2.1. Patients and tissue samples
49 samples of OC patients and 9 samples of ovarian benign tumor patients who performed surgery for the first time, without any radiotherapy, chemotherapy or neoadjuvant chemotherapy before surgery. Patient information is shown in Table S1. Exclusion criteria: patients with other malignant tumors or severe complicated diseases were excluded. The Ethics Committee of the Women’s Hospital, school of medicine, Zhejiang University approved this study (granted number: IRB-20210147-R), and the participants were all provided informed consent.
2.2.Cell lines and cell culture.
The human ovarian cancer cell lines (HO8910 and HO8910-PM) were provided by the Women’s Hospital, school of medicine, Zhejiang University, where they were tested and authenticated. All cells were cultured in RPMI 1640 (BI, Israel) medium containing 10% fetal bovine serum (FBS) in a 5% CO2 humidified incubator at 37℃.
2.3.RNA sequencing analysis.
Total RNAs of HO8910 and HO8910PM cells were prepared by Trizol Reagent (Invitrogen, USA). RNA quality was evaluated by Agilent 2200(Agilent, USA). And high-quality RNA was used for RNA sequencing. Firstly, the rRNA was removed and then RNA was fragmented to about 200 nucleotides lengths. Secondly, complementary DNA (cDNA) was synthesized and purified. Thirdly, the ends of the cDNA were repaired and the primers were added for PCR amplification and purification. Finally, after quality inspected, libraries were sequenced by illumine Hiseq Platform (RiboBio CO., LTD,China).
2.4. RNA extraction and RT-qPCR
Total RNA was extracted by TRIzol reagent. The nuclear and cytoplasmic fractions were isolated by PARIS Kit RNA Isolation System (Invitrogen, Thermo Fisher Scientific). PrimeScript RT reagent Kit with gDNA Eraser (Takara Bio Inc.) were used for synthesizing cDNA, and RT-qPCR was performed through TB Green Premix Ex Taq (Takara Bio Inc.). Primers’ sequences are provided in Table S2. Statistical analyses of the results were performed using the 2−ΔΔCT relative quantification method.
2.5. Northern blotting
Probes were prepared and digoxigenin-labeled by Sangon Biotech Co., Ltd. (Shanghai, China), whose sequences were shown in Table S3. Afterwards, 15 μg RNA was separated in 1% formaldehyde denatured gel after electrophoresis under the statement of 25 V constant pressure and low temperature overnight and subsequently transferred to HyBond N+ membrane by the upward capillary transfer method for 20 h. The membrane was prehybridized in a hybrid furnace at 50°C for 2 h and hybridized overnight in a 50°C hybridization apparatus. Following that, film was blocked for 1h and then reacted in antibody solution for 30 min. The final step is X-ray film exposure and records of results in the darkroom.
2.6. Transfection of siRNA and lentiviral vectors into ovarian cancer cells
SiRNAs purchased from GenePharm (Shanghai, China) were used to knock down RAD51B-AS1 or RAD51B, and efficiency had been evaluated by RT-qPCR. The sequences are represented in Table S4. Additionally, for the ectopic expression of RAD51B-AS1, the lentiviruses encoding the RAD51-AS1 sequence and the negative control lentiviruses (LV‐RAD51B‐AS1 and LV‐NC, respectively, GeneChem, Shanghai, China) were infected into HO8910 cells. After 72 hours of infection, the cells were selected by complete culture medium containing 2 μg/mL puromycin.
2.7. CCK-8 assay
Cell proliferation was assessed by the CCK-8 assay (Dojindo Laboratories, Japan). The cells were seeded at 5 × 103 cells/well into 96-well plates. CCK-8 solution (10 μL) was added to each well at hours 0h, 24h, 48h, 72h and 96h post transfection. After incubation at 37°C for 2 hours, the absorbance at 450 nm was measured by a microplate reader (SkanIt RE for Varioskan Flash 2.4.3).
2.8. Plate colony formation assay and soft-agar colony formation assay
For plate colony formation assays, 500 cells per well were resuspended and added to six-well plates and incubated at 37 °C for 2 weeks. For soft-agar colony formation assays, 5000 cells per well were resuspended by complete culture medium containing 0.3% agarose, and then added to six-well plates already solidified by the laid medium containing 0.5% agarose, and incubated for 3 weeks. Colonies were fixed and stained with 0.1% crystal violet for 20 min. Cell colonies(>50 cells) were counted and analyzed per well.
2.9. Transwell migration and invasion assay
Cell migration and invasion assays were conducted by 24-well transwell plates (8 μm pore, Corning Costar, USA). The transwell filter inserts were coated with (invasion) or without (migration) Matrigel (BD Biosciences, 1:12 diluted). 3× 105 cells (invasion) or 2× 105 cells (migration) suspended in 200μL OPTI-MEM were added to the upper chamber, and 500μL high concentration medium containing 18%FBS was added to the lower chamber. Air bubbles should be avoided between the upper and lower chambers. After incubated for 24h, the cells which traversed the bottom of the upper chamber were fixed in 0.5% crystal violet solution for 20 min. And cells remaining in upper chamber were removed by cotton swabs. Images were captured and number of cells were counted per field.
2.10. Establishment of anoikis model
Adherent ovarian cancer cells were digested with 0.25% trypsin and added into a six-well plate with ultra-low attachment surface (polystyrene, non-pyrogenic) purchased from Corning, with about 2.5× 105 cells each well. Under these conditions, and the morphology and growth of the suspended cells were observed by microscope. The culture medium was changed about once 2 or 3 days depending on the state of the cells (medium changing method: after centrifugation at 800rpm for 3min, supernatant was discarded and cells were resuspended by fresh complete medium and then added to a new culture plate with ultra-low attachment surface).
2.11. Flow cytometric analysis of apoptosis
Adherent cells were cultured in serum-free medium to induce apoptosis and harvested 72 hours after transfection. Both cells digested with 400 μl of accutase (Invitrogen) and the supernatant were collected into a 15-ml collection tube. Suspended cells and their medium were collected, too. The samples were centrifuged at 1000 rpm for 5 min, and the precipitate was collected and washed by PBS twice. For apoptosis analysis, the cells were stained by an Annexin V-FITC/ propidium iodide (PI) Apoptosis Kit (MULTI SCIENCES, China). Thereafter, the cell apoptosis ratio was measured by a BD FACSVerse.
2.12. RNA fluorescence in-situ hybridization (FISH) and immunofluorescence assay
FISH probes targeting for RAD51B‐AS1 were purchased from RiboBio CO., LTD (China). Briefly, HO8910 cells were grown on 4-chamber glass bottom dish (Cellvis, USA), fixed in a 4% formaldehyde solution for 30 minutes at room temperature, permeabilized in PBS containing 0.5% Triton X‐100 for 5 minutes at 4°C, and then prehybridized at 37°C for 1 hour. Hybridization was conducted with FISH probes overnight at 37°C in the dark. If immunofluorescence assay was followed, cells were then blocked with 3% BSA for 1 hour. Then cells were incubated with primary antibody anti-RAD51B (Santa cruz) at 4°C overnight and secondary antibody FITC labeled anti-mouse(multisciences) at room temperature for 1 h. After the cells were gradually washed, DAPI was counterstained to visualize the cell nucleus. Finally, the dish was observed by confocal laser scanning microscopy (Olympus).
2.13. Western blotting
Firstly, cell protein lysates were separated in 10% SDS-PAGE gel, and then transferred to polyvinylidene difluoride membrane. Secondly, western blotting analysis was carried out with anti-RAD51B (Santa cruz), anti-Bcl-2(Santa cruz) antibodies. Anti-β-ACTIN antibody (Fdbio science) was used as an internal control. Thirdly, the membrane was washed and subsequently incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, USA). Finally, complexes were visualized with Fdbio-Dura Enhanced Chemiluminescence kit and the expression levels of proteins were assessed by Image J.
2.14. Mouse xenograft assay
An in vivo model of ovarian cancer was established by subcutaneously injecting 3-week-old female BALB/c nude mice (Silaike Experiment Animal Co., Ltd., Shanghai, China) with a rate of 5× 106 HO8910 cells stably overexpressed RAD51B-AS1 per mouse (12 mice) and same number lentivirus infected negative control cells suspended in PBS (6 mice). After 1 week, tumors were formed in both groups of nude mice, the experimental group of LV-RAD51B-AS1 nude mice were randomly divided into two groups on average, one group was injected with si-RAD51B intratumorally every 3 days, the other group of nude mice and the control group of LV-NC nude mice were injected with the same amount of Si-NC every 3 days to control variables. The tumor volumes of the mice were measured every week and calculated by the following formula: tumor volume [mm3] = 0.5 × Length × Width2). After 5 weeks, nude mice were sacrificed, and the subcutaneous implanted tumors lesions were excised, weighed, measured and photographed. The half of the tumor tissues were used for RNA extraction and the other half were used for immunohistochemical staining. This experiment was carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the China Medical University Animal Care and Use Committee (Approval number: IACUC-20200506-07).
2.15. Immunohistochemistry (IHC) analysis
Tissue samples were fixed with 4% paraformaldehyde. In good fixed state, they were pruned, dehydrated, embedded, sliced, stained and sealed in strict accordance with pathological experiment detection standard operating procedure. Finally, the qualified samples were examined under microscopy. IHC analysis was performed with anti-RAD51B (Huabio), anti-Bcl-2(Abcam), anti-Ki-67(Abcam) antibodies.
2.16. Statistical analysis
SPSS 24 and Graphpad Prism 8 were used for all statistical analysis. Student's T test was used for comparison of data with normal distribution and homogeneity of variance, while Welch's Correction's unpaired T test was used for comparison of data with normal distribution but uneven variance. Nonparametric Mann-Whitney test was used for non-normal distribution data. Kaplan-Meier method was introduced for survival analysis. P < 0.05 indicated the significant difference.