The anti-LAPTM4B rabbit polyclonal [for immunohistochemistry (IHC), 1:100 dilution; for western blot analysis, 1:1,000 dilution; cat. no. PA5-43047] antibody was purchased from Thermo Fisher Scientific, Inc., and the anti-β-actin mouse monoclonal (1:1,000 dilution; cat. no. ab8226) was purchased from Abcam.

The following primers were used: LAPTM4B forward, 5′-GGAACTGCTACCGATACATCAA-3′ and reverse, 5′-TCACAGTGGCATCATCATACG-3′; β-actin forward, 5′-CAGCTCACCATGGATGATGATATC-3′ and reverse, 5′-AAGCCGGCCTTGCACAT-3′.

The pLVX shRNA plasmid of LAPTM4B (cat. no. 101111) was purchased from OriGene Technologies, Inc., and has the following targeted sequence: 5′-GGTCGCCTTCGGAGCGAAGGGTA-3′.

A total of 111 human bladder tumor tissues were collected from patients (mean age 67.5; 71 males and 40 females) with bladder cancer, and adjacent tissues (5 mm from the tumor tissues) following surgery at Liaocheng People's Hospital (Shandong, China) between September 2015 and June 2018. The clinicopathological features, including age, sex, tumor stage, tumor grade, lymph node metastasis and recurrence (tumor redevelops at the original site) were respectively recorded and analyzed at Liaocheng People's Hospital.

To further investigate the association between LAPTM4B expression level and bladder cancer, IHC was performed. In brief, the samples were fixed with 10% formalin for 24 h at 98°C, embedded with resin (Epoxy resin; Sigma-Aldrich; Merck KGaA), and divided into 5-µm thick sections. The sections were dewaxed with xylene at 65°C, then rehydrated in a gradient ethanol series. The samples were immersed in citrate buffer (pH, 6.0) at 98°C for 30 min and placed in a microwave for incubation for 10 min for antigen retrieval. Then, hydrogen peroxide was added to block endogenous peroxidase activity and the samples were incubated at room temperature for 10 min, followed by blocking with 2% BSA (Sigma-Aldrich; Merck KGaA) for 20 min at room temperature. Subsequently, the samples were incubated with the LAPTM4B antibody at room temperature for 2 h. Lastly, the samples were washed with PBS, 4 times and incubated with the secondary antibody (rabbit; 1:200 dilution; cat. no. ab205718; Abcam). Diaminobenzidine was used as a chromogen substrate. Images were captured using an Olympus inverted fluorescence microscope (IX71; Zeiss AG).

The proportion of positive stained cells was scored as follows: 0, negative; 1, 10–50% positive and 2, >50% positive staining. The staining intensity was assessed on a score of 0 (negative level staining), 1 (low level staining), and 2 (high level staining). The expression levels of LAPTM4B were further examined based on a staining index: Staining intensity score plus staining percentage score. Staining index, 0–2 indicated low expression, while 3/4 indicated high expression. The Kaplan-Meier survival analysis of overall survival (OS) and progression-free survival (PFS) rates was performed between LAPTM4B low and high expression groups. OS was defined as the time from recruitment into the study to death from any cause. PFS was defined as the period after treatment when the disease was stable and did not progress. The development of the disease was followed up every 6 months.

The T24 and 5637 bladder tumor cell lines were purchased from American Type Culture Collection. Both the cell lines were maintained in RPMI-1640 medium, supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and incubated at 37°C in a humidified incubator with 5% CO₂.

The shRNA plasmids of LAPTM4B were transfected into the bladder cancer cell lines using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 4 h. The scrambled control plasmid (5′-ATGGTACTGACCTCCAGAG-3′) was used as the negative control (NC). A total of 1×10⁵ cells per well were seeded into 6-well plates and a total of 2 groups were used: sh-LAPTM4B group (0.5 µg) and sh-NC group (0.5 µg). Knockdown efficiency was measured by both reverse transcription-quantitative PCR (RT-qPCR) and western blot analysis after 48 h. These cells were used to assess the association between LAPTM4B expression level and cellular processes. Subsequently the cells in which LAPTM4B was stably knocked down were screened and used for the in vitro and in vivo assays.

Total RNA was isolated from the T24 and 5637 cell lines or tumor tissues using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.). Subsequently, the total RNA was reverse transcribed using M-MLV reverse transcriptase at 42°C for 60 min (Promega Corporation). qPCR was conducted using SYBR PrimeScript RT-PCR kit II (cat. no. DRR083; Takara Biotechnology Co., Ltd.) and the relative expression levels of LAPTM4B was normalized to the mRNA expression levels of β-actin. The following thermocycling conditions were used: Initial denaturation at 95°C for 3 min; followed by 30 cycles of denaturation at 95°C for 30 sec, annealing at 58°C for 30 sec and extension at 72°C for 30 sec. The 2^−ΔΔCq method was used to quantify the results (21).

The bladder cancer cells or tissues were lyzed with lysis buffer [60 mM Tris-HCl (pH 6.8), 2% SDS, 20% glycerol, 0.25% bromophenol blue, 1.25% 2-mercaptoethanol and protease inhibitor cocktail]. Protein determination was performed using the BCA method. A total of 10 µg of each protein sample was loaded per lane and separated using 10% SDS-PAGE. Then, the proteins were transferred onto PVDF membranes (cat. no. IPSN07852; MilliporeSigma) and the membranes were blocked with 5% skimmed milk in TBS-Tween-20 (0.5%) buffer and subsequently incubated with the primary antibodies for 2 h at room temperature. Following which, the membranes were incubated with the secondary antibody (rabbit; 1:5,000 dilution; cat. no. ab205718; Abcam) for 45 min at room temperature. Each blot was subsequently visualized with an ECL kit (cat. no. RPN 2109; GE Healthcare).

Approximately 1,000 bladder cancer cells were added into 6-well plates and cultured at 37°C for 48 h after transfection. After 14 days, the cells were subsequently fixed with 4% paraformaldehyde for 30 min at room temperature and stained with 0.2% crystal violet at room temperature for 20 min, then washed with PBS for 4 times. Colony numbers were manually counted using an Olympus inverted fluorescence microscope and images captured (IX71; Zeiss AG). A colony was counted when it included >100. The number of colonies per visual field area visible under the microscope was counted.

The cells, transfected with shRNA plasmids, were added into 96-well plates for 3 days. Subsequently, the cells were treated with MTT for 3 h, then 200 µl dimethyl sulfoxide was added to dissolve the purple formazan and the OD value was measured using a microplate reader at 570 nm (22).

The T24 and 5637 cells, transfected with shRNA plasmid, were subsequently used for Transwell invasion assays. The upper chambers were coated with 20% Matrigel in RPMI-1640 medium and incubated at 37°C for 1 h. A total of 1×10⁵ cells in 150 µl serum-free medium was then added into the upper chambers of the inserts. Medium with 10% FBS was added into the bottom chamber to stimulate cell invasion. After 24 h incubation at 37°C, the cells were fixed with 4% paraformaldehyde for 25 min at room temperature and stained with 0.2% crystal violet for 15 min at room temperature. Then, images were captured and the number of invaded cells were counted using an Olympus inverted fluorescence microscope (IX71; Zeiss AG).

Both the T24 and 5637 cell lines were transfected with shRNA plasmids and cultured to form confluent monolayers. After the confluence reached 100%, the wound was created using a 20 µl pipette tip. Cell debris was washed with PBS 3 times. The serum-free culture medium was added to the cells and the cells were cultured. Images of the wounds were captured at 0 and 24 h, and the extent of healing was measured. Migration ability was measured using ImageJ software (v1.8.0; National Institutes of Health) and quantified as a percentage of wound width (post-healing wound width/pre-healing wound width).

All animal procedures were approved by the Institutional Animal Care and Use Committee of Liaocheng People's Hospital (Shandong, China). A total of 12 male nude BalB/c mice (8-weeks-old; 18–22 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and fed with food and water ad libitum and at specific pathogen-free conditions (20°C; 60% humidity and alternating 12-h light/dark cycles).

For the tumor growth assay, the T24 cell line was stably transfected with shRNA plasmids, then 3×10⁶ cells were inoculated into the Balb/c nude mice and tumors formed 2 weeks later. The tumor growth curves were analyzed 7 weeks later. The tumor size was calculated using the following formula: Tumor volume=length × width × width/2. The mice were euthanized with an intraperitoneal injection of 120 mg/kg sodium pentobarbital before the tumor was removed. The hearts of the mice were then monitored and death was confirmed by cardiac arrest. There were six mice in the control and LAPTM4B knockdown group.

To further detect the expression level of the LAPTM4B in the tumor tissues from the mice, IHC was performed as aforementioned. Subsequently, the staining intensity of LAPTM4B in the tumor tissues was measured using ImageJ software (v1.8.0; National Institutes of Health) and analyzed statistically using the staining intensity, which was presented as the median ± interquartile range, including the Q1/Q3 quartiles.

For the lung metastasis assay, 1×10⁶ T24 cells, transfected with shRNA plasmids, were resuspended in 150 µl PBS buffer and injected into the tail vein to stimulate lung metastasis. After 7 weeks, all the mice were sacrificed and the lungs were isolated and images were captured, and the metastasis degree was measured.

GraphPad v6.0 software (GraphPad Software, Inc.) was used for statistical analysis. The in vitro and in vivo results were presented as the mean ± standard deviation. The analysis between clinical features and LAPTM4B expression was performed using a χ² test. A Student's t-test was used for statistical comparisons. The survival rates between cancer progression and LAPTM4B expression was analyzed using the Kaplan-Meier method and the log-rank test. IHC staining intensity in animal experiments was presented as the median + interquartile range and analyzed using Mann-Whitney U test. Each experiment was repeated 3 times. P<0.05 was considered to indicate a statistically significant difference.

To investigate the potential role of LAPTM4B in bladder cancer progression, IHC, clinicopathological characteristic analysis and Kaplan-Meier analysis were all conducted. The expression level of LAPTM4B in human tumor tissues from patients with bladder cancer, who underwent surgical resection was detected using IHC. According to the IHC staining results, LAPTM4B was mainly expressed in the cytoplasm and membrane of human bladder cancer tissues (Fig. 1A). A total number of 111 surgical samples were classified into LAPTM4B low and high-expression groups according to the staining intensity (Fig. 1A). In comparison, corresponding non-tumor tissues showed low expression level of LAPTM4B (Fig. 1B). Based on the expression level in bladder tumor tissues, 34 patients showed low LAPTM4B expression, whereas 77 showed high expression (Table I).

The clinicopathological characteristics of patients with bladder cancer were analyzed between the LAPTM4B low and high expression level groups. Patient age, sex, tumor size, grade and lymph node metastasis, were recorded and analyzed. Based on the analysis results, no significant difference was found in these features between the LAPTM4B low and high expression groups (Table I). Notably, LAPTM4B expression level in the bladder tumor tissues was significantly associated with the tumor stage (P=0.004) and recurrence (P=0.014).

Kaplan-Meier survival analysis was used to investigate the association between LAPTM4B and the prognosis of patients with bladder cancer. Data showed that LAPTM4B expression was associated with OS and PFS rates (Fig. 1C). These results indicated that LAPTM4B was associated with poor prognosis in patients with bladder cancer.

To further investigate the mechanism of LAPTM4B regulation in the progression of bladder cancer, a shRNA targeting LAPTM4B was transfected into the two types of bladder cancer cell lines, T24 and 5637, to suppress the expression of LAPTM4B. qPCR (Fig. 2A) and western blot analysis (Fig. 2B) suggested that transfection with LAPTM4B shRNA effectively knocked down LAPTM4B expression in the T24 and 5637 cell lines.

To investigate the potential effect of LAPTM4B on the proliferation of bladder cancer, colony formation assays were conducted. It was found that LAPTM4B knockdown markedly inhibited the proliferation of the T24 and 5637 cell lines, from the decrease in cell colony numbers (Fig. 3A). Similarly, there was lower proliferation from the MTT assay, as shown from the lower OD values in Fig. 3B.

The effects of LAPTM4B on the migration and invasion of bladder cancer cells were also investigated. As expected, knockdown of LAPTM4B also slowed down the extent of wound closure in the two bladder cancer cell lines (Fig. 3C). In addition, T24 and 5637 cells exhibited a significantly lower invasive ability following LAPTM4B knockdown, with markedly decreased the number of invasive cell (Fig. 3D).

Taken together, it was found that LAPTM4B was associated with the regulation of bladder cancer cell proliferation, and migration and invasion in vitro.

According to the in vitro findings, knockdown of LAPTM4B reduced the proliferation, and migration and invasion of bladder cancer cell lines; therefore, the role of LAPTM4B in the growth and metastasis of bladder cancer was investigated further in mice.

The T24 cell lines, transfected with control or LAPTM4B shRNA plasmids, were inoculated into Balb/c nude mice and tumor formation began 2 weeks later. The notable low mRNA expression levels of LAPTM4B in the knockdown group (Fig. 4A), confirmed successful transfection. Representative images of the tumors were captured and are shown in Fig. 4B. The maximum volume of tumor in mice was 10 mm. According to the results, the volume of the tumors from the LAPTM4B knockdown mice was significantly smaller compared with that in the control mice (Fig. 4B). Furthermore, lung metastasis assay was performed in the mice and the volume of lung metastasis in the mice with LAPTM4B-knockout T24 cells was markedly decreased compared with that in the mice transfected with the control shRNA (Fig. 4C).

IHC further confirmed the knockdown of LAPTM4B expression in the tumor tissues (Fig. 4D). Therefore, all these data confirmed that LAPTM4B was associated with the regulation of bladder cancer growth and development in vivo.