Patient samples
Bladder cancer and para-cancerous tissues were obtained from patients with bladder cancer at Zhucheng people’s Hospital between 2018 and 2022. All participants were informed of the study before surgery and provided consent. All tissues were immediately snap-frozen in liquid nitrogen and stored at -80°C. This study was approved by the Institutional Review Board of Zhucheng people’s Hospital (No. 20181109).
Bioinformatics analysis
Publicly available bladder cancer tissue data were downloaded from The Cancer Genome Atlas (TCGA). The GEPIA database (http://gepia.cancer-pku.cn/) was used to analyse gene expression in bladder cancer tissue and obtain a survival curve. Spearman’s correlation analysis was conducted using the Gepia database.
Cell culture
The bladder cancer cell Lines T24, UM-UC-3, 5637, SW780 and 253J were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). T24 cells were cultured in RPMI 1640 medium (Gibco, USA) containing 10% foetal bovine serum (BI). UM-UC-3 cells were cultured in DMEM (Gibco, USA) containing 10% foetal bovine serum (BI). All cell lines were cultured in a 5% CO2 humidified incubator at 37°C.
RNA isolation and quantitative real‑time PCR (qRT‑PCR) analysis
TRIzol reagent (Invitrogen, USA) was used to extract total RNA from tissues and cell lines. Evo M-MLV RT Premix (Accurate Biology, China) was used to synthesize cDNA from total RNA. qRT‒PCR was performed using the Premix Pro Taq HS qPCR Kit (Accurate Biology, China) on a LightCycler 96 (Roche, USA). β-actin was used as an internal control for mRNA. For each sample three replicates were performed, and the data were analysed using the 2 − ΔΔCT method. All PCR primers were purchased from Accurate Biology (China) and are listed in the Supplementary Table 1.
Western blotting and immunoprecipitation
Total proteins were extracted from cells and tissues using PIPA buffer mixed with PMSF(Bioss, China) and quantified by bicinchoninic acid (BCA) analysis (Beyotime, China). Protein extracts were separated by 10% SDS‒PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The PVDF membranes were incubated with anti-TK1 antibody (1:1000, Abcam, USA), anti-TFDP1 antibody (1:1000, Abcam, USA), anti-β-catenin antibody (1:1000, Abcam, USA) and anti-β-actin antibody (1:5000, Bioss, China) at 4°C overnight. Then, the PVDF membranes were incubated with peroxidase (HRP)-conjugated secondary antibody (1:10000, Boster, China) for 1 h at room temperature. The proteins were detected by enhanced chemiluminescence system (ECL) reagent (Beyotime, China) on the AMERSHAM ImageQuant 800 system and analysed by ImageJ Software.
Immunohistochemistry (IHC)
The bladder cancer tissues, adjacent normal tissues and xenograft tumour tissues were collected in formaldehyde, paraffin embedded and cut into 4 µl-thick sections. Sections were treated with xylene and 100% ethanol, followed by decreasing concentrations of ethanol. After antigen retrieval, each section was incubated with anti-TK1 antibody (1:200, Abcam, USA), anti-Ki67 antibody (1:200, Bioss, China), anti-TFDP1 antibody (1:200, Abcam, USA) and anti-β-catenin antibody (1:200, Abcam, USA) at 4°C overnight, followed by secondary antibody incubation. Images were acquired through an inverted microscope (Nikon, Japan).
Cell Transfection and Stable Cell Line Construction
Lentiviral vectors encoding shRNA of TK1 and shRNA of TFDP1 were synthesized by GeneChem China. Cell lines with stable overexpression or knockdown of TK1 or TFDP1 were established with the lentivirus vector based on the manufacturer’s instructions. The infected cells were selected by puromycin (2 µg/ml) for further experiments.
The siRNAs for transfection were purchased from GenePharma (Shanghai, China) and transfected into the cells using Lipofectamine 3000 reagent (Invitrogen, USA) according to the manufacturer’s instructions.
All lentivirus constructs and siRNA sequences are listed in the Supplementary Table 2.
CCK-8 assay
The cells were seeded in 96-well plates at approximately 2000 cells per well. After the cells completely adhered to the wall, cell viability was measured using the Cell Counting Kit-8 (CCK-8) (Bioss, China). The absorbance was measured at 450 nm using a spectrophotometer (Tecan, Switzerland).
Flow cytometry analysis
Cells were washed with phosphate-buffered saline (PBS) and fixed with 75% cold ethanol at 20°C for 24 h. Then, 5 µL of propidium iodide (PI) (KeyGen BioTECH, China) was added to each cell suspension. The cell cycle of the cell suspension was analysed using a FACS Calibur Flow Cytometer (BD Biosciences, USA). The percentages of cells in the G0/G1, S, and G2/M phases were counted and compared.
Co-IP assay
Co-IP assays were performed with a protein binding immunoprecipitation kit (Absin, China) according to the manufacturer’s protocols.
Xenograft studies in nude mice
All animal studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Animal Care Committee of Shandong University. Six-week-old female BALB/c nude mice were purchased from Vital River Laboratories (Beijing, China) and preserved in an SPF-grade animal laboratory. Mice were randomly divided into four groups (five mice per group) and subcutaneously injected with approximately 1×107 stably transfected T24 cells. After 4 weeks of injections, the mice were euthanized, and the tumour tissues were removed and weighed.
Statistical analysis
Data were analysed using SPSS version 22.0 and presented as the means ± standard deviations (SD). Student’s t test was used to estimate the differences between the two groups. The Kaplan‒Meier method was used to evaluate overall survival (OS). All results were reproduced across triplicate experiments. Statistical significance was set at P < 0.05.