2.1 Study objects and tissue collection

A total of 86 BLCA patients who underwent surgical intervention at Longhua Hospital Shanghai University of Traditional Chinese Medicine between 2017 and 2024 were selected for this study. The inclusion criteria for participants were as follows: (1) fulfillment of the diagnostic criteria for BLCA [17]; (2) confirmation of diagnosis through histological and pathological examination; (3) presentation of the disease as a first occurrence; (4) absence of prior treatment with radiotherapy, chemotherapy, or immunotherapy; and (5) demonstrated compliance by the patient. The exclusion criteria included: (1) a history of other malignancies; (2) a history of other autoimmune disorders; (3) the presence of dysfunction in other organs; (4) incomplete clinical data; and (5) loss of follow-up information.

During surgical procedures, cancerous tissue samples were collected from patients, ensuring the avoidance of necrotic tissue, which was verified by pathologists as BLCA tissue. Additionally, normal bladder epithelial tissue was obtained from a site more than 3 cm away from the excised lesion, which was also confirmed by a pathologist as normal bladder epithelium. Each sample measured approximately 1 cm³. Following collection and cleaning, the samples were immersed in an RNA stabilization solution (Beyotime, China). The tissue samples were initially stored overnight at 4 °C before being transferred to a − 80 °C freezer for long-term preservation.

The collection, utilization, and research protocol for the samples received approval from the Ethics Committee of Longhua Hospital Shanghai University of Traditional Chinese Medicine. Prior to the commencement of the study, informed consent was obtained from both the patients and their families, with a signed consent form documenting their agreement.

2.2 Clinical data collection and follow-up survey

Demographic data of patients were collected utilizing conventional questionnaires, which encompassed variables such as age, gender, smoking history, and alcohol consumption patterns. Additionally, clinical data pertaining to disease characteristics were extracted from patient medical records, including tumor size, differentiation, TNM staging, lymph node metastasis (LNM), and tumor subtype.

All patients were followed up for 5 years through a combination of inpatient admissions, outpatient consultations, and electronic communication methods. The follow-up schedule consisted of visits every 3 months during the initial 2 years, with subsequent adjustments to every 6 months. Follow-up commenced 1-month post-discharge and continued until the occurrence of tumor recurrence, deterioration of the patient’s condition, metastasis of lesions, death, or reaches 5 years. Causes of death have been divided into two categories: tumor-related death (death caused by the direct progression of bladder cancer, tumor metastasis, or treatment-related complications) and non-tumor-related death (death caused by factors unrelated to bladder cancer, such as cardiovascular and cerebrovascular diseases, other systemic malignancies, accidents, etc.). Other endpoint events are defined as follows: tumor recurrence refers to newly formed tumor lesions confirmed by cystoscopy, pathological biopsy or imaging examination; deterioration of the condition refers to an increase in tumor grading, an increase in primary lesion volume by more than 20%, or an expansion of local tissue invasion; disease metastasis refers to the spread of tumor cells to regional lymph nodes or distant organs, confirmed by imaging or pathological examination. The recurrence-free survival rate was calculated based on the data obtained during the follow-up period.

2.3 Experimental cell line

The human normal ureteral epithelial immortalized cell line SV-HUC-1 served as the control. The human BLCA cell lines BFTC-905, RT-4, T24, J82, UMUC3, and 5637 were procured from Shanghai Yaji Biotechnology Co., Ltd., China.

The complete culture medium for SV-HUC-1 was composed of 89% Ham’s F-12 K medium (Thermo Fisher, USA), 10% fetal bovine serum (FBS, Thermo Fisher, USA), and 1% penicillin-streptomycin (P/S, Thermo Fisher, USA). For the BLCA cell lines BFTC-905, J82, and UMUC3, the complete culture medium was modified to consist of 89% DMEM medium (Thermo Fisher, USA), while maintaining the other components unchanged. In a similar manner, RT-4 and T24 were cultured in 89% McCoy’s 5 A medium (Thermo Fisher, USA), and 5637 was cultured in 89% RPMI 1640 medium (Thermo Fisher, USA).

Following the recovery of the purchased cell lines, the cells were evenly distributed into culture flasks, and 5 mL of the corresponding complete culture medium was added. After thoroughly mixing the cells, they were incubated in a culture incubator set at 37 °C with 5% CO2 and saturated humidity. The culture medium was replaced every 3 days until the cell confluence exceeded 85%.

2.4 Cell transfection

The small interfering RNA designed to down-regulate the DLGAP1-AS2 gene was designated as si-DLGAP1-AS2, while the corresponding control was referred to as si-NC. The miR-451a mimetics, miR-451a inhibitors, and their respective controls (NC-mimic and NC-inhibitor) were synthesized by Saides Biotechnology Co., Ltd., located in Hefei, China.

The UMUC3 and T24 cell lines, which exhibited optimal growth characteristics, were selected and evenly distributed onto a 6-well plate. Upon reaching a cell density of approximately 80%, 100 pmol of the aforementioned synthetics were transfected into the UMUC3 and T24 cell lines using Meilunbio® Liposomal gene transfection reagent (MeilunBio, China). According to the instructions, 2 µL aforementioned synthesis and 5 µL transfection reagent were diluted with 250 µL of serum-free medium and left to stand at room temperature for 5 min before mixing. This mixture was then incubated at room temperature for 20 min before being added to the culture well. It was subsequently incubated in a 37 °C, 5% CO₂ incubator for 6 h before being replaced with complete medium. Following the 48-hour incubation period, the cells were collected and subsequently stored at − 80 °C.

2.5 Quantitative polymerase chain reaction (qPCR)

The EasyPure® RNA Kit (TransGen, China) was employed to extract total RNA from frozen tissues and cells. Subsequently, 1 µL of DNase I (Beyotime, China) was added to the collected RNA solution and the mixture was incubated at 37 °C for 30 min. Then, 1 µL 50 mM EDTA solution was added and the mixture was heated at 65 °C for 10 min to terminate the reaction and ensure the effective removal of genomic DNA. The concentration and purity of the extracted total RNA were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher, USA). Agarose gel electrophoresis was used to evaluate RNA integrity. Results showed that all samples exhibited clear 28 S and 18 S ribosomal RNA bands without tailing, and that the brightness of the 28 S band was approximately twice that of the 18 S band. This indicates good RNA integrity and no obvious degradation. Meanwhile, NanoDrop detection showed that the RNA purity (A260/A280 ratio) was between 1.8 and 2.0, meeting the requirements for subsequent experiments. Total RNA served as the template for the synthesis of first-strand cDNA, utilizing the LunaScript® RT SuperMix Kit (NEB, USA).

The levels of DLGAP1-AS2 and miR-451a in the tissues and cells were quantified using the lnRcute lncRNA fluorescence quantitative detection kit (SYBR Green) and the miRcut enhanced miRNA fluorescence quantitative detection kit (SYBR Green), both from Tiangen, China. Primers for the amplification were synthesized by Jinsorui Biotechnology Co., Ltd. in Nanjing, China. The upstream primer for DLGAP1-AS2 was 5’-ACACGGTGAAACCTGTTC-3’, while the downstream primer was 5’-GGAGACAATGGCGTGATCT-3’. For miR-451a, the upstream primer was 5’-ACCATCCAGTGGGAAACCGTTACCATTACT-3’, and the downstream primer was 5’-CTGGTGTCGTGGAGTCGGCAA-3’.

Relative quantification was performed using GAPDH and cel-miR-39 as internal reference genes. The detection was conducted with a LightCycler 480 real-time fluorescence quantitative PCR instrument (Roche Applied Science, Switzerland). This study used a 20 µL reaction system comprising 1 µL of cDNA template, 10 µL of SYBR Green Mix, 0.5 µL of upstream primer (10 µM), 0.5 µL of downstream primer (10 µM) and 8 µL of RNase-free water. The reaction programmer was set to pre-denature at 95 °C for 10 min, then denature at 95 °C for 15 s, anneal at 60 °C for 30 s and extend at 72 °C for 30 s, with a total of 40 cycles. The relative levels were calculated using the 2−ΔΔCt method.

2.6 Luciferase report assay

The binding site between DLGAP1-AS2 and miR-451a was predicted utilizing the online database lncRNASNP2 (version 2.0, http://bioinfo.life.hust.edu.cn/lncRNASNP2/). When conducting ‘miRNA target’ analysis, set the parameters to minimum free energy ≤ − 20 kcal/mol and complementary pairing length ≥ 7 bp. The mutant (Mut) sequence of DLGAP1-AS2 was generated through a gene point mutation technique. Both the wild-type (Wt) and mutant sequences of DLGAP1-AS2 were subsequently cloned into the psiCHECK™-2 vector. This study designed of primers that incorporate specific enzyme cleavage sites, with the upstream primers facilitating Hind III cleavage sites (AAGCTT) and the downstream primers incorporating Xho I cleavage sites (CTCGAG). The psiCHECK™-2 vector was digested using the enzymes Hind III and Xho I (New England Biolabs, USA), and then ligated with the amplified fragments using T4 DNA ligase (Thermo Scientific, USA) to construct wild-type and mutant reporter vectors. The UMUC3 and T24 cell lines, which exhibit robust growth characteristics, were uniformly seeded in a 6-well plate. Co-transfection of Wt-DLGAP1-AS2 (or Mut-DLGAP1-AS2) with either a miR-451a mimic or a miR-451a inhibitor was performed using the Meilunbio® Liposomal gene transfection reagent in the UMUC3 and T24 cell lines. Following a 48-hour incubation period, luciferase activity was assessed using a dual luciferase activity detection kit (Promega, USA) and measured with a Fluoroskan™ Microplate Fluorometer (Thermo Fisher, USA).

2.7 Cell proliferation

A total of 100 µL of cells from each transfection group were transferred to a 96-well plate and cultured at 37 °C and 5% CO2 for durations of 24, 48, and 72 h, respectively. Subsequently, 10 µL of CCK-8 solution (Beyotime, China) was added to each well, and the plates were incubated in a dark environment for 2 h. The optical density (OD) at 450 nm was then measured using an enzyme-linked immunosorbent assay (ELISA) reader to assess the proliferative activity of the cells in each group.

2.8 Cell migration and invasion

Following a 48-hour transfection period, the cells from each transfection group were resuspended in FBS-free basal medium and inoculated into the upper chamber of a Transwell chamber (Corning, USA). Concurrently, 600 µL of complete culture medium was added to the lower chamber. After a 24-hour incubation at 37 °C, any residual cells on the upper membrane were carefully removed, and the cells adhered to the bottom of the membrane were fixed with 4% paraformaldehyde (Beyotime, China) for 20 min. The cells were then stained with 0.5% crystal violet (Solarbio, China) for 30 min. Following a wash with PBS, an inverted phase contrast microscope was employed to randomly capture images of 5 fields, and the number of cells was recorded. The procedures for the invasion and migration experiments were identical, with the exception that the upper chamber of the Transwell was pre-coated with Matrigel matrix adhesive.

2.9 Data analysis

For the analysis of quantitative data, a t-test was employed to compare 2 groups, while one-way ANOVA was utilized for comparisons involving multiple groups. The SNK-q test was subsequently applied for further pairwise comparisons between groups. For categorical data, the chi-square test was implemented for comparative analysis. Additionally, a Cox regression model was employed to assess risk factors, and the Kaplan-Meier (KM) curve was utilized for survival analysis. Pearson correlation analysis was conducted to evaluate the relationship between the levels of DLGAP1-AS2 and miR-451a.