Cell culture
All cell lines including A2780, CAOV3, CAOV4, OVCAR3, SKOV3, 293T cells were purchased from China Infrastructure of Cell Line Resource (Beijing, China). A2780 and OVCAR3 cells were cultured in Roswell Park Memorial Institute-1640 (RPMI-1640) supplemented with 10% fetal bovine serum (FBS) and antibiotics. CAOV3, SKOV3, 293T were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and antibiotics. CAOV-4 were cultured in DMEM supplemented with 20% FBS and antibiotics. All cell lines were maintained at 37°C in a 5% CO2 incubator.
Oc Patients, Oc Tissue And Blood Serum Samples
A total of 63 samples of human OC tissues, 65 samples of human OC blood serums and 39 samples of healthy individuals’ and benign ovarian tumor patients’ blood serums were collected at at the Departments of Gynecological Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital l & Shenzhen Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College after obtaining the subjects' informed consent from October 2017 to November 2020. For chemotherapeutic response, 26 (41.27%) patients were resistant to platinum-based chemotherapy and 37 (58.73%) were sensitive to the same treatment. PFS was calculated at the time from the date of surgery to the occurrence of progression, or relapse. OS was measured as the length of time from the initiation of surgery to death from any cause or until the most recent follow-up. PFS less than 6 months was defined as resistant to the last chemotherapy, and more than 12 months was defined as sensitivity to the last chemotherapy. Our study was approved by the Ethics Committee of Cancer Hospital Chinese Academy of Medical Sciences, and each clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki consent.
Microrna Array And Mrna Array
The Agilent/Affymetrix microarray was used for miRNA/mRNA expression profiles (CapitalBio, Beijing, China).
Rna Extraction, Rt-pcr And Quantitative Real-time Pcr
Total RNA was isolated from frozen fresh tissue or EOC cell lines by using NCMzol Reagent and RNAExpress Total RNA Kit (NCM Biotech, Suzhou, China). Serum miRNA was purified by adding 1mL NCMzol Reagent, 100uL serum and 1ng external control (Mus musculus lncRNA Gm13008-201 synthesized by in vitro transcription). RT-PCR and qPCR were conducted using EasyScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) and PerfectStart Green qPCR SuperMix purchased from TransGen Biotech (Beijing, China). Relative expression level was normalized to U6 levels for cellular miR-6836, external control (lncRNA Gm13008-201) for exosomal miR-6836 and GAPDH for cellular DLG2 mRNA. Bulge-Loop miR-6836 qRT-PCR Primer Set was purchased from RiboBio (Guangzhou, China) and other qPCR primers used are listed in Table S1.
Tumor Spheroids Formation Assays
EOC cells were seeded in ultra-low attachment 6 well plates (Corning, Kennebunk ME, USA) and cultured in serum free DMEM/F12 supplemented with 2% B27 Supplement (50×) (Gibco, Grand Island NY, USA), basic fibroblast growth factor (bFGF, Sigma-Aldrich, Saint Louis MO, USA) and epidermal growth factor (EGF, Sigma-Aldrich) with the final concentration of 20ng/µL at the temperature of 37°C in a 5% CO2 incubator. Spheroids were recorded and counted under microscope after 2–4 weeks.
Oligonucleotide Transfection
MiR-6836 mimic or inhibitor and siRNA for DLG2, and TEAD1 were purchased from RiboBio (Guangzhou, China). Cell transfection was performed using Lipofectamine 2000 reagent (Invitrogen, Carlsbad CA, USA).
Western Blotting
Western blot was performed as previously described [33]. The antibodies included anti-Oct4 (Bioss, Beijing, China, bs-0830R, 1:1000), anti-Nanog (Proteintech, Wuhan, China, 14295-1-AP, 1:1000), anti-CD44 (Proteintech, 15675-1-AP, 1:1000), anti-Sox2 (Proteintech, 11064-1-AP, 1:1000), anti-γ-H2AX (Abcam, Cambridge, UK, ab26350, 1:1000), anti-DLG2 (Affinity Biosciences, Suzhou, China, DF3995, 1:1000), anti-p-Yap1 (CST, Boston MA, USA, 4911S, 1:1000), anti-Yap1 (Proteintech, 13584-1-AP, 1:1000), anti-Bax (CST, B8429, 1:1000), anti-Bcl2 (Proteintech, 60178-1-Ig, 1:1000), anti-β-tubulin (Sigma-Aldrich, T5201, 1:1000), anti-Lamin B1 (Santa, Dallas TX, USA, sc-365962, 1:1000), anti-TEAD1 (ABclonal, Wuhan, China, A13366, 1:1000), anti-β-actin (Santa, sc-8432, 1:4000), CD63 (Proteintech, 25682-1-AP, 1:1000), and TSG101 (Proteintech, 28283-1-AP, 1:1000).
Transwell Migration/invasion Assays
Cell migration and invasion assays were performed as previously described [33].
Colony Formation Assay
Colony formation assay was conducted as previously described [33].
Drug Sensitivity Detected By Cck8 Assay
After 5×103 cells were seeded in 96-well plates for 12h, cells were treated with cisplatin at different concentration gradients for 24h. Then cell viability was determined by Cell Counting Kit-8 (NCM Biotech, Suzhou, China). The growth-inhibitory curves were charted by CCK8, and the half maximal inhibitory concentration (IC50) representing the cisplatin concentration when cell viability is 50% was calculated.
Apoptosis Detected By Flow Cytometry Assay
Cells were stained with AnnexinV and PI using Annexin V-FITC/PI Apoptosis Detection Kit (KeyGEN BioTECH, Nanjing, China), and then apoptosis ratio was analyzed using multicolor flow cytometer, LSRII (BD Biosciences, Franklin Lakes NJ, USA).
Immunofluorescence Assay
For immunofluorescence assay, primary antibodies included anti-γ-H2AX (Abcam, ab26350, 1:200), anti-53BP1 (Abcam, ab36823, 1:100), and Yap1 (Proteintech, 13584-1-AP, 1:50) and fluorescent secondary antibody included Alexa Flour 594 anti-mouse secondary antibody, Alexa Flour 488 anti-rabbit secondary antibody and Alexa Flour 594 anti-rabbit secondary antibody (ZSGB-BIO, Beijing, China). Immunofluorescence assay was conducted according to the manufacturer’s protocol (Abcam, Immunocytochemistry and immunofluorescence protocol). Images were collected using laser-scanning confocal microscope.
Retroviral Infection
The lentivirus for miR-6836 was purchased from GeneChem (Shanghai, China) and cells were infected based on the manufacturer’s instructions.
Animal Experiments
For spheroid cells tumorigenicity assay, four-week-old female NOD-SCID mice were purchased from Weitonglihua (Beijing, China), and experimental procedures were recorded in Result 3.
For subcutaneous or peritoneal metastatic model, five-week-old female BALB/c nude mice were purchased from Beijing HFK Bioscience (Beijing, China). Ctrl and Lv-miR-6836 CAOV3 cells were injected subcutaneously (1×106 cells per mouse) or intraperitoneally (2×106 cells per mouse) into nude mice. Mice were were sacrificed after 30 days and the volume of subcutaneous tumor tissues were measured. Tumor tissues and abdominal organs from intraperitoneal metastasis model were fixed in formaldehyde solution, embedded in paraffin and sectioned into slices for histological examination.
For drug combination animal model, antagomir-miR-6836 were purchased from RiboBio (Guangzhou, China). CAOV3 (2×106 cells per mouse) were injected subcutaneously into the left axilla of nude mice. After 10 days, the nude mice formed tumors with the approximate volume of 75 mm3. PBS or cisplatin were injected intraperitoneally and antagomir-NC or antagomir-miR-6836 were dosed through tail vein.
Plasmid Construction
The DLG2 3’UTR (WT) or mutant (MUT) with a predicted miR-6836 target sequence was inserted downstream of the firefly luciferase gene in the pmirGLO vector.
The miR-6836 promoter region (-2000 bp ~ 0 bp) was inserted upstream of the firefly luciferase gene in the pmirGLO vector.
Luciferase Reporter Assay
The Luciferase reporter assay was performed with the Dual-Luciferase Reporter Assay System (Promega, Madison WI, USA) as previously described [33].
Nuclear And Cytoplasmic Protein Extraction Assay
Nuclear and cytoplasmic protein extraction assay was conducted using Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China).
Chromatin Immunoprecipitation Assays (Chip) Assay
ChIP assays were performed using a PierceTM Magnetic ChIP Kit (Thermo ScientificTM, Shanghai, China). Immunoprecipitation was performed with anti-TEAD1 antibodies (ABclonal, A13366). Primers (Primer a, b, c) involved in qRT-PCR were listed in Table S1.
Exosome Purification, Identification And Internalization
Exosomes were isolated with ultracentrifugation method. Cells were cultured in exosome-free culture medium. The supernatant was collected after incubation for 48 h, centrifuged at 1 000 g for 10 min, 12 000 g at 4°C for 30 min and then ultracentrifuged at 4°C at 110 000 g for 90 min. Exosomes were identified by observation under transmission electron microscope (TEM-1400 Plus) and nanoparticle tracking analysis using NanoSight NS300 instrument (Malvern Instruments Ltd, UK).
In order to examine exosome internalization, biotin-miR-6836 was synthesized in Shanghai Generay Biotech (Shanghai, China). Donor cells were transfected with biotin-miR6836 and exosomes were collected from supernatant of donor cells 48h after transfection. Then exosomes were labeled with PKH67 (Sigma-Aldrich) according to manufacturer's protocols, and incubated with recepient cells for 12h. Recipient cells were fixed and stained with Streptavidin/RBITC (Bioss, bs-0437P-RBITC). Images were collected using laser-scanning confocal microscope.
Study Approval
This work was approved by the Cancer Hospital, the Chinese Academy of Medical Sciences, the National GCP Center for Anticancer Drugs, and the Independent Ethics Committee. The collection of all of the human samples used in the experiments was approved by the hospital. Informed consent to participate in this study was obtained from all patients. All experiments involving mice were approved by the Cancer Hospital, the Chinese Academy of Medical Sciences, and the Experimental Animal Ethics Committee and followed the Institutional Animal Welfare Guidelines.
Statistical analysis
The data were presented as means ± SEM and the differences between groups were analyzed using unpaired two-tailed Student’s t test using the GraphPad Prism 8. Spearman correlation was used to assess the correlation between two involved variables. The statistical analyses were carried out using GraphPad Prism 8 and SPSS 23.0 software. For the significance of difference, p < 0.05 represented a significant difference. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.