All cell lines used in this study were obtained from the Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (Shanghai, China) and were authenticated by short tandem repeat profiling and monitoring Mycoplasma contamination. 22RV1, LNCaP, PC3 and DU145 PCa cell lines were routinely cultured in RPMI-1640 medium. 293T cells were grown in DMEM medium. All mediums were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. All the medium was purchased from Gibco. All cell lines were incubated at 37°C with 5% carbon dioxide.
Cell counting Kit-8 (CCK-8) assay
The cells concentration was adjusted to around 2×103 cells/well, and the cells were seeded into 96-well plates in 100μl of culture medium, followed by incubation at 37°C in an atmosphere with 5% carbon dioxidecultivation. At various time points, 10μL of CCK-8 reagent (MCE, HY-K0301) at a 1:10 dilution with serum-free RPMI-1640 medium was added to each well, followed by a further 2h incubation. The cell viability was determined using the CCK-8 assay, and the optical density (OD) was measured at 450 nm. Each experiment was conducted in triplicates.
Plasmids, siRNA, and transfection
The pEX-1 plasmid vector used in this study carried an enhanced green fluorescence protein (eGFP), and HA-tag or FLAG-tag were added in front of the target gene as needed. The construction and quality inspection of the plasmid was completed by Sangon Biotech (Shanghai, China). The targeting plasmids were delivered into PC3 and DU145 cells using the Lipofectamine 2000 transfection reagent (Invitrogen, USA, #11668019) according to the manufacturer’s instructions. G418 (PC3 100μg /mL, DU145 120μg /mL, Gibco, USA) was used to screen individual clone expressing FBXW2 or vector control. siRNA designed to target FBWX2 were purchased from Sangon Biotech (Shanghai, China), including siRNA- FBXW2-#970 (5’-CCUCUUAAGUGCAGACAAATT-3’), siRNA-FBWX2-#1014 (5’-GGAGAGAAAUCAACUGUAATT-3’) and siRNA-FBWX2-#1174 (5’-CAU CAAGACUCCUGAGAUATT-3’). For transient transfection, control and FBWX2 siRNA (#970, #1014 and #1174) were mixed with Lipofectamine 2000 and then added to cell culture medium in 22RV1 and LNCaP cells according to the manufacturer's instructions. Finally, we used IB to evaluate the transfection efficiency.
RNA isolation, reverse-transcription, and qPCR
Total RNA was isolated by Trizol-Chloroform method and then transcribed into cDNA using reverse transcription kit (Takara, Japan) with an oligo (dT) 20bp primer. RT-qPCR were performed by using the SYBR green reagent (Takara, Japan) on Real-Time PCR System (Thermo Fisher, USA). The 2−ΔΔCt method was used to quantify the data, and the housekeeping GAPDH was used as loading control in the analysis. The sequences of primer sets were 5’-GGACATGCCTGAACACACTC-3’ and 5’-CCAGGACTGTGCAAGAGAGA-3’ for FBXW2; 5’-ACCCAGAAGACTG TGGATGG-3’ and 5’-TCAGCTCAGGGATGACCTTG-3’ for GAPDH. Each experiment was conducted in triplicates.
Placed the Culture-Insert (Ibidi, Germany) in the 12-well plate and made the sticky side stick to the bottom of the well. The cells were counted, diluted to 70-100×104 cells/mL, and 70μL suspension was added to the slots on both sides of the Culture-Insert. Vertically removed the Culture-Insert 24h later, and a straight scratch was formed between the cells in the slots. Plate was washed by PBS and the cells were continually incubated in serum free medium. Photographs of the scratch were taken at various time points by inverted microscope (Olympus, Japan). The mean value of each gap width was measured for statistical analysis. Each experiment was conducted in triplicates.
Matrigel (Sigma, USA) was diluted with 5 times serum-free cell medium, and 50μL of which was added to the bottom of each transwell chamber and incubated for 4h. The matrigel-coated chambers were washed with serum free medium for later use. Placing the chambers in a 24-well plate, in each of them 4×104 cells in 200μl medium containing 2% FBS free medium were plated, together with medium containing 10% FBS at the bottom of the well. Cells were incubated for 24-48h, and then fixed with 4% paraformaldehyde for 20 min. After washing with PBS for three times, cells were stained with 0.1% crystal violet blue (Beyotime, China) for 20 min, and then washed with PBS. Cells on the upper surface of the chamber were removed by cotton swab. The positively stained cells were photographed under the microscope, and multiple fields were taken for cell count and statistics. Each experiment was conducted in triplicates.
Apoptosis and cell cycle assays
Cells were synchronized at G0/G1 by serum starvation for 24h and then released by serum addition. For cell apoptosis detection, cells both in adherent and supernatant were collected and were stained in dark with 5μL Annexin v-fitc (Gibco, USA) for 10 min, and then labeled with 10μL FxCycleTM PI/RNase for 5min before flow cytometry. For cell cycle detection, cells were collected when cell density reached 70% to 90%. and fixed in 70% ice-cold ethanol overnight, labeled with 500μL FxCycleTM PI/RNase (Gibco, USA) for 15-30 min in dark at room temperature, and analyzed directly on an LSR II flowcytometer (BD Biosciences, USA). Each experiment was conducted in triplicates.
Immunoblotting (IB) and Immunoprecipitation (IP)
For direct IB analysis, cells were lysed in RIPA buffer with protease inhibitors and phosphatase inhibitors (Roche Life Science, Switzerland). The following primary antibodies were used: rabbit-FBXW2 (ab83467, Abcam; 1:1,000 overnight, 4°C); rabbit-FBXW2 (11499-1-AP, proteintech; 1:500 overnight, 4°C); rabbit-EGFR (#4267, CST; 1:1,000 overnight, 4°C); rabbit-caspase 3 (19677-1-AP, proteintech, 1:1000 overnight, 4°C); rat-HA (#11867423001, Roche Life Science, 1:2000 overnight, 4°C); mouse-FLAG (#F1804, Sigma, 1:2000 overnight, 4°C); mouse-His-Tag (66005-1-Ig, proteintech, 1:1000 overnight, 4°C); rabbit-AR (#5153, CST, 1:1000 overnight, 4°C); rabbit-p-AKT (#4060S, CST, 1:1000 overnight, 4°C); rabbit-AKT (#4691S, CST, 1:1000 overnight, 4°C); rabbit-Cyclin B1 (#12231S, CST, 1:1000 overnight, 4°C); rabbit-Cyclin D1 (#2978, CST, 1:1000 overnight, 4°C); rabbit-Cyclin E1 (11554-1-AP, proteintech, 1:1000 overnight, 4°C); rabbit-p-STAT3 (#9145, CST, 1:1,000 overnight, 4°C); mouse-STAT3 (ab119352, Abcam, 1:1,000 overnight, 4°C); rabbit-BAX (50599-2-Ig, proteintech, 1:1000 overnight, 4°C); rabbit-BCL2 (12789-1-AP, proteintech, 1:1000 overnight, 4°C); rabbit-PARP1 (13371-1-AP, proteintech, 1:1000 overnight, 4°C); rabbit-p27 (25614-1-AP, proteintech, 1:1000 overnight, 4°C); rabbit-p21 (103551-1-AP, proteintech, 1:1000 overnight, 4°C); mouse-GAPDH (60004-1-Ig, proteintech, 1:2000 overnight, 4°C); mouse-β-Actin (60008-1-Ig, proteintech, 1:5000 overnight, 4°C); mouse-α-Tubulin (66031-1-Ig, proteintech, 1:5000 overnight, 4°C). For immune-precipitation, cells were treated by MG132 (10 μg/mL, Beyotime, China) for 4h before lysed in IP-RIPA buffer with protease inhibitors, and Protein A/G plus Agarose (Santa Cruz, USA) were used to pull down the proteins. To immunoprecipitate endogenous proteins, whole cell extracts were pre-cleared with normal IgG-AC (Santa Cruz, USA) followed by overnight incubation at 4°C with antibody against HA-Tag. To immunoprecipitate exogenously expressed FLAG-Tag proteins, the pre-cleared cell lysates were incubated with HA-Tag antibody in a rotating incubator overnight at 4°C. The Protein A/G plus agarose were washed with NETN-100 and the co-precipitated proteins were assessed by IB. Each experiment was conducted in triplicates.
After gene manipulation, 20μg/mL cycloheximide (CHX, MCE, China) was added to the cell medium for inhibiting new protein synthesis. At the indicated time points, cells were harvested, lysed and subjected to IB analysis. Each experiment was conducted in triplicates.
The in vivo ubiquitylation assay
Briefly, PC3 cells were co-transfected with HA-FBXW2 and His-Ub to detect ubiquitination of endogenous EGFR. To detect ubiquitination of exogenous EGFR, 293T cells were co-transfected with HA-FBXW2, His-Ub, and FLAG-EGFR-WT, FLAG-EGFR-MU1 or FLAG-EGFR-MU2. Mock vector was used as a control for ubiquitination assay. Cells were lysed in buffer A (6M guanidine-HCl, 0.1M Na2HPO4, 0.007M NaH2PO4, and 5mM imidazole, 0.1% Triton X-100, 10mM β-mercaptoethanol, pH 8.0) and sonicated. The lysates were incubated with nickel-nitrilotriacetic acid (Ni-NTA) beads (QIAGEN, Germany) at 4°C overnight. The beads were washed respectively with buffer A and buffer B (8M urea, 0.1M Na2HPO4, 0.007M NaH2PO4, and 5mM imidazole, 0.1% Triton X-100, 10mM β-mercaptoethanol, pH 8.0), and then three times with buffer C (8M urea, 0.025M Na2HPO4, 0.075M NaH2PO4, and 5mM imidazole, 0.1% Triton X-100, 10mM β-mercaptoethanol, pH 6.3). The beads were boiled and the pull-down proteins were resolved with anti-HA or anti-FLAG antibody by subsequently IB assay. Each experiment was conducted in triplicates.
All experimental procedures using mice were performed in accordance with protocols approved by Laboratory Animal Welfare and Ethics Committee of Third Military Medical University of China. For xenograft model, 1×106 PC3 stable cells (Vector and HA-FBXW2) were mixed 1:1 with matrigel in a total volume of 0.2mL and were injected subcutaneously into both flanks of BALB/c athymic nude mice (nu/nu, male; 4–6wk old; 13-15g). The size of tumors and the weight of the mice were measured twice a week. Mice were killed by carbon dioxide asphyxiation on day 30 after tumor cell injection, when some of the tumors reached the size limit set by the Institutional Animal Care and Use Committee. Tumors were weighed and fixed with 4% paraformaldehyde after resection.
For intratibial injection model, 2.0×106 PC3 stable cells (Vector and HA-FBXW2) were resuspended in 100µL PBS and 10µL of cell solution was slowly injected NOD/SCID mice (male; 4–6wk old; 13-15g) with a 28.5-G needle into the tibia using a drilling motion. Osteolytic lesions were identified on radiographs as radiolucent lesions in the bone. Each bone metastasis was scored as follows : 0, no metastasis; 1, bone lesion covering less than 1/4 of the bone width; 2, bone lesion involving 1/4 to 1/2 of the bone width; 3, bone lesion across 1/2 to 3/4 of the bone width; and 4, bone lesion more than 3/4 of the bone width. The bone metastasis score of each mouse was the sum of all bone injuries. Mice were sacrificed dependent on survival time. Two hind limbs were dissected and fixed with 4% paraformaldehyde, which were subsequently used for H&E staining.
Clinical specimens and tissue microarray
All 9 prostate samples were collected from the urological specimen bank of the second affiliated hospital of the army medical university. The establishment of the specimen bank met the ethical requirements, and each sample had complete case information and pathological results. Three prostate hyperplasia samples were in the control group, three prostate cancer samples with Gleason score ≤ 7 without metastasis were in the PCa group, and three prostate cancer samples with Gleason score of > 7 and metastasis were in the M-PCa group. Tissue microarray was purchased from Servicebio (Wuhan, China), which provided chip information and completed immunohistochemical staining of the chips.
IHC staining of Ki-67, EGFR, p-AKT, p-STAT3, p27 and p21 proteins in tumors of the nude mice was performed as described previously[17, 20]. The tissues were paraffin-embedded and sectioned after formalin-fixation. Tissue sections were subjected to IHC by incubation with anti-Ki67 (#9449, CST), anti-EGFR, anti-p-AKT, anti-p-STAT3, anti-p27 and anti-p21 antibodies respectively after deparaffinizing and antigen repairing. The sections were then incubated sequentially with biotinylated secondary antibody and HRP (horseradish peroxidase)-conjugated streptavidin. Antigenic detection was performed using chromogenic substrate DAB (3,3-diaminobenzidine tetrachloromethane) and the sections were counter stained further by hematoxylin. The positively stained cells were photographed under the microscope, and multiple fields were taken for cell count and statistics.
Statistical methods and chart
All data were expressed as Mean ± SD, SPSS 16.0 software was used for statistical analysis, and GraphPad Prism 7.0 software was used to make statistical charts. The proliferation curve was analyzed by repeated measures of variance, and the other data were analyzed by independent sample t-test or one-way analysis of variance, followed by Newman-Keuls test for evaluating the significance of differences between groups. P-values less than 0.05 were defined as statistically significant.