Study population and tissue collection
Twenty normal pregnant (NP) women and 20 severe preeclampsia (sPE) patients were recruited from Ji'nan Maternity and Child Care Hospital between June 2016 and July 2017 in this study. All subjects were single pregnancies, and women suffered from chronic hypertension, diabetes mellitus, renal disease and other pregnancy related disorders were excluded. sPE was diagnosed according to the American College of Obstetricians and Gynecologists . By definition, the systolic pressure blood was ≥ 160 mm Hg or diastolic blood pressure ≥ 110 mm Hg on two occasions ≥ 4 h apart. Proteinuria was present as ≥ 3 grams in 24 h urine collection (≥ 2 + by dipstick). Sampling was carried out by scrubbing the uterus from the site of implantation as soon as the placenta was delivered by caesarean section using sterile gauze. The decidual tissues were washed with sterile saline to remove blood, snap frozen in liquid nitrogen and stored at -80°C for further processing. The clinical characteristics of the participants are summarized in Table S1.
Decidual tissues were embedded in O.C.T. compound (Sakura Tissue-Tek Xpress, Torrance, CA, USA) and cut into 5-µm thick cryosections. The labeled slides were fixed in ice-cold acetone for 10 min at -20°C, and rinsed in phosphate-buffered saline (PBS) at room temperature every five min with three changes of buffer . Thereafter, the sections were immersed in absolute methanol containing 3% H2O2 to eliminate endogenous peroxidase activity. The tissues were probed with polyclonal primary antibody against GLUT1 (sc-7903; Santa Cruz Biotechnology, CA, USA) at 1:100 dilution overnight at 4°C. Pre-immune IgG rather than primary antibody was used for negative control. After washing with PBS, the specimens were incubated with horseradish peroxidase-conjugated secondary antibodies diluted 1:200 at 37°C for 1 h. Following three additional 5 min washing in PBS, the sections were stained with diaminobenzidine (DAB) (ZLI-9033, ZSGB-BIO, Beijing, China), counterstained with hematoxylin, dehydrated through ethanols, and hyalinized in xylene, then the sections were mounted with neutral gum and examined using a Digital Scanning System (Pannoramic MIDI, 3DHISTECH, Budapest, Hungary) . Immunohistochemical staining was quantified with Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA), and for each group, total of 36 fields from 12 different women (each woman 3 fields) were quantified and analyzed.
Total RNA extraction and quantitative PCR
Total RNA was extracted from decidual tissues and human endometrial stromal cells (hESCs) using TRNzol reagent (Tiangen, Beijing, China) and animal total RNA isolation kit (Foregene, Chengdu, China) respectively according to manufacturer’s instructions. RNA samples (500 ng) were reverse transcribed into cDNA using FastQuant RT Kit with gDNase (Tiangen). As for miRNA, the synthesis of cDNA was performed using Mir-X™ miRNA First-Strand Synthesis Kit (Takara, Dalian, China). Quantitative PCR was carried out using SuperReal PreMix Plus Kit (Tiangen) in Light Cycler 96 system (Roche, Basel, Switzerland). Quantitative PCR was conducted using the following procedures: pre-denaturation for 5 min at 95°C, 40 cycles of 10 s at 95°C, 20 s at 58°C, 20 s at 72°C and a final extension for 5 min at 72°C. The primers used are listed in Table S2. The mRNA and miRNA levels were analyzed using the ΔΔCt method with normalization to reference genes ACTB and U6 respectively . All samples were performed in triplicate in one assay.
Protein extraction and Western blotting
Total protein was extracted using radio immunoprecipitation assay lysis buffer (Beyotime, Shanghai, China) with protease inhibitors (Sigma Chemical Co., St. Louis, MO, USA). Protein extracts (30 µg) were subjected to SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocked with 5% non-fat milk, the membranes were incubated overnight at 4°C with primary antibodies against GLUT1 (1:2000 dilution; sc-7903, Santa Cruz Biotechnology) or β-actin (1:5000 dilution; sc-47778, Santa Cruz Biotechnology). Subsequently, the membranes were probed with horseradish peroxidase-conjugated secondary antibodies at 1:5000 dilutions for 1 h at room temperature. At the end, the chromogenic reaction was performed by incubating the membranes with enhanced chemoluminescence (ECL) detection reagents (Millipore). The images were acquired with a Gel-Imaging Detection System (5500 Multi, Tanon, Shanghai, China). Densitometric analysis was conducted by using QuantiScan software (Biosoft, Cambridge, UK) .
Cell culture, decidualization in vitro and transfection
The immortalized hESCs were from the American Type Culture Collection (ATCCR CRL-4003TM). The cells were maintained in phenol red-free DMEM/F12 medium (Gibco, NY, USA) supplemented with 10% charcoal-stripped FBS (BI, Beit Haemek, Israel) at 37°C under 5% CO2. HESCs were treated with 0.5 mmol/L db-cAMP (Sigma Chemical Co.,) and 1 µmol/L P4 (Meilun Biotechnology Co., Dalian, China) simultaneously to induce decidualization in vitro. GLUT1 siRNA, miRNA mimics and inhibitor were synthesized by RiboBio (RiboBio Co., Guangzhou, China). When the cells reached 60% confluence, transient transfection was performed using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Subsequently, these cells were harvested for quantitative PCR and Western blotting analysis.
Glucose uptake and lactate production assay
Glucose uptake was measured using glucose oxidase method kit (GOD, Applygen Technologies, China) according to the manufacturers’ instructions. Briefly, the cells were grown in starvation medium overnight and were then stimulated with 1 µmol/L insulin for 30 min. 48 h later, the supernatants were harvested and the optical density was measured at 550 nm using microplate reader (Spectra Max M5, Molecular Devices, USA).
For lactate assay, the cell supernatants were harvest after transfection with GLUT1-siRNA. After centrifugation, 20 µL of supernatants were used to analyze the optical density at 530 nm using Lactic Acid assay kit (Jianchengbio, Nanjing, China) according to the manufacturers’ instructions.
Dual-luciferase reporter assays
GLUT1 3′-UTR wild-type fragment containing miR-140-5p binding sites was inserted into a pmirGLO dual-luciferase miRNA target expression vector (Promega, WI, USA). The cells were transfected with miR-140-5p mimics along with GLUT1 wild-type (WT) or mutant constructs (MUT) using lipofectamine 3000 reagent (Invitrogen, CA). Luciferase activity was assayed after 48 h transfection using dual-luciferase reporter assay system (Promega). The renilla luciferase activity was used to normalize the activity of firefly luciferase.
Data analysis was performed using SPSS 24.0 software (SPSS, Chicago, IL, USA). All data are expressed as mean ± SEM and all experiments were independently performed at least three times. Log transformation was applied to change the scattered distribution of miRNA data. Comparison between two groups was carried out using Student's t-test and differences with P < 0.05 were considered as statistically significant. In addition, Pearson correlation analysis was carried out to evaluate the relationship between the levels of miRNA and GLUT1.