Patient samples

Samples were obtained from the Department of Urology at The First Affiliated Hospital of Shenzhen University, with the approval of the hospital’s ethics committee. Patients were fully informed about their sample nature, associated risks, study objectives, and provided written informed consent before participating in the experiments.

Cell lines

Human epithelial cells SV-HUC-1 and BCa cells (TCCSUP, SW780, UMUC-3, T24, 5637, J82) were obtained from the American Type Culture Collection, while human fibroblast cells were from Saibaikon Biotech (Shanghai). SV-HUC-1 cells were cultured in Ham’s F-12 K medium, 5637 cells in 1640 medium, TCCSUP, UMUC-3, T24, SW780, and J82 cells in Dulbecco’s Modified Eagle’s Medium (Gibco, USA). Human fibroblast cells were grown in the PriMed-iCell-003 medium (Saibaikon). All cells were incubated at 37 °C in a humidified 5% CO2 atmosphere with 10% fetal bovine serum (FBS), 100 U/mL penicillin(Gibco), and 100 μg/mL streptomycin(Gibco).

Antibody

p-PI3K (CST 17366), TSG101 (Abcam, ab125011), Alix (Abcam, ab186429), CD9 (Abcam, ab263019), Calnexin (Abcam, ab22595), cyclin B1 (CST 12231), β-tubulin (Abcam, ab6046), H3 (Abcam, ab1791), α-SMA (Proteintech, 67735-1-IG), PI3K (Santa, sc-1637), p-AKT (CST, 13038), AKT (CST, 11E7), KPNA2 (Abcam, ab70160), and KIFC1 (Abcam, 172620), and Ki67 (CST, 9449).

Database mining

The TCGA database (https://portal.gdc.cancer.gov/) and the GEO dataset GSE13507 (https://www.ncbi.nlm.nih.gov/) were utilized to obtain mRNA expression levels in BCa and their corresponding normal tissues. The GEO dataset GSE236933 (https://www.ncbi.nlm.nih.gov/) was utilized to obtain miRNA expression levels in BCa and their corresponding normal tissues.

Screening for miRNAs targeting KPNA2

To identify differentially expressed miRNAs, we analyzed GEO’s miRNA expression data. We utilized online prediction tools, such as starBase (http://starbase.sysu.edu.cn/), miRTarBase (https://mirtarbase.cuhk.edu.cn/), and miRDB (https://mirdb.org/) to identify miRNAs targeting the KPNA2 gene for potential regulation of its expression.

In situ hybridization

Single-site tissue microarray (TMA) slides (HBlaU066Su01, Shanghai Outdo Biotech) were analyzed using the ISH detection kit mk11286-h and BOSTER probes (Wuhan). Staining intensity was categorized from 0 to 3 (negative to dark blue/purple) and confirmed by two pathologists.

Immunohistochemistry (IHC)

IHC staining on single spot TMA slides (HBlaU066Su01, Shanghai Outdo Biotech) was performed using KIFC1 and KPNA2 rabbit antibodies (1:1000, Abcam) with the rabbit streptavidin–biotin detection system (ZSGB-BIO). Staining was scored by proportion (1–4: 0–25%, 25–49%, 50–75%, and 75–100%, respectively) and intensity (0–3: negative, weak, moderate, and strong, respectively). Combined scores (0–12) categorized samples into high (7–12) and low (0–6) expression groups, confirmed by two pathologists.

Immunofluorescence

Cells were inoculated on cell slides in 24-well plates. After 2 days, they were washed with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde for 30 min. After rinsing with PBS and permeabilizing/blocking with 5% bovine serum albumin (BSA) and 0.5% Triton X-100 for 1 h, cells were incubated with primary antibody in 5% BSA at 4 °C overnight. Following PBS rinses, cells were incubated with a fluorescent secondary antibody (Servicebio) in 5% BSA for 1 h in darkness, stained with DAPI for 10 min, washed with PBS, and observed via fluorescence microscopy.

RT-qPCR

RNA was isolated from cultured cells and tissues using Trizol Reagent (Takara). Total RNA was quantified with a SYBR Premix Ex Taq™ II kit (Takara) on a LightCycler 480 (Roche) following the manufacturer’s protocol. Cycling threshold (CT) data were collected and averaged, with comparisons made to controls using the comparative CT method. U6 snRNA and human GAPDH were internal controls for miRNA and mRNA, respectively. Normalized quantities were calculated using the 2-ΔΔCT method and analyzed with GraphPad Prism 6. The primer sequences, synthesized by Sangon Biotech (Shanghai), are listed in Additional file 2: Table S1.

Western blot

Proteins were lysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer, separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were blocked with 5% skimmed milk for 2 h at room temperature, washed three times with tris-buffered saline with Tween 20 (TBST; 10 min each), and incubated overnight at 4 °C with the corresponding primary antibodies. GAPDH served as the control. After TBST washes, membranes were incubated with horseradish peroxidase (HRP)-labeled anti-rabbit or anti-mouse immunoglobulin G (IgG) for 1 h at room temperature. Signals were detected using an enhanced chemiluminescent substrate (Millipore) and a chemiluminescent imaging system (Tanon-5200Multi, Tanon).

Transient transfections

Cells in six-well plates were cultured to 30–50% confluence for transfection. Lipofectamine 3000 (Invitrogen, USA) was combined with the respective plasmids, mimics, inhibitors, or small interfering RNAs (siRNAs; GenePharma, China) in opti-MEM for 15 min, then added to each well. Transfection was performed for 24 h, followed by a 48-h recovery period, after which RNA, proteins, and cells were collected for analysis. The sequences of each target are listed in Additional file 3: Table S2.

Dual luciferase reporter assay

Next, we retrieved the complete KPNA2 sequence from the gene library and design primers to amplify the KPNA2 3′ untranslated region (UTR). Conducted XhoI and NotI double digestion of the psiCHECK2 vector, then ligated the KPNA2 3′ UTR to create the psiCHECK2-KPNA2 3′ UTR vector (Gene Pharma, China). The vector was transformed into competent Escherichia coli and the psiCHECK2-KPNA2 3′ UTR dual luciferase reporter plasmid was amplified. Lipofectamine 3000 (Invitrogen, USA) was used to transfect miRNA mimics and inhibitors (Gene Pharma, China) into cells for 48 h. Luciferase activity was assessed using the dual-luciferase reporter assay system (Promega, USA), calculating relative activity (Renilla/Firefly) with firefly luciferase as the reference for reporter gene expression.

Cell counting kit-8 (CCK-8) assay

Cells were seeded at 1500 cells per well in 96-well plates. Ten microliters of CCK-8 reagent were added at 0, 24, 48, and 72 h post-seeding, in darkness, according to the manufacturer’s instructions. After 3 h of incubation, optical density (OD) at 450 nm was measured using a microplate reader.

Colony formation assay

Cells were seeded in six-well plates at a density of 1500 cells per well and cultured for 10 days. After fixation with 4% paraformaldehyde and staining with 0.1% crystal violet for 30 min, the colonies were washed with PBS and imaged. The crystal violet stain was then eluted with 33.3% glacial acetic acid, and the absorbance was measured at 590 nm using a microplate reader.

Cell migration and invasion assay

In the wound healing assay, cells in six-well plates formed monolayers, after which wounds were created using a 200 µL pipette, and migration was assessed at 0 and 24 h. Transwell chambers (CoStar, USA) were utilized for the transwell assays following the manufacturer’s instructions. Cells were fixed and stained with 0.2% crystal violet to visualize migration and invasion. Absorbance was measured after elution of the dye with 33.3% glacial acetic acid and transferred to a 96-well plate.

Cell apoptosis assay

Cells were centrifuged, washed twice with precooled PBS at 4 °C, and resuspended in 500 μL of binding buffer to achieve a concentration of 1 × 107 cells/mL. Subsequently, 100 μL of this suspension was mixed with 5 μL of Annexin V-FITC (Dojindo) and 5 μL of Propidium iodide (PI), followed by a 15-min incubation at room temperature in the dark. Apoptosis data were analyzed via flow cytometry using FlowJo V10 software. Experiments were conducted in triplicate.

Cell cycle analysis

Cells were harvested and washed with ice-cold PBS twice. Subsequently, the cells were fixed by incubating in 1 ml of precooled 75% ethanol at 4 °C overnight. The target cells were stained with PI/RNase staining buffer (BD Biosciences) for 30 min in the dark at room temperature. The DNA content was assessed using flow cytometry, and the data was analyzed with FlowJo V 10 software. All experiments were conducted a minimum of three times.

Chemosensitivity assay

Cells were seeded into 96-well plates at 3000 cells per well to assess sensitivity to doxorubicin (Sigma). After an overnight incubation, the cells were exposed to different concentrations of doxorubicin in a culture medium. Cell viability was measured after 48 h using the CCK-8 assay. The experiment was independently repeated at least three times for reliability. Maintaining aseptic techniques and safe drug handling was essential.

HDOCK prediction

We obtained the PDB structures of the corresponding proteins from the PDB database, predicted the direct interactions between the two proteins using HDOCK protein–protein, and analyzed the free energy between the two proteins using PRODIGY. The predicted multimeric structures obtained were analyzed using pymol v2.5.0 to obtain amino acid pairs that can interact to form hydrogen bonds between proteins and proteins.

Coimmunoprecipitation

Coimmunoprecipitation was performed using an Invitrogen kit (USA) with proteins extracted in NP-40 lysis buffer (Beyotime, China). Cell lysates were incubated overnight at 4 °C with protein A/G agarose beads and either anti-KPNA2 or anti-KIFC1 antibodies. Immunoprecipitation was facilitated by magnetic beads, followed by washing with pre-chilled RIPA lysis buffer. Bound proteins were then boiled and subjected to SDS-PAGE. Western blotting followed, using anti-KPNA2 or anti-KIFC1 antibodies.

Tissue exosome isolation and protein profiling

To promote dissociation, 200 mg of tissue was sliced on dry ice and incubated at 37 °C for 10–15 min. Then, the tissue was filtered using a 70 μm mesh to remove debris and centrifuge at 300 × g for 10 min and at 10,000 × g for 20 min, both at 4 °C. The supernatant was filtered through a 0.22 μm filter. Ultracentrifugation was performed at 150,000 × g for 2 h at 4 °C. The pellet was resuspended in 1 mL PBS and purified using the Exospur® kit (Echobiotech, China). The exosomes were concentrated to 200 μL with a 100 kDa MWCO Amicon® Ultra filter (Merck, Germany). Exosomes were identified by transmission electron microscopy and analyze protein markers via western blot. Size distribution and concentration were measured using a Nano Flow Cytometer (Flow NanoAnalyzer, NanoFCM Inc, China). Purify and extract proteins from exosomes, quantify them, perform SDS-PAGE, and conduct enzymatic digestion. Analyze the samples with mass spectrometry as detailed in Additional file 4: Table S3.

Exosome tracing

Exosomes were purified and labeled with the PKH67 green fluorescent kit (Sigma-Mini67, Germany). The washed exosomes were suspended in Diluent C and gently mixed with PKH67, followed by the addition of an equal volume of FBS to bind excess dye. The labeled exosomes were incubated with target cells for 24 h. After nuclear staining, the uptake of exosomes by target cells was monitored with a confocal microscope (Sunyu, China).

In vivo xenograft model

The animal experimental protocol received approval from the animal ethics committee of the First Affiliated Hospital of Shenzhen University. We utilized two cohorts of 4–6 week-old Balb/c nude mice to assess the in vivo effects of the target genes. In the first cohort of 20 mice, we employed a random number generator to assign them to four groups: a lentiviral control group, a miR-26b-5p overexpression group, a KPNA2 overexpression group, and a miR-26b-5p plus KPNA2 group. Each mouse received a subcutaneous injection of 3.5 × 106 cells. Tumor diameters were measured with digital calipers, and mice were euthanized after 25 days for tumor photography and pathological examination. In the second cohort, mice were divided into two groups and injected with bladder cells either overexpressing or not expressing KIFC1. Tumor sizes were measured similarly, and mice were euthanized after 22 days.

Statistical analysis

The statistician performed data analyses independently. Correlations between KIFC1 and KPNA2 expression and clinical pathologic variables were analyzed using the χ2 test and Fisher’s exact test. For normally distributed samples, a two-tailed t-test was used to compare group means, while the Mann–Whitney U test was applied for non-normally distributed samples. One-way ANOVA, followed by Tukey’s test, was utilized to compare means among three or more groups, with p < 0.05 indicating statistical significance. All analyses were conducted using SPSS 20.0 and GraphPad Prism 6.