1.Experimental object and specimen collection
(1) Experimental object: The protocol governing the subjects of this study was approved by the Ethics Committee of the Affiliated Hospital of Guangdong Medical University(PJ2013014).Patients with unexplained recurrent abortion (URSA group) or unexpected pregnancy (<12 weeks) (NC group) were selected from the Outpatient Department of the Affiliated Hospital of Guangdong Medical University from 2016 to 2018. Each group entailed 25 cases of chorionic villi. The mean age of the URSA group was 33.73 ± 5.33 years (mean ± SD), and the mean gestational age was 7.82 ± 0.81 weeks. The mean age of the NC group was 31.23 ± 4.83, and the mean gestational age was 7.31 ± 0.95 weeks. All of the patients signed informed consent forms. The inclusion criteria for the URSA group were all of the patients who underwent 2 or more miscarriages, possessed excluded possible causes (chromosomal abnormalities, immunological factors, etc.), and were diagnosed as having early abortion villi of URSA (<12 weeks). For the NC group, chronologic and gestational age were similar, and the patients who voluntarily requested termination of pregnancy had a history of normal pregnancy and delivery before the current pregnancy, had no stillbirths, no history of spontaneous abortion, no medication use, and no history of viral infection. During the current pregnancy, there were no threatened abortion symptoms or signs, and B-ultrasonography suggested normal embryonic development.There was no statistical difference in age and gestational age between the two groups(p>0.05).
(2) Specimen collection: The villi were extracted immediately in the aseptic state, washed with sterile saline until no obvious blood was observed. The villi were divided into 3 parts (some of the villi were quickly placed into cryopreserved tubes containing 1 mL of Trizol reagent [Invitrogen,US]). Tissues were placed into a cryotube, quickly deposited into liquid nitrogen for quick freezing, and stored in a -80°C refrigerator (Invitrogen,US) for later extraction of tissue protein and RNA. Partial fixation in 4% (v/v) formaldehyde was used for immunohistochemistry.
2.Cell culture: HTR-8/SVneo or BeWo cells from the Stem Cell R & D and Clinical Transformation Center of the Affiliated Hospital of Guangdong Medical University were cultured in RPMI 1640 medium (Gibco,US) containing 10% (v/v) fetal bovine serum (FBS, Gibco,Australia) and placed in an incubator with a CO2 concentration of 5%, a humidity of 95%, and a temperature of 37°C. Different cell lines were developed to study trophoblast functions( cell fusion, migration and invasion) including BeWo and HTR-8/SVneo. Studies have shown that BeWo is positive for the trophoblast / epithelial marker CK7, while HTR-8 / SVneo cells contain almost no CK7 positive cell clusters(Abou-Kheir et al, Placenta. 2017).Studies have shown that BeWo is more used to study cell fusion, while HTR-8 / SVneo is inclined to invasion and migration functions.
3. Transfection: For HTR-8/SVneo or BeWo cells, Lipofectamine 3000 (Invitrogen,US) was used to transfect plasmids (an LIN28B overexpression plasmid was constructed with green fluorescent GFP, EX-Y3355-Lv105,GeneCopoeia,US). Transfection was carried out according to the specifications of the transfection reagent.Transfection efficiency was about 70%.
4.Immunohistochemistry: Immunohistochemical staining using primary antibody (LIN28B, Abcam,UK) was performed on sections of placental villi. According to the instructions for the SP Rabbit & Mouse HRP Kit (DAB) (CWBIO,China), biotinylated anti-rabbit/mouse universal second antibody was combined with the first specific antibody, and the second antibody was labeled with biotin combined with the streptavidin-labeled peroxidase (HRP). A streptavidin complex was thereby achieved by label with antigen-specific first antibody-biotinylated second antibody-HRP. Blank control: the primary antibody was replaced with PBS, other steps were as above (including endogenous peroxidase blocking and serum blocking).We used ImageJ software to carry out a sample image for immunohistochemistry and made quantitative comparisons using digital image analysis.
5. RNA and protein extraction: We used collected tissue or untreated cell samples to which Trizol (Invitrogen,US) was added and ground and homogenized to extract RNA, while the protein was extracted by homogeneous lysis of RIPA lysate (Beyotime,China) containing 1% PMSF (Beyotime,China).
6. Real-time quantitative PCR (qPCR): CDNA was synthesized according to the manufacturer’s instructions found in the PrimeScriptRT Reagent Kit using gDNA Eraser (TaKaRa,Japan) kit, and the TB Green chimeric fluorescence method (TaKaRa,Japan) was used for real-time quantitative PCR. An ABI7500 real-time qPCR system was used to perform all qPCR reactions, and the data were standardized to β-actin. The qPCR primer sequence is shown in Table 1. We analyzed relative gene expression using the semi-quantitative 2-△△Ct method.
7. Western blotting analysis: As mentioned above, total protein lysates were separated by polyacrylamide gel electrophoresis (PAGE), transferred to PVDF membranes (Millipore,US), and sealed at room temperature with 5% (w/v) milk (BD,US) for 2 h. Lysates were incubated with LIN28B (Abcam,UK), β-actin, Akt, p-Akt, Bad, p-Bad, or Bcl-2 primary antibodies overnight at 4°C, and then incubated with secondary antibody (CST,US) for 1 h at room temperature. The resulting immunoblot was scanned and quantitatively assessed using a Tanon 5200 imaging system.
8. Invasion and migration assays: HTR-8/SVneo or BeWo cells were seeded in a 6-well culture dish and transfected with a plasmid on the next day. After 24 h, the transfected cells were digested and resuspended in serum-free RPMI 1640 medium and inoculated to an upper chamber (Corning,US) that had been coated with matrigel (Corning,US); the lower chamber was placed in 10% (v/v) FBS. After 1 or 2 days, the non-invasive cells in the upper chamber were wiped with a swab, and the remaining invasive cells were then stained with crystal violet and imaged. We counted 5 fields of view in each cell, and the numbers of invasive cells were analyzed statistically. The steps with respect to migration were the same as for invasion, but the inoculated upper chamber did not need to be covered with matrigel.
9. Apoptosis assay: FACSCantoII flow cytometry (BD,US) was used to measure apoptosis according to the instructions in the PE Annexin V apoptosis kit maker (BD,US).Cells that are considered viable are PE Annexin V and 7-AAD negative; cells that are in early apoptosis are PE Annexin V positive and 7-AAD negative; and cells that are in late apoptosis or already dead are are both PE Annexin V and 7-AAD positive.
10. Evaluation of cell cycle kinetics: According to the manufacturer’s instructions found in the cell cycle and apoptosis detection kit (Beyotime,China), we fixed cells with 70% (v/v) ethanol for 4 h at 4°C, and then added pyridine iodide staining solution (for a 0.5 mL sample we used 25 μl of 20X staining buffer and 10 μl of 50X RNase A at 37°C for 30 min), and measured cell cycle parameters by FACSCantoII flow cytometry (BD,US) within 24 h.
11. Cell fusion assay: BeWo cells were inoculated in confocal dishes and transfected with plasmid when the cell density was 30-50%. After 12 h, 50 μM adenylate cyclase activator (forskolin, FSK, Beyotime,China) and 0.1% (v/v) DMSO were added consecutively. After continuous culture for 1 or 2 weeks, we immobilized the cells with 4% (v/v) paraformaldehyde, incubated them overnight with β-catenin (Abcam,UK) at 4°C, and then incubated them additionally with secondary antibody (CST) for 1 h at room temperature. After staining cells with DAPI, we used a laser confocal microscope (Olympus FV3000) to photograph and analyze results. The full field of view of the confocal dish was taken using a 20X objective, and the number of cells with ≥3 nuclear fusions was counted.
The results are expressed as mean ± standard error. The normality and variance equivalence of all data were tested to determine the appropriate statistical test. We used Student’s t test, x2, or Fisher exact-probability test to determine significant differences between groups. All statistical analyses were processed using GraphPad Prism 7 software (USA); and a difference of P < 0.05 was considered statistically significant, except where noted. The number of experimental replicates and the number of replicates within each experiment were both three times.