All subjects signed a written informed consent form. All study procedures were approved by the Institutional Review Board of the Tri-Service General Hospital, National Defense Medical Center (TSGH-IRB-098-05-221 and TSGH-IRB-099-05-165).
The study cohort comprised 284 RCC 
and 267 benign renal disease (BRD) patients selected from an initial population of 3,494 patients who were treated at the Tri-Service General Hospital from 1988–2006 and in whom the diseases were diagnosed histologically by examining specimens obtained by excisional biopsy or therapeutic surgical resection (summarized in Figure S1
). Clinical and histological information of patients was obtained from their charts and pathological reports, which are summarized in , , and S3. Tumor staging was performed according to the American Joint Committee on Cancer tumor-node-metastasis staging system, and histological grading was performed according to the World Health Organization classification criteria. All formalin-fixed, paraffin-embedded specimens were resected, stained with H&E, and re-examined by CPY who was blinded to the original diagnosis and subsequent outcome. Representative specimens were spotted for tissue microarray (TMA) construction. All specimens and clinical information of patients were obtained from the Tissuebank of Tri-Service General Hospital, National Defense Medical Center.
Association of RCC risk factor and WNT signaling molecules.
Univariate and multivariate analyses of prognostic factors and RCC survival.
Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and Quantitative Real-time PCR of WNT Family Genes
Total RNA of kidney or RCC cells and tissues was isolated using TRIzol (Invitrogen). RNA samples were treated with RQ1 RNase-free DNase (Promega), according to the manufacturer’s instructions, to remove any genomic DNA contamination. Five micrograms of treated RNA samples were reverse transcribed using SuperScript III (Invitrogen). RT-PCR was performed using 20 µL of reaction mixture containing 2 µL of cDNA, 5 pmol of each primer, 2 U of recombinant Taq DNA polymerase (Invitrogen), 1× reaction buffer, and 200 pmol of dNTPs. Amplification was performed for 35 cycles under the following conditions: denaturation at 94°C for 1 min, annealing at 55°C for 30 sec, extension at 72°C for 1 min, and final elongation at 72°C for 7 min. PCR products were separated by electrophoresis on a 2% agarose gel and visualized by staining with ethidium bromide. SYBR Green quantitative real-time PCR was performed using StepOne Real-Time PCR System (Applied Biosystems) with Maxima Hot Start PCR Master Mix (2×) (Fermantus). Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control. In addition to the analysis of melting curve, real-time PCR products were analyzed by gel electrophoresis to confirm single PCR products. Primer sets uesd in RT-PCR and real-time PCR are listed in Table S1
Human RCC cell line A498 (derived from papillary RCC); ACHN, Caki-1, and 786-O (all derived from clear cell RCC); and immortalized proximal tubule epithelial cell line (HK-2) derived from normal adult human kidney were obtained from Bioresource Collection and Research Center (BCRC, Taiwan). RCC-1 cell line (derived from clear cell RCC) was obtained from Dr. DS Yu (Division of Urology, Tri-Service General Hospital); this cell line was derived from primary culture. Collection and maintenance of primary tissue/cell culture were approved by the Institutional Review Board of the Tri-Service General Hospital, National Defense Medical Center (TSGH-IRB-098-05-221) under the project “Establishment and Maintenance of the Tissue Bank of Tri-Service General Hospital.” RCC-1 and A498 were maintained in Dulbecco’s Modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 1 µg/mL penicillin, and 1 µg/mL streptomycin at 37°C in humidified 5% CO2 atmosphere. ACHN, Caki-1, and 786-O were maintained in RPMI-1640 medium containing 10% FBS, and HK-2 was maintained in keratinocyte serum-free medium containing 0.1 ng/mL recombinant epidermal growth factor and 20 µg/mL bovine pituitary extract (cell culture mediums and supplements were all obtained from Invitrogen) under the same antibiotic and culture conditions as that used for RCC-1 and A498.
Transfection of pcDNA3-WNT10A Plasmids or siRNAs Containing WNT10A and β-catenin
Cell lines were seeded in a 6-cm dish at density of 5×105 cells/dish and incubated overnight. HK-2, 786-O, and A498 were prepared for gain-of-function design, and RCC-1 and Caki-1 were prepared for loss-of-function design. Three micrograms of pcDNA3-WNT10A plasmid (constructed by laboratory processing) or 1 µg each of WNT10A siRNA (s37228 and s37230; Invitrogen) and β-catenin siRNA (sc29209 and sc44252; Santa Cruz) were added to Opti-MEM with Lipofectamine 2000 (Invitrogen) for transfection, according to the manufacturer’s instructions. After 12 h of incubation, the old medium was replaced with fresh DMEM containing 10% FBS. Cells were harvested at 48 h after transfection for the pcDNA-WNT10A group and at 72 h after transfection for the WNT10A and β-catenin siRNA groups. Harvested cells were subjected to RT-PCR, real-time PCR, western blot analysis, and immunocytochemistry.
Western Blot Analysis
Transfected cells were washed twice with 1×phosphate buffered saline (PBS) and lysed in 100 µL of 1× RIPA lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS); Millipore) containing 1× protease inhibitor (Roche, #04693116001). Thirty micrograms of protein from the supernatant was loaded onto a SDS polyacrylamide gel, and western blot analysis was performed to detect WNT10A (Abcam, #ab62051), β-catenin (Epitomics, #1247-1), and cyclin D1 (Cell signaling DCS-6, #2926) levels. Immunoreactive bands were developed using ECL system (Millipore) and were quantified using UVP BioSpectrum Imaging System.
Immunohistochemistry and Immunocytochemistry
Constructed tissue microarray sections containing 100 individual 2-mm diameter samples per array were subjected to immunohistochemical analysis. Four micrometer TMA sections were blocked with 10% goat serum for 1 h and incubated with WNT10A (Abcam, #ab62051), β-catenin (Epitomics, #1247-1), c-myc (Cell signaling, D84C12 #5605), and cyclin D1 (Cell signaling, DCS-6 #2926) antibodies (1
200 dilution for each) for 2 h at room temperature. Slides were washed 3 times with 1× TBST (10 mM Tris, pH 7.4, 150 mM NaCl, and 0.1% Tween-20) for 10 min, processed using Super Sensitive™ Polymer HRP Detection System/DAB (Biogenex, #QD410-YAXE), and counterstained using hematoxylin. For confirming WNT10A immunohistochemical profiles, WNT10A antibody (sc-69135, 1
50 dilution) was used on serial TMA sections, and antibody ablation was performed using a competitive peptide (sc-69135P). Results were interpreted by an experienced researcher (JYH) and a senior pathologist (CPY) who were blinded to the experimental and the associated clinical pathological data. Positive cell percentage and intensity were recorded to calculate histoscore (histoscore
positive cell percentage × intensity; intensity is divided into 4 ranks: negative, weak, moderate, and strong) 
. Membranous, cytoplasmic, or nuclear staining scores were separately recorded once for each specimen. For immunocytochemical analysis, approximately 2×104
cells were seeded on an 18×18-mm cover glass. After transfection, cells were resuspended, counted, and reseeded on a cover glass and incubated for 16 h. Next, the cells were washed twice at the indicated time with 1×PBS (200 mM NaCl, 3 mM KCl, 10 mM Na2
, and 1.5 mM KH2
, pH 7.4), fixed in acetone/methanol (1
1) at −20°C for 30 min, and permeabilized with 0.1% Triton X-100 in 1× PBS at room temperature for 10 min. The cells were then washed 3 times with 1× TBST and blocked in 10% goat serum for 1 h. After incubation with the previously mentioned antibodies (1
200 dilution for each) for 2 h at room temperature, the cells were washed 3 times with 1× TBST for 10 min, stained using the Super Sensitive Polymer HRP Detection System/DAB (Biogenex), and counterstained with hematoxylin. For dual immunostaining, DAB (brown) was used as the chromogen for cyclin D1 and AEC was used as the chromogen for β-catenin.
TCF/LEF Reporter Assay
We used TCF/LEF reporter assay with luciferase reporter plasmids (Super 8× TOPFlash, containing wild-type TCF binding sites, and Super 8× FOPflash, containing mutant TCF binding sites; Addgene, #clones M50 and M51) to detect the activity of WNT/β-catenin signal transduction. pGL4.71 Renilla luciferase vector (Promega) was cotransfected to normalize transfection efficiency. Lipofectamine 2000 (Invitrogen) was used for transfection, as described above. Luciferase activity was assayed at 48 h after transfection by using a Dual-Luciferase Reporter Assay System (Promega). All the experiments were performed in triplicate.
Cell Viability Assay
Cells transfected with pcDNA3-WNT10A plasmid, WNT10A siRNA, β-catenin siRNA, vector control, and scrambled siRNA control were washed twice with PBS at the indicated time and subjected to 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay for the detection of cell viability. In brief, 100 µL of 2 mg/mL MTT reagent was added to each well and incubated at 37°C for 3 h, and absorbance was read at 570 nm. A570 was recorded at 0, 24, 48, and 72 h. For each indicated time and condition, reading was taken in 6 repeats.
For determining the effects of WNT10A on apoptosis, pcDNA-WNT10A-, pcDNA3.1 vector control-, and β-catenin siRNA-transfected 786-O (after reaching >70% confluence) were treated with 2 µM epirubicin or 10 µM cisplatin in a fresh culture medium. WNT10A siRNA-, β-catenin siRNA-, or scrambled siRNA control-transfected Caki-1 were also administered the same treatment. Since epirubicin is a water-soluble compound, the untreated controls were treated with equal DMSO concentration of cisplatin group. After 48 h, the treated cells were trypsinized, washed with 1× PBS, and resuspended. A total of 1×106 cells were fixed with 100% EtOH for 10 min and then incubated with 1 mg/mL propidium iodide (PI) for 10 min at room temperature. Cells were filtered using 40-µm Cell Strainer (BD Falcon) and analyzed using a BD FACSCalibur (BD Biosciences) within 20 min of staining.
Cell Migration and Invasion Assay
For wound-healing experiments, cells were plated in 6-well plates and cultured to 90% confluence. Cells were scraped using a p200 tip (time 0), and suspended cells were washed before obtaining each image. Migration distance was measured from images (5 fields) taken at each indicated time point. Transwell assay of Caki-1 and 786-O was assessed using 8-µm inserts (BD Biosciences). A total of 1×104 cells were suspended in 100 µL serum-free RPMI-1640 media and loaded into upper wells; lower chambers were filled with 500 µL of complete medium (RPMI-1640 supplemented with 10% FBS). For invasion assay, each insert was coated with 1 mg/mL Matrigel and incubated at 37°C for 5 h. A total of 2×104 cells were suspended in 100 µL serum-free RPMI-1640 media and loaded into coated inserts; lower chambers were filled with the complete medium. Migration and invasion chambers were incubated in a humidified 5% CO2 incubator at 37°C for 48 h. Cells were then fixed with 500 µL of methanol for 15 min. The inner surface of the upper chambers was wiped using cotton swabs to remove non-migrated cells in the migration assay or scrapes of Matrigel in the invasion assay. The chambers were then washed with 500 µL PBS and stained with 500 µL hematoxylin for 1 min at room temperature. After washing again with 500 µL PBS, the transwell membranes were torn and kept in slides. Stained cells were counted using the Image J software, and 5 random fields were counted at 100× magnification.
Colony Formation Assay
Transfected 786-O and Caki-1 were used in the colony formation assay. 786-O were transfected with pcDNA-WNT10A, vector control, or cotransfected with β-catenin siRNA or scrambled siRNA control; Caki-1 were transfected with WNT10A siRNA or scrambled siRNA control. In brief, 2 mL of 0.5% agarose in complete RPMI-1640 was used as the bottom agar in a 6-cm dish. A total of 2×104 cells were mixed with 0.3% agarose in complete RPMI-1640 after 48 h of transfection. Cells were maintained in a humidified 5% CO2 incubator at 37°C for 15 days, with the old medium being replaced by a fresh medium every 3 days. For prolonging the ectopic effects of WNT10A and WNT10A siRNA, pcDNA-WNT10A-transfected 786-O were treated with 200 µg/mL G418 (Invitrogen); moreover, cells were continuously treated with 10 nM of indicated siRNA in a soft agar medium. On the 15th day, the cells were stained with crystal violet for 1 min and destained with tap water for 15 min. For each dish, colonies were counted using the Image J software. Each measurement was performed in triplicate.
Histoscores of BRD, paratumoral, and RCC tissues were subclassified as high (histoscores > mean of total samples of BRD, RCC, and paratumoral tissues) and low (histoscores ≤ mean of total samples of BRD, RCC, and paratumoral tissues) expression levels (mean values are listed in ). Incidence risk was evaluated using logistic regression, and survival analysis was performed using Cox hazard regression model. Original real-time PCR data, western blot data, migration/invasion data, and colony formation data were recorded as continuous variables and analyzed using Student’s t-test or linear polynomial ANOVA with LSD post hoc examination. All the statistical analyses were performed using SPSS 16.0 and Excel 2007. All the statistical tests and P values were 2-sided, and the level of significance was set at <0.05 (*), <0.01 (**), or <0.001 (***).
Immunohistochemical staining of WNT10A, β-catenin, and cyclin D1 in kidney tissues.