wound-healing assay was performed. When the cell culture reached the fusion state, the medium was replaced with a serum-free medium, and cells were treated with 1 μg/mL mitomycin C (Sigma, MO, USA) for 1 h. Cells were scratched with a 200 μL pipette tip and photographed by a microscope (100×). The cells were cultured in an incubator with 5% CO₂ at 37°C for 24 h and then photographed to calculate the migration rate of the cells. The migration rate is calculated using the formula [Migration rate = (0 h average width of wound - 24 h width of the wound)/0 h average width of wound × 100%].

The cells were seeded in the 6-well plates until their density reached 90%. 200 μL of cell suspension (1 × 10⁵ cells/mL) was added to the Transwell chamber (Corning, NY, USA) containing Matrigel glue (BD Biosciences, CA, USA), and 800 μL of medium containing 10% fetal bovine serum (FBS) was added to the lower chamber. After 48 h, cells were fixed with 4% paraformaldehyde (Aladdin, Shanghai, China) for 20 min and stained with 0.5% crystal violet (Amresco, OH, USA) for 5 min. The cells were counted under an IX53 inverted microscope (Olympus, Tokyo, Japan).

Statistical analysis and graphical representation were performed using GraphPad Prism 8 software. Differences between two groups were analyzed by Student’s t-test, and differences among three or more groups were determined using the one-way analysis of variance. The data were presented as the mean ± standard deviation. The p < 0.05 was considered that the results have statistical significance.

Differently expressed genes in the GSE147776 dataset are presented in Fig. 1A. The significant-downregulated genes were enriched in GO terms (Fig. 1B). The 15 GO terms with the highest count of enriched genes in the biological process category were visualized. The genes enriched in the GO terms associated with typical cell biological behavior in PE are displayed in Fig. 1C. UCK2 was enriched in “in utero embryonic development” and “regulation of trophoblast cell migration”; however, its role in PE is still not clear. We further estimated the expression of UCK2 in the dataset, and the results suggested that UCK2 had significantly lower expression in the placenta tissues of patients with PE than that of the healthy controls (Fig. 1D).

Hypertension and proteinuria are important features of PE (Hauth et al., 2000; Sibai, 2003; Stefańska et al., 2020). During the establishment of the PE mouse model, we monitored their blood pressure and urinary protein levels. After L-NAME injection, the MAP was significantly upregulated compared with the control mice from day 12 to day 18 of pregnancy (Fig. 2A). The 24-h urine protein content of L-NAME-treated mice was significantly higher than that of the control group (Fig. 2B). The two images illustrated that the classical PE-like models were successfully established by L-NAME treatment. Moreover, the mRNA and protein expression of UCK2 was decreased in the placenta tissue of the PE mouse model (Figs. 2C and 2D). The above results demonstrated lowered expression of UCK2 in the placental tissues of the PE mouse model.

As shown in Figs. 3A and 3B, the results of real-time PCR and western blot clarified that the relative mRNA and protein expression of UCK2 was remarkably upregulated in the HTR-8/SVneo cells when UCK2 was overexpressed and was downregulated by the silence of UCK2. MTT results showed that the proliferation of trophoblast cells was enhanced by the overexpression of UCK2 and was suppressed by the siUCK2-1 and siUCK2-2 (Fig. 3C). When UCK2 was upregulated, the expression of cyclinD1 and cyclinE proteins increased significantly. When UCK2 was knocked-down, the expression decreased in the trophoblast cells (Fig. 3D). Results of flow cytometry verified that the cells in S and G2 phases were significantly increased by UCK2 overexpression, while the cells in the G1 phase were significantly decreased. After UCK2 was knocked down, the cells in the G1 phase were dramatically increased, and those in the S and G2 phases were remarkably reduced (Fig. 3E). These results indicated that UCK2 could promote the proliferation of trophoblast cells.

The apoptosis proportion of HTR-8/SVneo was detected by flow cytometry. When UCK2 was knockdown by siUCK2-1 and siUCK2-2, the apoptosis rate was significantly increased (Fig. 4A). UCK2-downregulation-induced apoptosis was also confirmed by the TUNEL assay (Fig. 4B). Moreover, the protein expression of cleaved-caspase-3 and cleaved-PARP was significantly increased after siUCK2-1 and siUCK2-2 treatment (Fig. 4C). Taken together, the knockdown of UCK2 enhanced the apoptosis of trophoblast cells.

As depicted in Fig. 5A, the wound healing assay demonstrated that the migration rate was significantly facilitated by UCK2 overexpression and was suppressed by siUCK2-1 and siUCK2-2 in HTR-8/SVneo cells. Furthermore, the results of the transwell assay suggested that the upregulation of UCK2 significantly facilitated the invasion ability of trophoblast cells. The silence of UCK2 remarkably reduced the number of invaded HTR-8/SVneo cells (Fig. 5B). Matrix metalloproteinases (MMPs) are reported to participate in the process of cell adhesion and invasion, and their tissue inhibitors (TIMPs) are involved in the regulation of cell migration and invasion through interaction with MMPs (Chen, 1992; Polette et al., 2004; Bhuvarahamurthy et al., 2006). Results shown in Fig. 5C suggest that the protein expression of MMP2 and MMP9 was significantly increased, and the TIMP1 and TIMP2 expressions were obviously declined in HTR-8/SVneo cells by the upregulation of UCK2. When UCK2 was knocked-down, the protein expression of MMP2, MMP9, TIMP1, and TIMP2 was opposite to that of the above situation. Based on these results, UCK2 was demonstrated to promote the migration and invasion of trophoblast cells.

STAT3 is a crucial mediator participating in the normal biological function of trophoblast cells (Ko et al., 2013). As shown in Fig. 6A, when UCK2 was overexpressed, the phosphorylated STAT3 (p-STAT3) expression was obviously upregulated, while there was no significant change in STAT3 expression. When UCK2 was knocked-down, the p-STAT3 expression was significantly decreased. To further investigate whether UCK2 regulates the biological behavior of trophoblast cells via the STAT3 signaling pathway, the STAT3 antagonist stattic was used to treat the UCK2-overexpressed trophoblast cells. Results of Fig. 6B reveal that the upregulation of p-STAT3 expression induced by UCK2 was suppressed by stattic. The UCK2-induced cell proliferation was also inhibited by stattic (Fig. 6C). The promotion of UCK2 on cell migration was dramatically inhibited after the addition of stattic (Fig. 6D). Besides, UCK2-induced invasion of HTR-8/SVneo cells was clearly rescued by static (Fig. 6E). Therefore, UCK2 facilitated cell proliferation, migration, and invasion of trophoblast cells partly through the activation of the STAT3 pathway.