Cell isolation and culture
Umbilical cords were collected from healthy women with full-term delivery. Endometrium samples were collected from patients with intramural or pedunculated subserosal hysteromyoma without endometrial abnormalities to obtain ESCs. None of the women had taken medication or had received hormonal therapy for at least 6 months before undergoing hysterectomy. The collection and use of human biological specimens were approved by the Ethics Committee of the Affiliated Hospital of Nantong University. WJ-MSCs and ESCs were isolated, cultured, and identified as previously described . WJ-MSCs and ESCs were cultured in Dulbecco’s modified Eagle’s medium F12 (DMEM/F-12; Hyclone, USA) containing 10% fetal bovine serum (FBS; Gibco, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a humidified 5% CO2 atmosphere.
ESCs treated with mifepristone and cocultured with WJ-MSCs
The damaged ESC model and the WJ-MSCs coculture system were developed according to our previous protocol. Briefly, the ESCs were dispensed into 24-well plates at a density of 1.5 × 104 cells/well and cultured in DMEM-F12 with 10% FBS. After 24 h, the medium was replaced with another medium containing 2% FBS. After 12 h, the cells were treated with mifepristone (#M8046; Sigma-Aldrich, USA) at the concentration of 60 μmol/L. The medium was then replaced with a fresh medium (DMEM-F12 with 2% FBS) after treatment for 48 h. The Transwell system (24 mm Transwell plate with a 0.4-μm pore polycarbonate membrane insert; #3412, Corning, NY, USA) was used to establish the coculture system. WJ-MSCs were seeded on top of the artificial basement membrane and placed in the upper part of the plate for coculture with damaged ESCs. The coculture was stopped after 48 h. ESCs were digested with 0.25% trypsin without ethylenediaminetetraacetic acid (EDTA) (Invitrogen, Carlsbad, CA, USA), and WJ-MSCs were digested with TrypLE Express (Thermo Fisher Scientific, USA).
deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining
Cells were fixed with 4% paraformaldehyde for 30 min at room temperature and washed twice with PBS. The cells were then incubated in the TUNEL solution containing FITC-dUTP for 60 min at 37 °C according to the manufacturer’s protocol (#C1088, Beyotime Biotechnology, China). Next, the cells were incubated in Hoechst 33342 for counterstaining of cell nuclei. Images were captured and analyzed using the EVOS M7000 Imaging System (Invitrogen, Thermo Fisher Scientific). The apoptosis rate was calculated as the ratio of FITC-positive cells (green cells) to total Hoechst 33342-positive cells (blue cells).
5-Ethynyl-2′-deoxyuridine (EdU) staining
DNA replication was detected by the EdU incorporation assay according to the manufacturer’s protocol (#C10310-3, RiboBio Co., China). Briefly, 1.5 × 105 cells were inoculated in a 24-well plate. A labeling medium was added to each well for 2 h at 37 °C under 5% CO2. Cell samples were fixed with 4% paraformaldehyde for 30 min, permeated with 0.5% Triton X-100 for 10 min, and washed twice with PBS. The cell samples were then incubated with Pollo™ 488 dye buffer in caliginous condition for 30 min at room temperature. Subsequently, Hoechst 33342 was added, and the cell samples were incubated for 30 min. Images were captured and analyzed using the EVOS M7000 Imaging System. The EdU incorporation rate was calculated as the ratio of EdU-positive cells (green cells) to total Hoechst 33342-positive cells (blue cells).
Cell apoptosis analysis by flow cytometry
ESCs from each group were collected and washed twice with cold PBS for apoptosis analysis. The cell apoptosis rate was determined using an annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (#KGA108, Keygen Biotechnology, China) according to the manufacturer’s instructions. Cells in each group were prepared in triplicate, and the stained cells were then sorted with a FACScan flow cytometer (Becton Dickinson, NJ, USA) within 1 h. The data were analyzed using Flow Jo software (Flow Jo, Ashland, OR, USA).
Western blot assay
Total protein was extracted with RIPA buffer containing a protease inhibitor cocktail (Thermo Fisher Scientific). The protein concentration was determined using a Coomassie (Bradford) protein assay kit (Thermo Fisher Scientific). Equal amounts of total protein (30 μg) were separated by 12% SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA), followed by blocking with 5% skim milk. The membranes were incubated with antibodies against VEGFA (#ab46154, Abcam), VEGF Receptor 1 (#ab32152, Abcam), VEGF Receptor 2 (#ab194806, Abcam), RAP1B (#ab154756, Abcam), and GAPDH (#ab8245, Abcam) overnight at 4 °C. The membranes were then washed three times with TBST followed by incubation with secondary antibodies for 2 h at room temperature. Bands were detected by a chemiluminescence imaging system (Molecular Imager, ChemiDoc XRS+, Bio-Rad, USA) with Western Bright ECL HRP substrate (#WBKL0100, Millipore, USA). Band intensities were quantified using Image J software (NIH, Bethesda, MD, USA).
RNA extraction and quantitative real-time PCR (qRT-PCR)
Total RNA containing small RNA was extracted using TRIzol (Invitrogen) according to the manufacturer’s instructions. RNA quality was analyzed with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). To determine miRNA expression, reverse transcription was performed using miRNA-specific primers and a miScript reverse transcription kit (Qiagen, Germany) with RNU6B expression as an endogenous control. SYBR Green PCR Master Mix (Qiagen) was used to determine mRNA expression, with GAPDH expression as an endogenous control. To determine circRNA expression, cDNA reverse transcription and amplifications were conducted using a circRNA fluorescence quantitative detection kit (#GS0201-1, Geneseed Biotechnology, Guangzhou, China) according to the manufacturer’s instructions. qRT-PCR was performed using the ABI 7500 Real-Time PCR system (Applied Biosystems, USA). The 18s rRNA gene served as an endogenous control for normalization. Each relative RNA expression level was calculated from three different experiments. The sequences of gene-specific qRT-PCR primers are provided in Additional file 2 Table S1. The primers were designed using the Primer-BLAST program based on the National Center for Biotechnology Information (NCBI) sequence data. The relative expression level was calculated by the 2-ΔΔCT method.
circRNA and miRNA microarray analysis
CircRNA microarray analysis was performed with Human CircRNA Array v2 microarray (Capital Biotechnology, China). miRNA microarray analysis was performed with Affymetrix GeneChip miRNA 2.0 platform (Capital Biotechnology, China). Data were analyzed with GeneSpring software V13.0 (Agilent). The results of sample analysis are provided in Additional file 1 Figure S1. To select differentially expressed genes, we used threshold values of ≥2 and ≤−2-fold change and a Benjamini-Hochberg corrected p-value of 0.05. The data were log2 transformed and median centered by genes using the Adjust Data function of CLUSTER 3.0 software and then further analyzed with hierarchical clustering with average linkage. A comparative analysis between the T (cocultured WJ-MSCs) sample and N (non-cocultured WJ-MSCs) sample was performed using fold-change. Hierarchical cluster analysis was performed using complete linkage and Euclidean distance as a measure of similarity.
circRNA-overexpressing plasmid construction and lentivirus infection
To generate WJ-MSCs with stable overexpressing circ6401, circ6401 cDNA was first synthesized and cloned into the pLC5-ciR vector (Geneseed) and confirmed by sequencing. Then, pLC5-circ6401 or pLC5-ciR empty vector was transfected into 293T cells by Lipofectamine 2000 (Invitrogen) to construct the lentivirus. The transfection efficiency was confirmed by qRT-PCR. After determining the viral titer, WJ-MSCs were infected by the obtained lentiviral particles.
Prediction of circRNA-miRNA and miRNA-mRNA interactions
The interaction between circ6401 and miRNA was predicted using miRanda and Targetscan (http://www.microrna.org/microrna/ and http://www.targetscan.org/, respectively). The miRNA-mRNA interactions were predicted by Targetscan, while the miRNA binding sites were predicted by miRcode software (http://www.mircode.org/). Filtering restrictions were as follows: Context score ≥ 140.
miRNA mimic or inhibitor transfection
All miRNA mimics, inhibitor, and their negative controls (miR-29b-1-5p mimics NC, miR-29b-1-5p inhibitor NC) were purchased from RiboBio and diluted to 50 nM. WJ-MSCs were transfected with miR-29b-1-5p inhibitor oligonucleotide, mimic oligonucleotide, or their negative controls by using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. The transfection efficiency was confirmed by qRT-PCR.
Double RNA fluorescence in situ hybridization (FISH)
Circ6401-overexpressing WJ-MSCs were transfected with miR-29b-1-5p mimics. After transfection, the cells were hybridized with a circ6401 probe (cy3-labeled) and an miR-29b-1-5p probe (FITC-labeled). Finally, the slides were stained with DAPI (Cell Signaling Technology, Boston, MA) and subjected to fluorescence signal detection under a Leica TCS SP8 laser confocal microscope (Leica). Sequences of FISH probes are shown in Additional file 2 Table S2.
Dual-luciferase reporter assay
To evaluate the direct binding of miR-29b-1-5p to circ6401 or the 3ʹ-UTR of RAP1B, the fragment of circ6401 or the 3ʹ-UTR of RAP1B was first amplified by PCR and then cloned downstream of the Renilla gene in the psiCHECK-2 vector (Promega, Madison, WI, USA), which was named as circ6401-wt or RAP1B-wt, respectively. To generate the circ6401, RAP1B 3ʹ-UTR mutant reporters, the seed regions of circ-001971 and the RAP1B 3ʹ-UTR were mutated to remove the complementarity to miR-29b-1-5p, and the resulting constructs were named as circ6401-mut and RAP1B-mut, respectively. Cells were seeded into a 24-well plate. Next, miR-29b-1-5p mimics or NC was co-transfected with the wild-type vector or the mutant vector. Forty-eight hours after transfection, alterations in the luciferase activity were evaluated by the Dual-Luciferase Reporter Assay System (Promega) using firefly luciferase activity for normalization.
Data were analyzed using GraphPad Prism software (GraphPad Software, La Jolla, CA, USA). Two-tailed Student’s t test was used to assess statistical significance in two groups, and one-way ANOVA with post hoc Bonferroni test was used in three or more groups. After comparison test, the differences were considered significant at p<0.05 or p<0.001.