The Cancer Genome Atlas (TCGA) database was utilized to obtain clinical information and Level 3 RNA sequencing (RNA-Seq) data. The search terms used in TCGA contained the following keywords: ‘cases’, ‘primary site’, ‘bladder’ and ‘TCGA-BLCA’. Based on the above search terms, the clinical information and Level 3 RNA-Seq data were obtained from 404 bladder cancer samples and 28 normal bladder tissue samples (11). TNFRSF14 expression levels in normal and bladder cancer tissue were analyzed using the limma package (version 3.32.8) from Bioconductor (12).

T24 and SW780 bladder cancer cell lines, and SV-HUC-1 normal human bladder epithelium cell line were purchased from the Shanghai Cell Bank at the Chinese Academy of Sciences (Shanghai, China). SV-HUC-1 cells were cultured in Corning™ cellgro™ F12K (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.). T24 and SW780 bladder cancer cell lines, and SV-HUC-1 cells were cultured in a humidified atmosphere of 5% CO₂ at 37°C. The EJ-M3 cell line was established from the EJ cell line, which has been demonstrated to be a T24 derivative/contaminants. EJ-M3 cells are essentially highly invasive T24 derivative cells (13) and were obtained from EJ cells (14). EJ-M3 cells were generated, according to the methods described by Girnita et al (15). Briefly, 24-well plates were filled with media containing chemokine, and Transwell inserts coated with 100 µg Matrigel were placed into the wells and 3×10⁵ EJ cells were added onto the membrane. After 7 h in culture, the insert was removed and cells invading the lower chamber were further cultured. The medium was changed after 4–6 days. After reaching lamellar fusion, the cells were digested and subcultured. This process was repeated three times and each time the amount of Matrigel coating on the insert membrane was increased by 50 µg. Cells that were retained following this selection process were identified highly invasive EJ-M3 cells. These cancer cells were cultured in RPMI-1640 (HyClone; GE Healthcare, Chicago, IL, USA) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO₂ until 70–80% confluence was reached. Non-adherent cells were washed away after 3 days of culture and adherent cells were fed with fresh complete medium. The cells were subcultured at a 1:2 split ratio after reaching confluence.

Cells were cultured in 6-well plates in medium without antibiotics for 24 h prior to transfection, resulting in 70–80% confluence. For transient transfection, the TNFRSF14 gene was subcloned into a pSG5-HA vector, generating pSG5-HA-TNFRSF14. The empty vector was used as a negative control for transfection. Cells were transfected by Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Specifically, cells were cultured in fresh medium without antibiotics, and when the T24 cells reached 70–80% confluence, 4 µg DNA (pSG5-HA-TNFRSF14) was combined with 250 µl Opti-MEM^® medium (Invitrogen; Thermo Fisher Scientific, Inc.) and 10 µl Lipofectamine^® 2000 was diluted in 240 µl Opti-MEM^®, which were incubated at room temperature for 5 min. DNA/Opti-MEM^® and Lipofectamine^® 2000/Opti-MEM^® were combined and incubated at room temperature for 20 min. A total of 500 µl plasmid/Lipofectamine^® complex was added to the cells, which were cultured at 37°C. Following 24 h, the transfection medium was replaced with fresh complete medium and cells were harvested for analysis of proliferation and other experiments.

siRNA against TNFRSF14 (si-TNFRSF14; cat. no. stQ0003897-1) and negative control (si-NC; cat. no. siN05815122147) were produced and purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). The negative control siRNA does not match any known mammalian GenBank sequences. Cells were seeded at a density of 3×10⁵/well in 6-well plates overnight and transfected with siRNA or si-NC at a final concentration of 100 nM using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Following 24 h of transfection, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to determine transfection efficiency.

RNA was extracted from cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was generated using PrimeScript RT reagent kit (cat. no. RR037A; Takara Biotechnology Co., Ltd., Dalian, China) at 37°C for 15 min. Synthesized first-strand cDNA was used as template, and GAPDH was applied for normalization. Primer sequences were used as follows: TNFRSF14 forward, 5′-CCAAGTGCAGTCCAGGTTAT-3′ and reverse, 5′-ATTGAGGTGGGCAATGTAGG-3′; GAPDH forward, 5′-GGTGTGAACCATGAGAAGTATGA-3′ and reverse, 5′-GAGTCCTTCCACGATACCAAAG-3′. The RT-qPCR reaction was performed at 95°C for 5 min, followed by 40 cycles of 95°C for 30 sec, 60°C for 45 sec and 72°C for 30 min using an ABI 7500 real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using SYBR Select Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol The relative expression levels of TNFRSF14 were analyzed and normalized to GAPDH using the 2^−ΔΔCq method (16).

The Cell Counting kit-8 (CCK-8) assay is a sensitive and accurate method for determining cell viability. In the present study, cell proliferation was evaluated using the CCK-8 assay at 24, 48 and 72 h after transfection, according to a previous study (17). Briefly, cells in the TNFRSF14 or si-TNFRSF14 transfection group and control transfection group were cultured in 100 µl RPMI-1640 supplemented with 10% FBS in 96-well plates and incubated at 37°C for 3 days. Subsequently, CCK-8 dye (BestBio, Shanghai, China), diluted 1:10 in cell culture medium, was added to each well and incubated for 2 h at 37°C. The absorbance was read using a microplate reader set at 450 nm. The 450 nm optical density value is proportional to the total number of live cells and was used to assess cell viability in transfected vs. control transfected cells. Triplicate wells were used and experiments were repeated at least three times.

Total protein was extracted from transfected and control transfected cells using 1 mM phenylmethylsulfonyl fluoride in 1 ml ice-cold radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China). Subsequently, protein centration was measured using the bicinchoninic acid assay. Denatured proteins (20 µg) were separated using 12% SDS-PAGE and transferred onto Immobilon-P polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked for 2 h with 5% skimmed milk powder in Tris-buffered saline containing 0.1% Tween-20 (TBST) and incubated overnight at 4°C with the following primary antibodies: Anti-protein kinase B (AKT; 1:1,000; cat. no. SAB4500797), anti-phosphorylated (p)-AKT (1:1,000; cat. no. SAB4301414), anti-P70 S6 kinase (P70; 1:1,000; cat. no. SAB4502683), anti-active caspase3-p17 (1:1,000; cat. no. SAB4503294) and anti-GAPDH (1:5,000; cat. no. SAB2108266; Sigma-Aldrich; Merck KGaA). Subsequently, membranes were washed three times with TBST at room temperature, followed by incubation with an horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1:5,000; cat. no. sc-2004; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at room temperature for 2 h. Membranes were washed a final time with TBST. GAPDH was used as the loading control. All bands were detected using Enhanced Chemiluminescent Western Blotting Detection kit (GE Healthcare Life Sciences, Little Chalfont, UK). Imaging and quantification of protein bands was conducted using the Bio-Rad Quantity One 1-D Analysis software version 4.6.9 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Following 24 h of transfection, cells were harvested by trypsinization, washed with pre-cooled PBS and fixed overnight with 70% ethanol at −20°C. T24 or EJ-M3 cells were washed twice, resuspended in PBS containing 0.1 mg/ml RNase A and 0.1% Triton X-100 in the dark for 30 min at 37°C. A total of 1×10⁶ cells were double stained with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) using the Apoptosis Detection kit (BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer's protocol. The stained cells were acquired on a FACScan flow cytometer (BD Biosciences) equipped with a 488 nm argon laser and the data were analyzed using Cell Quest software version 6.0 (BD Biosciences). Cells were categorized into viable (Annexin V-FITC⁻/PI⁻), dead (Annexin V-FITC⁻/PI⁺), early apoptotic (Annexin V-FITC⁺/PI⁻) or apoptotic cells (Annexin V-FITC⁺/PI⁺). The percentage of apoptotic cells in the experiment group was compared with the control transfection group. All the samples were measured in triplicate.

In the present study, SPSS software version 18.0 (SPSS, Inc., Chicago, IL, USA) was used to conduct all statistical analyses. Each assay was performed at least three times. The data are presented as the mean ± standard deviation. The means between two groups were compared using Student's t-test. One-way analysis of variance followed by Student-Newman-Keuls post hoc test was used to compare the means of multiple groups. The Kaplan-Meier method was used to evaluate the prognostic value of TNFRSF14 in bladder cancer, and the Mantel-Cox log-rank test was used to determine the statistical significance of difference between survival curves. P<0.05 was considered to indicate a statistically significant difference.

The association between TNFRSF14 expression levels and prognosis of bladder cancer was assessed using RNA-Seq data from the TCGA database. The results indicated that TNFRSF14 expression levels were downregulated in the bladder tissues of patients with bladder cancer compared with healthy controls (Fig. 1A; P<0.05). Based on the median value of TNFRSF14 expression, patients were divided into TNFRSF14 low and TNFRSF14 high expression groups. As shown in Fig. 1B, the overall survival was significantly longer in the TNFRSF14 high expression group compared with in the TNFRSF14 low expression group.

TNFRSF14 expression levels in three human bladder cancer cell lines (T24, SW780 and EJ-M3) and a normal human bladder epithelium cell line (SV-HUC-1) were measured by RT-qPCR. The results demonstrated TNFRSF14 expression levels were lower in all bladder cancer cell lines compared with in the SV-HUC-1 cell line (Fig. 2). Notably, the expression levels of TNFRSF14 in T24 cells were lower compared with in SW780 and EJ-M3 cells. Therefore, T24 cells were used to perform overexpression experiments, whereas EJ-M3 cells were used to conduct silencing assays.

To investigate the role of TNFRSF14 in T24 or EJ-M3 cell proliferation, overexpression and silencing experiments were conducted using pSG5-HA-TNFRSF14 and si-TNFRSF14, respectively. RT-qPCR analysis was conducted 72 h after transfection to measure transfection efficacy. The results in Fig. 3A indicated that TNFRSF14 expression levels were significantly increased in T24 cells after transfection (P<0.05). As demonstrated in Fig. 4A, TNFRSF14 expression levels were decreased in EJ-M3 cells after gene silencing.

Subsequently, the effect of TNFRSF14 overexpression on cell proliferation was evaluated using the CCK-8 assay at 24, 48 and 72 h after transfection. As demonstrated in Fig. 3B, TNFRSF14 transfection led to a reduction in cell viability of T24 cells, which was statistically significant compared with control transfection group after 72 h of transfection (P<0.05; Fig. 3B). The results indicated TNFRSF14 overexpression decreased cell proliferation of bladder cancer cells.

In addition, CCK-8 assay was used to measure the effect of TNFRSF14 silencing on cell proliferation of EJ-M3 cells after 24, 48 and 72 h. Compared with control transfected cells, TNFRSF14 silencing resulted in increased cell viability of EJ-M3 cells, particularly at 72 h after transfection (P<0.05; Fig. 4B). The results indicated that TNFRSF14 silencing increased cell proliferation of bladder cancer cells.

To evaluate the effect of alterations in TNFRSF14 expression levels on apoptosis of T24 or EJ-M3 cells, Annexin V-FITC and PI double staining was utilized to detect apoptotic cells, including early (Annexin V-FITC⁺/PI⁻) and late apoptotic (Annexin V-FITC⁺/PI⁺) cells. As presented in Fig. 5, TNFRSF14 overexpression promoted increased apoptosis in transfected cells compared with in control cells (5.52 vs. 1; P<0.05), indicating that TNFRSF14 overexpression may have a direct therapeutic effect against the human T24 bladder cancer cell line. As demonstrated in Fig. 6, TNFRSF14 silencing decreased apoptosis in transfected cells compared with in control cells (0.51 vs. 1; P<0.05). The results suggested that suppression of cell viability following TNFRSF14 overexpression was associated with induction of cell apoptosis.

To determine whether abnormalities in TNFRSF14 expression levels affect PI3K signaling, numerous proteins were detected using western blot analysis, including p-AKT, caspase3-p17 and P70. As demonstrated in Fig. 7, TNFRSF14 overexpression significantly increased caspase3-p17 expression levels in T24 cells (P<0.05), which was in line with the cell apoptosis results obtained in the present study. Furthermore, TNFRSF14 overexpression significantly decreased p-AKT and P70 expression levels in the T24 bladder cancer cell line (Fig. 7B, P<0.05). Conversely, TNFRSF14 silencing significantly decreased caspase3-p17 expression levels in EJ-M3 cells (P<0.05), which was again in line with the cell apoptosis results. Furthermore, TNFRSF14 silencing significantly increased p-AKT (P<0.01) and P70 (P<0.05) expression levels in the EJ-M3 bladder cancer cell line (Fig. 8).