Plasmids, Cell Lines, and Reagents
Human RCC lines (A498, 769-P) were obtained from the American Type Culture Collection (Manassas, VA, USA), maintained in our laboratory, and cultured under standard conditions. Cells were cultured in RPMI-DMEM medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 IU/mL), and streptomycin (100 µg/mL).
The MEK5 inhibitor BIX was purchased from MedChemExpress(Monmouth Junction, NJ, USA). Dimethyl sulfoxide (DMSO) was acquired from Sigma-Aldrich (St. Louis, MO, USA) and Cell Counting Kit-8 (CCK-8) from MedChemExpress (Monmouth Junction, NJ, USA). Antibodies to MEK5, p-mTOR, p-4EBP1, p-p70, and β-actin were obtained from Cell Signaling Technology (Beverly, MA, USA).
MEK5 knockdown and overexpression plasmid were purchased from the DNA library of Shanghai Jiao Tong University School of Medicine (Shanghai, China).
Real-time fluorescence quantitative PCR (RT-qPCR)
RT-qPCR was performed following the instructions of the TransStart Top Green qPCR SuperMix kit (TransGen Biotech, Beijing, China), and all the primers were synthesized by Sangon (Sangon Biotech Co., Ltd., Shanghai, China).
MEK5 forward:5’-CCTTCCAGTTGGAGAGTTCTCG
MEK5 reverse:5’-CGGCATTTCCATCATTGAACTGC
Actin forward:5’-CACCATTGGCAATGAGCGGTTC
Actin reverse:5’-AGGTCTTTGCGGATGTCCACGT
Western blotting
The whole-cell lysates were prepared by direct lysis with 1× SDS sample buffer and separated using 10% SDS-PAGE. The separated proteins were transferred to nitrocellulose (Bio-Rad, Hercules, CA, USA). After blocking the membrane with 5% non-fat milk prepared in +0.1% Tris-buffered saline, the membranes were incubated with primary antibodies overnight at 4°C. This was followed by an hour incubation with horseradish peroxidase (HRP)-linked secondary antibody (Cell Signaling Technology, Beverly, MA, USA). The protein bands were detected using a chemiluminescence phototope-HRP kit (Cell Signaling Technology).
Detection of apoptosis
The eBioscienceTM Annexin V-APC apoptosis detection kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to detect apoptosis. Briefly, ccRCC cells were treated with BIX solution for the indicated times, ccRCC cells (1 × 106) were harvested, washed with PBS, and resuspended in the binding buffer. Annexin V-APC (5 µL) and PI (5 µL) were sequentially added to the cell suspension and incubated for 15 min in the dark at room temperature. Cells were analyzed using a flow cytometer (Fortessa, San Francisco, CA, USA) and DIVA™ v8.0 software. Approximately 10,000 cells were analyzed from each sample.
Cell cycle assay
Cell cycle distribution was analyzed by estimating the DNA content using flow cytometry. Harvested cells were fixed with 75% ethanol at −20°C for 24 h. The samples were then incubated with 50 µg/mL RNase A for 30 min at 37°C, followed by 100 µg/mL propidium iodide (PI) at 37°C for 30 min. The DNA content was analyzed by a flow cytometer (Fortessa), and cell cycle distribution was estimated using FlowJo software.
Wound-healing assay
Cells were grown to confluence in 100 mm2 dishes, and artificial wounds were created using a sterile 200 µL pipette tip by scraping. The cells were allowed to close the wound and photographed at the indicated times.
Colony Formation Assay
The cells were subcultured until reaching the logarithmic growth phase, the stable transfected cells were digested with 0.25% trypsin and gently pipetted to make them single cells. The cells were counted and re-seeded at a density of 1000–2000 cells per well into a 6-well plate and incubated at 37°C with 5% CO2 in a humidified incubator for 10–15 days. When the cell formed visible clones, cells were terminated cultured, and washed twice with PBS, fixed using 4% paraformaldehyde for 15 min, and then stained using Crystal Violet for 5 min. The cells were then washed with PBS and dried.
Immunohistochemical analysis
Isolated tumors was formalin-fixed and paraffin-embedded and sectioned into slices. The tissue sections were subsequently stained with hematoxylin and eosin(HE) using standard techniques and used for the MEK5 and anti-Ki67 immunoassays.
Xenograft mouse model
The whole animal experimental protocol was approved by the Shanghai Jiao Tong University School of Medicine Institutional Animal Care and Use Committee(Shanghai, China). BALB/c nu/nu mice (female, aged 4 weeks) were purchased from Beijing Vital River Laboratory Animal Technology (Beijing, China) and kept in specific pathogen-free(SPF) condition in the Animal Center at Renji Hospital༈shanghai, China༉. A human ccRCC model was established by inoculating subcutaneously 1 × 106 A498 cells near the armpits of the forelimbs of experimental mice. The mice were randomly divided into the BIX and control groups when tumor masses were visible. BIX was intraperitoneally administrated at a dose of 30 mg/kg once a day for subsequent 10 days. The tumor growth was monitored by daily measurements by electronic cliper, in two dimensions(length(L) and width(W) ), and tumor volume(V) was estimated using with the formula: V=L/2 ×W2. After ten days treatments, the mice were sacrificed by cervical dislocation, and the tumor masses were removed, photographed and weighed.
Patient's Samples and Analysis
Under the supervision of the local ethics committee and pathologists, and without interfering with histological evaluation, fresh samples of four cases were obtained from ccRCC patients, diagnosed and treated at the Department of Urology, Renji Hospital. T test was used to evaluate the difference between MEK5 expression in ccRCC and adjacent normal tissues.
Statistical analysis
The statistically significant differences observed in drug-treated versus control cultures were determined using the Wilcoxon signed-rank test. The minimum level of significance was p < 0.05.