Clinical sample collection
Tumour tissues and adjacent normal tissues of 138 BUC patients who underwent tumour excision or tissue biopsy in the First Affiliated Hospital of Chongqing Medical University between March 2019 and February 2021 were collected. (Table 1) Patients with severe underlying diseases or other primary cancers were excluded. The collected clinical tissues were utilized to analyze the expression of lncRNAs and percentage of endothelial cells (ECs). All patients included in this study provided written informed consent, and this study was approved by the Medical Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (IRB:2021-085). All clinical data were reviewed according to medical records.
SV-HUC-1, BUC cell lines (T24, UM-UC-3, 5637, J82 and TCC-SUP) and HUVEC were purchased from the American Type Culture Collection (Manassas, Virginia, USA). Cells were cultured in DMEM (SV-HUC-1, UM-UC-3, T24 and HUVEC), McCoy's 5A (J82) and RPMI-1640 (5637 and TCC-SUP) basal medium (Gibco, Gaithersburg, MD, USA), which were supplemented with 10% fetal bovine serum (FBS, MilliporeSigma, Burlington, MA, USA), 100 U/ml penicillin and 0.1 mg/ml streptomycin (Beyotime, Beijing, China). Cells were incubated at 37°C in 5% CO2 incubator. The medium was changed every 1-3 days.
Bladder specimens were obtained fresh from the operating field where grossly apparent tumour tissue or adjacent tissue not grossly affected by tumour (Par-cancer tissue). These tissues were transported at room temperature immersed in the RPMI-1640 medium (Gibco, Gaithersburg, MD, USA) with 10% FBS (MilliporeSigma, Burlington, MA, USA). Once received, tumour tissues were divided into two parts, one of which was cut into approximately 1 mm3 pieces and enzymatically digested to single cell suspensions using MACS tumor dissociation kit (Miltenyi Biotec) for 1 h on a rotor at 37°C for further flow cytometry/FACS analysis, another part of tumour tissues and all par-cancer tissues were frozen in liquid nitrogen immediately and then stored at -80℃ until lncRNA extraction.
ECs percentage detection by Flow cytometry/FACS
The single cell suspensions were filtered with screen cloth and cell surface staining was performed in FACS buffer containing CD31 antibody (Cat# 303102, BioLegend, USA) on ice for 1 hour. Following washing twice with FACS buffer, the percentages of EC subtype (CD31 positive) in these single cell suspensions were detected using a FACS flow cytometry system (Cytoflex, Beckman Coulter, USA).
The siRNAs of AC005625.1 and AC008760.1 were used to silence the expression of AC005625.1 and AC008760.1. The sequences used were: si-AC005625.1 (sense:5′-GCUUCACAGCCACCAUCUATT-3′, antisense: 5′-UAGAUGGUGGCUGUGAAGCT T-3′), si-AC008760.1 (sense: 5′-GACAGGUAGUCACGACUAUTT-3′, antisense: 5′-AUAGUC GUGACUACCUGUCTT-3′). For transient transfection, T24 cells were added into 6-well plate (1×10⁵ cells per well). When cells grew to 50-60 % confluence, cells were transfected with 10 μl siRNAs (20 nM) using 5 μl Lipofectamine 3000 (Invitrogen, USA) for 48 h according to the manufacturer. Finally, we used RT-qPCR to evaluate the expression levels of lncRNAs.
Real-time quantitative PCR
We used Trizol (Takara) to extract total lncRNAs from tissues and cell lines under various experimental conditions. cDNA Synthesis Kit (Takara) combined with lncRNAs (1μg) was utilized to reverse transcribed cDNA. The quantitative polymerase chain reaction (qPCR) was performed on an ABI 7500 real-time PCR system (Applied Biosystems) by SYBR-Green method (Takara). The values of Ct were calculated with the 2−ΔΔCt method and normalized to the expression levels of β-actin. The expression levels of lncRNAs were relative to the fold change of their controls which were defined as 1. The primer sequences were shown in Table 2. Three assays were conducted per cDNA sample.
Tube formation assay
100μL ice‐cold Matrigel was added into a well of a 48‐wells plate for the HUVEC cells tube formation assay. 1 hour later, 1×10⁵ HUVEC cells resuspended in 100μL DMEM medium were added into the 48‐wells plate. After incubating at 37°C in 5% CO2 incubator for 4 hours, the tube formation status was photographed by light microscopy. The numbers of branch points were counted and analyzed by image J. 5 fields (200X) per chamber were observed for counting invaded cell numbers.
For the migration assay, 1×10⁵ HUVEC cells were suspended in 100μl medium without FBS and seeded into an upper chamber (Corning, USA). Then, 800μl complete DMEM medium containing 10% FBS was added to the lower chamber. For the invasion assay, Diluted Matrigel (1:5 dilution with the DMEM medium) was added into the cell culture inserts. Four hours later, 1×10⁵ HUVEC cells in 100μl non-FBS DMEM medium were added into the upper chamber, while the lower chamber was filled with 800μl of DMEM medium with 10%FBS. After culturing 24 hours for migration test and 48 hours for invasion test, the cells culture inserts were washed by PBS and stained with 4% paraformaldehyde. Then inserts were dyed by 0.1% crystal violet solution for 20 minutes. The dyed cells were photographed by light microscopy. 5 fields (200X) per chamber were observed for counting invaded cell numbers.
Total protein was extracted from HUVEC cells using Radio Immunoprecipitation Assay (RIPA) lysis buffer (Beyotime, Beijing, China) and 1% PMSF (Beyotime). Next, the protein concentration was determined by the BCA Kit (Beyotime). The protein was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and transferred onto 0.22μm polyvinylidene fluoride (PVDF) membranes (Merck Millipore). The membranes were blocked with 5% milk in TBST buffer, and then the membranes were incubated with primary antibodies against VEGFA (Cat# 65373, Cell Signaling Technology (CST), USA), CD31(Cat# ab9498, Abcam, UK), VIMENTIN (Cat# 5741, CST, USA), E-CADHERIN (Cat# 3195, CST, USA) and N-CADHERIN (Cat# 13116, CST, USA) and β-Actin (Cat# 4970, CST, USA) at 4℃ for 16 hours. After washing with TBST buffer for 30 minutes, the membranes were incubated with anti‐rabbit horseradish peroxidase‐conjugate secondary antibodies (Cat# 7074, CST, USA) at room temperature for 1 hour, and then washed by TBST 30 minutes. Signals were visualized by ECL Substrates (Beyotime). Three assays were conducted per protein sample. Protein bands were quantified by ImageJ software (U.S. National Institutes of Health). Target protein expression levels (band intensities) were expressed by relative to β-actin expression.
Immunohistochemistry (IHC) analysis was performed as described previously24. IHC was performed using antibodies against CD31 (Cat# ab9498, Abcam, UK). Images were scanned using Pannoramic SCAN.
BUC transcriptome data downloading and preprocessing
Transcriptome RNA-sequencing data of 414 BUC tumour tissues and 19 normal tissues were downloaded and extracted from The Cancer Genome Atlas (TCGA) data portal (https://portal.gdc.cancer.gov/). We excluded patients whose OS ≤ 30 days from this study because they might die of unpredictable factors such as hemorrhage and infection. The data utilized in the study were updated in March 21, 2021. Raw data of BUC patients were collected for further analyses. Transcriptome RNA-sequencing results and clinical data of BUC patients were combined into a matrix file by a merge script in the Perl language (http://www.perl.org/).
Identification of angiogenesis-related long non-coding RNAs (ARLNRs) and the survival-related ARLNRs (sARLNRs)
Angiogenesis-related genes (ARGs) were extracted from The Molecular Signatures Database v4.0 (ANGIOGENESIS M14493 and WP_ANGIOGENESIS M39556, http://www. broadinstitute.org/gsea/msigdb/index.jsp). Then the angiogenetic scores of these ARGs were calculated according to their expression levels in BUC tissues. To further identify the ARLNRs, we conducted the Pearson correlation analysis to clarify the correlation between angiogenetic score and the expression of lncRNA in BUC tissue. A standard of |r|>0.6 and P<0.05 was used to screen the ARLNRs. Besides, we selected the sARLNRs by univariate COX analysis and survival packages of R software (P<0.01). sARLNRs were further divided into deleterious and protective portions by the Hazard ratio (HR).
Establish angiogenesis-related risk score model (ARRSM)
Through multivariate COX regression analysis, we established the ARRSM based on the selected sARLNRs. The score in the ARRSM was calculated based on the expression of sARLNRs together with the Cox regression coefficients. The formula was as followed, [Expression levels of KIRREL1-IT1 * (0.266525)] + [Expression levels of AC005625.1 * (-0.135165)] + [Expression levels of AC018809.1 * (-0.170812)] + [Expression levels of AC008760.1 * (-0.133273)] + [Expression levels of AC083862.2 * (-0.221852)]. BUC patients were separated into the high-risk group and the low-risk group according to the median score.
Receiver operating characteristic (ROC) curves were used to assess the sensitivity and specificity of the ARRSM and drawn by survival ROC package of R software. Gene set enrichment analysis (GSEA) was used to detect the different pathways of ARRSM. We evaluated the survival probabilities of patients in different risk groups by Kaplan‐Meier survival curves. We conducted the univariate and multivariate Cox regression analyses to verify the independent prognostic factor of BUC. Nomograms were drawn to predict the survival probabilities of BUC patients by the rms package of R software.
Statistical analysis was conducted by SPSS21.0 software (SPSS Inc, Chicago, IL) and GraphPad Prism8 (GraphPad Software Inc, La Jolla, CA). Data were expressed as means ± SD. The correlations between average expression of sARLNRs and clinicopathological characteristics of patients were evaluated using Fisher’s exact probability method. Student T-test, ANOVA and post-hoc test (Boferroni method) were used for difference comparison of two or more groups. Pearson correlation analysis was utilized to analyze the correlation. P<0.05 was considered a significantly statistical difference.