br Gene knockdown RNA interference and plasmid transfection
2.2. Gene knockdown, RNA interference and plasmid transfection
Short hairpin RNA (shRNA) interference vector pLKO.1-GFP contain-ing an U6 promoter upstream of the shRNA, lentivirus packaging vector pVSVG-I and pCMV-GAG-POL were obtained from Shanghai Integrated Biotech Solutions Co, Ltd., (Shanghai, China). Green fluorescent protein (GFP) was used as an internal control with an independent promoter. The C4–2B or DU145 cell line was cultured in 6-well plates, inoculated at a density of 5 × 104 cells/mL, and transfected with the shRNAs-expressing lentivirus (Lv-shCDC20 or Lv-sh-β-catenin) or control lenti-virus at a multiplicity of infection (MOI) of 45. After 72 h transfection, they were observed and photographed under microscope. The se-quences for shRNA were shown in Supplementary Table S2.
The siRNA (si-CDC20) were purchased from Shanghai Integrated Biotech Solutions Co, Ltd., (Shanghai, China), and siRNA transfection was carried out using RNA iMAX reagents (L3000015, Invitrogen, Shanghai, China) according to the manufacturer's protocols. The se-quences for siRNA were presented in Supplementary Table S2. Transfection of CDC20 pcDNA 3.1-WT or -MT plasmid was carried out using Lipofectamine 3000 reagents (L3000015, Invitrogen) accord-ing to the manufacturer's protocols, the sequence of CDC20 plasmids were shown in Supplementary Table S2.
2.3. Real-time polymerase chain reaction (PCR)
Total RNA was prepared from cultured CHIR-265 from C4–2B or DU145 using RNAiso Plus (9109, Takara, Japan) according to the manufacturer's protocol. Reverse transcriptase PCR (RT-PCR) was performed using PrimeScript One Step RT reagent Kit (RR037B, Takara, Japan) in the presence of random primers. Amplification of the generated cDNA was carried out in SYBR Green Realtime PCR Master Mix (QPK201, Toyobo, Japan) with ABI PRISM 7300HT Sequence Detection System. Each
measurement was performed in triplicate and the results were normal-ized by the expression of the β-actin gene. Fold change relative to mean value was determined by 2–△△Ct. The primer sequences were presented in Supplementary Table S2.
2.4. Animal experiments
All experimental animal procedures were approved by the Animal Care and Use Committee of the Second Military Medical University (Shanghai, China). Nude mice (male, 6-weeks old) were purchased from the Shanghai Laboratory Animal Centre (SLAC, China) and housed under specific pathogen-free (SPF) conditions.
For the in vivo limiting dilution assay, the sorted C4–2B CD44+ and DU145 CD44+ cells with or without Lv-shCDC20 stable transfection were serially diluted to the appropriate cell dose. Cells were injected and the number of tumours formed from each cell dose injected was scored. The frequency of cancer stem cells had been calculated using the ELDA software provided by the Walter and Eliza Hall Institute (http://bioinf.wehi.edu.au/software/elda/index.html).
2.5. Statistical analysis
Numerical data were expressed as the Mean ± S.D. Statistical differ-ences between variables were analysed by chi-square test or Fisher's exact test for categorical/binary measures and ANOVA for continuous measures. Survival curve was plotted by the Kaplan-Meier method and compared using the log-rank analysis. Difference was considered significant at P b .05. All experiments for cell cultures were performed independently at least three times and in triplicate each time. Data anal-ysis was performed by the GraphPad Prism 5 and SPSS 22.0.
Supplementary information of materials and methods are described in Supplementary materials and methods.
3.1. Up-regulated CDC20 is associated with poor prognosis, and combina-tion of CDC20 and CD44 expression is a significant prognostic factor in pros-tate cancer patients
Previous study has indicated that CDC20 is up-regulated in prostate cancer tissues , and higher Gleason score tissues exert higher CDC20 expression level which was shown in our recent study . To confirm the fundamental role of CDC20 in prostate cancer progression, firstly, we detected the CDC20 expression level in different stages of prostate cancer development. Samples from locally advanced prostate cancer (LAPC), docetaxel treated prostate cancer exhibited higher CDC20 ex-pression than localised prostate cancers (Fig. 1a, b), Similarly, analysis of various types of prostate cancer cell lines demonstrated relatively higher expression of CDC20 in the more aggressive cell lines such as C4–2B and DU145 (Supplementary Fig. S1a). According to the expres-sion level of CDC20, all 121 prostate cancer patients were divided into CDC20low and CDC20high groups, as shown in Supplementary Table S1, the patients with high expression of CDC20 showed more aggressive features in superior Gleason score, higher T-stage and positive surgical margin, they presented much worse biochemical recurrence (BCR) (P b .001) and disease-free survival (DFS) (P = .0078) through Kaplan-Meier analysis (Fig. 1c, d), which further indicated that CDC20 may be used as a predictor for poor survival of prostate cancer patients.