Cell lines and cultures
Prostate cancer cell lines DU145 and VCaP were purchased from the Chinese Academy of Sciences, Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). These cells were maintained at 37 ˚C with 5% CO2 in an RPMI‑1640 and DMEM culture medium, respectively, containing 10% fetal bovine serum and 1% streptomycin/penicillin. The docetaxel resistant cell line DU145-DR and VCaP-DR were developed by the methods as described in our previous publication [15].
Transfection
The sequences of siRNAs of FOXM1 and KIF20A were as follows: the sense sequence of siRNA FOXM1: 5′-CUCUUCUCCCUCAGAUAUATT-3′, and the antisense sequence: 5′-UAUAUGAGGGAGAGTT-3′; the sense sequence of siRNA KIF20A: 5′-GCAGCAGGUUCCAUCUGAGTT-3′, and the antisense sequence: 5′-CUCAGAUGG
AACCUGCUGCTT-3′. The sequence of non‐targeting control siRNA is the 5′‐UUCUCCGAACGUGUCACGUTT‐3′. siRNAs were transfected to cells using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). In addition, pcDNA3.1/FOXM1 (pcFOXM1) and pc DNA3.1/KIF20A(pcKIF20A) plasmids were constructed according to standard cloning methods. Cells were transiently transfected with pcFOXM1, pcKIF20A, and their NC plasmids, using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Mock-transfected cells served as the control group (Ctrl). The transfection efficiency was evaluated after 48 h.
Stable transfection
Human lentivirus-shFOXM1 was purchased from GenePharma (Shanghai, China). The lentiviruses were ultracentrifuged, concentrated, validated and then added to the culture medium. After infection, the transduced cells were selected using puromycin (Gibco, Grand Island, NY, USA) for 2–3 weeks, and the surviving cells were continuously cultured and referred to as DU145-DR stably transfected siFOXM1.
Cell apoptosis analysis
Cells in an exponential growth phase were harvested and seeded onto six-well plates. Once the cells adhered to the plate, different treatments were performed. After the cells were cultured for 72 hr, an Annexin V-FITC/PI Apoptosis Assay Kit was used to measure the percentage of apoptotic cells via BD FACS Calibur flow cytometry (BD Biosciences). And the apoptotic rate was calculated as the amount of the percentages of both upper right quadrant and lower right quadrant.
Cell cycle analysis
After different treatments, the cells were harvested and fixed with 70% precooled ethanol for at least 4 hr. Then, the cells were incubated in PBS solution containing 50 mg/ml propidium iodide (PI) and 100 mg/ml RNase for 30 min at room temperature. Finally, the cell cycle was analyzed using BD FACS Calibur flow cytometry (BD Biosciences, San Jose, CA) within 1 hr.
Cell viability assay and colony formation assay
Cells were seeded into a 96-well plate at 5×103 cells/well and exposed to different treatments. MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) dye solution was added to each well and incubated for 4 h at 37 °C. The medium was aspirated, and 100 μL of DMSO was added to stop the reaction. Cell viability was assessed by measuring the absorbance at 590 nm in a spectrophotometer. For the colony formation assay, 500 cells were plated in 6-well plates and cultured at 37 °C for 14 days. The colonies were visualized by staining with 0.1% crystal violet in methanol for 30 min. The number of colonies/well were counted and analyzed.
Wound-healing assay
Cells in the exponential growth phase were seeded in 6-well culture plates. After reaching confluency, the cell layer was scratched with a sterile P-200 pipette tip. The cells were then cultured in complete medium containing docetaxel for 24 h before wound exposure. Photographs were taken (100× magnification), and cell migration distances were measured.
Cell invasion
Cell invasion assays were performed as previously described using a modified transwell chamber with a Matrigel-coated membrane (BD Biosciences Bedford, MA). Briefly, approximately 1 x 104 cells resuspended in serum-free medium were plated in 24-well plates and treated with the docetaxel, FOXM1-siRNA, pc-FOXM1, KIF20A-siRNA, and pc-KIF20A. At 24 h post-treatment, cell invasion was measured as per the manufacturer’s instructions.
Western blot analysis
Cells were lysed with radioimmunoprecipitation assay (RIPA) buffer. After centrifugation, 20 μg of cell lysate were electrophoresed in sodium dodecyl sulfate (SDS)-polyacrylamide gel (10% or 15%) and transferred to polyvinylidene fluoride(PVDF) membranes. After blocking with 5% skimmed milk in TBST (Tris-buffered saline with 0.1% Tween-20) at room temperature for 2 h, the membranes were incubated with the following primary antibodies: FOXM1 (proteintech, 13147-1-AP, Wuhan, China), and KIF20A (proteintech, 15911-1-AP,Wuhan,China), CyclinA2
(Abcam, ab181591, Shanghai, China), CyclinD1(Abcam, ab134175, ,Shanghai,China), CyclinE1(Abcam, ab33911, Shanghai, China), Bcl-2(Abcam, ab182858, Shanghai, China), PARP (proteintech, 66520-1-Ig, ,Wuhan, China) at 4 °C for overnight. After washing with TBST, the membranes were incubated with anti-rabbit IgG secondary antibody (Bioworld, BS10650, Dublin, OH, USA), diluted in blocking buffer for 1 h at room temperature. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; proteintech, 60004-1-Ig, Wuhan, China) was used as the loading control. The relative protein expression levels were quantified by densitometry using the Gel-Pro 32 software (Media Cybernetics, Inc., Rockville, MD, USA).
Quantitative real-time PCR
Total RNA from cells was extracted using TRIzol reagent following the manufacturer’s protocol. RNA samples were then reversed transcribed with a PrimerScript RT reagent kit (Vazyme, Nanjing, China). RT-PCR was performed on an Applied Biosystems 7500 RT-PCR system using Power SYBR Green PCR Master Mix (Vazyme, Nanjing, China).. The sequences of the primers for PCR amplification were the following: FOXM1-sense, 5′-TCCTCCACCCCGAGCAA-3′ and FOXM1-antisense, 5′-CGTGA GCCTCCAGG
ATTCAG-3′; KIF20A-sense, 5′- TGCTGTCCGATGACGATGTC-3′ and KIF20A-antisense, 5′-AGGTTCTTGCGTACCACAGAC-3′. All the experiments were at least performed in triplicate.
Mouse PCa xenograft model for in vivo study
All animal experiments were approved by the Hospital of Nanjing BenQ Medical Center Animal Care and Use Committee. Animal experiments were carried out following the guidelines in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Briefly, DU145-DR cells (5 × 106/100 μL) were subcutaneously injected into two flanks of 6-week old nude mice (BALB/C-nu/nu, SLAC Laboratory, Shanghai, China). When the size of the xenografts in mice reached 100 mm3, the mice were treated with docetaxel (10 mg/kg ) by i.p. injection twice a week or thiostrepton (TST,30mg/kg) by i.p. injection qod. The size and body weight of tumor xenografts were measured every 4 days, and the tumor volume was calculated using the following formula: tumor volume (mm3) = π/6 × (length) × (width)2. After one month, the mice were euthanized, and the tumors were weighed and prepared for subsequent experiments.
Luciferase reporter assay
VCaP-DR cells were co-transfected with the human KIF20A luciferase reporter (WT or MUT), siFOXM1 RNA and pRL-SV40 (Promega) using the TransIT-X2 Dynamic Delivery System (Mirus, Madison, WI, USA). The firefly/Renilla luciferase activities were detected by the Dual-Glo Luciferase reporter assay system (Promega) according to the manufacturer’s manual 48 h after transfection. Luminescence was then measured using spectraMax id3(Molecular Devices, MD, USA)
Chromatin immunoprecipitation (ChIP)-PCR
The treated VCaP-DR cells were cross-linked in 1% formaldehyde and lysed. The DNA fragment was sonicated to shear a mean DNA fragment. The chromatin–protein complex was incubated with FOXM1 antibodies overnight at 4°C, with IgG as the negative control. Some lysate was used for input control. The immune complexes were harvested with beads, and then de-crosslinked and the chromatin DNA fragments purified. The degree of FOXM binding to chromatin DNA was analyzed by quantitative RT-PCR. The primers used are: 5′-TTCCTTACGCGGATTGGTAG-3′ (KIF20A sense) and 5′- AGCCGCAGAG CACAACTC-3′ (KIF20A anti-sense); 5′-CCGCCTCCCTCT
TAGCATAA-3′ (control sense) and 5′-CAGGAAATTGCATCTCGGGG-3′(control anti-sense).
Statistical analyses
The results are presented as the mean ± standard deviation (S.D.). All statistical analyses were performed using GraphPad Prism 5.2 software. Differences in each group were analyzed by one-way analysis of variance (ANOVA). When the comparison involved only two groups, Student’s t-test was used. *P < 0.05 was considered statistically significant.