Cell lines and cell culture
RWPE-1, PC-3, DU145, C4-2B, VCaP and LNCaP cells were obtained from the ATCC (Manassas, VA, USA). RWPE-1 cells were cultured in defined keratinocyte-SFM (1×) (Invitrogen, Carlsbad, CA, USA). PC-3, C4-2B and LNCaP cells were cultured in RPMI 1640 medium (Invitrogen) supplemented with10% FBS (Invitrogen), while DU145 and VCaP cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with10% FBS.
Patient information and tissue specimens
A total of 116 paraffin-embedded and archived PCa samples were collected for this study, which had been diagnosed histopathologically. Clinical information on the samples is summarized in detail in Additional file 1: Table S1. The fresh tissues including eight paired PCa tissues and adjacent non-tumor tissues were obtained from individuals who were diagnosed with PCa. All samples were collected from the First Affiliated Hospital of Sun Yat-sen University. Prior patient's consents were obtained to use these clinical specimens for research purposes. Our study was approved by the Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University according to the 1975 Declaration of Helsinki.
Plasmids, virus constructs and retroviral infection of target cells
A human PHF21B cDNA clone (EX-T2701-Lv105), as well as short hairpin RNA (shRNA) expression clone (HSH001525-CU6), was purchased from GeneCopoeia (Guangzhou, China). SMARTpool siRNA against human SFRP1, SFRP2, and β-catenin was purchased from RiboBio (Guangzhou, China). The reporter plasmids containing wild-type (CCTTTGATC; TOP flash) or mutated (CCTTTGGCC; FOP flash) TCF/LEF DNA binding sites were purchased from Upstate Biotechnology (New York, USA). Transfection of plasmids or siRNA was performed using the Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction. Stable cell lines expressing PHF21B and PHF21B-shRNA were generated by retroviral infection and selected with 0.5 μg/ml puromycin for 10 days.
RNA extraction, reverse transcription (RT) and real-time PCR
Total RNA was extracted from cultured cells and PCa tissues using the Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. A total of 2 μg of RNA was reverse transcribed to cDNA with M-MLV Reverse Transcriptase (Promega, Madison, US). qRT-PCR analysis was performed on an ABI Prism 7500 Sequence Detection System (Applied Biosystems, Texas, US). Expression data were normalized to the housekeeping gene GAPDH. Relative expression levels were calculated as 2-[(Ct of gene) - (Ct of GAPDH)], in which Ct represents the threshold cycle for each transcript. The PCR primer sequences are listed in Additional file 2: Table S2.
Western blotting was performed as described previously , using anti-PHF21B (ab70435, 1:1000; Abcam, Cambridge, UK), anti-β-catenin (#9562, 1:1000; Cell Signaling, Danvers, MA, USA), anti-phospho-β-Catenin (Ser33/37/Thr41) (#9561, 1:1000; Cell Signaling), and anti-p84 (ab102684, 1:500; Abcam) antibodies. Blotting membranes were stripped and re-probed with anti-α-tubulin antibody (T6199, 1:2000; Sigma-Aldrich, St. Louis, MO, USA) as a loading control. Nuclear extracts were prepared using the Nuclear Extraction kit (Active Motif), according to the manufacturer's instructions.
Immunohistochemical (IHC) analysis was conducted to study protein expression in clinical specimens. The procedure was carried out similarly to previously described methods . After deparaffinization, the sections were immunohistochemically stained using anti-PHF21B (HPA053834, 1:2500; Sigma-Aldrich), and anti-β-catenin antibody (#9562, 1:200; Cell Signaling). The degree of immunostaining offormalin-fixed, paraffin-embedded sections was examined and scored independently by two pathologists. The scores were determined by combining the proportion of positively stained tumor cells and the intensity of the IHC signals. Tumor cell proportions were scored as follows: 0, no positive tumor cells; 1, 1%-25% positive tumor cells; 2, 25%-50% positive cells; 3, 50%-75% positive tumor cells; and 4, >75% positive tumor cells. Staining intensity was graded according to the following criteria: 0, no staining; 1, weak staining (light yellow); 2, moderate staining (yellow brown); and 3, strong staining (brown). The staining index (SI) was calculated as the staining intensity score × the proportion of positive tumor cells (ranging from 0 to 12). We evaluated protein expression by determining the SI. Samples with an SI ≥ 6 were classified as showing high expression, while samples with an SI < 6 were classified as showing low expression.
Enzyme-linked immunosorbent assay (ELISA)
Using a commercially available SFRP1 (E95880Hu) and SFRP2 (E95879Hu) ELISA Kit (USCN, Wuhan, China), the concentration of SFRP1 and SFRP2 in the conditioned media (CM) derived from the cells was determined. ELISA was performed according to the directions of the manufacturer.
Sphere formation assay
Cells (500/well) were seeded into 6-well ultra-low cluster plates (Corning, NY) and cultured in DMEM/F12 serum-free medium (Invitrogen) supplemented with 2% B27 (Invitrogen), 20 ng/ml EGF (PeproTech, Rocky Hill, NJ, USA), 20 ng/ml bFGF (PeproTech), 0.4% BSA (Sigma-Aldrich), and 5 μg/ml insulin (Sigma-Aldrich). After 10 days, the number of spheres was counted, and images of the spheres were photographed.
Luciferase activity assay
Thirty thousand cells were cultured in triplicate in 48-well plates for 24 h. Then, 100 ng of luciferase reporter plasmids containing the SFRP1 and SFRP2 promoter, TOP-Flash or FOP-Flash luciferase reporter, plus 3 ng of pRL-TK Renilla plasmid (Promega), were transfected into cells using the Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s recommendations. Luciferase and Renilla signals were measured 36 h after transfection using the Dual Luciferase Reporter Assay Kit (Promega) according to the manufacturer’s protocol.
Specific primers used for promoter luciferase reporter were presented in Additional file 3: Table S3.
The promoter sequences of SFRP1/2 were obtained from the UCSC Genome Browser (http://genome.ucsc.edu) referring the enrichment of H3K27Ac mark (often found near active regulatory elements) on seven cell lines as determined by ChIP-seq assays provided by the website. Each of the cell lines including GM12878, H1-hESC, HSMM, HUVEC, K562, NHEK, NHLF in this track is consistent across the ENCODE (Encyclopedia of DNA Elements) Regulation super-track.
Chromatin immunoprecipitation (ChIP)
The SimpleChIP Enzymatic Chromatin IP Kit with magnetic beads (#9003, Cell Signaling, Danvers, MA, USA) was used according to the manufacturer's protocol. Cells (4 × 106) transfected with Flag-tagged PHF21B (EX-T2701-Lv102; GeneCopoeia, Guangzhou, China) in a 100 mm culture dish were treated with formaldehyde to cross-link proteins to DNA. 1× Glycine was added to terminate cross-linking. A total of 5 μg of anti-FLAG antibody (SAB4200071; Sigma, St Louis, MO, USA),) or anti-IgG antibody was incubated with 10 μg of sheared chromatin overnight at 4 °C. ChIP-grade protein G magnetic beads were added and incubated for 2 h at 4 °C with rotation. Immunoprecipitated chromatin was then washed with low- and high-salt ChIP buffer. After reverse cross-linking of protein/DNA complexes to free DNA, PCR was performed. Primers used for ChIP are presented in Additional file 4: Table S4–S5.
Xenograft tumor model
All experimental procedures were approved by the Institutional Animal Care and Use Committee of Sun Yat-Sen University. Male BALB/c-nude mice (4–6 weeks old, 18–20 g) were purchased from the Slac-Jingda Animal Laboratory (Hunan, China) and housed in barrier facilities on a 12-h light/dark cycle. Mice were randomly allocated into groups (n = 6 per group). The indicated cells at three doses (1 × 106, 1 × 105, and 1 × 104) were inoculated subcutaneously with Matrigel (final concentration of 25%) into the inguinal folds of the mice. Tumor volume was determined using an external caliper and calculated using the equation (L × W2)/2. After mice were sacrificed, the tumors were excised, weighed and subjected to pathologic examination. The frequency of tumor-initiating cells (TICs) was calculated using the Extreme Limiting Dilution Analysis (ELDA) program (http://bioinf.wehi.edu.au/software/elda/) .
All statistical analyses were carried out using the SPSS 17.0 statistical software package (SPSS Inc, Chicago, IL, USA). The relationship between PHF21B expression and the clinicopathological characteristics was tested by the χ
2 test. Bivariate correlations between study variables were calculated using Spearman's rank correlation coefficients. Survival curves were plotted with the Kaplan-Meier method and compared by the log-rank test. Other comparisons were analyzed by unpaired two-sided independent Student’s t-test without equal variance assumption. P < 0.05 was considered statistically significant.