2.1. Ethics statement, patients, and samples
15 EOC tissue and 15 normal ovarian tissue specimens were obtained from the First Affiliated Hospital of Nanjing Medical University from September 2018 to February 2020. No patients received chemotherapy or radiotherapy before sample collection. Tissues were stored in liquid for further use. The study was approved by the Ethics Committee of The First Affiliated Hospital of Nanjing Medical University and all enrolled patients provided written informed consent for sample collection and analyses (approval number: 2017-MD-327.A1).
2.2 Ovarian cancer tissue microarrays
Tissue microarray (TMA, diameter, 1.5mm; depth, 4μm), which contained 157 formalin-fixed, paraffin-embedded ovarian cancer and 3 adjacent non-malignant ovarian tissues, was purchased from Shanghai Outdo Biotech (Shanghai, China). Immunohistochemistry was performed on TMA to analyze the KMT2A protein expression in the clinical specimens. Two ovarian cancer cases were excluded in statistical analyses due to severe detachment during immunohistochemistry. All tissue samples were obtained from patients having no anti-cancer treatment before surgery. The tissue specimens were histologically examined and classified according to the 2014 International Federation of Gynecology and Obstetrics (FIGO) staging system.
2.3 Immunohistochemistry (IHC) scoring system
The immunostaining intensity was accessed as following criteria: 0 (absent staining = green), 1 (weak staining = pale yellow), 2 (moderate staining = tan), 3 (strong staining = brown). The nuclear and cytoplasmic expression levels were evaluated using the H-score system which is a semi-quantitative method. The formula for the H-score is: H-score = (% of cells of weak intensity ×1) + (percentage of cells of moderate intensity ×2) + (percentage of cells of strong intensity ×3). The maximum score of H-score was 300, corresponding to 100% of cells with strong intensity. Scoring was independently calculated by two evaluators in a blind manner.
2.4 Cell lines and cell culture
Human ovarian cancer cell lines (A2780, HO-8910, OVCAR3) and human normal ovarian epithelial cell (IOSE-80) were purchased from the Chinese Academy of Sciences Cell Bank. SKOV3 were purchased from the American Type Culture Collection (ATCC, Manassas, USA). A2780, HO-8910, OVCAR3 and IOSE-80 were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific, USA) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific, USA), 100 μg/mL penicillin and 100 U/mL streptomycin. SKOV3 were maintained in MoCoy’s 5a medium supplemented with 10% FBS. All referred cell lines were maintained at 37°C in a humidified atmosphere containing 5% CO2.
2.5 RAN interference (RNAi)
According to the manufacturer’s protocol, SKOV3 and HO-8910 cells were transfected with KMT2A-specific small interference (siRNA) and the negative control siRNA by Lipofectamine 2000 (Thermo Fisher Scientific, USA), which were designed and synthesized by GenePharma (Shanghai, China). The sequences were as follows: si-KMT2A #1: sense 5'-CCAAGUGUGUUCGCUGUAATT-3' and antisense 5'-UUACAGCGAACACACUUGGTT-3', #2: sense 5'-GCUCUUUCCUAUUGGAUAUTT-3' and antisense 5'-AUAUCCAAUAGGAAAGAGCTT-3', #3: sense 5'-GCCAACCACCAUGUAACAATT-3' and antisense 5'- UUGUUACAUGGUCGUUGGCTT-3'. Negative control siRNA sequence was as follows: sense 5'-UUCUCCGAACGUGUCACGUTT-3' and antisense 5'-ACGUGACACGUUCGGAGAATT-3'.
2.6 Lentivirus production and cell transduction
Further validation of KMT2A overexpression phenotype in ovarian cell lines was carried out with in-fection of the lentivirus containing KMT2A or control lentivirus particles, which were obtained from Hanbio Biotechnology Co., Ltd. (Shanghai, China). Then, transfected cells were screened in medium containing puromycin.
2.7 Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from cells and tissues using Trizol reagent, and then reverse-transcribed in-to cDNA using Reverse Transcription kit (Thermo Fisher Scientific, USA), according to the manufa-cturer’s instructions. RT‑qPCR was performed using a SYBR Pre-mix Ex Taq™ II (Perfect Real Ti-me) kit (Takara, Japan) and Mastercycler® ep qrealplex thermal cycler (Eppendorf, German). β‑actin expression was applied for the normalization of KMT2A expression. The primers were synthesized by Thermo Fisher Scientific, and the sequences were as follows: KMT2A, forward 5'‑AGCGAGCATCCCCCAAAGTT-3', reverse 5'‑GGGCACGAAGGCTCATCATT-3'; β-actin, forward 5'-AGCGAGCATCCCCCAAAGTT-3', reverse 5'- GGGCACGAAGGCTCATCATT-3'. The qRT-PCR data were calculated using the 2-ΔΔCT method.
2.8 Western blot
Total protein was extracted from tissues and cell lines. Then, the protein lysates (30μg) were separated by electrophoresis in 4-12% SDS-PAGE, electrophoretically transferred to PVDF membranes, immunoblotted at 4 °C overnight with antibodies against KMT2A (Santa Cruz Biotechnology, USA),N-cadherin, Vimentin, E-cadherin, β-actin (all purchased from Proteintech, Wuhan, China), Bcl-2, cleaved caspase-3, cleaved PARP, phosphor-Smad2 (all purchased from Cell Signaling Technology, USA), and followed by incubation with the horseradish peroxidase-conjugated secondary antibodies. The protein bands were detected with enhanced chemiluminescence (ECL) detection reagents (Millipore, USA).
2.9 Cell proliferation assay
Cell Counting Kit-8 (CCK-8; Dojindo, Japan) was used to assess SKOV3 and HO-8910 cell proliferation according to the manufacturer’s instructions. Briefly, cells were seeded into 96-well plate at a 1000 cells/well ratio. Then, 10 μl CCK-8 and 90 μl FBS-free medium were added to each well at 24, 48 and 72 h. After 2 h incubation at 37 °C, the absorbance at wavelength of 450nm was measured using an enzyme-linked immunosorbent assay plate reader (Thermo Fisher Scientific, USA).
2.10 Wound healing assay
The cells were seeded into six-well plates and transfected with control siRNA, KMT2A siRNA-2, KMT2A siRNA-3, KMT2A-NC, KMT2A-1, KMT2A-2. After cells reached 90% confluence, artificial scratch wounds were generated on the confluent monolayer of cells using a 200 μl pipette tip, and non-adherent cells and debris were removed with PBS. Serum-free medium was added for 48h, and the wound margins were photographed every 24h in 5 randomly selected microscopic regions. The percentage of wound closure areas were calculated using the formula as follows: (wound width at 0 h-wound width at 48 h) / wound width at 0 h ×100%.
2.11 Cell migration and invasion assay
A total of 5×104 transfected cells were suspended in 200 μl serum-free medium and added to the upper chamber of 24-well transwell plates (8 μm, Corning, USA) precoated without (for migration) or with (for invasion) Matrigel (BD Biosciences, USA). Then, as a chemoattractant, medium containing 20% FBS was added to the lower chamber. After 24 h of incubation for the migration assay and 48 h of incubation for the invasion assay, the cells attached to the lower surface of the chamber were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and imaged. Migration and invasion were assessed by counting cell nuclei in 5 random fields under a light microscope (Olympus Corporation, magnification, 10×).
2.12 Immunofluorescence assay
Cells were seeded on chamber slides in 24-well plates with or without KMT2A siRNA transfection. The cells were fixed with 4% polyoxymethylene for 30 min at room temperature, permeabilized with 0.5% Triton X-100, and blocked with 5% bovine serum albumin (BSA) for 30 min. The samples were subsequently incubated using primary antibodies specific for N-cadherin, Vimentin, E-cadherin (all 1:100 dilution, Proteintech, China) overnight at 4°C. After 3 washes, the cells were incubated with CoraLite488-conjugated Affinipure Goat Anti-Rabbit IgG for 1 h. The nuclei were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI), and the cells were imaged under a fluorescence microscope.
2.13 Cell cycle analysis
Cell cycle distribution was examined by flow cytometry. At 48 h after transfection with siRNA, cells were trypsinized, harvested and then washed with cold phosphate-buffered saline solution (PBS). 70% cold ethanol was used to fix cells at 4°for at least 1 hour. After two washes, the cells were stained in a solution containing propidium iodide (PI, 50μg/ml) and RNaseA (50μg/ml) and incubated for 1 hours. After two washes, the cells were stained in a solution containing propidium iodide (PI, 50μg/ml) and RNaseA (50μg/ml) and incubated for 1 hours in the dark. The samples were then analysed by flow cytometry (BD Bioscience, USA).
2.14 Apoptosis analysis
Cells (SKOV3 and HO-8910) were transfected with KMT2A siRNA. At 48 h after transfection, cells were harvested, washed twice with cold PBS, and then stained with 5 μl Annexin V-FITC and 5 μl PI using an Annexin V-FITC/PI-staining kit (Becton Dickinson, USA). The cells were placed at room temperature for 15 min in the dark and then measured by flow cytometry (BD Bioscience, USA).
2.15 Animal studies
All animal operational procedures were approved by the Nanjing Medical Animal Care Committee (PZW2023034). For xenograft tumor growth formation, female nude mice aged 4-6 weeks were randomly divided into two groups (si-Cont and si-2). The SKOV3 cells (5×106 in 100 μL PBS) were injected subcutaneously into the left flank of each mouse. Control siRNA or KMT2A siRNA-2 were injected subcutaneously into the mice twice a week for 4 weeks. Tumor volume (width2×length)/2 was measured weekly after injection. Mice were sacrificed four weeks after injection and tumors were obtained for weighting and photographing. Meanwhile, the tumour tissues were IHC stained with KMT2A, E-cadherin, N-cadherin and Vimentin.
2.16 Statistical analysis
All data were analyzed using SPSS 22.0 (SPSS Inc., Chicago, Illinois, USA) and presented as the mean± standard deviation (SD) based on at least three independent experiments. Student’s t-tests were performed to compare two independent groups of data. Chi-square tests were applied to analyze the association between KMT2A expression and clinicopathologic features of ovarian cancer patients. Survival curves were plotted using the Kaplan-Meier method and were compared using the log rank test. Univariate and multivariate analysis were done using the Cox regression model. P values less than 0.05 was considered statistically significant.