Ethics statement
Based on the guidelines of the National Science Council of China, all study animals were maintained in an aseptic condition with a laminar airflow cabinet. All experimental procedures conformed to the guidelines of the Beijing Medical Experimental Animal Care Commission. The present study was approved by the Laboratory Animal Ethics Committee of the Air Force Medical University.
Cell culture
PC3 (androgen-independent, ATCC Cat# CRL-1435, RRID: CVCL_0035), LNCaP (androgen-sensitive, ATCC Cat# CRL-1740, RRID: CVCL_1379), LNCaP AI+F (androgen-independent, gift from Zhihua Tao’s Lab)(16), and normal prostate stroma myofibroblast cell line WYMP-1 (ATCC Cat# CRL-2854, RRID:CVCL_3814) were used in this study. Cells were grown in RPMI-1640 medium (Gibco), supplemented with 10% fetal bovine serum (Gibco), or Charcoal-stripped fetal bovine serum (CS-FBS) (GeneTex, Inc. San Antonio, Texas), 100 IU penicillin and 0.1 mg/ml streptomycin (Sigma) at 37 °C in a humidified environment with 5% CO2.
Tissue microarray
The tissue microarrays were obtained from CRBRDI (Shaanxi Chaoying Biotec Co. LTD). The PCa microarray included 11 normal prostate samples (13.8%), 8 clinical-stage I samples (10%), 25 stage II samples (31.3%), 15 stage III samples (18.8%), and 21 hyperplasia samples (26.3%).
Plasmid Generation
A 2.0 kb sequence from the human TFDP3 promoter was generated by PCR using genomic DNA as a template that was extracted from the PC3 cell line. Primer sequences containing the NheI and HindIII restriction enzyme sites were used in the PCR (primer sequences are listed in Supplementary Table 1). Following enzymatic digestion with the restriction enzymes (NEB, New England Biolabs), the amplicon was sub-cloned into the pGL3-basic vector (Promega). In addition, we generated the promoter-CREB-mut construct, which contained the mutant type of the TFDP3 promoter. Sequencing was performed to verify all constructs before transfection. The pcDNA3.1-CREB (Invitrogen) was then generated in our lab. The primers for CREB used for PCR are listed in Table 1. A total of 6 different sets of siRNA sequences (home-317, home-434, home-719, home-577, home-725, home-1038) for CREB and TFDP3 were purchased from Shanghai GenePharma LTD. The sequences for CREB siRNAs are listed in table 1. pcDNA3.1-TFDP3 was generated in our laboratory as previously described (2).
Luciferase assays
A total of 1×106 cells (PC3, LNCaP, or WYMP-1) were seeded in 6 well plates and grown overnight before treatment. Co-transfection of expression plasmids with the TFDP3 core promoter was performed using Lipofectamine 3000 (Invitrogen) reagent according to the manufacturer’s protocol. For promoter activity assessment, each well was transfected with 900 ng of the reporter constructs and 100 ng of a pRL-CMV plasmid vector encoding Renilla luciferase (Promega). Renilla luciferase served as the internal control for transfection efficiency. Following 48 h of incubation, the cells were harvested and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocol. Each experiment was repeated at least 3 times, and each sample was tested in triplicate.
Chromatin immunoprecipitation assay
ChIP assays were performed using the ChIP kit (Millipore) according to the manufacturer’s protocol. A total of 4 to 6×106 PC3 cells were washed and protein–DNA complexes were cross-linked in 1% formaldehyde in PBS for 10 min at 37 °C. Subsequently, the cells were collected and lysed in SDS lysis buffer (1% SDS, 10mM EDTA, 50 mM Tris-HCl, pH 8.1, and freshly added protease/phosphatase inhibitors). The chromatin was sonicated to shear the DNA to an average length of 300 to 700 bp using the Branson Low Power Ultrasonic Systems 2000 LPt/LPe sonicator. The DNA fragments were subsequently immunoprecipitated with 2μg antibody against CREB (Cell Signal Technology) or IgG. The precipitated DNA was extracted and amplified by PCR. The primer pairs were designed to bind to the CREB element in the TFDP3 promoter region. Primer sequences used in PCR are shown in Table 1. The soluble chromatin before immunoprecipitation was used as the input control. The PCR products were electrophoresed on a 3% agarose gel.
Western blotting analysis
Briefly, WPMY-1, PC3, and LNCap cells were lysed in RIPA lysis buffer (Beyotime) and total protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Thermo). A total of 30 µg of protein was separated using a 10% SDS–PAGE, and then transferred onto PVDF membranes (Invitrogen). After incubation in blocking buffer (Invitrogen) for 2 h at room temperature, the membranes were incubated with antibodies for CREB (1:800, Abcam Cat# ab32515, RRID:AB_2292301), TFDP3(1:500, Abcam Cat# ab57342, RRID:AB_2202697) or β-actin(1:1000, A2228, Sigma) followed by incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signal Technology). The catalog numbers of the primary antibodies are shown in Supplementary Table 2. Specific protein bands were detected using the Bio-Rad Image Lab system. Each band density was measured using the Image J software (java, National Institutes of Health: NIH) and normalized using the internal controls.
Immunohistochemistry (IHC) staining of tissue microarrays
Tissue slides were washed 2 times for 5 min in PBS and then blocked for endogenous peroxidases using 3% H2O2-methanol for 10 min at room temperature. Subsequently, primary antibodies against TFDP3 (1:50, Abcam Cat# ab57342, RRID: AB_2202697) and/or CREB (1:200, Abcam Cat# ab32515, RRID: AB_2292301) were incubated at 4 °C overnight. After washing, the membranes were incubated with anti-mouse/rabbit-HRP secondary antibody at room temperature for 30 min. Finally, DAB was used to visualize protein expression levels. Immunoreactivity scores (IRS) were calculated using the 0–3+ scale, where 0 indicated no staining; 0–1+ indicated trace staining that was weaker than 1+ and more intense than 0; 1+; 2+, and 3+ indicated increased intensities of staining. IRS was scored independently by two pathologists who were blinded to the clinical data. The subregions, excluding necrosis, macrophages, and infiltrating neutrophils and lymphocytes were selected and scored. The intensity score for an array spot was the average of all sub-regions.
Immuno-cytofluorescence (ICF) staining of PCa cells
For ICF experiments, cell-coated slides were washed with 0.01 M PBS and fixed with 4% cold paraformaldehyde in phosphate buffer (pH 7.4). The slides were blocked with 0.01 M PBS containing 3% BSA and 0.3% Triton X-100 for 30 min and then incubated with rabbit anti-CREB (1:800, Abcam Cat# ab32515, RRID: AB_2292301) and mouse anti-TFDP3 (1:500, Abcam Cat# ab57342, RRID: AB_2202697) at 4°C overnight. Following three washes with 0.01 M PBS, the slides were incubated with the corresponding secondary antibodies conjugated with FITC (1:100, BBI Life Sciences) and/or Cy3 (1:400, BBI Life Sciences) for 1 h at room temperature. Nuclei were counterstained using DAPI (1:500, Beyotime). Sections were examined and photographed using a confocal microscope (FV1000, Olympus).
Apoptosis assay
PC3 cells (6×105 cells/well) were stained with Annexin V-FITC using the Annexin V FITC Apoptosis Detection Kit (Millipore). The cells were then washed twice in PBS and resuspended in binding buffer. 50 µl of the cell suspension was then stained with 10 µl of Annexin V-FITC and gently vortexed and incubated in the dark for 15 min at room temperature. Finally, 5 µl of Propidium Iodide (PI) was added, and the suspension was incubated in the dark for an additional 5 min at room temperature. Following the addition of 400 ul of binding buffer to each tube, cells were analyzed by flow cytometry (CantoII, BD).
TUNEL staining was performed according to the Dead END™TUNEL system instruction manual (Promega, Catalog number selected: G3250) to determine the percentage of cellular apoptosis. Briefly, PC3 cells were fixed to a microscope slide by immersing in 4% methanol-free formaldehyde in PBS for 25 minutes at 4°C. Then, cell sections were permeabilized with 0.2% Triton® X-100 (Sigma) for 5 min and rinsed 2 times with PBS. The cells were then covered with 100µl of Equilibration Buffer and incubated at room temperature for 5–10 min. The Equilibration Buffer was then removed and 50µl of rTdT incubation buffer was added to the cells covering a 5cm2 area. The slides were then covered with aluminum foil and incubated at 37°C for 60 min inside a humidified chamber. The reaction was terminated by immersing the slides in 2X SSC for 15 min at room temperature. The slides were then stained by immersing in 40ml of propidium iodide solution diluted to 1µg/ml in PBS for 15 minutes at room temperature in the dark. Samples were immediately analyzed under a fluorescence microscope for green fluorescence of fluorescein at 520 ± 20nm, red fluorescence of propidium iodide at >620nm, and blue DAPI at 460nm. Nuclei were counterstained with Hoechst33342 (1:5000, Sigma).
Cell Proliferation Assay
Cell proliferation was measured using the Cell Counting Kit-8 (CCK-8) (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s protocol. Briefly, the cells were seeded in 96-well plates (2×103 cells /well) in triplicate and incubated for 12 h. The cells were then transfected with pcDNA3.1-CREB and/or siRNA-CREB for 24, 48, and 72 h, followed by the addition of 10 μl of CCK-8 solution to each well. Following incubation at 37 °C for 1.5 h, the absorbance of each sample at 450 nm was measured using a microplate reader (Tecan, Austria).
Cell cycle analysis
The effect of CREB and TFDP3 on LNCaP and LNCaPAI+F on cell cycle progression were determined on a fluorescence-activated cell sorter (FACS) using the Cell Cycle Assay kit (Fluorometric-Green, ab112116) according to the manufacturer's instructions. A total of 1×106 cells were collected in flow tubes, washed with PBS, and then stained with Nuclear Green CCS1. After cells were incubated in the dark for 15 minutes, ten thousand cells were analyzed by flow cytometry (CantoII, BD). Cells stained with Nuclear Green CCS1 were monitored using a flow cytometer set at an Ex/Em of 490 nm/520 nm. All experiments were performed in triplicate.
Construction of CREB1/TFDP3 knockout cell lines using CRISPR / cas9
sgRNAs targeting CREB1 and TFDP3 were designed using CRISPR DESIGN (http://crispr.mit.edu/). The transfer plasmid lentiCRISPR-CREB and lentiCRISPR-TFDP3 were obtained from TsingKe Biotechnology Co., Ltd. Briefly, three targets for CREB1 and TFDP3 were designed. The target sequences were as follows: Target1: GGGCAGACAGTTCAAGTCCA, Target2: GGGCTTGAACTGTCATTTGT and Target3: GGAGCCGAGAACCAGCAGAG for CREB1; Target1: GCCGGGCAGCACAACAGGAA, Target2: GCCGTCTTTCCATGAAGGTC and Target3: GGAGGTGTGTTCACGACGGC for TFDP3. The corresponding lentiCRISPR vectors were constructed to synthesize oligos for lentiCRISPR-CREB-T1, lentiCRISPR-CREB-T2, lentiCRISPR-CREB-T3 and lentiCRISPR-TFDP3-T1, lentiCRISPR-TFDP3-T2, lentiCRISPR-TFDP3-T3, respectively. CRISPR / cas9 activity was measured using Luciferase SSA. The strongest fluorescence vectors were selected for Lentivirus packaging. The specific target sequences were amplified and cloned into a Lentivirus plasmid packaging system. DNA sequence analysis was used to verify the sequences.
Animal experiments
Six-week-old female BALB / C nude mice (from Vital River, Beijing, China) were randomly divided into three groups according to weight (n=10 per group). PC3, PC3 with lentiCRISPR-CREB1, or PC3 with lentiCRISPR-TFDP3 cell lines (1×107) were suspended in 0.1 ml of PBS and injected subcutaneously into the right abdominal flank of mice using 26G needles. The tumor volume was observed daily. The tumor volume was calculated using the following formula: volume(mm3)=(width)2(mm2)×length(mm)/2. Relative tumor volume (RTV): RTV= Vt / V0, where V0 is the starting tumor volume and Vt is the tumor volume at each measurement. On day 19, tumors were harvested from the right abdominal flank of the mouse, weighed, photographed, fixed in formalin, and embedded in paraffin for sectioning.
Statistical analyses
Statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software Inc., La Jolla, CA, USA) and IBM SPSS software 21.0. Statistical differences between groups were determined using one-way ANOVA and unpaired t-tests. Correlation was analyzed using the Spearman's rank test. Results were presented as mean (central values) ± SD (error bars). P values of less than 0.05 were considered significant. Three levels of significance (*P<0.05; **P<0.01; ***P<0.001) were applied for all tests.