A total of 30 HGSOC tissues and 20 normal ovarian epithelium tissues were collected from the First Affiliated Hospital of Shandong First Medical University. None of patients was treated with chemotherapy or radiotherapy before surgery. Samples were washed with sterile phosphate buffered saline and then frozen in liquid nitrogen. Tumor stage was determined according to the FIGO staging system. This study was approved by the Ethic Committee of the First Affiliated Hospital of Shandong First Medical University.
Cell culture and transfection
Human EOC cell lines A2780 and SKOV3 were obtained from American Type Culture Collection (ATCC) and maintained using Dulbecco’s modified Eagle’s medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS). All cells were incubated in a humidified atmosphere containing 5% CO2 at 37 ℃. Vectors that stably overexpressed lncRNA SHNG7, siRNA targeting lncRNA and SHNG7, miR-34a-5p mimics, miR-34a-5p inhibitor and controls were transfected into cells using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.
Quantitative RT-PCR analysis
Total RNA was extracted from tissues and cells using Trizol reagent (Invitrogen) according to the manufacturer's protocol. Extracted RNA was reversely transcribed into cDNA using a PrimeScriptTM RT Reagent Kit (TaKaRa Bio, China) following the manufacturer's instructions. qRT-PCR was carried out using a SYBR Premix Ex Taq™ Kit (Takara). U6 or GAPDH was used as an internal control. The relative expression was determined using the 2−∆∆CT method.
Cell proliferation was detected using Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) according to the manufacturer's protocol. Cells were seeded into 96-well plates. After incubation for the indicated time, 20 µL CCK-8 solution was added into each well and incubated for 2 h. Absorbance values at 490 nm were measured on a Microplate Reader (Bio-Rad).
Cell cycle assay
Cell cycle analysis was performed using flow cytometry. Cells were collected and wash twice with PBS (1×), and fixed with 70% ethanol at -20°C for 24 h. Cells were incubated with RNase A at 37°C for 30 min, and then stained with 400 µL propidium iodide on ice for 30 min. Cell cycle distribution was analyzed using a BD FACSCalibur™ flow cytometer (BD Biosciences).
Cell migration and invasion were completed with a Transwell system (Corning, NY, USA). A total of 104 cells in 200 µL of serumfree medium were added to the upper chamber. For invasion assay, Matrigel was additional coated on the upper chamber. The lower chamber was filled with 600 µl medium with 20% FBS. After incubation, cells on the upper chamber were removed and the cells on the lower surface were fixed with 20% methanol and stained with 0.1% crystal violet. The cells were observed under a microscope.
Cells were collected and lysed in RIPA buffer containing protease inhibitors. Proteins were separated by 8% SDS-PAGE then transferred onto PVDF membranes (Millipore, USA). Then the membranes were blocked with 5% nonfat milk for 1 h at room temperature, and incubated with primary antibodies overnight at 4°C. Subsequently, the membranes were incubated with secondary antibodies for 1.5 h. the protein bands were detected using the Pro-lighting horseradish peroxidase (HRP) agent. The expression of β-actin was used as the internal control.
The dual-luciferase miRNA target expression vector pmirGLO (Promega, Madison, Wisconsin) was used to generate luciferase reporter constructs. Wild-type (SHNG7-WT and Notch1-WT) and mutant-type (SHNG7-MT and Notch1-MT) vectors were constructed. Cells were cotransfected with miR-34a-5p mimics (or NC) and wild-type (or mutant-type) vectors using Lipofectamine 2000. Luciferase activity was examined using a luciferase reporter assay kit (Transgen Biotech, Beijing, China). Each group was performed in triplicate.
Isolation of exosomes
Exosomes were isolated from cell culture medium by differential centrifugation. Cells and other debris were removed by centrifugation at 300 g and 3,000 g. Shedding vesicles were removed from supernatant by centrifuged at 10,000 g. Finally, supernatant was centrifuged at 110,000g and exosomes were obtained. Isolation of exosomes from serum was performed using ExoQuick Plasma prep and Exosome precipitation kit (SBI, USA).
Tumor formation assay in nude mice
NCG (NOD-Prkdcem26Cd52 IL2rgem26Cd22/Gpt) mice were purchased from NBRI of Nanjing University (Nanjing, China), which were maintained in a pathogen-free facility. Cells overexpressing SHNG7 were trypsin digested, washed with PBS, and then resuspended in PBS. Then 200µl of the suspended cells (1×107) were injected into the armpit or peritoneal cavity of each mouse.3-4 weeks later, the mice were sacrificed and tumor weight or metastasis number were examined. All the animal experiments were performed with the approval of the Shandong First Medical University Animal Care and Use Committee.
Each experiment was repeated in triplicate independently. Values were shown as mean ±SD. Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software, USA). P values<0.05 were considered statistically significant.