The placental tissues from the normal and GDM groups were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin wax and serially sectioned at a thickness of 4 μm. Subsequently, the sections were subjected to antigen repair with citric acid, blocked with 10% normal goat serum blocking solution, and incubated with primary antibodies (mouse anti-human MUC1 monoclonal antibody, ab218464, 1:100; rabbit anti-human GLUT4 monoclonal antibody, ab33780, 1 µg/ml) overnight at 4 °C. The sections were stained with fluorescent secondary antibodies (anti-rabbit IgG/FITC or goat anti-mouse IgG/FITC) (Bioss, bs-0295G-FITC, 2 mg/ml). The nuclei were stained with DAPI (Invitrogen). The sections were photographed using a fluorescence microscope (Olympus BX53, Tokyo, Japan). A minimum of 6 random images from 3 samples were analyzed per group.

Western blotting

Proteins were isolated from the placenta of normal and GDM and HTR-8/SVneo cells using RIPA (Sigma, USA) buffer containing protease and phosphatase inhibitors. Protein concentrations were quantified by BCA assay (Thermo Fisher, A23227, USA). Extracted proteins were separated by 10% SDS‒PAGE and transferred to polyvinylidene difluoride membranes (Millipore, 88,518, USA). The membrane was blocked with 5% nonfat milk, incubated overnight at 4 °C with primary antibody in TBST buffer and shaken gently. After incubation with the secondary antibody, i.e., either horseradish peroxidase goat anti-rabbit IgG (1:3000; Cell Signaling Technology, AB_2099233, USA) or horseradish peroxidase goat anti-mouse IgG (1:3000; Cell Signaling Technology, AB_330924, USA), the samples were treated with SuperSignal™ West Femto chemiluminescent substrate (Thermo Fisher, 34,096, USA) and then photographed by the Gel Doc™ XR + System (Bio-Rad, USA). ImageJ software was used to capture the chemiluminescent signals and analyze the data. The following primary antibodies were used: MUC1 (1:1000, ab109185, Abcam, USA), GLUT4 (1 µg/ml, ab33780, Abcam, USA), INSR (1: 1000, ab227831, Abcam, USA), Bcl-2 (1:1000, ab32124, Abcam, USA), Caspase-3 (1:500, ab13847, Abcam, USA), β-Catenin (1:5000, ab32572, Abcam, USA), p-β-Catenin (1:500, ab75777, Abcam, USA), GSK-3β (1:1000, ab93926, Abcam, USA), p-GSK-3β (1 µg/ml, ab107166, Abcam, USA), Wnt3a (1:1000, ab219412, Abcam, USA), TCF4 (1:10000, ab217668, Abcam, USA), c-Myc (1:1000, ab32072, Abcam, USA), CyclinD1 (1:100, ab16663, Abcam, USA), and β-actin (1:3000, #4970, CST, USA). All experiments were performed with at least three replicates.

Cell culture and transfection

The human trophoblast cell line HTR8/SVneo was purchased from Zhongqiao Xinzhou Biotechnology Co., Ltd. (Shanghai, China). HTR-8/SVneo cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin‒streptomycin solution (HyClone) at 37°C with 5% CO2. HTR-8/SVneo cells were treated for 72 hours with 30 mM D-glucose as the high glucose (HG) group, and normal medium (5.5 mM D-glucose) was used as a control group. HTR-8/SVneo cells (2 × 105 cells/mL) were seeded into 6-well plates and incubated for 24 h until the cells reached 60–70% confluence. Then, the cells were transfected with negative control (sh-NC group) and MUC1-shRNA (sh-MUC1 group) plasmids purchased from Shanghai GeneChem Co., Ltd. (Shanghai, China). The target sequence for MUC1 was as follows: shMUC1: 5’-CCGGCCGGGATACCTACCATCCTATCTCGAGATAGGATGGTAGGTATCCCGGTTTTTG-3’, and the negative control shRNA (sh-NC) sequence was 5’-TTCTCCGAACGTGTCACGT-3’. The vector map and sequence result of the sh-MUC1 plasmid are presented in Supplementary file 1: Results S1-2. The transfection assay was performed according to the manufacturer’s protocol for Lipofectamine 2000. FH535 was purchased from EMD Millipore (Billerica, MA, USA).

In vitro experiments Flow cytometry (cell apoptosis)

After transfection and treatment, HTR-8/SVneo cells (1 × 106) were collected by centrifugation at 1,000 rpm for 5 min and resuspended in 1 mL of cold PBS. The cells were then stained with an Annexin V-FITC apoptosis kit (88-8005-72, Thermo Fisher, USA) and detected using a FACScan flow cytometer (Becton-Dickinson, San Jose, USA). The acquired data were analyzed using FCS-Express software version 3.0 (De Novo). Three independent experiments were performed.

Flow cytometry (cell cycle)

Cell cycle analysis was also detected by flow cytometry. After transfection and treatment, HTR-8/SVneo cells (1 × 106) were collected and washed with cold PBS and then centrifuged, and the supernatant was discarded. Then, 1 ml of -20 °C 70% ethanol was added, gently mixed and fixed overnight at -20 °C. After overnight incubation at -20 °C and washing with PBS, the cells were stained with PI and subjected to flow cytometry. Then, the distribution of cells in the G1, S, and G2/M phases of the cell cycle was determined. Three independent experiments were performed.

Wound healing assay: The in vitro migration ability of cells was assessed by the wound healing assay. After transfection, 5 × 105 HTR-8/SVneo cells were seeded in 6-well plates and grown to reach confluent monolayers. Then, a 2 µl pipette tip was used to create scratches. Images of migrated cells were recorded at 0–24 h and exported to TIFF with ZEN 2.3 software. The experiments were repeated at least three times. The cell-free area was calculated as the wound area in captured images using ImageJ. The percentage of wound closure was expressed according to the following Eq.  [48]:

$$}\,}\,}\,\left( }_} - }_}} \right)/}_} \times 100\%$$

A0h is the area of the wound measured immediately after scratching (0 h).

A24h was the area of the wound measured 24 h after the scratch was performed.

CCK-8 assay

Cell viability was measured using a cell counting kit-8 (CCK8, ab228554, Abcam, UK). Cells from the Con, sh-NC and sh-MUC1 groups were cultured in 96-well plates (2.5 × 104 cells/ml) and treated in the absence or presence of 50 mM D-glucose for 12, 24, 48 and 72 h. Briefly, 10 µl of CCK8 reagent (Dojindo, Kumamoto, Japan) was added to 96-well plates and incubated continually for 2 h at 37 °C. The absorbance values were measured at 450 nm using a Bio-Rad Model 450 microplate reader (Bio-Rad, Hercules, CA, USA).

Statistical analysis

Construction of statistical charts was performed using the GraphPad Prism 8 software package (GraphPad Software, CA, USA). Statistical analysis was performed using the SPSS 23.0 statistical package program. A comparison of the means between two groups was performed using the Mann‒Whitney U or Student’s t test to estimate the differences. Multiple group comparisons of the means were performed by one-way analysis of variance (ANOVA). Blinded outcome assessment was implemented. All values are presented as the mean ± SD (standard deviation). All statistical data (including n number for the data) are presented in Supplementary file 1: Table S3. P < 0.05 was considered statistically significant.