Clinical specimens were collected from patients with BC (n=32) who had undergone cystectomy (n=6) or transurethral resection of their bladder tumors (n=26) at the Kagoshima University Hospital (Kagoshima, Japan) from 2004 to 2013. normal bladder epithelial (NBE; n=12) specimens were derived from patients with non-cancerous diseases. The specimens were staged according to the American Joint Committee on Cancer (AJCC)/Union Internationale Contre le Cancer (UICC) tumor-node-metastasis classification system and were histologically graded. This study was approved by the Bioethics Committee of Kagoshima University; the study numbers were H27-104 and H27-105. Written informed consent and approval were obtained from all patients prior to obtaining the samples. The clinicopathological characteristics of the patients are presented in Table I.

In addition, we used two human BC cell lines: Invasive T24 cells, which were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA: cat. no. HTB-4) and BOY cells, which were established in our laboratory from an Asian male patient, 66 years of age, given a diagnosis of stage III BC with lung metastasis. These cell lines were maintained in the recommended media containing 10% fetal bovine serum (FBS) in a humidified atmosphere of 95% air at 37°C. Eagle’s minimum essential medium (MEM) was used for the culture medium for the BOY cells. These cell lines were subjected to the experiments at passage numbers between 10 and 20. Regularly, mycoplasma testing was carried out for the cell lines and no infection was confirmed. We found no information regarding misidentified or cross-contaminated cell lines the all cell lines used were cross-checked from the International Cell Line Authentication Committee (http://iclac.org/databases/cross-contaminations/) and ExPASy Cellosaurus databases (https://web.expasy.org/cellosaurus/).

Tissues were treated with RNAlater (Thermo Fisher Scientific, Waltham, MA, USA) and stored at -20°C until RNA extraction. Total RNA, including miRNAs, was extracted from the tissues and the cells using the mirVana miRNA Isolation kit (Thermo Fisher Scientific) and Isogen (Nippon Gene, Tokyo, Japan), respectively following the manufacturer’s protocol.

Stem-loop RT-PCR (TaqMan MicroRNA Assay for miR-223; P/N 002295; Applied Biosystems, Foster City, CA, USA) was used for RT-qPCR to evaluate miRNA expression levels according to previously published methods (11). We used human RNU48 (P/N 001006; Applied Biosystems) as an internal control and employed the ΔΔCq method to calculate the fold changes in expression (12). For WDR62 and glucuronidase beta (GUSB) expression, we applied a SYBR-Green qPCR system using the following primers: WDR62 forward, 5′-GCCTTCTCACCCAA TATGAAGC-3′ and reverse, 5′-GCCTTCTCACCCAATATGA AGC-3′; and GUSB forward, 5′-CGTCCCACCTAGAATCT GCT-3′ and reverse, 5′-TTGCTCACAAAGGTCACAGG-3′. We also performed RT-qPCR analyses of the 11 genes that were significantly upregulated in the TCGA cohort of BC. The primers for detecting expressions of those genes are described in Table III. For the reverse transcription step, 500 ng total RNA was reverse transcribed into cDNA using the High Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific, Inc.) under the incubation conditions of 25°C for 10 min, 37°C for 120 min and 85°C for 5 min. qPCR was performed using a Power SYBR-Green Master Mix (cat. no. 4367659) on a 7300 Real-time PCR System (both Applied Biosystems; Thermo Fisher Scientific, Inc.). Furthermore, the initial step time for activation was 10 min at 95°C, followed by 40 cycles between a denaturation step for 15 sec at 95°C and an annealing/extension step for 1 min at 60°C.

The specificity of amplification was monitored according to the dissociation curve of the amplified product. All expression values were normalized to those for GUSB, and the ΔΔCq method was employed to calculate the fold change values (12). All experiments were performed in triplicate.

As previously described (11), the T24 and BOY cells were transfected with 10 nM mature miRNA, siRNA or anti-miRNA using Lipofectamine RNAiMAX transfection reagent and Opti-MEM (both from Thermo Fisher Scientific). Mature miRNA molecules, Pre-miR™ miRNA precursors (miR-223, hsa-miR-223-3p, P/N PM12301; Thermo Fisher Scientific) and negative control miRNA (P/N AM17111; Thermo Fisher Scientific) were used for the gain-of-function experiments, whereas two different WDR62 siRNA (P/N HSS138565 and HSS138567; Thermo Fisher Scientific), negative control siRNA (P/N D-001810-10; Thermo Fisher Scientific), mirVana^® miRNA inhibitor (anti-miR-223, hsa-miR-223-3p, P/N MH12301; Thermo Fisher Scientific) and negative control miRNA inhibitor (P/N AM17010; Thermo Fisher Scientific) were used for the loss-of-function experiments. For co-transfection, the BC cells were simultaneously transfected with 10 nM miR-223 and 10 nM anti-miR-223 using Lipofectamine RNAiMAX and Opti-MEM (both from Thermo Fisher Scientific).

In order to investigate the functional significance of miR-223 and WDR62, we performed cell viability, migration and invasion assays using the T24 and BOY cells transfected with 10 nM miRNA or siRNA by reverse transfection. The cells were seeded in 96-well plates at 3×10³/well for XTT assays. After 72 h, cell viability was determined using the Cell Proliferation kit II (Roche Diagnostics GmbH, Mannheim, Germany) as previously described (11). The cell migration ability was evaluated using wound healing assays. Cells were plated in 6-well plates at 2×10⁵/well, and after 48 h of transfection, the cell monolayer was scratched using a P-20 micropipette tip. The initial gap length (0 h) and residual gap length at 24 h after wounding were calculated from photomicrographs by using an OLYMPUS CK2 microscope (Olympus Optical Corp., Tokyo Japan) as previously described (13). Cell invasion assays were assessed by modified Boyden chambers consisting of Transwell pre-coated Matrigel membrane filter inserts with 8-micrometer pores in 24-well tissue culture plates (BD Biosciences, Bedford, MA, USA). At 72 h following transfection, the cells were seeded in the upper chamber of 24-well plates at 1×10⁵/well with serum-free Eagle’s MEM. After 24 h, 37°C incubation. MEM containing 10% FBS in the lower chamber served as the chemoattractant, as previously described (11). The cells were stained by Diff-Quick (a modified Giemsa stain) (Richard Allan Scientific, San Diego, CA, USA). Briefly, the cells were fixed with pure methanol for 2 min, followed-by stained by the dye I and dye II each for 2 min. The number of the cells on the surface of the chamber was counted by using OLYMPUS BX41 (Olympus Optical Corp.). All experiments were performed in triplicate.

For colony formation assays to examine effects of WDR62 knockdown, the cells transfected with 10 nM siRNA were seeded into 10-cm dish at a density of 1,000 cells/well. Following 7 days of incubation in the MEM containing 10% FBS in a humidified atmosphere of 95% air at 37°C, the resulting colonies were fixed with 4% paraformaldehyde phosphate buffer solution (Nacalai Tesque, Kyoto, Japan) and stained with 0.04% crystal violet (Nacalai Tesque) for 10 min at room temperature. The numbers of colonies were counted. The average colony density was calculated and expressed as the relative percentage of the mock group (only with opti-MEM Lipofectamine RNAiMAX).

The BC cell lines were transfected with reagent only (mock), miR-control, miR-223, siRNA-control or si-WDR62 at the concentration of 10 nM in 6-well tissue culture plates, as described previously (14). The cells were harvested by trypsinization at 72 h following transfection and washed in cold phosphate-buffered saline. For the apoptosis assays, double staining with FITC-Annexin V and propidium iodide was performed using the FITC Annexin V Apoptosis Detection kit (BD Biosciences) according to the manufacturer’s recommendations and analyzed within 1 h by flow cytometry (CytoFLEX analyzer; Beckman Coulter, Brea, CA, USA). Cells were identified as viable, dead, early apoptotic or apoptotic cells using CytExpert™ 1.2 software (Beckman Coulter), and the percentages of early apoptotic and apoptotic cells in each experiment were then compared.

Caspase-3/7 activity was measured using CellEvent™ caspase-3/7 Green Detection Reagent (Invitrogen/Thermo Fisher Scientific). BC cell lines in a 96-well plate were transfected with mature miRNAs and siRNAs as described above. After 72 h, 5 µM CellEvent™ caspase-3/7 Green Detection Reagent were added to each well and incubated at 37°C for 30 min. The fluorescence was then measured in each well. For densitometric analysis, the total fluorescence was quantitated using the BZ-II Analyzer (Keyence, Osaka, Japan). Experiments were performed in triplicate.

For cell cycle analyses, the cells were fixed in 70% aqueous ethanol and stained with propidium iodide at 4°C for 30 min following treatment with RNase A as previously described (15). The data were analyzed using the CytoFLEX analyzer (Beckman Coulter). The percentages of cells in the G0/G1, S and G2/M phases were determined and compared. Experiments were performed in triplicate. We used 1 µg/ml nocodazole (P/N ab120630; Abcam, Cambridge, UK) to induce G2/M arrest in each cell line as previously described (16). Experiments were done in triplicate.

Following 3 days of transfection, total protein lysate was prepared with a radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific, Inc.) containing protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA). The protein concentrations were determined using the Bradford assay (17). Protein lysate (20 µg) per lane was loaded in NuPAGE on 4-12% bis-tris gel (Invitrogen/Thermo Fisher Scientific) and transferred into a polyvinylidene fluoride membrane. Following transfer, the membranes were blocked in washing buffer (0.35 M NaCl, 10 mM Tris-HCl, pH 8.0, and 0.05% Tween-20) containing 3% skim milk for 2 h at room temperature, followed by an overnight incubation at 4°C with rabbit anti-WDR62 antibodies (1:500, P/N GTX119724; GeneTex, San Antonio, TX, USA), anti-cleaved poly (ADP-ribose) polymerase (PARP) antibodies (1:1,000, P/N 5625), anti-cleaved caspase-3 antibodies (1:1,000, P/N 9664) (both from Cell Signaling Technology, Danvers, MA, USA) and anti-β-actin antibodies (1:1,000, P/N bs-0061R; Bioss, Woburn, MA, USA). The secondary antibodies were peroxidase-labelled anti-rabbit IgG (1 h at 25°C; 1:5,000; cat. no. 7074S; Cell Signaling Technology, Inc.). Specific complexes were visualized using an echochemiluminescence detection system (GE Healthcare, Little Chalfont, UK) as previously described (13).

A tissue microarray containing 78 urothelial cancers and 20 normal bladder tissues was obtained from US Biomax, Inc. (product ID: BL 1002; Rockville, MD, USA). Detailed information on all tumor specimens can be found at http://www.biomax.us/index.php. The tissue microarray was immunostained following the manufacturer’s protocol with an Ultra Vision Detection System (Thermo Fisher Scientific). Primary rabbit polyclonal antibodies against WDR62 (P/N GTX119724; GeneTex) were diluted 1:1,000. Immunostaining was evaluated according to the scoring method described previously (13). Each case was scored on the basis of the intensity and area of staining. The intensity of staining was graded on the following scale: 0, no staining; 1+, mild staining; 2+, moderate staining; and 3+, intense staining. The area of staining was evaluated as follows: 0, no staining of cells in any microscopic field; 1+, <30% of cells stained positive; 2+, 30-60% stained positive; 3+, >60% stained positive. The immunostaining scores (intensity + extent) were combined and analyzed. All samples were independently scored by two of the authors (T.S. and K.M.), who were blinded to the patient status.

In silico analysis was performed to identify target genes of miR-223 using the TargetScan database Release 7.1 (http://www.targetscan.org/vert_71/). Additionally, the Gene Expression Omnibus (accession nos. GSE11783 and GSE31684) and The Cancer Genome Atlas (TCGA) databases were used to identify upregulated genes in BC specimens. We merged these datasets and selected possible miR-223 target genes.

Partial sequences of the wild-type WDR62 3′-UTR or the WDR62 3′-UTR with deletion of the miR-223 target site (position 62-69 of the WDR62 3′-UTR) and the miR-223 target site sequence (position 62-69 of the WDR62 3′-UTR) were inserted between the XhoI and PmeI restriction sites within the 3′-UTR of the hRluc gene in the psiCHECK-2 vector (P/N C8021; Promega, Madison, WI, USA). The T24 and BOY cells were transfected with 50 ng vector and 10 nM miR-223 using Lipofectamine 2000 (Thermo Fisher Scientific) and Opti-MEM (Thermo Fisher Scientific). The activities of Firefly and Renilla luciferases in the cell lysates were determined using a dual luciferase assay system according to the manufacturer′s protocol (P/N E1960; Promega). Normalized data were presented as Renilla/Firefly luciferase activity ratios. Experiments were performed in triplicate.

BC samples (n=407) and normal bladder samples (n=19) from the TCGA database were used to analyze the WDR62 mRNA expression levels in BC compared with normal and BC tissues. BC samples from the TCGA database were used to determine the associations between the expression of miR-223 or WDR62 and clinicopathological factors. Gene and miRNA quantification were performed using RNA-seq expression data (normalized RSEM) and miRNA-seq data (reads per million mapped reads) (18). Whole-exome sequencing data were available for the 407 BC samples and 19 normal samples. Full sequencing information and clinical information were acquired using the cBio Portal (http://www.cbioportal.org/public-portal/) and TCGA (https://tcga-data.nci.nih.gov/).

The differences between 2 groups were analyzed using Mann-Whitney U tests. The differences between 3 variables and numerical values were analyzed using Bonferroni-adjusted Mann-Whitney U tests. The overall survival of patients with BC from the TCGA cohort was evaluated by the Kaplan-Meier method. Patients were divided into 2 groups according to the median miR-223 or WDR62 expression level, and differences between the 2 groups were evaluated by log-rank tests. All analyses were performed using Expert StatView software, version 5.0 (SAS Institute Inc., Cary, NC, USA). All data are expressed as the means ± standard deviation. P-values <0.05 were considered to indicate statistically significant differences.

First, we evaluated the expression levels of miR-223 in BC samples (n=32) and NBE samples (n=12) in our facility by RT-qPCR. Patient details and clinicopathological characteristics are summarized in Table I. The expression level of miR-223 was significantly lower in the BC tissues than in the NBE tissues (P=0.013; Fig. 1A). miR-223 expression was also decreased in the T24 and BOY BC cell lines compared with the NBE samples.

We evaluated the association between miR-223 expression and the patient clinicopathological parameters or overall survival. Among the BC cohort of TCGA, we evaluated 407 patients with available miR-223 expression, clinicopathological and survival data. Among these, data of lymphovascular invasion and distant metastasis were available for 278 and 202 patients, respectively. The expression level of this miRNA was significantly lower in patients with BC with lymphovascular invasion (LVI) (P=0.0316; Fig. 1B, left panel) and had a tendency to be lower in patients with BC with distant metastasis compared with their counterparts (P=0.0796; Fig. 1B, right panel). Next, the cohort was divided into 2 groups according to the median miR-223 expression level. Kaplan-Meier analysis revealed no differences in the overall survival rate between patients with a high (n=203) and patients with a low miR-223 expression (n=203) (P=0.2866) (data not shown).

To investigate the functional roles of miR-223, we performed gain-of-function analyses using miRNA-transfected T24 and BOY BC cell lines. XTT assays revealed a significant inhibition of the viability of the T24 and BOY cells transfected with miR-223 in comparison with the mock or miR-control transfectants (each P<0.0001; Fig. 2A). Wound healing assays demonstrated that the migration activity was significantly suppressed in the miR-223 transfectants compared with the mock or miR-control transfectants (each P<0.0001; Fig. 2B). Matrigel invasion assays also demonstrated that the number of invading cells was significantly decreased in the miR-223 transfectants compared with the mock or miR-control transfectants (T24 cells: P=0.0002, BOY cells: P<0.0001; Fig. 2C).

As it has been previously reported that miR-223 promotes the apoptosis of acute myeloid leukemia cell lines (22), in this study, we performed flow cytometric analyses to determine the number of apoptotic cells following the restoration of this miRNA into BC cell lines. The numbers of apoptotic cells (apoptotic and early apoptotic cells) were significantly higher in the miR-223 transfectants than in the mock or miR-control transfectants (each P<0.0001; Fig. 2D). Moreover, in a caspase-3/7 activity assay, the fluorescence intensity was significantly increased in the miR-223 transfectants compared with the mock or miR-control transfectants (T24 cells: P=0.0013, BOY cells: P=0.0002; Fig. 2E).

To identify the target genes of miR-223, we performed in silico analysis. Fig. 3 shows our strategy used to narrow down possible target genes of miR-223. Candidate targets of miR-223 with one or more conserved miR-223 binding sites (489 genes) were identified using the TargetScan database, release 7.1 (http://www.targetscan.org). Among these candidate genes, 24 genes were upregulated in the GEO database (accession nos. GSE11783 and GSE31684). Moreover, the TCGA database was used to identify genes with a higher expression in the BC samples than in the normal bladder samples. The analysis of the TCGA database revealed that the mRNA expression levels of 11 genes were significantly upregulated in BC. Subsequently, we performed RT-qPCR analyses of these 11 genes to confirm which genes have a lower mRNA expression in the miR-223 transfectants compared with the mock transfectants (Table II). Table II shows that the 11 genes were candidates which could possibility be targets of miR-223 in clinical BC. Finally, we focused on WDR62 as, to date, to the best of our knowledge, there have been no reports on this gene as a target of miR-223, while the direct regulation of integrin alpha 3 (ITGA3) by miR-223 has been previously reported in prostate cancer (23).

We performed RT-qPCR and western blot analyses to investigate whether the restoration of miR-223 expression downregulates WDR62 expression in BC cells. The WDR62 mRNA levels were significantly decreased in the miR-223 transfectants compared with the mock or miR-control transfectants (T24 cells: P=0.0002, BOY cells: P=0.0012; Fig. 4A). The WDR62 protein levels were also decreased in the miR-223 transfectants compared with the mock or miR-control transfectants (Fig. 4B). In addition, we performed dual-luciferase reporter assays in the BC cells to determine whether WDR62 was regulated by miR-223 directly. As the TargetScan database predicts a binding site for miR-223 in the 3′-UTR of WDR62 mRNA (position 62-69), we constructed vectors encoding a partial wild-type WDR62 mRNA sequence including the miR-223 binding site in the 3′-UTR. We found a significantly reduced luminescence intensity following the co-transfection of miR-223 and the vector carrying the wild-type sequences at position 62-69 of the WDR62 3′-UTR (each P<0.0001), whereas no reduction in luminescence was observed following transfection with a vector in which the binding site had been deleted (Fig. 4C). These data suggest that miR-223 binds directly to the specific site of the 3′-UTR of WDR62 mRNA.

In a cohort of BC samples (n=407) and normal bladder samples (n=19) from the TCGA database, the mRNA expression level of WDR62 was significantly higher in the BC samples than in the normal bladder samples (P<0.0001; Fig. 5A). In addition, we evaluated the associations between WDR62 expression and the patient clinicopathological parameters or overall survival. Among the BC cohort of TCGA, we evaluated 403 patients with available WDR62 expression, clinicopathological and survival data. Among these, data of pathological subtype, tumor grade, distant metastasis and clinical were available for 396, 399, 203 and 400 patients, respectively. The mRNA expression level of WDR62 was significantly higher in the non-papillary BC (P<0.0001), high-grade tumors (P<0.0001) and high-stage tumors (P<0.01; Fig. 5B). The higher expression level exhibited a trend towards significance in patients with BC with distant metastasis compared with their counterparts (P=0.0528; Fig. 5B). Subsequently, the cohort was divided into 2 groups according to the median WDR62 expression level. Kaplan-Meier analysis revealed no difference in the overall survival rate between patients with a high (n=201) and those with a low WDR62 expression (n=202) (P=0.5194) (data not shown). In the BC samples (n=32) and normal bladder samples (n=12) from our facility, WDR62 mRNA expression was significantly higher in the BC samples than in the normal bladder samples (P<0.0095; Fig. 6A). However, there was no significant association between WDR62 expression and the patient clinicopathological parameters, such as non-papillary BC, high-grade tumors and high-stage tumors in clinical specimens from our facility (data not shown). Our cohort was too small to evaluate the precise statistical value.

We examined the expression level of WDR62 in BC specimens by IHC staining. WDR62 was strongly expressed in several tumor lesions (Fig. 6B, left panel), whereas a low expression was observed in the normal tissue (Fig. 6B, middle panel). Tissue microarray analysis revealed that the IHC score of the tumor tissues was significantly higher than that of the normal tissues (P<0.0001; Fig. 6B, right panel).

To investigate the functional role of WDR62 in BC cells, we performed loss-of-function experients in the T24 and BOY cell lines transfected with 2 siRNA constructs targeting WDR62 (si-WDR62_1 and si-WDR62_2). The results of RT-qPCR and western blot analyses revealed that both siRNAs effectively decreased WDR62 expression in both cell lines (Fig. S1). XTT assays then revealed a significant inhibition of viability in the si-WDR62 transfectants compared with the mock or siRNA-control transfectants (each P<0.0001; Fig. 7A). In addition, colony formation assay confirmed that there was a significantly lower number of surviving BC cell colonies among the si-WDR62 transfectants than among the mock or siRNA-control transfectants (each P<0.0001; Fig. 8). Wound healing assays also demonstrated that the cell migratory activity was significantly suppressed in the si-WDR62 transfectants compared with the mock or siRNA-control transfectants (T24 cells: each P<0.0001; BOY cells: P=0.0005 and P<0.0001, respectively; Fig. 7B). Matrigel invasion assays demonstrated a significantly decreased number of invading cells among the si-WDR62 transfectants compared with the mock or siRNA-control transfectants (T24 cells: each P<0.0001; BOY cells: P=0.0277 and P<0.0001, respectively; Fig. 7C).

We performed flow cytometric analyses to determine the number of apoptotic cells following the siRNA-mediated knockdown of WDR62 expression. There was a significantly greater number of apoptotic cells among the si-WDR62 transfectants than among the mock or siRNA-control transfectants (T24 cells: each P<0.0001; BOY cells: P<0.0001 and P=0.0147, respectively; Fig. 7D). In addition, a caspase-3/7 activity assay revealed that the fluorescence intensity was significantly increased in the si-WDR62 transfectants than in mock or siRNA-control transfectants (T24 cells: P=0.0095 and P=0.0017; BOY cells: P=0.0045 and P=0.0001, respectively; Fig. 7E). Moreover, the results of western blot analysis confirmed that cleaved caspase-3 and cleaved PARP expression increased in the si-WDR62 transfectants in comparison with the mock or siRNA-control transfectants (Fig. 9).

To investigate the cell cycle effects, we performed flow cytometric analyses using miR-223 or si-WDR62 transfectants. Cell cycle assays revealed no fixed tendency in either transfectant (data not shown).

To confirm the roles of miR-223, we performed functional analyses using BC cells co-transfected with miR-223 and anti-miR-223. The results of RT-qPCR and western blot analyses revealed that co-transfection with anti-miR-223 and miR-223 recovered the WDR62 mRNA and protein expression levels that were suppressed by transfection miR-223 with alone (Fig. 10A and B, respectively). XTT assays revealed that cell viability was restored in the cells co-transfected with anti-miR-223 and miR-223 in comparison with that of the cells transfected with miR-223 alone (each P<0.0001; Fig. 10C). Wound healing and Matrigel invasion assays also demonstrated that the migratory and invasive activity was significantly restored in the cells co-transfected with anti-miR-223 and miR-223 in comparison with the cells transfected with miR-223 alone (each P<0.0001; Fig. 10D and E, respectively). These findings suggest that miR-223 suppresses cell viability, migration and invasion through WDR62 knockdown in BC.