Clinical study

According to the diagnostic criteria of the International Association of Diabetes and Pregnancy Research Groups (IADPSG), whole blood samples from normal pregnancies, patients with GDM, gestational obesity, and GDM with obesity were collected. All subjects (aged 24–30) attended Changsha Maternity and Child Health Hospital from January 2022 to January 2025 according to the IADPSG. During the experiment, all subjects informed consent to the conduct of this experiment. Clinical umbilical cord blood and placental samples from the normal pregnancies, GDM, gestational obesity, and GDM with obesity patients were collected for subsequent testing.

Isolation, culture, and treatment of primary trophoblast cells from placental tissue

Under sterile conditions, chorionic villus tissue between the decidua layer and amniotic membrane was carefully excised. PBS was added to remove blood clots from the tissue surface. The tissue fragments were meticulously dissected using ophthalmic scissors and digested with 6 mL of trypsin (0.25%, AWC0232, Abiowell, Changsha, China). Digestion was terminated by adding 5 mL of complete medium. The cell suspension was filtered through a 40-μm strainer, followed by centrifugation at 1000 rpm for 5 min to collect the cell pellet. The cells were resuspended in D/F12 medium containing 10% fetal bovine serum + 1% penicillin and streptomycin, and cultured in T25 flasks (DH-160I, SANTN, Shanghai, China) in a 37°C, 5% CO2, and saturated humidity incubator. After passaging, third-generation cell climbing slices were identified by immunofluorescence (CK7) for subsequent experiments. For LPS treatment, 10 mg of the LPS (L2630, Sigma, St. Louis, MO) was added to 2 mL of H2O and dissolved to obtain the mother liquor with a concentration of 5 mg/mL. When in use, just dilute it by the corresponding multiple. For high glucose (HG) treatment, 20 mg of glucose was added to 4.440 mL of culture medium and dissolved to obtain the working solution with a concentration of 25 mM. Cells were treated with 200 ng/mL LPS + 25 mM HG induction + co-transfection for 48 h. Decorin recombinant protein (DCN-r) was administered at a concentration of 5 µg/mL for 48 h of co-treatment (Adam et al. 2012).

For transfection, the si-negative control (si-NC), si-DCN (Sense: GACUUUAUCUGUCCAAGAA/dT//dT/, Antisense: UUCUUGGACAGAUAAAGUC/dT//dT/), and si-ADAMTS12 (Sense: GGGUUGUUCCAUAACCCAA/dT//dT/, Antisense: UUGGGUUAUGGAACAACCC/dT//dT/) were provided by Sangon Biotech. Cells (1 × 105 cells/well) were transfected with 3 µg of si-DCN or si-ADAMTS12 through 5 µL of Lipofectamine 2000 (11668019, Invitrogen, Carlsbad, CA).

RT-qPCR

Total RNA was extracted from tissues using Trizol (15596026, Thermo, Waltham, MA). cDNA was synthesized using an mRNA reverse transcription kit (CW2569, Cowin Biotech, Beijing, China). The expression of target genes was detected using the UltraSYBR Mixture kit (CW2601, Cowin Biotech) and a real-time PCR system (PIKOREAL96, Thermo). The 2−ΔΔCt method was employed to calculate the relative expression of target genes (Table 1).

Western blot

The cells or tissues were lysed with radioimmunoprecipitation assay (RIPA) buffer (AWB0136, Abiowell), and protein concentrations were determined using the BCA method. A total of 200-μg protein samples were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred onto polyvinylidene fluoride (PVDF) membranes activated with methanol and blocked with 5% skim milk (AWB0004, Abiowell), then dried at room temperature for at least 1 h. Subsequently, the membranes were incubated overnight at 4°C with primary antibodies anti-DCN (14667-1-AP, Proteintech, Chicago, IL), anti-ADAMTS12 (24934-1-AP, Proteintech, USA), and β-actin (66009-1-Ig, Proteintech) followed by incubation with secondary antibodies HRP goat anti-mouse IgG (1:5000, SA00001-1, Proteintech) and HRP goat anti-rabbit IgG (1:6000, SA00001-2, Proteintech) at 37°C for 90 min. Finally, protein bands were visualized and analyzed using ECL Plus ultrasensitive luminescence solution (AWB0005, Abiowell). The exposed photos were analyzed using quantity one professional gray-scale analysis software. The gray value of the target protein is divided by that of the internal reference protein for normalization processing to calculate the relative expression level.

CCK8

The cells were seeded in a 96-well plate at a density of 5 × 103 cells/well (100 μL per well). Then, 10 μL/well of CCK8 solution (prepared with complete medium) was added to each well, followed by incubation at 37°C with 5% CO2 for 4 h. Finally, the absorbance was measured using a microplate reader (MB-530, Huisong, Shenzhen, China).

Transwell

To detect the migration ability of cells, 500 μL of complete medium containing 10% fetal bovine serum was placed in the lower chamber. Cells were adjusted to a density of 2 × 106 cells/mL, and 100 μL of the cell suspension was added to each well. The plate was placed in a 37°C incubator for 48 h. The upper chamber was removed and transferred to a new well containing PBS. The upper chamber was washed three times with PBS. Non-migrated cells on the upper surface of the chamber were gently wiped off using a cotton swab. Cells were fixed with 4% paraformaldehyde (HY-Y0333, MCE, Monmouth Junction, NJ) for 20 min. The membrane was carefully detached from the chamber. The membrane was stained with 0.1% crystal violet for 5 min. The membrane was placed on a glass slide. Migrated cells on the outer surface of the upper chamber were observed and counted under an inverted microscope (DSZ2000X, Beijing Zhongxian Hengye Instruments, Beijing, China).

To detect the invasive ability of cells, 100 μL of ice-cold, serum-free DMEM-diluted Matrigel (200 μg) was added to each well and incubated at 37°C for 30 min. Subsequently, the transwell chamber (3428, Corning, NY) was used to repeat the above experimental steps for the analysis of cell invasive ability.

ELISA

According to the manufacturer’s instructions, the level of DCN in the umbilical cord blood serum of subjects was detected using the DCN (CSB-E16522h, Wuhan Huamei Bioengineering Co., Ltd, Wuhan, China) kit.

N6-methyl-adenosine (m6A) assay

According to the manufacturer’s instructions, changes in intracellular m6A levels were detected using the m6A (ab185912, Abcam, Cambridge, UK) assay kit.

Data statistics

The statistical analysis in this study was performed using GraphPad Prism 8.0 software. Measurement data are expressed as mean ± standard deviation. Normality and homogeneity of variance were tested first. For data conforming to normal distribution with equal variances, an unpaired t-test was used for intergroup comparisons, while one-way ANOVA or repeated measures ANOVA was employed for multigroup comparisons, followed by Tukey’s post hoc test. A p-value < 0.05 was considered statistically significant.