Patients and tissue samples
The OC patients’ tumor tissues and paired non-tumor tissues were obtained from patients who underwent curative surgery at General Hospital of Northern Theater Command. The specimens were frozen in liquid nitrogen immediately and then stored at -80°C. Overall survival (OS) was defined as the time interval between the date of surgery to the date of death or the last follow-up. Cumulative recurrence was defined as the time interval between the dates of surgery to the date of diagnosis of recurrence. A total of 90 tissue samples were also used for clinical prognosis analysis. Patient informed consent was also obtained and the procedure of human sample collection was approved by the Ethic Committee of General Hospital of Northern Theater Command.
Cell lines and culture
HO8910 and HGSOC cells were purchased from Chinese Academy of Sciences, Shanghai, China. The ovarian cancer cells were cultured in RPMI-1640 medium supplemented with 10 % fetal calf serum (FCS; Invitrogen, Carlsbad, CA, USA) at 37°C in a 5% CO2 incubator. The cultured cells were trypsinized with 0.5% trypsin and moved to a new six-well plate three times a week.
HO8910 or HGSOC cells were seeded into a six-well plate until they reached 60-70% confluence. The cells were infected with shTHOR lenti-virus and control virus as described previously (12). The sequence of shTHOR is as follows: 5'- GGUGAACACAAUCGAGCAATT-3'. The shRNA lenti-virus was purchased from Shanghai GenePharma (Shanghai, China).
HO8910 shTHOR or HGSOC shTHOR cells and their control cells were treated with S3I-201 (100 nM) or not and then subjected to CCK8, Transwell (24 h) and invasion assays (48 h).
Cell proliferation assays
HO8910 shTHOR or HGSOC shTHOR cells and their control cells were seeded in 96-well plates (3x103 cells/well). ATP activity was measured using CCK8 assays at the indicated time points. The procedure was as follows: The cell suspension (100 μl/well) was inoculated in a 96-well plate, and the plate was pre-incubated in a humidified incubator at 37°C for 1 hour. This was followed by the addition of 10 μl of the CCK-8 solution to each well of the plate, and incubation of the plate for 1 h in the incubator. Finally, the absorbance was measured at 450 nm using a microplate reader (Synergy H1; BioTek Instruments, Inc., Winooski, VT, USA).
Colony formation assay
For colony formation assays, the HO8910 shTHOR or HGSOC shTHOR cells and their control cells were seeded in 12-well plates (3000 cells/well). The cells were incubated for 7 days and then fixed with 10% neutral formalin for more than 4 hours. The cells were dyed with crystal violet (Beyotime, Haimen, China). The cells were then photographed under a microscope (Olympus, Tokyo, Japan).
EdU immunofluorescence staining
For cell EdU immunofluorescence staining, HO8910 shTHOR or HGSOC shTHOR cells and their control cells were seeded into 96-well plates and examined using the EdU kit (RiboBio, Guangzhou, China). The results were quantified with a Zeiss Axiophot photomicroscope (Carl Zeiss, Jena, Germany) and Image-Pro Plus 6.0 software.
Cell migration assays
For cell migration experiments, 2x105 OC cells were seeded into the upper chamber of a 24-well polycarbonate transwell in serum-free DMEM. The lower chamber was supplemented with DMEM containing 20% FBS as chemoattractant. The cells were incubated for 24 hours, and the chamber was fixed with 10% neutral formalin for more than 4 hours. The cells were dyed with crystal violet (Beyotime). Then the cells were counted under a microscope (Olympus), and the cell number is expressed as the average number of the cells in 5 fields.
Cell invasion assays
For cell invasion experiments, 2x105 OC cells were seeded into the upper chamber of a Matrigel-coated Boyden chamber in serum-free DMEM. The lower chamber was supplemented with DMEM containing 20% FBS as a chemoattractant. The cells were incubated for 48 hours, and the chamber was fixed with 10% neutral formalin for more than 4 hours. The cells were dyed with crystal violet (Beyotime). Then the cells were counted under a microscope (Olympus), and the cell number is expressed as the average number of cells in each field.
Spheroid formation assay
HO8910 shTHOR or HGSOC shTHOR cells and their control cells were cultured in a 6-well or 96-well ultra-low attachment culture plate for one week, and the total number of spheres was counted under the microscope.
Real-time PCR
The total RNA from OC cells or the OC patient tissues was extracted by using TRIzol reagent (Invitrogen, 15596-018). Total cDNAs were synthesized by a ThermoScriptTM RT-PCR system (Invitrogen, 11146-057). The total mRNA amount present in the cells was measured by RT-PCR using the ABI PRISM 7300 sequence detector (Applied Biosystems). The THOR primer sequences were as follows: forward: 5'-ACAATCGAGCAAGGCAGTGA-3', reverse: 5'-TGGCCAAGACCTGCTGTTAG-3'. β-actin was used as reference for relative expression calculation and its primer sequences were as follows: forward: 5'-GGCCCAGAATGCAGTTCGCCTT-3', reverse: 5'-AATGGCACCCTGCTCACGCA-3'. The PCR cycling conditions were as follows: 94℃ degeneration for 10 min; 94℃ modification for 30 sec, 60℃ annealing for 30 sec, 72℃ extension for 40 sec, total of 40 cycles, 72℃ terminal extension for 10 min.
Western blotting assays
The OC cells or OC patients’ tissues were lysed with cell lysis buffer (Beyotime) followed by supersonic splitting as described previous (13). The total protein was quantified using a BCA Protein Quantification kit. A total of 25 μg of protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes. The membranes were blocked with 10% non-fat milk and incubated with primary antibodies overnight. The protein band, specifically bound to the primary antibody, was detected using an IRDye 800CW-conjugated secondary antibody and the LI-COR imaging system (LI-COR Biosciences, Lincoln, NE, USA). The primary antibodies were p-AKT (1:1000; #4060, Cell Signaling Technology), p-STAT3 (1:1000; #9145, Cell Signaling Technology), p-MEK (1:1000; #9127, Cell Signaling Technology) p-SMAD3 (1:1000; #9520, Cell Signaling Technology), and GAPDH (1:5000; #5174, Cell Signaling Technology).
In vivo animal models
The NOD-SCID mice were purchased from Slake Company, Shanghai academy of sciences. All mouse experiments were performed according to the guidelines of the animal care and use committees at Shanghai Baoshan District Hospital of Integrated Chinese and Western medicine.
For xenograft formation assay, HO8910 shTHOR or its control cells (2X106) were injected subcutaneously into nude mice. Mice were sacrificed six weeks post inoculation and tumors were collected and examined.
For pulmonary metastasis assay, HO8910 shTHOR or its control cells (2 X106) were injected into the tail vein of nude mice. The mice were sacrificed 12 weeks post inoculation and consecutive sections of the whole lung were subjected to hematoxylin and eosin (H&E) staining. All metastatic foci in the lung were calculated microscopically to evaluate the development of pulmonary metastasis.
For in vivo limiting dilution assay, HO8910 shTHOR cells and its control cells were mixed with Matrigel (BD) at a ratio of 1:1 and injected subcutaneously at various cell doses per mouse. The tumor formation was evaluated at 8 weeks.
Statistical analysis
GraphPad Prism (GraphPad Software, Inc., La Jolla, USA) was used for all statistical analyses. Statistical analysis was carried out using t tests or Bonferroni multiple comparison tests: *p<0.05. A p value of less than 0.05 was considered statistically significant.