Cell culture
Endometrial cancer cell lines, including RL95-2 cells and Ishikawa cells were cultured with Dulbecco's modified Eagle's medium (DMEM) which contained 1% penicillin‑streptomycin solution (Gibco; Thermo Fisher Scientific Inc.) and 10% FBS. The cultured condition was at 37˚C in 5% CO2 humidified atmosphere.
Animals
Female nude mice (20g, 7-8 weeks) were used for the research and were kept in standard environment with the unified breeding. Mice were sacrificed by carbon dioxide asphyxiation. All experiments conform to the ethical standards for animals.
MTT assay
A total of 2000 RL95-2 cells and Ishikawa cells were plated into 96-well plates each well. The cells were treated without or with metapristone (50μM) for 5 days. The blank were the non-cell wells with medium. 20μl MTT (5 mg/ml) were added to each well and after four hours 150μl of DMSO were added. Cell numbers were counted at a wavelength of 570 nm by the Model 680 Microplate Reader (Bio-Rad Laboratories).
Cell colony formation assay
A total of 800 RL95-2 cells and Ishikawa cells were plated to 6-well plates each well with or without the treatment of metapristone (50μM). Two weeks later, cells were fixed with formaldehyde after being washed with phosphate-buffered saline (PBS) three times, and stained with Giemsa staining solution. Clones were counted by an Olympus inverted microscope.
Western Blot
The RIPA buffer (Cell Signaling Technology) were used to lyse all the cells sample. Total protein (30mg) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked at room temperature for 1 h with 5% non-fat milk in Tris-buffered saline with Tween 20(TBS-T). Then, the membranes with antibodies were incubated at 4℃ overnight. The primary antibodies were as followed: Cleaved-Caspase3 (dilution,1:1000; Cell Signaling Technology, 9661), Cleaved-Caspase-9 (dilution, 1:1000, Cell Signaling Technology, 20750), Bax (dilution, 1:1000, Cell Signaling Technology, 2774), Bcl-2 (dilution, 1:1000, Cell Signaling Technology, 15071), Klf5 (dilution, 1:1000, Cell Signaling Technology, 51586) and Nrf1 (dilution, 1:1000; Santa Cruz, sc-28379). Horseradish peroxidase-conjugated secondary antibodies were as followed: rabbit-HRP (easybio, 1:20000; BE0101-100). The results were detected by a chemiluminescence detection system (Immobilon Western Chemiluminescent HRP Substrate, MA).
Quantitative real-time polymerase chain reaction (q-PCR)
The total RNA of cells were extracted with Trizol(Invitrogen), and then had the process of reverse transcription to cDNA. Q-PCR was performed with SYBR Green PCR reagents on a Real-Time PCR System (Carlsbad, USA), and analyzed with StepOne Software.
Drug administration
Mifepristone was purchased from Shanghai New Hualian pharmaceutical Co., with purity >98%. Metapristone was synthesized, using mifepristone as the starting material as described previously[7].
MiR Mimics
MiR-492 mimic and miR-NC were purchased from Genepharma (Shanghai, China). MiR-492 mimic or miR-NC was transfected into RL95-2 cells and Ishikawa cells in 100nM for 48h.
Immunohistochemistry
The tissue sections were divided into 5μm thickness and the dehydration was reached with the different concentration ethanol after dewaxed in xylene. The sections were oven heating in 0.01 M sodium citrate buffer (pH 6.0) for 10 min to antigen retrieval. 3% hydrogen peroxide and 5% BSA were used to block endogenous peroxidase activity and nonspecific staining for 20 min and 1 h. the primary antibody of anti-Klf5 (1:100, Cell Signaling Technology, 51586) and anti-Nrf1 (1:1000, Santa Cruz, sc-28379) were incubated with tissue sections for overnight in 4 ◦C and incubated with secondary for 1 h. and cultured the section with streptavidin peroxidase for 10 min and 3,3- Diaminobenzidine tetrachloride (DAB) were used to detect peroxidase activity. All the experimental processes were performed at room temperature and the section were washed with PBS for three times. At last, sections were dehydrated in alcohol and cleared in xylene.
Tumor xenograft model
Two million of RL95-2 cells and Ishikawa cells were diluted in 0.1ml of saline and injected subcutaneously into 7-week old female nude mice. Mice were intraperitoneally injected with metapristone (45mg/kg, three times for one week) during two weeks. Tumor xenografts were measured during the experiment and were separated at the end of the experiment.
EdU Assay
A total of 5000 RL95-2 cells and Ishikawa cells were plated to 6-well plates each well and treated with metapristone (50μM, 48h). EdU staining proliferation kit was purchased from Abcam (ab219801). The EdU assay was performed as the instruction described.
Statistical analysis
All the results were analyzed with Mean ± S.D. or ±S.E.M. The significance analysis of all the experiments by student’s two-tailed non-paired t-test. P < 0.05 was considered to have statistical significance.