Patients and sample collection
Placentas were collected immediately after delivery at the Second Affiliated Hospital of Harbin Medical University. A total of 40 placentas were used in the study, 23 from normal and 17 from preeclamptic pregnancies. Diagnostic criteria for study participants were used as previously described21. Normal pregnancy was defined as pregnancy with a blood pressure < 140/90 mmHg, and without proteinuria or obstetrical and medical complications21. Diagnosis of preeclampsia was defined as follows: a sustained systolic blood pressure of ≥ 140 mmHg or a sustained diastolic blood pressure of ≥ 90 mmg on two separate readings; a proteinuria measurement of 1 + or more on a dipstick; or ≥ 300 mg of protein in a 24-h urine specimen21. Smokers were excluded. The demographic data including maternal age, body mass index, gestational age at delivery, blood pressure, and infant birth weight, are summarized in Table 1.
Placenta collection was approved by the Ethical Committee for the Use of Human Samples of Harbin Medical University (# 82001577). All the participants signed a written informed consent for study enrollment. All the experiments were performed in accordance with the relevant guidelines and regulations of ethics committee of Harbin Medical University.
Placental trophoblasts were isolated by trypsin digestion, further purified by Percoll gradient centrifugation and cultured in six-well plates (5 × 106 cells per well) in Dulbecco’s modified Eagle medium supplemented with fetal bovine serum and antibiotics. The trophoblasts were maintained at 37 °C and 5% CO2 for at least 24 h to spontaneously differentiate into syncytiotrophoblasts prior to further analysis.
Cell culture and treatment
The immortalized human trophoblast cell line HTR-8/SVneo (BeNa Cultrue Collection, Beijing, China) was cultured in DMEM/F12 medium supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin. All the cells were cultured under standard conditions in 5% CO2 at 37 °C, and the medium was replaced every 2 days. 1,25(OH)2D3 was used as bioactive vitamin D, and CoCl2 was used as an inducer of hypoxia that causes oxidative stress in placental trophoblasts. HTR-8/SVneo cells were treated with CoCl2 at concentrations of 10, 50, 100, and 250 μM or with 1,25(OH)2D3 at 10, 50, 100, and 250 nM. In the experiment to test the anti-inflammatory role of vitamin D under oxidative stress, CoCl2 at a concentration of 250 μM and 1,25(OH)2D3 at a concentration of 100 nM were used. At the end of each experiment, the total cellular protein or RNA was extracted and used to determine protein expression or mRNA expression.
A standard immunohistochemistry staining procedure was performed as previously described. After blocking, placental tissue sections were incubated with primary antibodies specific for hum VDR (Affinity Biosciences, Jiangsu, China), PGIS (Affinity Biosciences, Jiangsu, China) or COX-2 (Bimake, Shanghai, China) overnight at 4 °C. The corresponding biotinylated secondary antibodies and ABC staining system was subsequently used according to the manufacturer’s instructions. Slides stained with the same antibody were all processed at the same time. The stained slides were reviewed under a microscope and images were captured by a digital scanning microscopy imaging system (PreciPoint, Germany).
Western blot analysis
Placental tissue and trophoblast protein expression of VDR, COX-2 and PGIS was examined by Western blot. An aliquot of 10 μg of total protein was subjected to electrophoresis and then transferred to a polyvinylidene fluoride membrane. After blocking, the membranes were probed with primary antibodies against VDR, COX-2, or PGIS followed by the corresponding secondary antibodies (Bimake, Shanghai, China). The bound antibody was visualized with an enhanced chemiluminescencent detection kit (Yeasen, Shanghai, China). The bands for VDR, COX-2 and PGIS were detected at 48KD, 69KD and 57KD, respectively. The band density was analyzed by ImageJ software (National Institutes of Health, USA). β-actin expression was used as the loading control for each sample.
miR-26b mimic and VDR siRNA transfection
miR-26b overexpression was achieved by transfection of miR-26b mimic, and VDR downregulation was achieved by transfection of VDR siRNA into HTR-8/SVneo cells using the Lipofectamine 2000 transfection reagent. miR-26b mimic and VDR siRNA were purchased from GenePharma and Sangon Biotech (Shanghai, China), respectively. Transfection was performed when the cells reached 60–70% confluence. Total RNA was extracted with TRIzol reagent approximately 48 h after transfection, and miR-26b-5p expression was determined by quantitative PCR. Total cellular protein was collected to determine COX-2 expression. The medium was also collected to determine PGE2 and PGI2 production.
Quantitative polymerase chain reaction (qPCR)
Total RNA was extracted from placental tissue or HTR-8/SVneo cells with TRIzol reagent. cDNA was synthesized using the Mir-X miRNA First Strand Synthesis Kit (Takara, Japan) following the manufacturer’s instructions. miR-26b-5p expression was determined by qPCR. The qPCR was performed in 20 μL solutions using the SYBR Premix Ex Taq II Kit (Takara, Japan). The expression of U6 snRNA was determined and served as the endogenous control for the expression of miRNA-26b-5p. The relative expression values were calculated by the ΔΔCT method of relative quantification using an Applied Biosystems 7500 Real-Time PCR System. The primer for miR-26b-5p was 5′-UUCAAGUAAUUCAGGAUAGGU-3′, which was synthesized by Sangon Biotech (Shanghai, China).
PGE2 and PGI2 concentrations were measured using enzyme-linked immunosorbent assay (ELISA). The ELISA kits for the detection of human PGE2 and PGI2 were purchased from Elabscience Biotechnology (Wuhan, China). The sensitivity of the ELISA kits for detection of PGE2 and PGI2 was 18.75 pg/mL. The assay was carried out according to the manufacturer’s instructions. Both PGE2 and PGI2 standards were serially diluted, with ranges of 3.9 ~ 500 pg/mL. An aliquot of 50 μL of each sample was assayed in duplicate. After reaction, the plates were read at 450 nm by an autoplate reader (Molecular Devices, USA). The Within-assay variations were < 8% for all the assays.
Data presentation and statistics
Data are presented as mean ± SEM. Statistical analysis was performed with unpaired t-test to compare two groups. Ordinary one-way ANOVA followed by Tukey’s post hoc test was performed to compare multiple groups using GraphPad Prism 8 software. A probability level < 0.05 was considered statistically significant.