This study was approved by the ethics committee of Peking University People's Hospital (Beijing, China). All patients provided written informed consent.

The clinical samples, including 77 bladder cancer specimens, 3 cystitis glandularis tissues and 4 normal tissues, were obtained from patients who underwent surgical resection of primary tumors at Peking University People's Hospital between April 2011 and August 2012. Patients who received preoperative radiotherapy or adjuvant chemotherapy were excluded from this study. The biopsy tissues for immunohistochemical staining were fixed immediately in 10% buffered formalin and, 24 h later, were dehydrated in increasing concentrations of alcohol, coagulated and embedded in paraffin.

The bladder cancer cell line 5637 was obtained from American Type Culture Collection (Manassas, VA, USA). The bladder cancer cell lines BIU87 and EJ were obtained from the urology department of Peking University First Hospital (Beijing, China). The cells were cultured in RPMI-1640 medium (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone; GE Healthcare Life Sciences) and incubated at 37°C in a humidified atmosphere containing 5% CO₂.

RP215 was provided by Professor Gregory Lee of the University of British Columbia in Vancouver, Canada. The 5637, BIU87 and EJ cells were seeded into 12-well plates upon reaching 60–70% confluence and were maintained in an incubator at 37°C containing 5% CO₂. The cells were fixed in acetone for 5 min at room temperature, after which they were blocked with 10% normal goat serum (Hyclone; GE Healthcare Life Sciences) for 30 min and incubated with 12.5 µg/ml purified RP215 as a primary antibody at 4°C for 45 min. The cells were then incubated with the fluorescein isothiocyanate-conjugated goat anti-mouse polyclonal secondary antibody (1:400; cat. no. ab97022; Abcam, Cambridge, UK) at 4°C for 30 min. Images were captured using an inverted fluorescence microscope subsequent to mounting with 50% glycerin.

Tissue sections (4-µm) from the clinical samples were deparaffinized, rehydrated and then heated in 10 mmol/l citrate buffer (pH 6.0) for antigen retrieval. Subsequently, the sections were washed in PBS, blocked with 10% normal goat serum for 30 min and incubated with 7.5 µg/ml purified RP215 in a humidified chamber overnight at 4°C. Inmunodetection was performed using the Envision™ ABC kit (GeneTech Co., Ltd., Shanghai, China). After staining with hematoxylin, the tissues were dehydrated and mounted. A pathologist independently evaluated the extent and intensity of RP215 staining and was blinded with respect to the clinical data.

The relative number of positive cells and the intensity of staining were assessed in five random 200x microscopic fields. The percentage of stained cells per field was scored as follows: 0, 0% (negative); 1, 1–25%; 2, 26–50%; and 3, 51–100%. The staining intensity was scored on a 4-tiered scale, as follows: 0, absence of signal; 1, low-intensity signal (light brown); 2, moderate-intensity signal (brown); and 3, high-intensity signal (dark brown). The percentage score and intensity score were multiplied to obtain the score for each field, and the final score for each case was the average score of the five fields. The score for RP215 staining was described as follows: negative (−) when the score was 0–1; ‘low expression’ (+) when the score was 2–3; and ‘high expression’ when the score was 4–6 and 7–9 (++ and +++, respectively). All evaluations were conducted using a Leica DM4000B/M microscope (Leica Microsystems, Inc., Buffalo Grove, IL, USA).

Protein was extracted using lysis buffer containing radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc., Rockford, IL, USA), 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 0.15 M NaCl and 10 mM Tris (pH 7.2). The protein concentration was assessed using a Pierce Bicinchoninic Acid Assay kit (Thermo Fisher Scientific, Inc.). Protein samples (50 µg) were separated by 12.5% SDS-PAGE and transferred onto a nitrocellulose membrane (EMD Millipore, Bedford, MA, USA). The membrane was blocked with 5% non-fat milk for 1 h and then incubated with the RP215 and anti-GAPDH (cat. no. TA-08; OriGene Technologies, Inc., Rockville, MD, USA) primary antibodies (7.5 µg/ml) overnight at 4°C. The blots were then incubated with horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (1:3,000; cat. no. ab97040; Abcam) for 1 h at room temperature. Immunoreactive bands were visualized using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Inc.).

Total RNA was extracted from the bladder cancer cell lines EJ, 5637 and BIU87 using TRIzol reagent (Thermo Fisher Scientific, Inc.), treated with DNase (Magen, Guangzhou, China), and then reverse transcribed using the RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The primers for the constant region of the IgGH were as follows: Forward, 5′-CAGGACTGGCTGAATGGC-3′ and reverse, 5′-GGCGTGGTCTTGTAGTTGTT-3′. The primers for GAPDH were: Forward, 5′-CAAGGTCATCCATGACAACTTTG-3′; and reverse, 5′-GTCCACCACCCTGTTGCTGTAG-3′.

In order to amplify the human IgVH gene by nested PCR, the first round of PCR was performed using upstream primers to VH1 (5′-GAGGTGCAGCTCGAGGAGTCTGGG-3′), VH2 (5′-CAGGTGCAGCTCGAGCAGTCTGGG-3′), VH3 (5′-CAGGTACAGCTCGAGCAGTCAGG-3′) and VH4 (5′-CAGGTGCAGCTGCTCGAGTCGGG-3′), coupled with a CH1 region primer (5′-ACACCGTCACCGGTTCGG-3′). PCR was performed using 2X Taq PCR Master Mix kit (Biomed, Beijing, China). The reaction conditions of the first round of PCR were as follows: Pre-denaturation at 94°C for 5 min; followed by denaturation at 94°C for 30 sec, annealing at 59–47°C for 30 sec for the first 18 cycles (with decreases of 2°C increments at each step); then 20 cycles of denaturation at 94°C for 30 sec, annealing at 47°C for 30 sec and polymerization at 72°C for 30 sec with a final elongation step for 7 min at 72°C. A total of 38 cycles was performed. For the second round of PCR, an upstream primer targeting the framework 2 region [5′-TGG(A/G)TCCG(A/C/G) CAG(G/C)C(T/C)CC(A/C/G/T)GG-3′], coupled with a JH primer (5′-AACTGCAGAGGAGACGGTGACC-3′), was used. The reaction conditions for the second round of PCR were as follows: Pre-incubation at 95°C for 15 min, followed by 38 cycles of denaturation at 94°C for 1.5 min, annealing at 57°C for 1.5 min and polymerization at 72°C for 3 min, with a final elongation step for 5 min at 72°C. PCR products were subjected to electrophoresis on 1.5% agarose gels containing 0.5 µg/ml ethidium bromide.

PCR products were cloned into a pGEM-T Easy Vector (Promega Corporation, Madison, WI, USA) and sequenced on an ABI 3100 Genetic Analyzer (Applied Biosystems; Thermo Fisher Scientific, Inc.). The V_HDJ_H sequences were compared with those found in the Basic Local Alignment Search Tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and the International Immunogenetics information system (http://www.imgt.org/) databases in order to identify the best matched germline gene segments and V(D)J junctions. V gene sequences belonging to a set of V_HDJ_H recombinants were defined on the basis of identical VH, DH and JH gene usage and V-D and D-J junction sequences. The repertoire of the cancer-derived Ig V genes was compared with that of published B-cell-derived Ig V genes (17).

siRNAs against the constant region of the Ig γ-chain (siRNA1, 5′-GGUGGACAAGACAGUUGAG-3′; and siRNA2, 5′-AGUGCAAGGUCUCCAACAA-3′) and the non-silencing control RNA (NC, 5′-UUCUCCGAACGUGUCACGU-3′) were produced by Shanghai GenePharma Co. Ltd. (Shanghai, China). The siRNAs and NC were transfected into the 5637 and BIU87 cell lines using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The knockdown efficiency of IgG was verified by western blot analysis.

The 5637 and BIU87 cells were transfected with siRNA (50 µg/ml) in 96-well plates and incubated at 37°C. Cell proliferation was analyzed using the Cell Counting kit-8 (CCK8) Cell Proliferation Assay (Invitrogen; Thermo Fisher Scientific, Inc.). Briefly, CCK8 reagent (5 µl) was added to each well for 2 h, after which the number of viable cells was calculated by measuring the absorbance at 450 nm. Each condition was assessed in triplicate.

Migration assays were performed in 24-well Transwell plates (Corning Incorporated, Corning, NY, USA) containing a polycarbonate filter (8-µm pore size). In total, 10⁵ 5637 and BIU87 cells from each group were added to the Transwell chamber. Conditioned medium supplemented with 10% FBS was added to the bottom of the chamber. The cells were incubated in an incubator containing 5% CO₂ at 37°C for 24 h. Following incubation, the cells in the upper chamber that were attached and had not migrated were removed, and the cells that had migrated to the bottom of the filter were fixed in methanol and stained with hematoxylin. The number of cells was counted in at least 6 randomized fields under a light microscope. The results were obtained from at least three individual experiments.

Invasion assays were performed in 24-well invasion chambers (Corning Incorporated) containing a polycarbonate filter (8-µm pore size). In total, 10⁵ 5637 and BIU87 cells from each group were added to the invasion chamber. Conditioned medium supplemented with 10% FBS was added to the bottom portion of the chamber. The cells were incubated in an incubator with 5% CO₂ at 37°C for 24 h. Following incubation, the cells in the upper chamber that were attached and had not migrated were removed, and the cells that had migrated to the bottom of the filter were fixed in methanol and stained with hematoxylin. The number of cells was counted in at least 6 randomized fields under a light microscope. The results were obtained from at least three individual experiments.

Data are presented as the mean ± standard error of the mean. Statistical analyses were performed using the χ² test and Student's t-test. P<0.05 was considered statistically significant. All statistical evaluations were performed using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA).

In the present study, IgG expression was first detected in bladder cancer cell lines (EJ, 5637 and BIU87) by immunofluorescence. IgG staining was observed in the cell cytoplasm, filopodia-like structures and the extracellular area (Fig. 1A). Furthermore, IgG expression was also detected in these bladder cancer cell lines by western blotting (Fig. 1B). Subsequently, immunohistochemistry demonstrated that IgG immunoreactivity was recurrently localized to the cytoplasm of tumor cells in bladder cancer tissues (58/77, 75.3%) and to the cytoplasm of cells in cystitis glandularis tissues (3/3, 100%), but not to the transitional epithelial cells of normal tissues (0/4) (Fig. 1C).

In order to determine whether IgG was produced by the cancer cells themselves and was not the result of uptake from the extracellular environment, the transcription of the IgGH in EJ, 5637 and BIU87 cells was examined by RT-PCR using primers targeting both the constant and V regions. The results showed that the transcript of the IgGH was expressed in these three cancer cell lines (Fig. 2A). Subsequently, the sequences of these V_HDJ_H rearrangements were analyzed by a comparison with the best matched functional germline IgVH, IgDH and IgJH genes. The results demonstrated that, similar to the B-cell-Igs, all of the bladder cancer-IgG transcripts displayed classical and functional V_HDJ_H rearrangement patterns. However, unlike B-cell-derived IgVH, which has great diversity, several sets of V_HDJ_H rearrangements were frequently shared among these cell lines, and even among different cell lines (VH3-30/D6-6/JH4 was shown in 3/6 5637 cells, VH3-30/D1-26/JH5 was shown in 2/6 5637 cells and VH3-30/D5-5/JH4 was shared between 3/4 BIU87 cells and 3/5 EJ cells). The bladder cancer-V_HDJ_H rearrangements showed restricted VH, DH and JH usage, and a unique V_HDJ_H pattern, including the VH3 region, which was dominantly used (14/16 V_HDJ_H rearrangements analyzed in this study). In particular, the VH3-30 region was dominantly expressed in 5637 (8/9), BIU87 (3/4) and EJ (3/5) cells. Furthermore, among the germline IgHJ1-6 genes, only IgHJ4 (11/16) and IgHJ5 (5/16) were frequently used. Conversely, IgDH showed diversity within each cell line, as follows: DH6-6, DH6-19 and DH1-26 were shown in 5637 cells, DH5-5 and DH6-19 were shown in BIU87 cells, and DH5-5, DH3-16 and DH3-9 were shown in EJ cells. However, DH6-19 was shown in 5637 cells and in BIU87 cells, whereas DH5-5 was shown in BIU87 and EJ cells (Table I, Fig. 2B).

To enhance IgG affinity, the B-cell-derived IgVH of IgG is usually hypermutated (25). Therefore, the present study analyzed the mutation pattern in bladder cancer-derived IgVH and compared the sequence homology among V_HDJ_H rearrangements from three cancer cell lines. It was demonstrated that the bladder cancer-derived IgVH showed only a low frequency of mutation (Table I). Furthermore, the same mutations were frequently present among different V_HDJ_H rearrangements, which resulted in a high homology among V_HDJ_H rearrangements in IgVH (Fig. 2). These results suggested that the conservative domain of IgVH, as well as IgHJ4 and IgHJ5, may exert a similar framework effect in different V_HDJ_H rearrangements in bladder cancer cells, but that IgDH determines the unique biological activity of each V_HDJ_H rearrangement.

In order to investigate whether IgG has a role in the proliferation of bladder cells, two siRNAs to knockdown IgG expression in bladder cancer cell lines were designed and the proliferation of these cells was analyzed following transfection. The results showed that the ability of these cells to proliferate was significantly downregulated following the knockdown of IgG heavy chain expression in 5637 and BIU87 cells (P<0.05; Fig. 3).

The present study investigated whether IgG is involved in the migration and invasiveness of bladder cancer cells. Migration and invasion assays were performed in 24-well invasion chambers using the same number of 5637 and BIU87 cells in each group. It was demonstrated that the migration and invasion of 5637 and BIU87 cells was significantly decreased following knockdown of IgG expression (P<0.05; Figs. 4 and 5).

Next, the present study aimed to determine whether the expression of IgG was correlated with a high histological grade, the size of the tumor and/or the recurrence of bladder cancer. A high positivity of IgG staining was found in patients with high-grade bladder cancer (32/36, 88.9%) compared with patients with low-grade bladder cancer (63.4%, 26/41), and this difference was statistically significant (P=0.045; Table II). Following a comparison of the frequency of positivity between larger tumors (>3 cm) and smaller tumors (≤3 cm), it was found that the rate of IgG positivity in patients with large tumors was 94.1% (16/17), which was significantly higher than patients with smaller tumors (42/60, 70.0%) (P=0.003; Table II). Furthermore, IgG expression was significantly associated with the recurrence of bladder cancer (P=0.012). The rate of positivity (++ and +++) in patients who experienced bladder cancer recurrence was 62.9% (17/27), whereas the rate of positivity (++ and +++) in patients with non-recurrence was 22% (11/50) (P=0.012; Table II).