Patients
The ovarian cancer tissues and adjacent tissues (normal fallopian tube tissues not invaded determined by pathology) of 119 patients with ovarian cancer who underwent surgery in Affiliated Tumor Hospital of Nantong University from January 2013 to January 2016 were collected. Inclusion criterias: ⑴Epithelial ovarian cancer was diagnosed by pathology. ⑵The clinical data and follow-up data were completed. Exclusion criterias: ⑴Combined with or secondary to other malignant tumors. ⑵The clinical data and follow-up data were deficient.
Platinum sensitive recurrence is defined as the recurrence occurring more than 6 months after drug withdrawal, and platinum resistant recurrence is defined as the recurrence occurring within 6 months after drug withdrawal. Overall survival (OS) was defined as the time from the beginning of treatment to the end of death or follow-up. Progression free survival (PFS) was defined as the time between the beginning of treatment and the observation of disease progression or death from any cause. The deadline for follow-up was December, 2020.
Immunohistochemical staining
The chips were incubated at 85°C for 1 h. All chips were dewaxed and dehydrated. The alkaline repair solution was heated for antigen repair for 3 min, cooled to room temperature, blocked by peroxidase blocker at room temperature for 20 min, and the primary antibodies (dilution concentration was 1:50), Gab2(Abcam, ab235932) and CrkII(Abcam, ab45136), were 4℃ overnight. The next day, rewarming to room temperature, incubating the secondary antibody at room temperature for 20 min, and flushing with PBS buffer 3 times for 5 minutes each time. DAB coloration for 2 min, hematoxylin staining for 30s, ethanol dehydration, xylene dewaxing, neutral gum sealing. All chips were re-read by 2 senior pathologists.
The positive standard of staining is cytoplasmic staining, and the negative standard is no cytoplasmic staining, regardless of whether the cell membrane and nucleus are stained or not. According to staining intensity, they were divided into non staining, light yellow, yellow and brown. The scores were 0, 1, 2 and 3. The percentage of positive cells were < 20%, 21% ~ 50%, 51% ~ 75% and > 75%, and the scores were 1, 2, 3 and 4. The comprehensive score is the product of the two indicators.
Cell culture and transfection
Ovarian cancer cells (SKOV3, A2780 and HO8910) and normal ovarian cells (IOSE80), were obtained from National Collection of Authenticated Cell Cultures. SKOV3 and A2780, HO8910 and IOSE80 cell lines were cultured in DMEM medium, RPMI-1640 medium, supplement with 10% FBS, 100 µg/ml streptomycin and 100 µg/ml penicillin in 5% CO2 at 37°C. Gab2 overexpression virus and CrkII knockdown virus were synthesized by GENE (Shanghai, china). Gab2 shRNA and CrkII overexpression plasmid were synthesized by OBiO (Shanghai, china). The vector was transfected into ovarian cancer cells A2780 and SKOV3 with Lipofectamine™ 2000 (Thermo Fisher Scientific).
Western blot
Collect ovarian cancer cells, add protein lysate to the cells, lyse them on ice for 5 min, absorb the supernatant and store it at -20℃. The concentration of the extracted protein samples was determined by BCA method. Assemble SDS-PAGE electrophoresis device and configurate gel for electrophoresis. Protein sample was incubated in boiling water bath for 5 min. The sample loading amount in each hole is 15 µl protein, electrophoresis at constant pressure of 80 V for 1.5 h. The PVDF membrane was cut to a proper size, soaked with methanol, and then placed in ddH2O, soak in buffer for 10 min. Turn the film at 300 A for 90 min. Hold PVDF membrane was placed in skimmed milk powder solution and incubated in a shaking table at room temperature for 2 h; Then the PVDF membrane was placed in the primary antibody and combined at 4℃ for overnight; PVDF membrane was placed in secondary antibody and combined at room temperature for 2 h. Color rendering. The primary antibodies of Gab2 and CrkII were diluted at 1:1000 ,GAPDH (proteintech, 60004-1-1g) was diluted at 1:2000 and the secondary antibodie, Mouse(Absin, abs20039) and Rabbit(Absin, abs20040), were diluted at 1:2000.
Cell proliferation assay
Cells (5×103) were cultured into 96-well plates and incubated for 24 h, 48 h or 72 h. Then, CCK-8 solution was added into 96-well plates. After 2 h, the OD in each well was determined at 450 nm.
Migration assay
1×105 cells were seeded into the upper chamber of a transwell chamber in basal medium. The lower chamber was supplemented with medium containing 10% FBS. The cells were cultured for 72 h, and the chamber was fixed with 4% paraformaldehyde for 30 min. The cells were stained with 1% crystal violet for 15 min. Then the number of invaded cells was counted under a microscope.
Cell viability assay
Cells (5×103) were cultured into 96-well plates. After the cell adheres to the wall, the cell were treated with different concentrations of carboplatin (100, 300, 500, 700, 900, 1200 ug/ml) and cultured for 48 h. Then, CCK-8 solution was added into 96-well plates. After 2 h, the OD in each well was determined at 450 nm.
Immunoprecipitation
Collect the cells, add cell lysate (including protease inhibitor), lyse on ice or 4℃ for 30 min, centrifuge at 12000 rpm/30min, and take the supernatant after centrifugation. Take a small amount of lysate for western blot. Add 1µl of corresponding antibody and 10–50 µl of protein A/G to the remaining lysate, and shake slowly for 4℃ overnight. After immunoprecipitation reaction, centrifugation was carried out at 3000rpm/5min at 4℃, and protein A/G was centrifuged to the bottom of the tube, suck up the supernatant. Protein A/G was washed 3–4 times with 1ml lysate; Finally, add 15µl of 2×SDS sampling buffer and cook for 10 min. Western blot analysis was performed.
Statistical analysis
Statistical analysis were performed using SPSS 26.0. McNemar’stest was used to analyze the expression differences of Gab2 and CrkII in ovarian cancer tissues. The associations between Gab2 and CrkII and clinical parameters were measured by Chi-square test. Cox regression model was used to analyze the risk factors affecting the prognosis of patients. Kaplan-Meier method and Log-rank test were used to draw the survival curve. T-test was used for comparison between the two groups. In vitro experiments were repeated at least three times and data were presented as mean ± SD. P < 0.05 was considered statistically significant.