From April 2017 to December 2018, we identified 20 parturient women diagnosed with GDM and 20 parturient healthy control women in the obstetrics department of the Changzhou Maternal and Child Health Care Hospital, an affiliated hospital of Nanjing Medical University. Placenta tissues were obtained within 10 min after delivery, while the maternal plasma specimens were collected on an empty stomach early in the morning on the day of hospitalization during the third trimester (37–40 weeks). After collection, the plasma and placenta samples were stored at −80°C. All GDM and control women were matched by age and body mass index. The diagnosis of GDM was conducted by 75-g oral glucose tolerance test at 24–28 weeks. Exclusion criteria included multiple births, premature delivery, delivery age < 20 years old or > 40 years old, diabetes, chronic liver and kidney diseases, thyroid and other endocrine diseases, and hypertension prior to pregnancy. Informed consent was obtained from each participant and this study was approved by the ethics committee of the hospital.
Cell culture and transfection
Human trophoblast HTR-8/SVneo cells were cultured in 1640 medium supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin at 37°C with 5% CO2. Cells (2 × 105) were seeded in 6-well plates and transfected with small interfering RNAs (siRNAs) targeting hsa_circ_0005243 using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s protocol. The knockdown efficiency of siRNAs was determined by quantitative reverse transcription (qRT) PCR using the following primers: siRNA-1, 5'-UGA CCA UCA UCU ACA ACA UTT-3', 5'-AUG UUG UAG AUG AUG GUC ATT-3'; siRNA-2, 5'-CCA UGA ACC CGC ACG ACA UTT-3', 5'-AUG UCG UGC GGG UUC AUG GTT-3'; siRNA-3, 5'-CCU ACA AGG UCU AUG CUG ATT-3', 5'-UCA GCA UAG ACC UUG UAG GTT-3'. All siRNAs and negative controls (NCs) were obtained from RiboBio (Guangzhou, China).
Cells were trypsinized and seeded into 96-well cell culture plates (Corning Inc., Corning, NY, USA) at a concentration of 3 × 103 cells/mL. Cell viability was measured after culture for 24, 48, and 72 h by adding 10 μL of CCK8 reagent (DOJINDO Laboratories, Kumamoto, Japan). Cells were then incubated at 37°C for 3 h, after which the optical density at 450 nm (OD450) was assessed using a microplate reader (BioTek, Winooski, VT, USA).
Colony formation assay
Cells in logarithmic phase growth were trypsinized, re-suspended, and inoculated in 6-well cell culture plates containing 2 mL of medium. After plating, the culture plates were gently shaken to ensure even distribution of the cells within the wells and placed in an incubator at 37°C and 5% CO2 for 24 h until full adherence was obtained. After 12 days, the medium was discarded, and the cells were carefully soaked twice with phosphate-buffered saline (PBS). Cells were then fixed for 15 min with 5 mL of absolute ethanol. After discarding the fixative solution, the cells were treated with Giemsa dye solution (Thermo Fisher Scientific, Waltham, MA, USA) for 10–30 min, followed by slow washing with running water. Finally, cells were air dried, photographed, and counted.
To evaluate the proliferation ability of trophoblast cells, an EdU assay was performed using a keyFluor 555 Click-iT EdU imaging detection kit (Keygen Tec, Nanjing, China) according to the manufacturer’s protocol. Briefly, cells were fixed with 4% paraformaldehyde, then incubated with 2 mg/mL glycine for 5 min, followed by 200 µL of 1× Apollo staining solution for 30 min in a bleached shaker at room temperature, away from light. Cells were then washed with PBS, after which 100 µL of penetrant agent (0.5% Triton X-100 in PBS) was added. Cell nuclei were stained with Hoechst 33342, and the cells were photographed with a high-content imaging system (MD Micro Solutions, Gloucester, MA, USA).
For the in vitro transwell migration assay, the transfected cells were trypsinized, adjusted to a density of 1 × 105 cells/mL, and 100 µL of cell suspension and 700 µL of medium containing FBS were added to the upper and lower chambers of a transwell plate (Corning Inc.). The cell culture plates were then placed in an incubator at 37°C with 5% CO2 for 24 h. Cells in the upper chamber were removed using a cotton swab, while the cells on the lower surface of membranes were fixed with formaldehyde and stained with 0.1% crystal violet (Sigma-Aldrich, St. Louis, MO, USA). After incubation at 37°C for 30 min, cells were washed with PBS, and three to five fields were randomly selected and photographed, with the number of migrated cells counted under an inverted microscope (Olympus, Tokyo, Japan).
For the wound-healing assay, cells in logarithmic growth phase were trypsinized and inoculated into a 6-well plate. After 24 h, when the cell aggregation reached ~60%, a sterile nozzle was used to evenly draw lines in the plate. Floating cells were removed by washing with PBS, and then fresh medium added for further culture. After 24 h, the cells were taken out and photographed (200× magnification), and the migration distance of cells was measured.
Enzyme-linked immunosorbent assay
Cells were seeded in 6-well plates (Corning Inc.) and transfected as described above, after which the medium was collected and replaced with fresh culture medium. The culture medium was then centrifuged for 20 min at 1000 × g to remove cell debris and impurities. The concentrations of tumor necrosis factor alpha (TNF-α) and IL-6 in the medium were detected using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Mlbio, Shanghai, China) according to the manufacturer’s protocol. The absorbance (OD450) of each group was measured using a microplate reader (MD SpectraMax M3; Molecular Devices, San Jose, CA, USA).
Transfected cells were harvested and lysed in lysate buffer containing protease inhibitors. Protein concentration was determined using a BCA kit (Thermo Fisher Scientific). After denaturation, the proteins were separated using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then transferred to a polyvinylidene fluoride membrane (Merck Millipore, Darmstadt, Germany) and blocked with 5% skim milk. Membranes were then incubated in the presence of primary antibodies at 4°C overnight at the following concentrations: anti-c-myc (1:2000), anti-cyclinD1 (1:3000), anti-survivin (1:3000), anti-β-catenin (1:1000), anti-p65 (1:2000), anti-laminin B (1:3000), and anti-β-actin (1:3000). All antibodies were purchased from Abcam (Cambridge, UK). The membranes were then washed with tris-buffered saline–Tween 20 (TBST) and incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Beyotime, China) for 1 h. Membranes were washed again in TBST, incubated with enhanced chemiluminescence reaction reagent (BeyoECL Plus; Beyotime), and visualized using a luminescence imaging system (Tanon, Shanghai, China).
Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific). The expression of circRNA and GAPDH were detected using the SYBR Premix Ex Taq system (Takara, Madison, WI, USA) following the manufacturer’s instructions. RT-PCR was performed using the following primers: hsa_circ_0005243, forward, 5'-TTATCTACATGCACCTGCGCT-3', reverse, 5'-AAGTGACAAGCTAGCCCTCAT-3'; GAPDH, forward: 5'-CAAATTCCATGGCACCGTCA-3', reverse: 5'-AGCATCGCCCCACTTGATTT-3'. PCR reactions were conducted as follows: denaturation at 95°C for 10 min, amplification at 40 cycles of 95°C for 10 s and 58°C for 15 s, followed by elongation at 70°C for 30 s. Relative expression levels were determined by comparing the Ct values of the target genes to those of the GAPDH gene.
To assess cell apoptosis, transfected cells were digested with 0.25% trypsin (without EDTA) and collected, washed twice with PBS, stained using an annexin V–FITC apoptosis detection kit (Beyotime), and analyzed by flow cytometry (FACSCalibur; BD, Franklin Lakes, NJ, USA). The number of apoptotic cells was determined by counting and expressed as a ratio relative to live cells.
Cells were fixed in 4% polyformaldehyde, washed with PBS, and treated with 0.5% Triton X-100, after which the slides were blocked with 5% bovine serum albumin for 30 min. Cells were then incubated with antibodies against β-catenin and p65 (1:100; Abcam) at 4°C overnight. Next, slides were washed three times in PBS, after which FITC-conjugated secondary antibody (1:100; Abcam) was added and incubated at 37°C for 1 h in the dark. Finally, slides were stained with DAPI for 5 min, and the expression of protein in cells was observed under a laser confocal microscopy (LSM710; Zeiss, Oberkochen, Germany). Three photographs were randomly taken per slide.
All data were analyzed using SPSS ver. 22 software (IBM Corp., Armonk, NY, USA), with continuous variables expressed as the mean ± standard error. A two-tailed Student’s t-test was used to compare the mean of the two sets of samples. The diagnostic value of hsa_circ_0005243 for GDM was established by a ROC curve and the AUC was calculated (adjusted by BMI and age). P values < 0.05 were considered statistically significant.