A total of 136 BLCA tissue and adjacent normal tissue samples (>5 cm away from the tumor edge) were collected from randomly selected patients undergoing surgical resection between May 2008 and July 2013 at The First Affiliated Hospital of Dalian Medical University (Dalian, China). After surgical resection, all samples were routinely processed and paraffin-embedded. The inclusion criterion was primary and metastatic BLCA tissues of patients. Tissue specimens from patients who received radiotherapy or chemotherapy before surgical resection were excluded. The age range of the patients was 34–82 years with a mean age of 58.8 years. The ratio of male to female was 97:39. Tumors were staged according to the 7th Edition of the American Joint Committee on Cancer (19). Of the 136 BLCA tissue, 39 were determined as early stage (I/II) and 97 as late stage (III/IV). The present study was approved by the Institutional Research Ethics Committee of the First Affiliated Hospital of Dalian Medical University (approval no. LCKY2015-08; Dalian, China), and written informed consent was provided by the patients with BLCA who underwent surgery.

SV-HUC-1 normal bladder epithelial cell, 5637 and T24 BLCA cell lines were purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. SV-HUC-1 normal bladder epithelial cells were cultured in F12/DMEM (Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Thermo Fisher Scientific, Inc.). The 5637 and T24 BLCA cell lines were cultured inRPMI-1640 medium (Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Thermo Fisher Scientific, Inc.). All cell cultures were supplemented with 1% penicillin/streptomycin, and cells were incubated with 5% CO₂ at 37°C.

TCGA (https://cancergenome.nih.gov/; Project ID:TCGA-BLCA) project provided mRNA expression profile data on 408 BLCA cases: 433 samples, including 19 normal and 414 BLCA samples. Some patients provided both cancer tissue and adjacent normal tissue and some patients provided >2 cancer tissues. Therefore, the number of patient cases was less than the tissue samples obtained from the TCGA database. The ‘edgeR’ package (http://www.bioconductor.org/help/search/index.html?q=edger/; release number 3.13; Bioconductor) in R software (https://www.r-project.org/; release number 3.6.1) was used to identify the differentially expressed mRNAs of glycosyltransferases in BLCA tissues compared with normal bladder tissues. The following thresholds were used to identify significant differentially expressed genes: False discovery rate (FDR)<0.001 and |log₂(fold change)|>2. After screening of differentially expressed genes, the differentially expressed gene ID were obtained. The gene ID were annotated using ENSEMBL (https://www.ensembl.org/) to get the differentially expressed gene symbol. Then, the expression levels of ST8SIA1 in BLCA and normal tissue were assessed based on the data obtained from GEPIA (http://gepia.cancer-pku.cn/).

BLCA and adjacent normal tissue were fixed with 4% paraformaldehyde at room temperature for 24 h. After paraffin embedding, 4 µm-thick sections were cut and mounted onto slides. Next, slides were deparaffinized with xylene, rehydrated by a series of graded alcohol solutions as follows: 100, 100, 95, 85 and 75%. The antigen was retrieved with 10 mM sodium citrate buffer at 95°C for 20 mi and immersed in 3% hydrogen peroxide in methanol at room temperature for 15 min to block the endogenous peroxidase activity. The slides were washed with PBS twice, blocked with 5% goat serum (Beijing Zhong Shan-Golden Bridge Biological Technology Co., Ltd.) at room temperature for 20 min and incubated with a primary anti-ST8SIA1 antibody (1:80; cat. no. 24918-1-AP; ProteinTech Group, Inc.) at room temperature for 1 h. Following washing with PBS, the PBS surrounding the tissue was wiped dry and the samples were incubated with a secondary horseradish peroxidase (HRP)-conjugated IgG antibody (1:100; cat. no. sc-2357; Santa Cruz Biotechnology, Inc.) at room temperature for 45 min. The sections were then treated with 3,3′-diaminobenzidine at room temperature for 30 sec, counterstained with hematoxylin at room temperature for 5 min, dried, sealed with a neutral gum and observed under a light microscope. The immunostaining intensity was then evaluated. The immunostaining for ST8SIA1 expression was scored using ‘−’, ‘+’, ‘++’ and ‘+++’, to indicate a positive staining percentage of <5, 5–25, 25–50 and >50%, respectively. All specimens were examined by 2 pathologists of The First Affiliated Hospital of Dalian Medical University (Dalian, China), who were blinded to the clinical data.

T24 and 5637 cells were transfected with the recombinant pcDNA3.1/ST8SIA1 or mock vectors. Non-transfected and mock vector transfected cells were used as the control. The pcDNA3.1/ST8SIA1 and mock vectors were synthesized by Public Protein/Plasmid Library (http://www.geneppl.com/index.php; pcDNA3.1/ST8SIA1, cat. no. BC046158; mock vector, cat. no. V790-20). Cells were transfected with a mixture of 5 µg plasmids and 10 µl Lipofectamine^® 2000 transfection reagent (Thermo Fisher Scientific, Inc.) at 37°C for 7 h. To select a stable population of transfected cells, the culture medium was replaced with complete medium containing 600 mg/ml G418 (Merck KGaA) after 48 h. After 2 months of screening with 300 mg/ml G418, two cell lines stably expressing ST8SIA1 (ST8SIA1-1 and ST8SIA1-2) were established. The modified expression was confirmed by reverse transcription-quantitative PCR (RT-qPCR) and western blotting.

Total RNA was extracted from the tissue samples and cultured cells using TRIzol^® reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. RNA was subjected to reverse transcription to generate cDNA using a PrimeScript RT reagent kit (Takara Bio, Inc.) for 2 min at 42°C, 15 min at 37°C and 5 sec at 85°C and stored at −20°C and amplified by subsequent qPCR using a qPCR SYBR GreenSuperMix (TransGen Biotech Co., Ltd.). The amplification conditions were as follows: 94°C for 30 sec, followed by 94°C for 5 sec and 60°C for 34 sec for 40 cycles. Relative changes in gene expression were analyzed using the 2^−ΔΔCq method (20). GAPDH was used as an internal control. Specific primers for GAPDH and ST8SIA1 were purchased from Shanghai GenePharma Co., Ltd. The primer sequences used were as follows: ST8SIA1 forward, 5′-CATTGAAGAAATGCGCGGTGG-3′ and reverse, 5′-GTTCTGAAACCTTTGCCGAATTATG-3′; GAPDH forward, 5′-TCCAAAATCAAGTGGGGCGA-3′ and reverse, 5′-AAATGAGCCCCAGCCTTCTC-3′.

Total protein was extracted from the cultured cells and specimens using RIPA buffer (Beyotime Institute of Biotechnology) and PMSF (dilution, 1:100; Beyotime Institute of Biotechnology). A bicinchoninic acid assay kit (Beyotime Institute of Biotechnology) was used for quantification. Equal amounts of protein (30 µg/lane) were separated using 12% SDS-PAGE and transferred onto PVDF membranes (Pall Life Sciences). The membranes were blocked at room temperature for 2 h with 5% fat-free milk prepared in 1XTris-Buffered Saline, 0.1% Tween^® 20 Detergent (TBST). Western blot analysis was performed using phosphorylated (p-)STAT3 (dilution, 1:500; cat. no. 39596; ProteinTech Group, Inc.), STAT3 (dilution, 1:500; cat. no. AP0366; Bioworld Technology, Inc.), ST8SIA1 (dilution, 1:500; cat. no. 24918-1-AP; ProteinTech Group, Inc.), Janus kinase 2 (JAK2; dilution, 1:500; cat. no. 17670-1-AP; ProteinTech Group, Inc.), p-JAK2 (dilution, 1:500; cat. no. AP0531; ABclonal Biotech Co., Ltd.), cyclin D1 (dilution, 1:500; cat. no. 26939-1-AP; ProteinTech Group, Inc.), MMP2 (dilution, 1:500; cat. no. 10373-2-AP; ProteinTech Group, Inc.), proliferating cell nuclear antigen (PCNA; dilution, 1:500; cat. no. 10205-2-AP; ProteinTech Group, Inc.), Bcl2 (dilution, 1:500; cat. no. 12789-1-AP; ProteinTech Group, Inc.) and GAPDH (dilution, 1:500; cat. no. 10494-1-AP; ProteinTech Group, Inc.) antibodies. The membranes were incubated with primary antibodies at 4°C overnight. After three washes with TBST, the membranes were incubated with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (dilution, 1:5,000; cat. no. SA00001-2; ProteinTech Group, Inc.) at room temperature for 1 h. The immunoreactivity was detected using an electrochemiluminescence kit (Advansta, Inc.) and processed using Image Lab software v.5.2 (Bio-Rad Laboratories, Inc.).

Cell proliferation was determined using aCCK-8 assay (Dojindo Molecular Technologies, Inc.). BLCA cells were seeded into a 96-well plate at a density of 5×10³ cells per well in triplicate. Cells were incubated at 37°C for 12, 24, 36 or 48 h, and 10 µl CCK-8 reagent was added into each well. Cells were incubated at 37°C with 5% CO₂ for 2 h. A microplate reader (Thermo Fisher Scientific, Inc.) was used to measure the optical density values at 450 nm.

BLCA cells in the logarithmic growth phase were digested into a single-cell suspension. Cells were seeded into 6-well culture plates (Corning, Inc.) at a density of 200 cells per well and incubated at 37°C with 5% CO₂ for 14 days. Following three washes with PBS, cells were fixed with paraformaldehyde (4%) at room temperature for 15 min and stained with 0.1% crystal violet at room temperature for 30 min. The colonies were imaged using a light optical microscope and the number of colonies of >50 cells was counted using ImageJ software v.4.0 (National Institutes of Health).

T24 and 5637 cells were collected as a single cell suspension and fixed with 75% alcohol at 4°C overnight. The cells were centrifuged for 5 min at 1,000 × g at room temperature and then washed with ice-cold PBS. Cells were stained by incubation with propidium iodide (50 µg/ml; Beyotime Institute of Biotechnology) combined with RNase A (50 µg/ml; Beyotime Institute of Biotechnology) for 30 min at 37°C. Cells were detected using a flow cytometer (Accuri C6 Cytometer; BD Biosciences) and then analyzed with FlowJo software v.10.2 (TreeStar, Inc.). Signals were acquired from at least 1×10⁶ cells per sample.

Cell migration was analyzed using a wound healing assay. Non-transfected T24 or 5637, mock, ST8SIA1-1 and ST8SIA1-2 cells (2.5×10⁶) were respectively transferred to a six-well plate and allowed to grow to 100% confluence in complete medium. A 200-µl sterile pipette tip was used to scratch a gap through the monolayer. The cellular debris was dislodged by washing with PBS thrice. The cells were then washed and imaged under a light microscope at a magnification of ×10. Following cell culture with the serum-free medium for 12 h, the same areas were imaged again. Cell mobility was assessed by calculating the remaining open area of the wound using ImageJ software v.4.0 (National Institutes of Health) and analyzed with the following formula: (Width 0 h-Width 12 h)/Width 0 h ×100%.

A total of ~2×10⁴ cells in 150 µl RPMI-1640 medium without FBS were added to the upper compartment of a 24-well Transwell chamber (pore size, 8.0 µm; diameter, 6.5 mm; Corning, Inc.), and 500 µl RPMI-1640 medium with 20% FBS was added to the lower compartment. Following incubation at 37°C for 48 h in a 5% CO₂ incubator, the cells on the upper surface of the filter were completely removed using a cotton swab. The 24-well Transwell chambers were coated with 40 µl Matrigel^® for 30 min at 37°C on the upward-facing side of the polycarbonate membrane for the invasion assay. Subsequently, 4×10⁴ cells in 150 µl serum-free medium were added to the upper chamber and 500 µl medium containing 20% FBS was added into the lower chambers. After incubation for 48 h at 37°C, the cells were fixed with 4% paraformaldehyde at room temperature for 15 min and stained with 0.1% crystal violet at room temperature for 10 min. The cells were imaged using a light microscope and counted using ImageJ software v.4.0 (National Institutes of Health).

All experiments were repeated at least three times. Quantitative data are presented as the mean ± SD. Statistical analysis was performed using GraphPad Prism 5.0 software (GraphPad Software, Inc.). Differences between two groups were analyzed using Student's unpaired t-test, whereas those among multiple groups were analyzed using one-way ANOVA followed by the post hoc Tukey's test. The χ² test was performed for the comparisons of categorical variables. P<0.05 was considered to indicate a statistically significant difference.

To identify the STs associated with the malignant progression of BLCA, TCGA (https://cancergenome.nih.gov/) was utilized to analyze the differentially expressed glycosyltransferse genes between 414 BLCA and 19 normal tissue samples. Using FDR<0.001 and |log₂(fold change)|>2 as the selection criteria for the standardized data, 13 glycosyltransferases, including only 1 ST (ST8SIA1) with low expression levels in BLCA, were identified (Fig. 1A and B). The expression levels of ST8SIA1 were further assessed based on the data obtained from GEPIA (http://gepia.cancer-pku.cn/). Decreased ST8SIA1 expression was identified in BLCA tissues compared with normal tissues (Fig. 1C). These results suggested that ST8SIA1 may serve an important role in the development of BLCA.

To validate the results obtained using TCGA, immunohistochemical staining and western blot analysis were used to detect the expression levels of ST8SIA1 in BLCA. The data indicated that ST8SIA1 protein showed cytoplasm staining and the staining intensity of ST8SIA1 in high-grade cancer tissues was lower compared with that in low-grade cancer and adjacent normal tissues (Fig. 2A). The expression levels of ST8SIA1 in 8 pairs of BLCA and adjacent normal tissues were detected by western blot analysis, and the results revealed that the protein expression levels of ST8SIA1 were significantly reduced in the tumor tissue samples (Fig. 2B). Furthermore, ST8SIA1 expression was detected in two BLCA cell lines (5637 and T24) and a normal bladder epithelial cell line (SV-HUC-1). The relative protein expression levels of ST8SIA1 were significantly lower in the BLCA cell lines compared with the normal bladder epithelial cell line (Fig. 2C). The association between ST8SIA1 protein expression and major clinicopathological characteristics was further analyzed in 136 patients with BLCA. ST8SIA1 expression was negatively associated with tumor grade and invasiveness (Table I). These data indicated that ST8SIA1 may act as a tumor suppressor in BLCA progression.

To explore the roles of ST8SIA1 in the malignant progression of BLCA cells, T24 and 5637 cell lines stably overexpressing ST8SIA1 (ST8SIA1-1 and ST8SIA1-2) were established. RT-qPCR and western blot analysis demonstrated that T24 (Fig. 3A and B, top panel) and 5637 (Fig. 3A and B, bottom panel) cell lines with a stable overexpression of ST8SIA1 were successfully constructed using molecular cloning and liposome transfection technology. To investigate the effects of ST8SIA1 upregulation on the proliferation and colony formation of BLCA cells, CCK-8, colony formation and flow cytometry assays were performed. The CCK-8 assay revealed that the proliferation of T24 (Fig. 3C, left panel) and 5637 (Fig. 3C, right panel) cells stably overexpressing ST8SIA1 (ST8SIA1-1 and ST8SIA1-2) was significantly reduced compared with that of mock and non-transfected cells. To further confirm this result, a colony formation assay was performed and demonstrated that ST8SIA1 overexpression decreased the number of T24 (Fig. 3D, top panel) and 5637 (Fig. 3D, bottom panel) cell colonies. The flow cytometry results revealed that ST8SIA1 overexpression markedly arrested T24 (Fig. 3E, top panel) and 5637 (Fig. 3E, bottom panel) cells in the S phase of the cell cycle. These results suggested that ST8SIA1 downregulated the proliferation ability of 5637 and T24 cells.

To further verify the effects of ST8SIA1 on the malignant progression of BLCA cells, wound healing and Transwell assays were used to analyze migration and invasion following ST8SIA1 overexpression in 5637 and T24 cells. The results of the wound healing assay demonstrated that the motility of T24 (Fig. 4A, top panel) and 5637 (Fig. 4A, bottom panel) cells stably overexpressing ST8SIA1 (ST8SIA1-1 and ST8SIA1-2) was markedly reduced compared with that of mock and non-transfected cells within 12 h. Furthermore, the frequencies of T24 (Fig. 4B and C, top panel) and 5637 (Fig. 4B and C, bottom panel) cells that migrated and invaded into the lower chamber were significantly decreased following ST8SIA1 overexpression. These data indicated that ST8SIA1 attenuated the migration and invasion ability of 5637 and T24 cells.

To explore the molecular mechanisms of the antitumor effect of ST8SIA1 in BLCA cells, the activation and expression levels of JAK/STAT, as well as the involved signaling molecules were detected by western blot analysis in 5637 and T24 cells following ST8SIA1 overexpression. The results demonstrated that ST8SIA1 overexpression in T24 (Fig. 5A) and 5637 (Fig. 5B) cells markedly reduced the ratio of the phosphorylated JAK2/total JAK2 and the phosphorylated STAT3/total STAT3, as well as the expression levels of the downstream targets of JAK/STAT signaling pathway, such as MMP2, PCNA, cyclin D1 and Bcl2 compared with mock and non-transfected cells. These results suggested that ST8SIA1 inhibited the activation of JAK2/STAT3 signaling pathway.