2.1 BC patients and tissue specimens

From 2019 to 2021, sixty pairs of tumor tissues and adjacent tissues were totally collected from patients with bladder cancer at Harbin Medical University Cancer Hospital (Harbin, China). All specimens were pathologically confirmed as bladder cancer and did not receive preoperative radiotherapy or chemotherapy. Histological diagnosis of all cases was evaluated according to criteria established by the World Health Organization (WHO). All samples were immediately frozen in -80 °C after surgical resection. Written informed consent was obtained from each participant and with approval from the ethics committee of Mudanjiang Medical University (No.2018-MYGZR01).

2.2 RNA-Seq and bioinformatics analysis

RNA-seq of T24 and SV-HUC-1 cell lines were performed using an Illumina HiSeq™ 2000 (Illumina, USA). Quality control was performed using Fast QC and clean reads were aligned to the human genome(hg19) using Tophat. Differentially expressed lncRNA were identified by DEseq2, with P value < 0.05 and |log2(foldchange)|> 1 as the thresholds to evaluate the statistical significance of the lncRNA expression differences.

Three bioinformatics databases including miRDB (http://mirdb.org/), miRcode (http://www.mircode.org/) and Starbase (https://starbase.sysu.edu.cn/) were employed to predict the potential bindings of miRNAs to individual lncRNA. In addition, three algorithms including miRanda, RNA22 and microT were adopted to analyze possible target mRNAs for miRNAs. The binding sites were predicted using Starbase.

2.3 Cell culture

BC cell line T24, human normal uroepithelial cell line SV-HUC-1 and human renal epithelial cell line 293 T were purchased from China Center for Type Culture Collection (WuHan, China), and were allowed to stand in McCoy'5A medium (Termo Fisher Scientifc, 16600082), Roswell Park Memorial Institute 1640 medium (RPMI-1640; Termo Fisher Scientifc, 11875119) and Dulbecco modifed Eagle Medium (DMEM; Termo Fisher Scientifc, 11966025), respectively. All media were supplemented with 10% fetal bovine serum (FBS; VivaCell, Shanghai, China, C04001) and 1% penicillin—streptomycin at 37 °C with a 5% CO2 atmosphere.

2.4 RNA extraction and quantitative reverse transcription PCR (qRT-PCR)

Total RNA from cells and tissues were extracted using Trizol reagent (Invitrogen, USA) following the manufacturer’s instructions. For lncRNAs and mRNAs, Transcriptor First Strand cDNA synthesis Kit (Roche, 0489703000) was applied for reverse transcriptions. qRT-PCR was performed using FastStart Universal SYBR Green Master (Rox) (Roche, 4913850001) on the instrument of ABI 7500 (Thermo, USA). For miRNAs, SanPrep Column miRNA Extraction Kit(Sangon Biotech, B518811), miRNA First Strand cDNA Synthesis (Sangon Biotech, B532453) and miRNA qPCR Kit (Sangon Biotech, B532461) were adopted. Relative gene expressions were determined using the 2–ΔΔCT method. The primers were described in Table 1.

Table 1 Primer sequences used for qRT-PCR2.5 Cell transfection

When cell confluence reached 60%, cell transfection was performed using RNA TransMate (Sangon Biotech, E607402). The siRNAs were synthesized by Sangon (Shanghai, China) and sequences were listed as Table 2.

Table 2 Primer sequences used for cell transfection2.6 Cell proliferation assay

Cell proliferation was examined using Cell Counting Kit-8 (CCK-8; BioSharp, China) based on the the provided guidelines. Briefly, transfected cells (5 × 103 cells/well) were seeded into 96 well plates, and 10 μl of CCK-8 was added to each well. The optical density (OD) was read at 450 nm at different time points (24, 48, 72 h).

2.7 Cell migration and invasion assay

Stably transfected cells were inoculated into six well plates (5 × 105 cells/well). When 90% confluence was reached, a wound was created by a 10 µl pipette tip. The movement of cells was captured by a phase-contrast microscope at different time points (0,6,12 h). A total of 200 μl cells suspension (1 × 105 cells) filled with serum-free medium was added into the upper chamber with polycarbonate membrane coated with Matrigel in advance, while the bottom chamber was filled with 600 μl medium with 20% FBS. After incubating for 24 h, the invasive cells on the outer membrane were immobilized by paraformaldehyde and stained with crystal violet solution, and five different visual fields were randomly selected to be photographed under a microscope.

2.8 Luciferase reporter assay

Dual-Luciferase reporter vector with the wild-type (WT) or mutated (MUT) versions of lncRNA XIST and TNFSF10 that containing binding sites or mutant sites with miR-129-5p were constructed based on the pmiR-GLO luciferase Vector (Promega, USA). The miR-129-5p or NC mimics and vectors were co-transfected into 293 T cells for 48 h. Then the Dual-Luciferase Reporter Assay System (Promega, USA) was utilized to evaluate the luciferase activity of each group according to the provided protocol, with Renilla Luciferase activity being used as a normalization control.

2.9 Statistical analysis

SPSS 25.0 and GraphPad Prism 8.0 were utilized for statistical analysis, with P < 0.05 as the significance threshold. Statistical comparisons were performed using Student’s t-tests, Wilcoxon signed-rank tests or Pearson chi-squared tests as appropriate. The diagnostic indexes of each biomarkers were performed using receiver operating characteristic (ROC) curves, and the optimal cut-point value was determined according to the Youden index. To evaluate the variance of various biomarker combination, binary logistic regression and ROC curves were adopted.