From April 2017 to December 2018, 20 parturient women diagnosed with GDM in the obstetrics department of Changzhou maternal and child health hospital affiliated to Nanjing medical university and 20 normal adjust control women were selected. The diagnosis of GDM was conducted by 75 g glucose tolerance test at 24-28 weeks, excluding multiple births, premature delivery, delivery age less than 20 years old or over 40 years old, pre-pregnancy patients with hypertension, diabetes, chronic liver and kidney diseases, thyroid and other endocrine diseases.
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 cell line HTR-8/SVneo were cultured in 1640 supplemented with 10% 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_hsa_circ_0005243 using Lipofectamine 3000 (Invitrogen, USA) according to the manufacture’s protocol. The knockdown efficiency of siRNAs was determined by qRT-PCR, their sequences were listed as: 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 the siRNAs and negative controls were obtained from RiboBio (Guangzhou, China).
Cells were digested and seeded into 96-well cell culture plate (Corning, USA) with a concentration of 3× 103 cells/mL. Cells viability was measured after culture for 24hr, 48hr and 72hr by adding 10 μL CCK8 reagent (DOJINDO Laborataries, Japan). After incubated at 37℃ for 3 hr, the OD value at 450 nm was detected by a Microplate Reader (BioTek, Winooski, VT, USA).
Colony formation assay
The cells in logarithmic phase were digested into suspension and inoculated in 6-well cell culture plates containing 2 mL medium, shake the culture plates gently to make the cells disperse evenly. Then the cell culture plates were transplanted into a CO2 incubator, with 37℃ and 5% for 24 hours until it adhered to the wall. After 12 days, the medium was discarded and the culture was terminated, carefully soaked twice with PBS, fixed for 15 minutes with 5 mL absolute ethanol. After discarding the fixative solution, add Jimsa dye solution (ThermoFisher, USA) for 10-30 minutes, then wash the dye solution slowly with running water and dry the air, after photographed the clones were counted.
In order to evaluate the proliferation ability of trophoblast cells, EdU assay was performed using keyFluor555 Click-iTEdU imaging detection Kit (Keygentec, Nanjing, China) according to the manufacturer's protocol. Cells were fixed with 4% paraformaldehyde, then incubated with 2 mg/mL glycine for 5 min, 200 mul of 1×Apollo® staining solution was added to each well and incubated in a bleached shaker for 30 min at room temperature, away from light, after that washed with PBS, 100µL penetrant agent (0.5% TritonX-100in PBS) was added. After nucleus of the cells was stained with Hoechst 33342, cells are photographed by a high-content imaging system(MD Micro, USA).
For the in vitro Transwell migration assay, the transfected cells were digested and adjusted to a density of 1 ×105/mL, 100 µL cell suspension was added to the upper chamber(Corning Incorporated, USA), and 700 µL medium containing FBS was added to lower chamber. Cell culture plate was then placed in 37 C, 5% CO2 incubating for 24 hr. Wiped off the cells in the upper chamber using a cotton swab, while the cells on lower surface of membranes were fixed with formaldehyde and stained with 0.1% crystal violet(Sigma, USA). After incubated in 37℃ for 30 min, take it out and washed with PBS, 3-5 fields were randomly selected and photographed and the migrated cells were counted under an inverted Microscope(Olympus, Japan).
For the wound healing assay, cells in logarithmic growth phase were digested and inoculated into a six-well plate. After 24hr, when the cell aggregation reached about 60%, the sterile nozzle was used to evenly draw lines in the plate. The floating cells were washed with PBS, and then fresh medium was placed in a cell culture box for further culture. After 24 hours, the cells were taken out and photographed (with a magnification of 200×), and the migration distance of cells was measured.
Cells were seeded in a 6-well plate (Corning Incorporated), after transfection, the medium was replaced with new culture medium. The culture medium was then centrifuged for 20 minutes at 1000 ×g to remove cell debris and impurities. The concentrations of TNF-α and IL-6 in the medium was detected by Elias kit (Mlbio, Shanghai, China) according to the manufacturer's protocol. The absorbance (OD value) of each group was measured at 450 nm by a Microplate Reader (MD SpectraMax M3, USA).
The transfected cells were harvested and lysed in the lysate containing protease inhibitors. Then protein concentrations were determined by the BCA kit (Thermo Fisher Scientific, USA). After denaturation, the proteins were separated by 12% SDS-PAGE, then transferred to the PVDF membrane (Merck Millipore, Darmstadt, Germany). After blocked by 5% defatted milk, the membranes with proteins were incubated with diluted primarily antibodis at 4℃ overnight as follows: 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 purchased from Abcam (Cambridge, UK). The membranes were then washed by TBST and incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies (Beyotime, China) for 1 hr. After washed by TBST the membranes were incubated with enhanced chemiluminescence reaction reagent (BeyoECL Plus, Beyotime, China) and photographed by Luminescence imaging system (Tanon, Shanghai, China)
Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). The expression of circRNA and GAPDH was detected using SYBR Premix Ex Taq system (Takara, Madison, WI, USA) based on the manufacturer’s instructions. RT-PCR was performed using primer with the sequences listed as follows: hsa_hsa_circ_0005243, forward, 5'-TTATCTACATGCACCTGCGCT-3', reverse, 5'-AAGTGACAAGCTAGCCCTCAT-3', GAPDH, forward: 5'-CAAATTCCATGGCACCGTCA-3', reverse: 5'-AGCATCGCCCCACTTGATTT-3'. The PCR reactions were conducted as follows: denaturation at 95°C for 10 min, amplification 40 cycles of 95°C for 10 s and 58°C for 15 s, followed by treatment at 70°C for 30 s. The relative expression levels were determined by comparing the Ct values of the target genes to those of the GAPDH gene.
For the cell apoptosis analysis, after transfection cells were digested with 0.25% trypsin (without EDTA) and collected, washed twice with PBS and stained using Annexin V-FITC Apoptosis Detection Kit (Beyotime, China) and analyzed by flow cytometry (FACS Calibre, BD, USA) . The ratio of apoptotic cells was counted and obtained.
Slides of cells were fixed by 4% polyformaldehyde, washed by PBS, then treated with 0.5% Triton X-100, after that the slides were blocked by 5% BSA for 30 min. Cells were then incubated with antibodies against β-catenin and p65 (1:100, abcam) at 4°C overnight .After 3 washes with PBS, FITC-conjugated secondary antibody (1:100, abcam) was added and incubated at 37℃ for 1 hr away from light. Slides were stained by DAPI for 5 min, then the expression of protein in cells were observed under a laser confocal microscopy (Zeiss LSM710, Germany), 3 three photographs were randomly taken.