All cell lines were obtained from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). According to the American Type Culture Collection (ATCC) (Manassas, VA, USA), 786-O and 769-P were classified as ccRCC cell lines and ACHN was classified as the RCC cell line. OS-RC-2 was the RCC cell line and was maintained as previously described (26). The human embryonic kidney 293T (HEK 293T) cell line and Madin-Darby canine kidney (MDCK) cell line were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (all from HyClone, Logan, UT, USA). The ccRCC cell lines 786-O and 769-P and RCC cell line ACHN were cultured in RPMI-1640 (HyClone) supplemented with 10% FBS and 1% penicillin-streptomycin. The cells were incubated in 5% CO₂ at 37°C.

The rabbit polyclonal anti-CRB3 antibody was purchased from Sigma-Aldrich which was used for immunohistochemistry (IHC) (1:200 dilution, HPA013835; St. Louis, MO, USA). The following antibodies were used for western blot analysis: rabbit polyclonal anti-CRB3 antibody (1:200 dilution, SC-292449; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), mouse monoclonal anti-E-cadherin antibody (1:500 dilution, ab1416; Abcam, Cambridge, MA, USA), mouse monoclonal anti-N-cadherin antibody (1:1,000 dilution, 610920; BD Biosciences, Bedford, MA, USA), rabbit polyclonal anti-vimentin antibody (1:2,000 dilution, 10366-1-AP) and mouse monoclonal anti-GAPDH antibody (1:5,000 dilution, HRP-60004) (both from Wuhan Sanying Biotechnology, Hubei, China). Anti-rabbit and anti-mouse HRP-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (1:3,000 dilution, SC-2004 and SC-2005).

The 786-O and HEK 293T cells were seeded in a 6-cm dish one day before transfection. Then the 786-O cells were transfected with GV168-CRB3 plasmid using TurboFect Transfection Reagent (Thermo Scientific, Rockford, IL, USA) to overexpress CRB3 according to the manufacturer’s instructions. GV168-CRB3 plasmid was purchased from Genechem Co., Ltd. (Shanghai, China). The HEK 293T cells were transfected with siRNA using Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA) to knock down CRB3 expression according to the manufacturer’s instructions. The siRNA was purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). Target sequences of oligonucleotides used were as follows: CRB3, 5′-UGG CAC UGU UGG UGC GGA ATT-3′; control (non-targeting), 5′-UUC UCC GAA CGU GUC ACG UTT-3′. The cells were harvested 48 h after transfection.

The human study was approved by the Ethics Committee on Human Research of the First Affiliated Hospital of Xi’an Jiaotong University (Shaanxi, China). Written informed consent was obtained from all patients. Tissue microarrays (TMAs) were obtained from Shanghai Biochip Co., Ltd. (Shanghai, China). The TMAs contained 142 pairs of ccRCC and adjacent normal kidney tissues. Six pairs of tissue samples were excluded from the study due to lack of sufficient tumor tissue in the samples or fixation artifacts. The remaining 136 pairs of tissue samples consisted of tissues from 85 male and 51 female patients with a mean age of 59 years (range, 29–82 years). One-hundred and five patients were followed up for 2–74 months, with a median follow-up of 63 months. The follow-up data were summarized by the end of September, 2012. OS was defined as the period from the surgery to death or to the last known follow-up. Seventy-seven patients were still alive by the end of the follow-up.

The paraffin-embedded TMA slides were deparaffinized in xylene and rehydrated in ethanol with decreasing concentrations (100, 95, 90 and 85%, 5 min each). After the slides were washed five times (3 min each time) with phosphate-buffered saline (PBS), 3% H₂O₂ was used to block the endogenous peroxidase activity for 20 min. After being rinsed, the slides were placed in 0.01 M sodium citrate in a microwave oven (pH 6.0, 100 W, 6 min; 50 W, 13 min) for antigen retrieval. Then slides were cooled at room temperature and added with goat serum to block non-specific binding sites for 15 min. Without being washed, the slides were incubated with the primary antibody in a moist box at 4°C overnight. After being rinsed, the slides were incubated with the streptavidin-peroxidase staining system kit according to the manufacturer’s instructions (SP-9001; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China). Finally, the slides were stained by using the 3,3′-diaminobenzidine (DAB) liquid chromogen substrate kit (ZLI-9017; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) and counterstained with hematoxylin, followed by observation with Leica Microsystems GmbH (SCN 400; Mannheim, Germany). The results of IHC were evaluated individually in a blinded manner by three investigators who had no access to the clinical or clinicopathological status of the specimens. The intensity of immunohistochemical staining was scored as 0 (negative), 1 (weakly positive), 2 (moderately positive) or 3 (strongly positive). The extent of immunohistochemical staining was assessed according to the percentage of positive cells: 0 (negative), 1 (1–25%), 2 (26–50%), 3 (51–75%) and 4 (76–100%). The staining score for each field was calculated as the product of the intensity and extent of the staining. The final staining score for each sample was the mean of the staining scores of three randomly selected fields. The expression level was considered negative if the final score was 0–2 and positive if the final score was 3–12. The expression level was considered low if the final score was 3–5 and high if the final score was 6–12.

CRB3 is not expressed in the adipose tissue according to the data provided in EBI Expression Atlas database (27). We performed IHC of the adipose tissue using the primary antibody rabbit polyclonal anti-CRB3 antibody and found that CRB3 protein was not expressed in the adipose tissue. CRB3 was expressed in the ccRCC tissues incubated with rabbit polyclonal anti-CRB3 antibody, and not expressed in the ccRCC tissues incubated with CRB3 recombinant protein and rabbit polyclonal anti-CRB3 antibody. Therefore, the ccRCC tissue incubated with CRB3 recombinant protein and primary antibody, and the adipose tissue were used as negative controls in the study. Tissues incubated with PBS in place of the primary antibody were also used as negative control to verify the specificity of the IHC results.

Total RNA was extracted from cells by using the E.Z.N.A.^® Total RNA kit I (Omega Bio-Tek, Inc., Norcross, GA, USA) according to the manufacturer’s instructions. Reverse transcription was performed by using the PrimeScript™ RT Master Mix [Takara Biotechnology (Dalian) Co., Ltd., Liaoning, China] according to the manufacturer’s instructions. Real-time polymerase chain reaction (real-time PCR) was performed at an annealing temperature of 60°C using the SYBR-Green I kit [Takara Biotechnology (Dalian) Co., Ltd.]. The following primers were used for real-time PCR reaction: CRB3 forward, 5′-CTT CTG CAA ATG AGA ATA GCA CTG T-3′ and reverse, 5′-GAA GAC CAC GAT GAT AGC AGT GA-3′; GAPDH forward, 5′-CTC CTC CAC CTT TGA CGC TG-3′ and reverse, 5′-TCC TCT TGT GCT CTT GCT GG-3′. Results were analyzed using the comparative C_t method with the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control.

Proteins were extracted from cells using RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, and 1% sodium deoxycholate) supplemented with a protease inhibitor cocktail (Roche Diagnostics, Ltd., Basel, Switzerland). Cell lysates were centrifuged at 12,000 rpm for 20 min at 4°C. Proteins were separated by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinyl difluoride (PVDF) membranes (0.22 μm; Millipore, Billerica, MA, USA) by electroblotting. The membranes were blocked with 5% skimmed milk containing Tris-buffered saline and 0.1% Tween-20 (TBST) for 2 h, followed by incubation with the primary antibody at 4°C overnight. After the membranes were washed with TBST, they were incubated with the secondary antibody. Immunoreactions were visualized using the chemiluminescence reagent (Millipore) according to the manufacturer’s instructions. GAPDH was used as a protein loading control.

786-O and HEK 293T cells were transfected with GV168-CRB3 plasmid and siRNA, respectively. Cells were harvested 24 h after transfection and plated on 24-well cell culture plates. After 12 h, scratch wounds were created manually in the monolayer of cells with a P200 pipette tip, washed twice with PBS, and incubated with fresh medium. The plates were photographed at the identical locations 0 and 24 h after incubation by Phase Contrast Microscope (20x; Nikon, Tokyo, Japan). Migration was determined by comparing the two sets of images.

Migration and invasion assays were performed in 24-well Transwells with polycarbonate filters (8 μm pore size; Corning, Inc., Corning, NY, USA). The Transwell for the invasion assay was coated with Matrigel (1:5 dilution; BD Biosciences), while the Transwell for the migration assay was not coated. 786-O and HEK 293T cells were transfected with GV168-CRB3 plasmid and siRNA, respectively. The cells were harvested 24 h after transfection. 786-O cells were trypsinized and suspended with RPMI-1640 without FBS. 786-O cells, 2×10⁴ (for migration assay) or 2×10⁵ (for invasion assay), were added to the upper wells and 600 μl RPMI-1640 medium containing 10% FBS was added to the lower wells. Cells in the wells were incubated in 5% CO₂ at 37°C for 24 h for migration assay and 48 h for invasion assay. After incubation, cells in the upper wells were gently removed by scrubbing, fixed in 95% ethanol for 15 min and stained with 0.4% crystal violet for 30 min. The number of transmembrane cells was calculated as the mean of the cell numbers in three randomly selected microscopic fields (20x; Nikon). Migration and invasion assays of HEK 293T cells were performed following the same procedure except that HEK 293T cells were maintained with the DMEM medium and 5×10⁴ cells were added for migration assay and 5×10⁵ cells for invasion assay.

Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) software (version 13.0; SPSS Inc., Chicago, IL, USA). The χ² test was used to investigate the possible relationships between CRB3 expression and clinicopathological characteristics. The Mann-Whitney U test was used to compare differences between CRB3 protein expression in paired ccRCC and adjacent normal kidney tissues. Survival analysis was done by using the Kaplan-Meier curve and the log-rank test. Multivariate analysis was performed using the Cox proportional hazard regression model to explore the effects of CRB3 expression and ccRCC clinicopathological variables on survival. In cellular studies, values are expressed as mean ± SEM. Comparisons between two groups were conducted using the Student’s t-test. All experiments were done in triplicate. All statistical tests were two-sided. P<0.05 was considered statistically significant.

We first examined CRB3 mRNA and protein expressions in the normal canine kidney epithelial cell line MDCK, HEK 293T cell line, ccRCC cell lines 786-O and 769-P, and the RCC cell lines ACHN and OS-RC-2. Real-time PCR and western blot analyses showed that MDCK had the highest mRNA and protein expression levels of CRB3 among the cell lines (Fig. 1A and B). The mRNA and protein expressions of CRB3 in ccRCC and RCC cell lines were relatively lower than those in MDCK.

Immunohistochemical analysis of TMAs was performed to investigate whether CRB3 was differently expressed in paired ccRCC and adjacent normal kidney tissues. The TMAs contained 136 pairs of ccRCC and adjacent normal kidney tissue samples. Due to the small sample size, low and high CRB3 expressions were classified as positive expressions. The clinicopathological features of the ccRCC patients and their CRB3 expression levels are listed in Table I. The specificity of the primary antibody rabbit polyclonal anti-CRB3 antibody was validated by using three negative controls (Fig. 2A). The results showed that CRB3 was not expressed in the adipose tissue and the ccRCC tissues incubated with CRB3 recombinant protein or PBS, which indicated that the primary antibody used for IHC was specific and the IHC results are sound. We found that CRB3 protein was predominantly expressed in the membrane and cytoplasm of renal tubular epithelial cells (Fig. 2B and C). CRB3 protein was strongly expressed in adjacent normal kidney tissues and weakly expressed in ccRCC tissues (Fig. 2B and C). Sixty-nine percent of ccRCC tissues showed complete loss of CRB3 expressions (n=136, 69%, P<0.001, Table II). The expression level of CRB3 was significantly correlated with histologic grade and TNM stage (P<0.05, Fig. 2D and E, Table I), but not with age, gender, tumor size and site, and capsule invasion (Table I).

The significant differences in the CRB3 expression between the pairs of ccRCC and adjacent normal kidney tissues inspired us to consider whether CRB3 could be used as a prognostic predictor for ccRCC patients. The association of CRB3 expression with OS was assessed by univariate analysis using the Kaplan-Meier curve and the log-rank test. No significant difference was detected in OS among patients with negative, low and high CRB3 expressions (data not shown). Due to the small sample size, low and high CRB3 expressions were classified as positive expressions. Of the 105 patients followed up for survival, 30 patients showed positive expression of CRB3 while the other 75 patients showed negative expression. Patients with negative CRB3 expressions had a significantly shorter OS than patients with positive expression (P=0.015, log-rank test, Fig. 3A). The result indicated that CRB3 may be a prognostic indicator for patients with ccRCC. Univariate analysis also showed that patients with different histologic grades or TNM stages showed significantly different OS (data not shown). Here, due to the insufficient sample size, we divided the histologic grades or TNM stages into low histologic grade or TNM stage (histologic grade I-II or TNM stage 1–2) and high histologic grade or TNM stage (histologic grade III-IV or TNM stage 3–4). Log-rank test showed that high histologic grade or TNM stage was significantly associated with shorter OS (histologic grade I-II vs. III-IV, P=0.001, Fig. 3B; TNM stage 1–2 vs. 3–4, P=0.003, Fig. 3C).

Multivariate analysis was performed using the Cox proportional hazard regression model to further determine the effects of CRB3 expression and clinicopathological parameters on the prognosis of ccRCC patients. The multivariate Cox proportional hazard regression model mainly considered CRB3 expression and clinicopathological parameters, including age, gender, histologic grade, TNM stage, tumor size and site, and capsule invasion. Results showed that positive CRB3 expression was linked to longer OS [B=−1.288, Exp(B)=0.276, P=0.037, Table III], which indicated that positive CRB3 expression may be an independent favorable prognostic factor for OS and may be used to predict OS of ccRCC patients. Table III also shows that histologic grade may be a hazardous factor [B=1.250, Exp(B)=3.490, P=0.001, Table III]. Age, gender, TNM stage, tumor size and site, and capsule invasion were not independent indicators for clinical outcome (Table III).

Invasion and metastasis are the hallmarks of malignant tumor progression and the main causes of deaths in cancer. Here, cell migration, invasion and wound-healing assays were conducted to analyze the invasive and metastatic behavior in vitro. Since the 786-O cell line had a low protein expression of CRB3 and is a suitable host for transfection, 786-O cells were transfected with GV168-CRB3 plasmid to overexpress CRB3. Cell migration and invasion assays revealed that transfected 786-O cells showed reduced migration and invasion, when compared with control cells (Fig. 4A and B). Wound-healing assay showed that the wound of control cells disappeared, but the transfected cells failed to efficiently migrate to fill the void, indicating that the migration rate of 786-O cells was reduced after transfection with GV168-CRB3 plasmid (Fig. 4C). The results indicated that CRB3 overexpression significantly inhibited the migration and invasion of 786-O cells.

To investigate the effect of the loss of CRB3 on cell invasion and migration, HEK 293T cells were transfected with siRNA to knock down the CRB3 expression. The transfected HEK 293T cells showed increased migration and invasion, when compared with control cells, indicating that loss of CRB3 expression may promote the migration and invasion of HEK 293T cells (Fig. 4D and E). Unfortunately, wound-healing assay was not successful for HEK 293T cells, probably due to the poor anchorage-dependent growth of the cells.

To investigate the roles of CRB3 in epithelial-mesenchymal transition (EMT), we examined the expression levels of the mesenchymal markers vimentin and N-cadherin and the epithelial marker E-cadherin in 786-O cells transfected with GV168-CRB3 plasmid and HEK 293T cells transfected with siRNA. Western blot analysis revealed an increased E-cadherin expression and concomitant decreased vimentin and N-cadherin expressions in transfected 786-O cells (Fig. 4F). However, transfection with siRNA induced decreased E- and N-cadherin expressions and an increased vimentin expression in HEK 293T cells (Fig. 4F). The expression changes of the mesenchymal and epithelial markers indicated that CRB3 may be associated with EMT.