Participants and Samples
Samples were collected after patients gave written informed consent. The study was approved by the ethics committee of Zhongshan Hospital, Fudan University (Shanghai, People's Republic of China) and was performed in compliance with the Population and Family Planning Law of the People's Republic of China.
The endometrium for this study was collected from 3 participants who underwent in vitro fertilization (IVF) for an indication of tubal factor infertility at the Reproductive Center, Zhongshan Hospital, Fudan University.
Endometrial specimens were obtained from pipe suction curettage on day LH + 7 in a natural cycle. The next cycle included three patients who received the antagonist stimulation protocol. Briefly, on cycle day 3, ovarian stimulation was started by the daily injection of recombinant FSH (r-FSH) (Gonal-F; Merck Serono). Gonadotropin-releasing hormone (GnRH) antagonist (Ganirelix, 0.25 mg; MSD) injection was started on day 5 or 6 of stimulation. When at least three follicles had reached 17 mm or two follicles had reached 18 mm in diameter, an intramuscular injection of 5,000 IU of human chorionic gonadotropin (hCG) was used to trigger final oocyte maturation. Vaginal utrogestan (Capsugel) at 600 mg/d and oral dydrogesterone (Abbott) at 20 mg/d were given for luteal support beginning on the day of oocyte retrieval. The endometrium was collected on the 5th day after oocyte retrieval from patients. All specimens were frozen at –80°C until subsequent analysis.
Small RNA sequencing analysis
The quality of the original sequencing data was assessed by FastQC, cutadapt was used to remove the adaptor, trimmomatic was used to remove the low-quality bases at both ends, and reads were filtered. Then, blastn was used to compare reads with sRNA, tRNA, snRNA, and snoRNA sequences in the Rfam database, count the number and percentage of reads on the alignment, and filter out the reads on the alignment. The bowtie program was used to compare the reads with the exon and intron sequences of the species as well as compare the number and percentage of reads on the comparison and filter out the reads on the exon of the comparison that cannot be compared with the intron. Again, bowtie was used to align the reads with the species reference genome sequence, count the number and percentage of reads on the comparison, and filter out the reads that cannot be compared. Sequencing was performed by the Sangon Biotech (Shanghai) Co., Ltd.
Ishikawa cells were purchased from the Cell Bank of the Chinese Academy Sciences (Shanghai, China). The cells were cultured with DMEM high glucose medium containing 10% FBS and 1% antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) at 37°C in a humidified atmosphere containing 5% CO2. When the fusion degree reached 90%, the cells were subcultured.
Quantitative real-time polymerase chain reaction (qRT-PCR)
The total RNA were isolated from Ishikawa cells according to the manufacturer’s instruction of TRIzol® Reagent (Invitrogen, Carlsbad, CA, USA). According to the manufacturer's instructions, 1 μ g of total RNA was reverse transcribed into cDNA using primescript RT Master Mix kit (RR036A, Takara, Dalian, China). To quantitate ENST00000391318 and CDH11 expression, qPCR was carried out with a SYBR® Premix Ex Taq II Kit (RR420A, Takara, Dalian, China). The expression levels of ENST00000391318 and CDH11 were normalized to GAPDH expression. QPCR was performed using the miScript SYBR Green PCR Kit (Qiagen, Hilden, Germany) to determine miR-3928-5p expression. The expression level of miR-3928-5p was normalized to U6 expression. All of the data were analyzed by the 2−ΔΔCt method.
Cell proliferation assay
Ishikawa cells were seeded into 96 well culture plate according to the density of 4000 cells/ well. Ishikawa cells were divided into four groups: pLVX-vector group, pLVX- ENST00000391318, pLKO.1-vector group and pLKO.1- ENST00000391318, the activity of Ishikawa cells was detected by CCK-8 at 12h, 24h, 36h and 48h after inoculation, and 10 μl CCK-8 solution was added to each well. After incubation in 37 ℃ incubator for 2h, the absorbance at 450nm was detected by microplate reader.
Wound healing assay
The Ishikawa cells were seeded into 6-well culture plate according to 50000 cells/well. When the cells grew to 100% healing degree, they were scratched with 1 ml blue tips. After the scratch, the medium and suspension cells were sucked off, and then the serum-free medium was replaced and take photos at 0 h. Then the cells were cultured for 24 h to take photos. The cell mobility was calculated by comparing the healing degrees of 0 h and 24 h.
Cell invasion assay
After mixing DMEM medium and Matrigel 1:1, 50 μ l was evenly spread in Transwell chamber and placed in 37 ℃ incubator for 45 min. The cells were divided into four groups: pLVX-vector group, pLVX- ENST00000391318, pLKO.1-vector group and pLKO.1- ENST00000391318. Cells were inoculated into Transwell upper chamber according to the density of 20000 cells/well. The upper chamber of Transwell was serum-free medium, and 700 μ l medium containing 5% serum was added into the lower chamber. The cells in the upper chamber were removed after 12 h in the incubator. The cells at the bottom of Transwell were stained with crystal violet and counted after taking pictures.
Cell cycle analysis
The cell cycle distribution was determined by using the Cell Cycle Analysis Kit (Beyotime, China) according to the instructions. Cells were seeded onto 6-well plates and transfected with NC shRNA or ENST00000391318 shRNA. After 48 h, the cells were harvested and fixed with precooled 70% ethanol at 4 °C for 12 h. The fixed cells were suspended in staining buffer, mixed with 25 μL of propidium iodide staining solution and 10 μL of RNase A and incubated in the dark for 30 min. Flow cytometry detection was immediately performed.
The cell protein was extracted from RIPA protein lysate and lysed on 30min, then centrifuged at 1000r/min, and the supernatant was extracted. The protein concentration was detected by BCA solution and 10% SDS-PAGE gel was distributed. The protein was transferred to PVDF membrane and sealed with 5% BSA for 1 h. then the antibody was incubated at 4 ℃ overnight. After tbst cleaning, HRP labeled antibody was incubated for 1 h. tbst was washed and then developed. Finally, the protein bands were visualized using an ECL Kit (Solarbio, China). GAPDH served as an internal control. LIF antibody (#ab113262, Abcam, 1:1000), an ITGB3 antibody (#ab119992, Abcam, 1:1000), a DKK1 antibody (#ab109416, Abcam, 1:1000), and a claudin-4 antibody (#ab53156, Abcam, 1:1000) were used.
Statistical analysis was performed using GraphPad Prism 8.0 software. All results are expressed as the mean ± standard deviation (SD). p < 0.05 indicated statistical significance. The experiments were repeated at least three times.