Bioinformatics analysis
The clinical information and transcriptomic data of BLCA patients were retrieved from The Cancer Genome Atlas (TCGA) database. The expression levels of FSCN1 and TLN1 mRNA in BLCA patients, and the associated survival data were downloaded from the GEPIA database. The GSE130001 single cell transcriptomic dataset of BLCA was downloaded from the Gene Expression Omnibus (GEO) database. The AverageExpression function was used to calculate the average expression values in the different cell subsets, and the corresponding pathway scores for each cell subset were calculated and grouped using the GSVA package. The data was visualized with pheatmap. ClusterProfiler R package was used for data analysis.
Rip-sequencing
The FSCN1-bound RNAs were isolated from the immuno-precipitate of anti-FSCN1 antibody using TRIzol (Invitrogen). Complementary DNA (cDNA) libraries were prepared using the KAPA RNA Hyper Prep Kit (KAPA, KK8541) according to the manufacturer’s instructions. High-throughput sequencing of the cDNA libraries was performed on the Illumina X10 platform for 150 bp paired-end Sequencing.
Collection Of Tissue Specimens
Paired tumor and para-tumor tissue specimens were obtained from 16 patients with muscle-invasive bladder cancer and 4 patients with non-muscle-invasive bladder cancer at the Institute of Urology, The First Affiliated Hospital of China Medical University.
Cell Culture
The T24, UM-UC-3 and H293T cell lines were purchased from Chinese Academy of Sciences Type Culture Collection Cell Bank (Shanghai, China). The H293T and UM-UC-3 cells were grown in high-glucose DMEM (Hyclone, GE Healthcare) supplemented with 10% fetal bovine serum (FBS; Biological Industries, Beit-HaEmek, Israel), and the T24 cells were cultured in RPMI medium (Hyclone, GE Healthcare). All cells were cultured at 37°C under 5% CO₂.
Transfection
The siRNAs specific for FSCN1, TLN1, METTL14 and METTL3 were from JTS-BIO Co. (China). Plasmids expressing FCSN1 shRNA and TLN1 sh-RNA and the negative controls were purchased from GeneChem (Shanghai, China). Lentiviruses expressing METTL3 shRNA and overexpressing FSCN1 were also purchased from GeneChem (Shanghai, China). The cells were transfected with the different constructs using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). The sequences of the siRNAs are shown in Supplementary Table S1.
Western Blotting
Total protein was extracted from cells or tissues using RIPA lysis buffer containing 1% phenylmethylsulfonyl fluoride (PMSF), and quantified by the bicinchoninic acid (BCA) assay kit (Beyotime, China). Equal amount of proteins per sample were separated by SDS/PAGE and transferred to PVDF membranes, which were then blocked with 5% skimmed milk and incubated overnight with the diluted primary antibodies at 4°C. The following day, the membrane was incubated with horseradish-peroxidase-conjugated secondary antibodies for 1 hour at 37°C. The protein bands were detected using the EasySee Western Blot kit (Beijing Transgen Biotech, Beijing, China) and the chemiluminescence system (Bio-Rad, CA, USA). The intensities of the protein bands were calculated using ImageJ. The antibodies are listed in Supplementary Table S2.
Immunohistochemistry (Ihc) Assay
The tissue specimens were fixed with 4% neutral formalin, embedded in paraffin, and cut into 7µm-thick sections. After deparaffinization and dehydration at room temperature, the sections were immersed in 3% H₂O₂ for 20 min to quench endogenous peroxidases, and then incubated overnight with the primary antibodies at 4°C. The following day, the sections were incubated with biotinylated goat anti-rabbit or anti-mouse antibodies for 30 min, and the color was developed using DAB kit (Beyotime, Shanghai, China). The sections were counterstained with hematoxylin (Maixin Biotechnology) and observed under a microscope.
Rna Extraction And Qrt-pcr
Total RNA was extracted from the suitably treated cells using RNAiso Plus (Takara Biotechnology, Dalian, China), and reverse transcribed to cDNA using Prime Script RT Master Mix (Takara Biotechnology, Dalian, China). QRT-PCR was performed using the Sybr Premix Ex Taq TM Kit (Takara Biotechnology, Dalian, China) on the LightCyclerTM 480 II system (Roche, Basel, Switzerland). The primer sequences are shown in Supplementary Table S3.
Cell Migration And Invasion Assay
The cells were seeded into the upper chambers of 8µm-pore transwell inserts in 24-well plates (Corning Costar, Corning, NY, USA) at the density of 8000 cells/well in 200µl serum-free medium. For the invasion assay, the chambers were pre-coated with Matrigel (BD, San Diego, CA, USA). The lower chambers were filled with 600 µl of medium containing 10%-FBS. After culturing for 24h, the cells remaining in the upper chamber were washed off, and the cells that adhered to the lower surface of the membrane were stained with crystal violet. The migrated/invaded cells were counted under 40X magnification with an inverted microscope (EVOS XL system, AMEX1200; Life Technologies Corp, Bothell, WA, USA) using Image J software.
Wound-healing Assay
The cells were seeded in 6-well plates and grown till > 90% confluent. The monolayers were scratched longitudinally with a sterile 1 mL pipette tip. The dislodged cells were rinsed and fresh serum-free medium was added. The wound regions were photographed at 0h and 24h under 40X magnification with an inverted microscope. The wound coverage was calculated to determine the migration rate.
5-ethynyl-2′-deoxyuridine (Edu) Assay
EdU assay was performed to detect UM-UC-3 cells proliferation with the use of an EdU kit (BeyoClickTM, EDU-488, China) following the manufacture’s protocol. Fluorescence images were captured by a fluorescence microscope (Olympus Corporation, Japan) and cell counting was performed by the software ImageJ.
Molecular Docking
The ZDOCK module of Discovery Studio 4.0 software was used for molecular docking analysis. ZDOCK, a rigid protein docking algorithm based on the fast Fourier transform correlation technique, was used to search the translational and rotational spaces of the protein system, and these binding configurations were scored using an energy scoring function.
Co-ip Assay
Briefly, 2 x 107 cells were lysed in Co-IP lysis buffer (Beyotime) supplemented with protease inhibitors. The lysates were incubated overnight with anti-FSCN1 antibody, anti-METTL3 antibody or negative control IgG (1:100; 2729; Cell Signaling Technology) at 4°C, and then with 60µL Dynabeads for 2h at 4°C with constant rotation. The test tubes were placed against a magnet, and the supernatant was removed. The Dynabeads-antibody-antigen complex was gently washed five times with 500µL washing buffer, and the supernatant was removed after each wash in a magnetic field. The complex was then resuspended in SDS buffer, denatured by heating at 100°C for 15 min, and analyzed by western blotting as described.
Rip Assay
RIP experiments were performed using an RIP kit (Bes5101, BersinBio, China) and anti-FSCN1, anti-METTL3 or anti-eEF-2 antibody according to the instructions. QRT-PCR was performed as described.
Rna Pull-down Assay
An RNA pulldown kit (Bes5101, BersinBio, China) and a probe specific for the 3'UTR region of TLN1 mRNA (Gene Pharma, Shanghai Gene Pharma Co.) were used to perform the assay, according to the manufacturer's instructions.
Six short probes specific for the CDS region of TLN1 mRNA and negative control probes were synthesized by Gene Pharma (Shanghai Gene Pharma Co.). The cells were transfected with the respective probes and lysed. The probe-enriched RNA-protein conjugates were analyzed by immunoblotting[18]. The probe sequences are shown in Supplementary Table S4.
Immunofluorescence Assay
The suitably treated UM-UC-3 cells were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.5% Triton X-100 for 10 min. After washing with PBS, the cells were incubated with goat serum for 40 min to block non-specific binding, and then overnight with anti-FSCN1 antibodies at 4°C. Following five washes with PBS for 5 min each, the cells were incubated with fluorophore-conjugated secondary antibody for 2h at room temperature. The cells were then gently washed with PBS, counterstained with DAPI for 10 min, and washed again. The cells were incubated with TLN1 RNA probe (Gene Pharma, Shanghai Gene Pharma Co.) using an immunofluorescence kit (Gene Pharma, Shanghai Gene Pharma Co.).
Animal Experiments
An in vivo model of lung metastasis was constructed by injecting 6-week-old female BALB/c nude mice (Beijing HFK Bioscience, Changping, Beijing, China) with suitably transfected T24 cells via the tail vein. Each mouse was injected with 1 × 106 cells. Fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) was performed after 30 days by the Laboratory Animal Department of China Medical University. Lungs of mice were excised and stained by Hematoxylin and Eosin (HE).
Protein Stability
To measure protein stability, UM-UC-3 FSCN1-knockdown and control cells were treated with cycloheximide (CHX, final concentration 100 µg/ml) during indicated times. The expression of TLN1 protein was measured through western blot.
Statistical analysis
Data was presented as the mean ± standard deviation of at least three independent experiments. GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA) was used for statistical analysis. Two groups were compared using t-test. P < 0.05 was considered statistically significant.