Cell lines and cell culture
Human bladder mucosal epithelial HCV29, human benign non-muscle-invasive BLCA KK47, and highly malignant invasive BLCA YTS-1 cell lines were kindly donated by Dr. S. Hakomori (The Biomembrane Institute, Seattle, WA, USA) . Human uroepithelial SV-HUC-1 and transitional carcinoma T24, J82, 5637, and RT4 cell lines were from the Cell Bank of the Chinese Academy of Sciences (Shanghai). SV-HUC-1 cells were cultured in F12K medium (Invitrogen; Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Biological Industries; Beit Haemek, Israel) and 1% antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin sulfates) at 37 °C in 5% CO2 atmosphere. Bladder cell lines were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% antibiotics at 37 °C in 5% CO2 atmosphere. All cells were used for the experiments between passages 3 and 15.
Tissue microarray (TMA) analysis
TMA analyses were performed as described previously . TMAs containing BLCA and paracarcinoma tissues were from Shanghai Outdo Biotech Co. In brief, TMAs were deparaffinized, rehydrated, subjected to antigen retrieval, blocked, incubated with biotinylated lectin or primary antibody overnight at 4 °C, rinsed with 1× PBS (0.01 mol/L phosphate buffer containing 0.15 mol/L NaCl, pH 7.4), incubated with HRP-streptavidin or secondary antibody for 1 h at 37 °C, visualized with DAB reagent, stained with hematoxylin, and photographed. Mean optical densities of the TMA staining signals were calculated using Image-Pro Plus software (Media Cybernetics; Silver Spring, MD, USA).
Total protein extraction
Monolayer cells at 70–80% confluence were rinsed with 1× PBS, added with RIPA buffer (50 mM Tris, pH 7.2, Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 10 mM MgCl2, 5% glycerol) containing protease inhibitor (NCM Biotech; Suzhou, China), and incubated for 30 min on ice. BLCA tissues were homogenized in a tissue homogenizer (Jingxin Technology; Shanghai) with 500 μL T-PER Tissue Protein Extraction Reagent (Thermo Scientific; San Jose, CA, USA) containing protease inhibitor. Cell and tissue lysates were sonicated and centrifuged, and supernatants were collected. Protein content was determined by BCA assay (Beyotime Institute of Biotechnology; Haimen, China).
Western blotting and lectin blotting
Western and lectin blotting were performed as described previously . Proteins were separated by SDS-PAGE and transferred onto PVDF membranes (Bio-Rad; Hercules, CA, USA). Membranes were blocked, probed with primary anti-C1GALT1 antibody (Santa Cruz Biotechnology; Santa Cruz, CA, USA), anti-FGFR3 antibody (Proteintech; Wuhan, China), anti-MUC16 antibody (Proteintech; Wuhan, China), anti-GAPDH antibody (Sigma-Aldrich; St. Louis, MO, USA) or biotin-conjugated T-antigen-reactive lectin (Vector Laboratories; Burlingame, CA, USA) overnight at 4 °C, and incubated with appropriate HRP-conjugated secondary antibody/ streptavidin. Bands were visualized using enhanced chemiluminescence kit with luminescent imaging (Tanon; Shanghai).
Enzyme-linked immunosorbent assay (ELISA)
ELISA was performed as described previously . In brief, 96-well ELISA plates (Jet Biofil; Guangzhou, Guangdong, China) were coated with serum samples from BLCA patients, blocked with BSA, rinsed with 1× PBS containing 0.05% Tween-20 (PBST), incubated with biotin-labeled peanut agglutinin (PNA), which specifically recognizes T antigen (Vector Labs), added with VECTASTAIN ABC reagent (Vector Labs), and visualized using TMB substrate kit (Beyotime). Optical density at wavelength 450 nm was determined by microplate reader.
O-glycan analysis by mass spectrometry (MS)
Monolayer cells at 70–80% confluence were rinsed with 1× PBS, scraped, and centrifuged. Pellets were added with lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 1% SDS, 1% protease inhibitor), sonicated on ice, centrifuged, and supernatants were collected. Proteins were desalted in a dialysis bag (molecular weight cutoff 8–14 kDa) for 72 h at 4 °C, and lyophilized. Lyophilized proteins were dissolved with 1 mL of 0.1 M NaOH, and incubated with 1 mL of 1 M NaBH4 for 15 h at 50 °C. The mixture was acidified to pH 4.0 with 50% acetic acid, desalted with HyperSep Hypercarb SPE Cartridges  (Thermo Scientific), and O-glycans were subjected to liquid chromatography/ electrospray ionization/ MS (LC-ESI-MS) (Thermo Scientific). ESI-MS data were acquired as described previously . Samples were injected into ESI source in a 50% methanol stream; parameters: flow rate 100 μL/min, spray voltage 4000 V, sheath gas flow rate 20 arb, auxiliary gas flow rate 5 arb, capillary voltage 37 V, tube lens voltage 250 V, capillary temperature 300 °C.
Knockdown of C1GALT1 and cHP1BP3
shRNA targeting C1GALT1 or backsplice junction of cHP1BP3 were respectively subcloned into lentivirus vector (Tet-pLKO-puro vector, Addgene; Cambridge, MA, USA) to construct sh-C1GALT1 and sh-cHP1BP3 vector, and shRNA sequences were listed in Table S1. Constructed lentiviral vector and two assistant vectors (pMD2.G and psPAX2) were transfected into HEK293T cells with PEI MAX 40000 (Polysciences; Warrington, PA, USA). Virus particles were collected after 2 days, applied to infect YTS-1 cells, and stable transfectants were selected using puromycin. Transient silencing of C1GALT1 and cHP1BP3 in T24 was performed by transfection of constructed vectors. Silencing of C1GALT1 and cHP1BP3 was induced by doxycycline (Doxy).
Complementary single-strand DNA oligos encoding pre-miR-1-3p and their flanking 100 sequences were synthesized, annealed, and ligated into Tet-pLKO-puro vector (Addgene; Cambridge, MA, USA) for construction of miR-1-3p overexpression vector. Stable YTS-1 transfectants were established as described in the preceding section. Transient overexpressing of miR-1-3p in T24 was performed by transfection of constructed vectors. Overexpression of miRN1–1-3p was induced by doxycycline (Doxy).
C1GALT1 overexpression was performed as previously described . Briefly, C1GALT1 was cloned into pLVX-AcGFP1-N1 (Takara; Shiga, Japan) lentiviral vector. Lentivirus was packaged in HEK293T cells and collected. HCV29 cells were established by infecting lentivirus into cells, and stable transfectants were selected by puromycin selection.
Cell proliferation was assayed as described previously . For Edu assay, cells treated with/ without Doxy were incubated with 50 μM Edu for 6 h, fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, stained with iClick EdU solution (GeneCopoeia; Rockville, MD, USA), rinsed with 1× PBS, and subjected to flow cytometry. For CCK8 assay, cells in 96-well plates were incubated with CCK8 solution (Beyotime) for 4 h, and absorbance at 450 nm was determined using a microplate reader.
Colony formation assay
Colony formation was assayed as described previously . In brief, cells in 6-well plates (1000 cells/ well) were cultured for 1–2 wk. until colonies were clearly observable. Colonies were rinsed with 1× PBS, fixed with 4% paraformaldehyde, stained with crystal violet solution, and photographed. Acetic acid solution was added to dissolve crystal violet, and absorbance at 595 nm was determined.
This assay was performed as described previously . 104 cells suspended in FBS-free medium were inoculated into upper chamber, and medium supplemented with 10% FBS was added into lower chamber. After 24 h culture, cell migrated across the membrane were fixed, stained with crystal violet, and photographed under microscope.
Sera and tissues from normal subjects and BLCA patients were obtained from the Third Affiliated Hospital of Xi’an Jiaotong University. Written informed consent was obtained from all patients, in accordance with the Declaration of Helsinki. Experiments using human tissues were approved by the Research Ethics Committee of Northwest University.
RNA sequencing analyses
For circRNA sequencing analysis, total RNAs were isolated from BLCA and adjacent normal tissues using TRIzol reagent kit (Invitrogen) as per manufacturer’s instructions, treated with RNase R, and purified using RNeasy MinElute Cleanup Kit (Qiagen; Valencia, CA, USA). Strand-specific library was constructed using VAHTS Total RNA-seq Library Prep Kit for Illumina, as per manufacturer’s instructions. In brief, circRNAs were enriched by removal of rRNAs, fragmented into short fragments, and reverse-transcribed into cDNA. cDNA was further amplified, and sequenced using Illumina HiSeq 2500 by Gene Denovo Biotechnology (Guangzhou).
For miRNA sequencing analysis, isolated total RNAs (size range 18–30 nt) were enriched by PAGE. 3′ and 5′ adapters were ligated to RNAs, and ligation products were reverse-transcribed by PCR amplification, enriched to generate a cDNA library, and sequenced as above.
Fluorescence in situ hybridization (FISH)
FISH assay was performed as described previously . FITC-labeled cHP1BP3 and CY5-labeled miR-1-3p probes were designed and synthesized by GenePharma (Shanghai). Hybridization was performed overnight using cHP1BP3 and miR-1-3p probes as per manufacturer’s instructions. Images were acquired by laser confocal microscopy (model TCS SP8 (Leica; Weztlar, Germany). Sequences of cHP1BP3 and miR-1-3p probes for FISH are listed in Table S1.
RNase R treatment
RNAs from YTS-1 cells were treated with 3 U/μg RNase R (Geneseed; Guangzhou), incubated for 30 min at 37 °C, reverse-transcribed with primers, and detected by reverse transcription quantitative real-time PCR (RT-qPCR), and primers were listed in Table S1.
Biotin-coupled miRNA capture
Biotin-coupled miRNA pulldown assay was performed as described previously [27, 28]. In brief, 3′-end biotinylated miR-1-3p mimics (GenePharma) were transfected into YTS-1 cells using GP-transfect Mate reagent (GenePharma). Cells were lysed with lysis buffer (20 mM Tris (pH 7.5), 100 mM KCl, 5 mM MgCl2, 0.3% NP-40, 50 U of RNase OUT) (Sangon Biotech; Shanghai). Biotinylated RNA complex was pulled down by incubating lysates with streptavidin-coated magnetic beads (Invitrogen) pre-blocked with 1 mg/mL yeast tRNA (Beyotime) and 1 mg/mL BSA. cHP1BP3 levels in bound fractions were evaluated by RT-qPCR.
Luciferase reporter assay
Sequences of predicted miR-1-3p binding sites of circRNAs and C1GALT1 3′-untranslated regions (3′-UTRs), and corresponding mutants (listed in Table S1), were designed, synthesized, and inserted into luciferase reporter vector psiCHECK-2 (Promega; Madison, WI, USA). Constructed plasmids were co-transfected together with miR-1-3p mimics to YTS-1 cells. Relative luciferase activity was determined using Dual Luciferase Assay Kit (Promega) as per manufacturer’s instructions.
Identification of proteins with T antigen
Proteins were denatured, reduced, alkylated, digested, and desalted as described previously . Lyophilized peptides (150 μg) were dissolved with 50 mM NH4HCO3, and incubated with 75 μL PNA-agarose (Vector Labs) overnight at 4 °C. The mixture was rinsed with PBS, and boiled for 10 min to release peptides. Glycopeptides with T antigen were collected by centrifugation and purified using Oasis HLB cartridges (Waters; Milford, MA, USA). Two-dimensional LC-MS/MS and data analysis were performed using LTQ Orbitrap MS (Thermo Fisher), and MaxQuant software program with T antigen as variable modification, as described previously [29, 30].
Tumor formation in mice
Animal experiments were performed in accordance with guidelines of the Animal Care and Use Committee of Northwest University. Effects of C1GALT1 on BLCA tumor proliferation and metastasis were studied using mouse subcutaneous (s.c.) xenograft, trans-splenic metastasis and popliteal lymphatic nodes (LNs) models.
For mouse s.c. xenograft model, YTS-1 cells with silencing of C1GALT1 or circHP1BP3, or overexpression of miR-1-3p, were grown to 70–80% confluence, suspended in RPMI-1640 medium without FBS (107 cells /mL), and 0.2-mL aliquots were transplanted s.c. into 8-wk-old male BALB/c-nu mice. Tumor size was measured every other day for 3 wk., after which tumors were excised and weighed.
The role of C1GALT1 in BLCA based on blood metastasis to liver was investigated by injecting BLCA cells (105 cells in 100 μL PBS) into spleens of 4- to 6-wk-old mice as described previously . After 4 wk., mice were euthanized, and tumors were excised and weighed.
Tumors, spleens, and livers from each type of experiment were dissected, fixed, and paraffin-embedded for histopathological analysis.
PLNs metastasis model was performed by the injection of 100 μL PBS suspensions of BLCA cells (105 cells in 100 μL PBS) into footpads of 4- to 6-wk-old mice as described previously . After 4 wk., popliteal lymphatic nodes were dissected, fixed, and paraffin-embedded for histopathological analysis.
Patient-derived xenografts (PDXs) models
PDX tumor was derived from a male patient with papillary urothelial carcinoma. Briefly, three serial passage xenograft tumor pieces of ~ 60 mm were subcutaneously grafted into the 4- to 6-wk-old mice. When tumors grown to ~ 200 mm, the mice were randomly divided into vehicle or itraconazole (ITZ) subgroups. Solvent control or ITZ (20 mg/mL, twice daily) was given orally. Tumor size was measured, after which tumors were excised, weighed and subjected to histopathological analysis.
Data are presented as mean ± SD from three independent experiments unless specified otherwise. Two-tailed Student’s t-test was used for comparison of data sets between two groups, and differences with p < 0.05 were considered statistically significant. Notations in figures: *p < 0.05; **p < 0.01; ***p < 0.001.