Cell lines and cultures
Prostate cancer cell lines DU145 and VCaP were obtained from the Chinese Academy of Sciences, Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). DU145 cells were kept at 37 ˚C with 5% CO2 in RPMI 1640 media. VCaP cells were kept at 37 ˚C with 5% CO2 in DMEM medium. Media contained 10% fetal bovine serum and 1% streptomycin/penicillin.
Docetaxel-resistant cell lines DU145-DR and VCaP-DR were established as described previously [18]. Briefly, resistant PCa cells (DU145-DR and VCaP-DR) were generated by continuously exposing cells to increasing concentrations of docetaxel: 2 nM to 100 nM (DU145-DR) or 2 nM to 60 nM (VCaP-DR), over a 10-month period.
Transfection
Sequences corresponding to FOXM1 and KIF20A siRNAs were: 5′-CUCUUCUCCCUCAGAUAUATT-3′ (FOXM1 siRNAs; sense sequence), 5′-UAUAUGAGGGAGAGTT-3′ (FOXM1 siRNAs; antisense sequence), 5′-GCAGCAGGUUCCAUCUGAGTT-3′ (siRNA KIF20A; sense), 5′-CUCAGAUGGAACCUGCUGCTT-3′ (siRNA KIF20A; antisense). The non‐targeting siRNA control sequence was 5′-UUCUCCGAACGUGUCACGUTT-3′. siRNAs were transfected using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). pcDNA3.1/FOXM1 (pcFOXM1) and pc DNA3.1/KIF20A(pcKIF20A) plasmids were constructed using 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 a control (Ctrl). Transfection efficiency was evaluated after 48 h.
Stable transfection
Human lentivirus-shFOXM1 was obtained from GenePharma (Shanghai, China). Lentiviruses were ultracentrifuged, concentrated, validated, and added to the culture medium. After infection, transduced cells were selected using puromycin (Gibco, Grand Island, NY, USA) over 2–3 weeks, and the surviving cells were continuously cultured. The resulting cell was referred to as DU145-DR stably transfected siFOXM1.
Cell apoptosis analysis
Cells in an exponential growth phase were harvested and then seeded in six-well plates. Once cells became adherent, different treatments were undertaken. After cells had been cultured for 72 hr, an Annexin V-FITC/PI Apoptosis Assay Kit was used to assess the percentage of apoptotic cells using BD FACS Calibur flow cytometry (BD Biosciences). The experiments were performed in triplicate.
Cell cycle analysis
After treatment, cells were harvested and fixed for at least 4 hr using 70% precooled ethanol. Cells were then 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 status was analyzed using BD FACS Calibur flow cytometry (BD Biosciences, San Jose, CA), within 1 h.
Cell viability assay and colony formation assay
Cells were seeded into a 96-well plate at 5×103 cells/well and then treated. 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 added to halt the reaction. Cell viability was measured spectrophotometerically using absorbance at 590 nm. For the colony formation assay, 500 cells were added to 6-well plates and cultured at 37 °C for 14 days. Colonies were staining with 0.1% crystal violet in methanol for 30 min. The number of colonies per well was then counted and analyzed.
Wound-healing assay
Cells in the exponential growth phase were added to 6-well culture plates. After reaching confluence, the cell layer was scratched with a sterile P-200 pipette tip. The cells were then treated with docetaxel for 24 h. Photographs were taken (100× magnification), and cell migration distances measured.
Cell invasion
Cell invasion assays were undertaken using a modified transwell chamber with a Matrigel-coated membrane (BD Biosciences Bedford, MA), as described previously. Briefly, approximately 1 x 104 cells were re-suspended in serum-free medium, and then plated onto 24-well plates. Re-suspended cells were treated with docetaxel, FOXM1-siRNA, pc-FOXM1, KIF20A-siRNA, and pc-KIF20A. At 24 h post-treatment, cell invasion was measured, following manufacturer’s instructions.
Quantitative real-time PCR (qRT-PCR)
RNA was extracted from cells using a TRIzol reagent, following manufacturer’s instructions. RNA samples were then reversed transcribed using a PrimerScript RT reagent kit (Vazyme, Nanjing, China). RT-PCR was performed with an Applied Biosystems 7500 RT-PCR system using a Power SYBR Green PCR Master Mix (Vazyme, Nanjing, China). Primer sequences used for the PCR amplification were:
5′-TCCTCCACCCCGAGCAA-3′ (FOXM1-sense);
5′-CGTGAGCCTCCAGGATTCAG-3′ (FOXM1-antisense);
5′-TGCTGTCCGATGACGATGTC-3′ (KIF20A-sense);
5′-AGGTTCTTGCGTACCACAGAC-3′ (KIF20A-antisense).
All experiments were performed at least three times.
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 undertaken following the Guide for the Care and Use of Laboratory Animals, as published by the National Institutes of Health. Briefly, DU145-DR cells (5 × 106/100 μL) were subcutaneously injected into the two flanks of 6-week old nude mice (BALB/C-nu/nu, SLAC Laboratory, Shanghai, China). When the dimensions of a mouse xenograft reached 100 mm3, the subject was administered docetaxel (10 mg/kg) by i.p. injection twice a week or thiostrepton (TST, 30mg/kg) by i.p. injection every-other-day. The size of tumor xenografts and the corresponding mouse body mass were measured every 4 days. The nominal tumor volume was calculated thus: tumor volume (mm3) = π/6 × (length) × (width)2. After one month, mice were euthanized. Tumors were removed by dissection, weighed, and prepared for subsequent experiments.
Luciferase reporter assay
VCaP-DR cells were co-transfected with 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 48h after transfection using the Dual-Glo Luciferase reporter assay system (Promega), following manufacturer’s instructions. Luminescence was then measured using a spectraMax id3 three-mode microplate reader (Molecular Devices, MD, USA)
Chromatin immunoprecipitation (ChIP)-PCR
Treated VCaP-DR cells were cross-linked using 1% formaldehyde and then lysed. The DNA fragment was sonicated to shear a mean DNA fragment. Potential chromatin–protein complexes were isolated by shearing into short, mean-lengthed DNA fragments using sonication. The resulting complexes were incubated with FOXM1 antibodies overnight at 4°C, with IgG as a negative control. Some lysate was used for input control. Immune complexes were harvested using beads, before crosslinks were broken and DNA fragments purified. FOXM binding to chromatin DNA was quantified using qRT-PCR. The primers used were: 5′-TTCCTTACGCGGATTGGTAG-3′ (KIF20A sense); 5′-AGCCGCAGAGCACAA
CTC-3′ (KIF20A anti-sense); 5′-CCGCCTCCCTCTTAGCATAA-3′ (control sense); and 5′-CAGGAAATTGCATCTCGGGG-3′ (control anti-sense).
Statistical analyses
Results are presented as mean ± standard deviation (S.D.). All statistical analyses were undertaken using GraphPad Prism 5.2 software (GraphPad Software, San Diego, USA). Differences between groups were analyzed using one-way analysis of variance (ANOVA). When a comparison involved two groups only, the Student t-test was used. Survival was analyzed using Kaplan-Meier curves. A log-rank test was used to determine statistical significance. A threshold of *P < 0.05 was set for statistical significant.