Patients and tissue samples
This retrospective study included 151 cases of HGSOC and 33 cases of fallopian tube (FT) which were collected in Qilu hospital from November 2005 to December 2012. The HGSOC specimens were obtained from primary ovarian cancer patients and the FT tissues were from patients suffered from other benign pathologic changes or uterine disease. All participants in this study gave written informed consent as delineated by the protocol which was approved by the Ethics Committee of Shandong University.
Tissue microarray construction and immunohistochemistry
Tissue microarrays (TMAs) were constructed according to the method described previously . Immunohistochemistry (IHC) staining was performed on formalin-fixed paraffin-embedded HGSOC samples and xenograft tumors. Briefly, tissue slides were deparaffinized and rehydrated in a graded series of ethanol. After that, antigenic retrieval was proceeded by microwave heating. Nonspecific antigens were blocked with the 1.5% normal goat serum. Then they were incubated at 4 °C in a moist chamber overnight with antibodies against human Rad50 (1: 300, Abcam, Cambridge, MA, USA) and Ki67 (1:150, DAKO, Tokyo, Japan). The next day, the slides were incubated with the corresponding secondary antibody. The staining was detected with DAB detection system basing on the Biotin-Streptavidin HRP Detection Systems (Zhongshan Biotechnology Company, China). The final score of each sample was assessed by two independent pathologists based on intensity and extent of staining across the section. Staining intensity was graded according to the following standard: no staining (score 0); weak staining (score 1); moderate staining (score 2); strong staining (score 3). The proportion of every score was estimated for every sample as: 0 (0%), 1 (< 25%), 2 (25–50%), 3 (50–75%) and 4 (> 75%). The final staining index (SI) was calculated as the staining intensity score multiplied by the proportion of stained tumor cells. The cases were then divided into low (SI ≤ 6) and high (SI > 9) groups.
Cell lines and cell culture
HO8910 cells were obtained from China Type Culture Collection (CTCC, Shanghai, China); A2780, SKOV3 and HEK293T cells were originally purchased from American Type Culture Collection (ATCC, VA, USA). HO8910 and A2780 cells were routinely cultured in RPMI-1640 medium (Gibco, NY, USA). SKOV3 cells were cultured in McCoy’s 5A medium (Gibco, NY, USA) and HEK293T cells were cultured in DMEM (Gibco, NY, USA). All media were supplemented with 10% FBS (Gibco, NY, USA), 100 U/ml penicillin and 100 µg/ml streptomycin. All the cells were cultured at 37 °C, 5% CO2 in a humidified incubator (Thermo Fisher Scientific, IL, USA).
Constructs and lentivirus infection
Rad50 overexpression and knockdown plasmids were obtained from Origene (Origene, MD, USA). Lentivirus was produced in HEK293T cells. For stable infection, 1 × 105 cells were plated in 6-well plates with 2 ml medium without antibiotics. After overnight incubation, the medium was replaced by 1 ml Opti-MEM Reduced-Serum Medium (Gibco, NY, USA) containing 50 µl of concentrated lentiviral particles and 8 µg/ml of polybrene per well. Fresh medium containing 2 µg/ml of puromycin (Invitrogen, USA) was added to each well 24 hours later and then selected for two weeks.
Western blot and immunoprecipitation
Western blot and immunoprecipitation were performed as described. The primary antibodies included anti-Rad50 (Abcam, USA), anti-CARD9 (Protein Tech, USA), anti-p-p65 (Ser536), anti-p65, anti-p21Cip1, anti-p27Kip1, anti-Cyclin D1 (Cell Signaling Technology, USA),anti-MYC (Cell Signaling Technology, USA),anti-GAPDH(Zsbio, China) and anti-β-actin (Sigma-Aldrich, USA). Anti-E-cadherin, anti-N-cadherin, anti-Vimentin, anti-Twist and anti-Snail antibodies were all from the Epithelial-Mesenchymal Transition (EMT) Antibody Sampler Kit (Cell Signaling Technology, USA).
RNA isolation and real-time PCR
Total RNA from cells was extracted with TRIZOL reagent (Invitrogen, USA) according to the manufacturer’s protocol. RNA was reverse transcripted to cDNA using PrimeScript RT reagent Kit (Takara, JAPAN). Real-time PCR was performed with SYBR Green mix and detected by the Bio-Rad CFX96. GAPDH was served as an endogenous control. Primer information was shown in Table S2.
Cellular proliferation assay
Cell proliferation was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich) assay. Cells were plated in 96-well plates (2–3 × 103 per well) for continual 1–5 days. After incubation at designated times, 20 µl of MTT (5 mg/ml in PBS) was added to each well, and cells were incubated for another 4 h at 37 °C. The supernatants were carefully removed and 200 µl of dimethyl sulfoxide (DMSO, Sigma-Aldrich) was added to each well. The absorbance of each samples was measured at 490 nm with a Microplate Reader (Thermo Fisher Scientific, IL, USA).
Cells were seeded into 6-well plates (300–500 cells/well) and cultured for 2–3 weeks. The colonies were fixed with methanol and stained with crystal violet. Colonies containing more than 50 cells were counted. For soft agar colony formation, 3–8 × 103 cells were suspended in the upper layer (0.7%) of the two layers agar (0.7 and 1.0%) in 6 cm dish. After 3 weeks, the colonies were stained by MTT and the images of colonies were photographed.
Cell cycle analysis
Cells (2–3 × 106) were cultured overnight in medium without FBS. Then the cells were cultured in complete medium and harvested at different times. Cell suspensions were incubated with 1 ml PBS containing 50 µg/ml PI with RNase A and permeabilization buffer for 30 min. The DNA content was analyzed by flow cytometer with a FACScalibur (BD Biosciences, USA). The results were analyzed with the ModFit software (Becton-Dickinson, USA).
Invasion and migration assay
Invasion and migration assays were performed in 24-well transwell chambers (BD Biosciences, USA) system with 8 µm pores coated with or without diluted matrigel (BD Biosciences, USA). Briefly, 1.5-2 × 105 cells were seeded into the upper chambers in certain medium containing no FBS, and lower chambers were filled with culture media containing 10–20% FBS as a chemo-attractant. After incubating at 37 °C for 12 to 48 h depending on the cell lines, cells that penetrated through the membrane were fixed with methanol and stained with crystal violet.
Cells were seeded on sterile glass coverslips and cultured overnight at 37℃. Cells were washed with PBS and then fixed for 20 min with 4% paraformaldehyde. Following blocking with 10% goat serum in PBS for 2 h at room temperature, the cells were incubated with primary antibodies (anti-N-cadherin, anti-E-cadherin and anti-p65) overnight at 4℃. Cells were then incubated with Rhodamine/FITC-labeled secondary antibody (Beyotime Biotechnology, China) for 1 h at room temperature in the dark. Cells were washed with PBS three times before counterstaining with 1 µg/ml DAPI for 5 min in the dark. Finally, the slides were mounted with antifading reagent and examined with a fluorescence microscope (Olympus, Japan).
Chromatin immunoprecipitation (ChIP)
ChIP assay was conducted using EZ-Magna ChIP A/G Chromatin Immunoprecipitation Kit (Millipore) following manufacturer’s instructions. Briefly, cells were cross-linked using formaldehyde. Then the cells were lysed and DNA was sheared to 200–500 bp fragments by sonication. The cell lysate was incubated with the Myc antibody (CST 9402S) and magnetic beads overnight at 4 °C with rotation. DNA associated with MYC is represented as fold change of normal IgG, calculated byΔCq method. Primer sequences were shown in Supplementary Table S1.
RAD50 wild type or truncated mutant promoter was cloned into pGL4.26 plasmid (Promega). Then RAD50 promotor report vector was co-transfected with pRL-TK and MYC plasmids into HEK293T cells. Luciferase activity was measured 24 hours after transfection using Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocol.
Xenograft tumor model
Female BALB/c nude mice (aged 4- 6weeks) were injected subcutaneously in bilateral flanks with cell suspensions (5 × 106 cells in 100 µl PBS). Tumor size was measured every three days after 1 week, and mice were killed with anesthesia after three weeks. Tumor volume was calculated according to the formula TV (cm3) = a × b2 × π/6, where ‘a’ is the longest diameter, and ‘b’ is the shortest diameter. Hematoxylin and eosin (H/E) staining and immunohistochemistry were performed on sections from embedded samples. All animal experiments were performed with the approval of Shandong University Animal Care and Use Committee.
The software GraphPad Prism 5 was used for statistical analysis. The two-tailed Student's t-test and one-way ANOVA analysis were used to analyze the differences. The relationship between the expression of Rad50 and clinical pathological factors was analyzed by the chi-squared test. All error bars represent the standard error of three separate experiments. Differences with P < 0.05 were considered significant.