Cell culture
Cancer cell lines, including RL95-2 cells (ATCC® CRL-1671), Ishikawa cells (ECACC, 99040201), MCF7 cells (ATCC® HTB-22), BT-474 cells (ATCC® HTB-20), A549 cells (ATCC® CCL-185) and MGC-803 cells (BLUEFBIO, BFN608007257) were obtained from American Type Culture Collection (Manassas, VA, USA) and European Collection of Authenticated Cell Cultures (Porton Down, UK). These cells were cultured with Dulbecco's modified Eagle's medium (DMEM) which contained 1% penicillin‑streptomycin solution (Gibco, USA) and 10% FBS. The cultured condition was at 37˚C in 5% CO2 humidified atmosphere (HERAcell 150i/240i, Thermo, USA). STR profiling and mycoplasma contamination were performed to keep the authenticity of cell line on regular basis.
Transfection
The sequence of miR-492 was obtained from National Center for Biotechnology Information (NCBI). The small interfering RNA (siRNA) targeting miR-492 (si-miR492) and negative control (si-NC), miR-492 mimic and negative control (miR-NC) were purchased from Genepharma (Shanghai, China). Thereafter, both of these plasmids were transfected into RL95-2 cells and Ishikawa cells in 100 nM for 48h by use of lipofectamine 2000 (Invitrogen, Carlsbad, CA). The sequences were shown in Table 1
MTT assay
For the cell proliferation assay, the total of 2000 RL95-2 cells and Ishikawa cells were plated into 96-well plates each well. The cells were treated with ethanol or metapristone (50 μM), transfected with miR-NC or miR-492 mimic, or transfected with si-NC or si-miR-492 for 5 days. The blank were the non-cell wells with medium. The culture medium wad removed and 20μl 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg/ml) were added to each well for four hours at 37℃. Therefore, 150μl of DMSO were added and incubate for 15 min. Cell numbers were counted at a wavelength of 570 nm by the Model 680 Microplate Reader (Bio-Rad Laboratories, USA). The six replicates were in each treatment and three times were repeated for MTT assay[29].
Cell colony formation assay
A total of 800 RL95-2 cells and Ishikawa cells were plated to 6-well plates with soft agar each well with the treatment of ethanol or metapristone (50 μM), transfected with miR-NC or miR-492 mimic, or transfected with si-NC or si-miR-492. Two weeks later, cells were fixed with formaldehyde after being washed with phosphate-buffered saline (PBS) three times, and stained with Giemsa staining solution for 30 min under room temperature. Visible clones were counted by an inverted microscope (Olympus, Japan)[30]. Each experiment was repeated three times.
EdU assay
A total of 5000 RL95-2 cells and Ishikawa cells were plated to 6-well plates each well and treated with ethanol or metapristone (50 μM, 48h), transfected with miR-NC or miR-492 mimic (100 nM, 48h), or transfected with si-NC or si-miR-492 (100 nM, 48h). EdU staining proliferation kit was purchased from Abcam (ab219801). The plate was added with EdU solution and incubate for 3 hours and then treated with 4% formaldehyde. After the process, the cells were stained with DAPI and performed as the instruction described by inverted microscope (Olympus, Japan). Each experiment was repeated three times.
Luciferase reporter assay
Luciferase reporter assay was performed as the papers described[30, 31]. In brief, the wild type or mutanted 3’-UTR sequence of Klf5 and Nrf1 was cloned into pGL3-luc vector (Promega, USA). A total of 3*105 RL95-2 or ISK cells were plated in 24-well plates 24h prior to transfection. For each well, the reporter constructs (500 ng) and the miR-NC or miR-492 (100 nM) were co-transfected into RL95-2 or ISK cells by using Lipofectamine 2000. After transfection for 48h, the cells were lysed and the relative luciferase activity was measured by using dual-luciferase reporter assay system (Promega, USA). The miR-492 reporter activity was normalized to empty vector control. Each experiment was repeated three times.
Western Blot
The RIPA buffer (Beyotime, China) were used to lyse all the cell samples and tumor tissues and total protein amounts were determined by BCA protein assay (Pierce Biotechnology, USA) and then denatured with Laemmli buffer (Sigma, USA) at 95°C for 10 min. Total protein (30 mg) was separated by 10%-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Millipore, USA). 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: Caspase3 (dilution,1:1000; Cell Signaling Technology, 9662), Cleaved-Caspase-3 (dilution,1:1000; Cell Signaling Technology, 9654), 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), Nrf1 (dilution, 1:1000; Santa Cruz, sc-28379) and GAPDH (dilution, 1:1000; easybio, BE0023). Horseradish peroxidase-conjugated secondary antibodies were as followed: rabbit-HRP (easybio, 1:20000; BE0101-100). The results were detected by ECL Western Blotting Substrate (Pierce, Thermo Fisher, USA) and captured with the ImageQuant LAS 400 imaging system (GE Healthcare Life Sciences, USA). Each experiment was repeated three times.
Quantitative real-time polymerase chain reaction (qRT-PCR)
The total RNA of cells was extracted with Trizol (Invitrogen, USA), and then had the process of reverse transcription to cDNA Reverse Transcriptase M-MLV (TakaRa, Japan). QRT-PCR was performed with SYBR Green PCR reagents (CoWin Biosciences, China) on a Real-Time PCR System (Carlsbad, USA), and analyzed with StepOne Software (Thermo Fisher, USA). mRNA expression was normalized to GAPDH[30]. The primers were performed in supplementary table. The three replicates were in each treatment and three times were repeated for qRT-PCR.
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[29]. Metapristone was dissolved in ethanol, so that ethanol was used as negative control.
Immunohistochemistry
The tissues were fixed with formalin and embedded with paraffin. 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, Beyotime China) for 10 min to antigen retrieval. 3% hydrogen peroxide and 5% BSA (Amresco, USA) were used to block endogenous peroxidase activity and nonspecific staining for 20 min and 1 h, respectively. The primary antibody of anti-Ki-67 (1:1000, Cell Signaling Technology, 9449), anti-Cleaved-caspase 3 (1:1000, Cell Signaling Technology, 9661), anti-Klf5 (1:1000, Abcam, ab137676) and anti-Nrf1 (1:1000, Santa Cruz, sc-28379) were incubated with tissue sections for overnight in 4℃ and incubated with secondary for 1 h in room temperature, and the cultured section with streptavidin peroxidase for 10 min and 3,3- Diaminobenzidine tetrachloride (DAB, TIANGEN China) were used to detect peroxidase activity[32].
Tumor xenograft model
Female Balb/c nude mice (20g, 7-8 weeks) were obtained from Vital River Laboratory Animal Technology (Beijing, China) and were kept in standard environment with the unified breeding. Mice were sacrificed by carbon dioxide asphyxiation. All the experiment was approved by the Animal Research Committee of the Beijing Friendship Hospital Affiliated to Capital Medical University and conform to the ethical standards with the guidelines of the National Animal Care and Ethics Institution. Two million of RL95-2 cells and Ishikawa cells were diluted in 0.1 ml of saline and injected subcutaneously into 7-week old female nude mice. After the cell injection, the experimental group mice (more than 6 mice per trial) were intraperitoneally injected with metapristone (45mg/kg, three times for one week) during two weeks and the control were intraperitoneally injected with vehicle at the same time points. Tumor xenografts were measured using a caliper during the experiment. The tumor volume was calculated by this formula: volume = (length × width2)/2. After sacrificed, the tumors were separated for future analysis. Each group contained more than 6 mice[33].
Statistical analysis
For all studies, statistical analyses were conducted using the GraphPad Prism software. All the results were analyzed with Mean ± S.D. 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. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.