LINC00525 drives aggressive phenotypes in bladder cancer via YAP stabilization-mediated transcriptional activation

Abstract

LINC00525, a long noncoding RNA (lncRNA), has been implicated in the regulation of cancer progression across various types. However, its role in bladder cancer (BLCA) remains unconfirmed. In this study, we observed upregulation of LINC00525 expression in both bladder cancer tissues and cell lines compared to normal controls. Among the 12 pairs of collected tissue samples, LINC00525 exhibited higher expression levels in muscle-invasive bladder cancer (MIBC) tissues than in non-muscle-invasive bladder cancer (NMIBC) tissues, indicating a positive correlation between LINC00525 levels and bladder cancer progression. In vitro experiments demonstrated that knockdown of LINC00525 significantly inhibited proliferation, migration, and invasion of bladder cancer cell lines; conversely, overexpression of LINC00525 had the opposite effect. Bioinformatic analysis revealed an association between LINC00525 and YAP, which was further confirmed by western blotting and PCR analysis using patient tissues. Mechanistically, we found that LINC00525 reduced phosphorylation of YAP at serine 127 (S127), promoting its nuclear import to exert transcriptional regulatory effects on target genes. Additionally, LINC00525 inhibited YAP ubiquitination by acting on YAP lysine 321 (K321), thereby increasing its stability to prevent degradation. Through in vivo and in vitro experiments, we demonstrated the YAP-mediated promoting effect of LINC00525 on bladder cancer cells and tumor growth. Our study reveals the involvement of the LINC00525/YAP axis in regulating bladder cancer development, suggesting a potential therapeutic strategy for malignant tumors characterized by high levels of LINC00525 expression.

Introduction

BLCA, being a prevalent malignant tumor, poses a significant threat to the urinary system’s well-being worldwide. By 2023, it is estimated that there will be approximately 82,290 BLCA cases and 16,710 fatalities in the United States [1]. Currently, surgical intervention, chemotherapy regimens, and immunotherapy approaches serve as the primary pillars of BLCA treatment. Despite scientific and technological advancements laying the groundwork for BLCA discovery and management, challenges persist in combating metastasis and drug resistance during later stages of the disease. Urgent development of novel therapeutic strategies is imperative to overcome this predicament.

Yes-associated protein (YAP) functions as a transcriptional coactivator within the Hippo tumor suppressor pathway, playing a crucial role in tumorigenesis. In the core cascade of the Hippo pathway, upstream Mammalian Ste20-like kinases 1/2 (MST1/2) activate Large Tumor Suppressor 1/2 (LATS1/2) through phosphorylation. Subsequently, activated LATS1/2 phosphorylates downstream effector YAP. Phosphorylation of YAP at the S127 site leads to its binding with adapter protein 14-3-3 and subsequent retention in the cytoplasm, resulting in loss of its transcriptional activity. Conversely, dysregulation of the Hippo pathway facilitates nuclear translocation of YAP, where it forms complexes with TEAD transcription factors to induce target gene expression and influence various cellular processes [2]. Additionally, K48-linked polyubiquitination at specific lysine sites (K76, K204, K252, K254, K321 and K497) can modulate YAP protein stability and regulate its levels accordingly [3, 4].

The length of lncRNAs exceeds 200 nucleotides, resulting in their complex structures and potential interactions with proteins that influence cellular functions. Previous studies have demonstrated the role of LINC00525 in promoting lung adenocarcinoma and colorectal cancer progression, but its impact on BLCA remains uncertain [5, 6]. In our study, we observed elevated expression of LINC00525 in BLCA, which positively correlated with YAP levels. Mechanistically, LINC00525 activates YAP by inhibiting phosphorylation at the S127 site and promotes nuclear translocation, thereby affecting downstream transcription factors. Additionally, LINC00525 enhances YAP stability by inhibiting ubiquitination at the K321 site. These dual mechanisms synergistically upregulate YAP levels to promote BLCA progression. Importantly, YAP blockade inhibited the growth of LINC00525-overexpressing cells and tumors. This finding provides a basis for potential treatment strategies targeting bladder tumors and other malignancies characterized by elevated LINC00525 expression.

Methods and materials

Patient samples and information

In this study, we collected 12 pairs of bladder cancer tissues and adjacent non-cancerous tissues from cystectomy specimens obtained at the Department of Urology, the Second Hospital of Tianjin Medical University (Tianjin, China), without any prior history of radiotherapy or chemotherapy. All collected tissue samples were meticulously examined by certified pathologists. The collection process was ethically approved by the Ethics Committee of the Second Hospital of Tianjin Medical University and adhered to the principles outlined in the Helsinki Declaration on Human Rights. Written informed consent forms were obtained from all bladder cancer patients to facilitate sample collection (number: KY2021K192). Patient and tumor characteristics are detailed in Table S6.

Antibodies information

The following related antibodies are required in western blot (WB), immunohistochemical staining (IHC), immunofluorescent staining (IF), co-immunoprecipitation (IP) and other experiments: anti-YAP: Affinity Biosciences, DF3182, WB (1:1000), IHC (1:100), IF (1:200); anti-YAP: Abcam, abB52771, IP (1:20); anti-GNPAT: Affinity Biosciences, DF3993, WB (1:1000); anti-GAPDH: Affinity Biosciences, AF7021, WB (1:1000); anti-TEAD1: Affinity Biosciences, DF3141, WB (1:1000); anti-TEAD1: Cell Signaling Technology, #12,292, IP (1:100); anti-CTGF: Affinity Biosciences, DF7091, WB (1:1000); anti-CYR61: Affinity Biosciences, AF6250, WB (1:1000); anti-ANKRD1: Affinity Biosciences, AF0677, WB (1:1000); anti-MYC-tag: Affinity Biosciences, T0052, WB (1:1000); anti-KI67: Affinity Biosciences, IHC (1:100); anti-Phospho-YAP (Ser127), Affinity Biosciences, AF3328, WB (1:1000), IHC (1:100); anti-Ubiquitin: Affinity Biosciences, AF0289, WB (1:1000); anti-HA: Affinity Biosciences, T0008, WB (1:100), IP (1:20); anti-Lamin B1, Affinity Biosciences, AF5161, WB (1:1000); anti-Rabbit IgG, Affinity Biosciences, S0001, WB (1:3000), IHC (1:200); anti-Mouse IgG, Affinity Biosciences, S0002, WB (1:3000), IHC (1:200).

Cell lines and culture

The human bladder cancer cell lines (T24, 5637) and the human bladder epithelial immortalized cells (SV-HUC-1) were procured from the ATCC Cell Bank (USA). The human bladder cancer cell lines (BIU87, 253 J, EJ) were obtained from the University of Copenhagen. T24, BIU87 (87), 5637, 253 J (253), and EJ cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin under a CO2 concentration of 5%. SV-HUC-1 (SV) cells were cultured in F12K medium supplemented with serum at a concentration of 10% under a CO2 concentration of 5%.

Plasmids, lentivirus and reagents

The Eukaryotic expression vectors were generated by inserting PCR-amplified fragments into pcDNA3.0 (Invitrogen). The plasmid expressing the truncated version of HA-YAP protein was kindly provided by Dr. Ding Ai from Tianjin Medical University (China) [7]. The K321R mutant of YAP was obtained from the laboratory of Dr. Ting Zhuang from Xinxiang Medical University (China) [4]. GenePharma (Shanghai, China) supplied all the small interfering RNAs (siRNAs), which were transiently transfected into cells using Lipofectamine RNAi MAX Transfection Reagent (Thermo Scientific). Two different short hairpin RNAs (shRNA) targeting LINC00525 were ligated into puromycin-resistant lentiviral vectors and transfected using LipofectamineTM 2000 (Invitrogen). Subsequently, the cells were cultured and selected with medium containing 1 mg/ml puromycin. The sequences for siRNA and shRNA can be found in Table S7. GA-017 and Cytochalasin D are activator and inhibitor of YAP respectively, both obtained from MCE (HY15147) (HYN6682) and diluted into different concentrations using DMSO for subsequent experiments. All reagents were diluted with DMSO to different concentrations for subsequent experiments.

Bioinformatics and data extracting

We screened differentially expressed genes against the BLCA database in The Cancer Genome Atlas (TCGA). The mRNA expression profiles and clinical information of BLCA were obtained from the TCGA database (https://www.cancer.gov/aboutnci/organization/ccg/research/structural-genomics/tcga). Significant differentially expressed genes (|log2FoldChange|>1 & padj < 0.05) were identified and selected using R’s DESeq2 software package, among which LINC00525 showed differential expression between normal tissues and BLCA tissues. All differentially expressed genes in normal and BLCA tissues are provided in the supplementary material (Table S1), while further analysis of LINC00525 expression in normal and BLCA tissues is presented in Table S2 and S3. The relationship between LINC00525 and YAP was predicted and analyzed using R language. Interaction prediction between LINC00525 RNA and YAP protein used Random Forest (RF) and Support Vector Machine (SVM) via the RPISeq tool (http://pridb.gdcb.iastate.edu/RPISeq/index.html), with thresholds set as RF > 0.5 and SVM > 0.5 for significance.

Immunohistochemical staining

The Urology Department of the Second Hospital of Tianjin Medical University provided 12 pairs of bladder cancer and paracancerous tissues, with the informed consent of the patients and approval from the hospital’s experiment review board. Subcutaneous tumors were obtained from nude mice and removed under sterile conditions. Tumor specimens were fixed in formalin solution, embedded in paraffin, and sliced into 4 μm sections. Deparaffinization and rehydration procedures were performed on tissue sections, followed by treatment with 3% hydrogen peroxide (H2O2) for 15 min to inhibit endogenous peroxidase activity. Heat-induced epitope retrieval was then conducted using a microwave in a 10 mM citrate buffer [pH 6.0] for 30 min. The slides were incubated overnight at 4 °C with a pre-diluted primary antibody. On the following day, the slides were warmed up and subjected to secondary antibody incubation, followed by staining with 3,3’-diaminobenzidine (DAB) and hematoxylin. Finally, YAP expression, pYAPS127 expression, and KI67 expression were observed under a Zeiss microscope (100X or 200X).

Immunofluorescence staining

Cells were cultured on LabTek II-CC2 chamber slides (Nunc) placed within 24-well plates. To stabilize the cells for immunofluorescence assessment, a fixative solution of 4% paraformaldehyde was utilized. Following fixation, the cells were washed with phosphate buffered saline (PBS) and then permeabilized with 0.1% Triton X-100 for 10 min. Subsequently, the cells underwent an overnight incubation at 4 °C using the primary antibody diluted at a ratio of 1:100. The next day, the cells were treated with the corresponding secondary antibody along with DAPI staining. YAP’s cellular localization was visualized using a fluorescence imaging system.

Western blot and co-immunoprecipitation

For western blot, RIPA buffer (MCE) supplemented with Protease Inhibitor Cocktail (MCE) and Phosphatase Inhibitor Cocktail II (MCE) was used at a dilution of 1:100 (v/v). The protein concentration was further measured using the BCA protein assay kit (Thermo Scientific). Protein samples were separated by gel electrophoresis and transferred onto a polyvinylidene difluoride (PVDF) membrane. Following blocking of the PVDF membrane with 5% nonfat dry milk TBST buffer, it was incubated overnight at 4 °C with the appropriate western blot antibody. On the subsequent day, the membrane was incubated with the corresponding anti-rabbit/mouse IgG secondary antibody. Specific bands were detected using ECL chemiluminescence liquid (Theromo Scientific) and luminescence imaging workstation.

For immunoprecipitation, cells were lysed using Pierce immunoprecipitation lysis buffer containing phosphatase and protease inhibitors. The cell lysates containing 1 mg protein were then incubated with 2 µg of the primary antibody at 4 °C overnight. On the following day, Pierce Protein A/G agarose beads (Thermo Scientific), totaling to 20–40 µl in volume, were added to the protein-antibody mixture and incubated for 2 h at 4 °C. Subsequently, five washes with PBS were performed on the beads before discarding the supernatant. Finally, heating of the beads in a solution consisting of 50 µl of 1× SDS at a temperature of 100 °C for duration of ten minutes took place prior to conducting protein immunoblotting.

RNA pulldown and RIP assay

For RNA pulldown, we conducted RNA pulldown assays using the Pierce™ Magnetic RNA-Protein Pull-Down Kit (Thermo Scientific). Biotin-labeled lncRNA LINC00525 was synthesized in vitro with the Biotin RNA Labeling Mix and T7 RNA polymerase, followed by incubation of biotinylated RNA with streptavidin magnetic beads overnight at 4 °C. Fresh cell lysate was prepared and added to the binding reaction, resulting in a final concentration of 1X. All buffers used in the preceding steps were supplemented with RNase inhibitor, protease inhibitor, and phosphatase inhibitor. Eluted proteins were detected through western blotting.

RIP assay was performed using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer’s instructions. Briefly, T24 cells were collected and lysed using RIP lysis buffer. Subsequently, they were incubated with magnetic beads coated with anti-YAP and anti-IgG antibodies for immunoprecipitation purposes. The co-precipitated RNAs were then extracted and quantified via qRT-PCR.

RNA scope assay

The LINC00525 probe (5’-TGAATGGTCAATGTCTGGTAGTTGGACGG-3’) was synthesized by Advanced cell (ACD lnc.). The expression level of LINC00525 in the control group and bladder cancer tissues was detected following the instructions of RNAscope 2.0 HD Reagent Kit-Brown (ACD, catalog number 322310). Firstly, the tissue sections were dewaxed and subjected to target restoration agent for 15 min at boiling temperature, followed by protease digestion at 40 °C for 30 min. Subsequently, hybridization with Probe-LINC00525 was performed at 40 °C for 2 h. After amplification, DAB and hematoxylin were used to develop the probe and nuclei respectively.

RNA extraction and RT-PCR

According to the experimental protocol, the total RNA in cells, clinical samples and mouse tumors was extracted using TRIzol reagent (Thermo Scientific), and further reverse-transcribed into cDNA by SuperScript III (Thermo Scientific). RT-PCR was performed on the LineGene 9600 Plus RT-PCR System (Bioer) using PowerTrack SYBR Green (Thermo Scientific). The expression levels of lncRNA and mRNA were calculated using the standard 2−ΔΔCt method. GAPDH was chosen as a normalization control. The RT-PCR primer sequences are listed in Table S7.

MTT assay

We transfected T24 and 87 cells with siYAP, shLINC00525 lentivirus, and plasmids of various designs for a duration of 48 h. Approximately 2.5 × 103 cells were seeded in each well of a 96-well plate and incubated at 37 °C for 1–4 days. At a fixed time every day, we added 30ul of MTT reagent (Thermo Scientific, ) to each well and incubated them at 37 °C for 2 h. Subsequently, the MTT reagent was removed, followed by the addition of 15ul DMSO to each well which was then incubated at 37 °C for an additional period of 15 min. Finally, we measured the absorbance of the dissolved formazan at a wavelength of 490 nm using a microplate reader as a reference for cell density analysis. The obtained data was graphed and analyzed in GraphPad Prism8.

Transwell and migration

We transfected T24 and 87 cells with lentivirus and plasmids of different designs for 48 h. The cells were suspended in 200ul of 1640 medium and seeded in the top chamber of a 24-well plate (Corning, pore size: 8 μm). The bottom chamber was filled with 1640 medium containing 10% FBS. After incubation at 37 °C for 48 h, the cells were fixed with a solution of paraformaldehyde (4%) and stained using crystal violet. Migration assays were performed by observing and counting migrated cells under a light microscope. Migrated cells were counted in 5 random high-power fields (HPF) per filter and averaged. Further analysis was conducted using ImageJ software and GraphPad Prism 8. The relative fold changes were determined through normalization of cell counts to those of the control/sh control group, so as to assess the impacts of various experimental treatments on in vitro cell invasion capabilities.

Clone formation assay

We transfected T24 and 87 cells with lentivirus and plasmids of various designs for a duration of 48 h. A total of 2 × 103 cells were seeded in 6-well plates and cultured in 1640 medium supplemented with 10% FBS at a temperature of 37 °C for a period of 7 days. Subsequently, the cells were fixed using a solution containing 4% paraformaldehyde and stained with crystal violet. The number of colonies was then observed and quantified using ImageJ software (Minimum colony size threshold: ≥50 cells).

Wound healing assay

We transfected T24 and 87 cells with lentivirus and plasmids of different designs for 48 h. After 24 h of transfection, a channel was created on the transfected cells grown to 90% confluence in a 6-well plate by scraping with the tip of a 10ul pipette tip, and images were taken as 0 h images. The cells were then washed with serum-free medium to remove debris and cultured for an additional 24 h before capturing images under a microscope. The wound healing rate was calculated by comparing it with the wound width at 0 h to determine the effect of different treatment factors on migration ability.

Animal studies in vivo

Approximately 2 × 106 purified cells, which were either subjected to specified experimental treatments or transfected with plasmids/viruses, were suspended in a mixture of 200 µl Matrigel (Yeasen) and 1640 medium at a ratio of 1:1. Subsequently, the cell suspension was inoculated subcutaneously into nude mice (5-week-old, male Bab1/c mice, Hfk Biosicence, Beijing). Tumor volumes were measured accordingly throughout the study period. At the endpoint, all nude mice were euthanized via intraperitoneal injection of sodium pentobarbital (100 mg/kg). Death was confirmed by the absence of heartbeat, respiratory motion, and pupillary reflexes, followed by physical method (cervical dislocation) to ensure irreversibility. Subcutaneous tumors were excised, preserved in formalin for subsequent comparison between groups by measuring their differences. Following measurement procedures, IHC staining was performed on sliced tumor samples. All procedures involving mice were approved by the University Committee on Use and Care of Animals at Tianjin Medical University and complied with all regulatory standards. The permit number for mouse experiments is SYXK (Jin) 2019-0004.

Statistical analysis

The statistical analysis was performed by applying the Student’s t-test to evaluate differences between the two groups. For comparisons involving more than two groups, one-way or two-way ANOVA and Tukey’s multiple comparison test were employed to determine significance. In this study, a significance threshold of p < 0.05 was utilized for establishing statistical significance. GraphPad Prism 8 and ImageJ software tools were utilized for conducting the statistical analysis procedures.

Results

LINC00525 is upregulated in bladder cancer and positively correlated with progression

To identify lncRNAs associated with the initiation and progression of BLCA, we analyzed gene expression profiles in BLCA using TCGA. Our analysis revealed that LINC00525 was significantly upregulated in BLCA tissues compared to normal tissues (Fig. 1A; Table S1). Although the oncogenic role of LINC00525 has been extensively studied in lung adenocarcinoma, colorectal cancer, and oral squamous cell carcinoma [5, 6, 8], there is a lack of relevant experimental evidence in BLCA. To address this gap, we conducted follow-up experiments focusing on LINC00525 as our research target and explored its potential value in BLCA. In order to validate the findings from TCGA, we collected 12 pairs of BLCA tissue samples and quantified LINC00525 expression using RT-PCR. Our results confirmed the elevated expression of LINC00525 in BLCA tissues (Fig. 1B). Consistent outcomes were also observed through further TCGA and GEPIA analysis (Fig. 1C and D and Figure S1A; Table S2). Additionally, RNA scope analysis demonstrated a significant increase in LINC00525 expression within BLCA tissues compared to normal tissues (Fig. 1E). To further investigate the relationship between LINC00525 and BLCA progression, we divided the 12 pairs of BLCA tissues into non-muscle-invasive bladder cancer (NMIBC) (n = 6) and muscle-invasive bladder cancer (MIBC) groups according to the degree of tumor invasion into the bladder wall. We then performed staining analysis and RT-PCR on these samples (Fig. 1F). The results indicate that LINC00525 exhibits higher expression levels in MIBC, which is characterized by greater invasiveness and elevated KI67 levels, compared to NMIBC. Therefore, our findings suggest that LINC00525 may play an important role in the progression of BLCA.

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The LINC00525 gene is documented to have only one annotated transcript in the National Center for Biotechnology Information database (https://www.ncbi.nlm.nih.gov/; Figure S1B). By utilizing the open reading frame finder (https://www.ncbi.nlm.nih.gov/orffinder/; Figure S1C) results, we can infer that LINC00525 has limited protein-coding capacity. Subsequently, we assessed the coding potential of LINC00525 based on factors such as open reading frame length and GC content (LGC), coding potential calculator (CPC), and PhyloCSF codon substitution frequency analysis [9, 10, 11]. These findings strongly support the notion that LINC00525 functions as a non-coding RNA (Figures S1D-S1F).

LINC00525 promotes proliferation, migration, and invasion in bladder cancer cells

To investigate the biological function of LINC00525 in BLCA cells, we assessed the expression levels of LINC00525 in immortalized human bladder epithelial cells (SV) and various BLCA cell lines (EJ, 87, T24, 5637, 253). Consistent with previous findings, we observed an upregulation of LINC00525 in BLCA cells compared to normal cells (Fig. 2C). Notably, highly invasive bladder cancer T24 cells exhibited significantly higher expression of LINC00525 than other BLCA cell lines, whereas superficial bladder cancer 87 cells displayed the lowest level of LINC00525 expression among all tested BLCA cell lines. Subsequently, our focus shifted towards investigating T24 cells (with the highest LINC00525 expression) and 87 cells (with the lowest LINC00525 expression), which were transfected with two shLINC00525 viruses exhibiting different knockout efficiencies (sh1/sh2) as well as normal dosage (3 µM)/double dosage (6 µM) of LINC00525 plasmid (wt1/wt2) for further analysis (Fig. 3A F; Figures S2A and S2F). The changes in cell growth, migration and invasion abilities were then evaluated. Our results from MTT and colony formation assays demonstrated that knockdown of LINC00525 effectively inhibited proliferation in both T24 and 87 cells (Fig. 3B C; Figures S2B and S2C). Additionally, knockdown of LINC00525 also led to a significant reduction in cell migration and invasion capacities (Fig. 3D and E; Figures S2D-S2E). Conversely, overexpression of LINC00525 resulted in enhanced proliferation as well as increased migration and invasion abilities within these cell lines (Figures G-J; Figures S2G-SJ). Collectively, these findings strongly suggest that elevated levels of LINC00525 can promote proliferation, migration and invasion capabilities in BLCA cells.

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LINC00525 interacts with YAP and facilitates YAP nuclear translocation

The Yes-associated protein (YAP), a core factor in the Hippo tumor suppressor pathway, plays a pivotal role in the initiation and progression of BLCA [12, 13]. To investigate whether the regulation of LINC00525 on BLCA is mediated by YAP, we utilized the TPM data of BLCA from the TCGA database. The data was categorized into high and low expression groups for the target genes (LINC00525/YAP) based on median values. Differential gene analysis between these two groups was performed using the limma package with criteria set as|logFC| > 0.5 and FDR < 0.05. This analysis identified differentially expressed genes for both LINC00525 and YAP. The intersection of these gene sets was then selected, and a Venn diagram was generated to visually represent the overlapping genes (Fig. 2A; Table S4). 74 overlapping genes demonstrated a correlation between LINC00525 and YAP, which was subsequently verified. We transfected T24 and 87 cells with shLINC00525 lentivirus and a standard dose (3 µM) of LINC00525 plasmid, respectively. By western blotting experiments, we observed that knockdown of LINC00525 inhibited YAP protein levels, while overexpression of LINC00525 resulted in their upregulation (Fig. 2B). Additionally, using the RPISeq tool, we performed interaction prediction between the RNA sequence of LINC00525 and the protein sequence of YAP using two machine learning algorithms: RF and SVM. The results demonstrated a significant likelihood of interaction between LINC00525 and YAP, with prediction scores exceeding the default threshold (RF > 0.5, SVM > 0.5) (Table S5). The relationship between LINC00525 and YAP expression remains consistent in BLCA cell lines, including SV, EJ, 87, T24, 5637, and 253. Notably, the T24 cell line exhibits the highest expression of both LINC00525 and YAP, while the 87 cell line shows the lowest expression (Fig. 2C). Subsequently, we performed RT-PCR and western blot detection on collected 12 pairs of BLCA tissues and adjacent normal tissues further confirming the positive correlation between LINC00525 and YAP (Fig. 2D). The RNA sequence of LINC00525 was retrieved from NCBI, and using lncLocator (a predictive tool for lncRNA subcellular localization), we determined that LINC00525 was predominantly localized to the cytoplasmic compartment (Fig. 2E). Consequently, our hypothesis suggests an association between LINC00525 and the nuclear translocation of YAP activated in the cytoplasm. To validate this hypothesis, we conducted RNA pull-down and RIP assays to investigate the potential binding between LINC00525 and YAP (Fig. 2F). Furthermore, immunofluorescence and western blot results demonstrated that LINC00525 facilitates the nuclear translocation of YAP. Depletion of LINC00525 resulted in an augmented cytoplasmic retention of YAP (Fig. 2G and H). Importantly, depletion of LINC00525 leads to elevated phosphorylation at S127 on YAP, resulting in decreased expression levels of its target genes. Conversely, overexpression of LINC00525 reduces YAP phosphorylation while elevating levels of its target genes (Fig. 2I). We treated T24 cell line with knocked down expression level of LINC00525 and 87 cell line with overexpressed level with GA-017 and Cytochalasin D respectively. Western blot analysis demonstrates that activation of YAP effectively counteracts sh-00525-induced suppression on downstream target genes; conversely, inhibition on YAP reverses enhancement effects induced by overexpression level changes in LINC00525 on downstream targets (Fig. 2J). LATS1, acting as an upstream regulator of YAP, exerts inhibitory effects on YAP function by facilitating the phosphorylation of the S127 site on YAP. TEA domain family member 1 (TEAD1) is a pivotal transcription factor that interacts with YAP in the nucleus, and numerous biological effects of YAP rely on its association with TEAD1. To further investigate the regulatory mechanism of LINC00525 on YAP, we conducted relevant co-immunoprecipitation experiments which revealed that overexpression of LINC00525 enhances the interaction between YAP and TEAD1 while attenuating its interaction with LATS1 (Fig. 2K). These results confirm the interaction between LINC00525 and YAP, activating the signaling pathway by promoting YAP nuclear translocation.

LINC00525 promotes proliferation, migration and invasion by increasing YAP expression in bladder cancer cells

To investigate the regulatory role of LINC00525 in BLCA, we generated stable BLCA cell lines with knockdown or overexpression of LINC00525, as well as co-transfection with YAP/siYAP. Functional analysis revealed that the promotion of cell proliferation induced by LINC00525 overexpression could be suppressed by YAP knockdown, while upregulation of YAP expression successfully reversed the inhibition caused by LINC00525 knockdown on cell proliferation (Fig. 4A, B, E and F; Figures S3A-S3B and S3E-S3F). Similarly, in cell migration and invasion assays, we obtained consistent results where down-regulation of YAP inhibited the enhancement of cell migration and invasion mediated by LINC00525, whereas knockdown of LINC00525 and upregulation of YAP showed opposite effects (Fig. 4C, D, G and H; Figures S3C-S3D and S3G-S3H). Overall, our findings suggest that LINC00525 promotes BLCA progression through activation of the YAP signaling pathway.

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LINC00525 inhibits ubiquitination-mediated degradation of YAP

Through RT-PCR analysis, we observed that LINC00525 regulates the protein level of YAP without affecting its mRNA expression (Figure S4A). We speculate that LINC00525 functions by regulating the stability of YAP protein. To investigate this possibility, we conducted cycloheximide (CHX) chase assays and found a significant reduction in YAP protein half-life upon LINC00525 knockdown compared to control cells (Fig. 5A). Furthermore, treatment with the proteasome inhibitor MG132 reversed the effects of LINC00525 knockdown or overexpression on YAP levels by inhibiting its ubiquitination and degradation (Fig. 5B). By measuring the ubiquitination level, we observed the inhibitory effect of LINC00525 on YAP ubiquitination (Fig. 5C). These findings suggest that LINC00525 activates YAP signaling by promoting nuclear translocation and inhibiting phosphorylation and ubiquitination.

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To identify the specific region of YAP responsible for binding to LINC00525, we conducted predictive analysis using the Bioinformatics website (CISBP-RNA and catRAPID). The results obtained from catRAPID indicated that LINC00525 exhibited a tendency to bind to amino acid residues 304–355 of the YAP protein (Figure S4B-S4D). In order to validate this finding, we generated a series of YAP domain deletion mutants. The RNA pull-down assay revealed that LINC00525 was unable to interact with the YAP protein when the CC (coiled-coil motif) domain, which encompasses amino acids 305–355, was deleted (Fig. 5D). Subsequently, T24 cells were transfected with the YAP-mut1 plasmid and subjected to Western blotting and ubiquitination assays. The results demonstrated that under conditions where the CC domain was deleted, LINC00525 lost its ability to activate and deubiquitinate YAP (Fig. 5E and F). Previous studies have suggested that ubiquitination of YAP is associated with its K321 site [3]. Based on this information, we hypothesized that K321 might be crucial for mediating the interaction between LINC00525 and YAP as well as inhibiting its ubiquitination. To test this hypothesis, we introduced a mutation in which lysine at position K321 was replaced by arginine. We observed that this mutation led to a decrease in YAP ubiquitination while also abolishing the impact of LINC00525 knockdown on both YAP ubiquitination and its signaling pathway (Fig. 5G). Furthermore, upon mutating K321, LINC00525 lost its regulatory function in modulating interactions between YAP/LATS1 and YAP/TEAD1 proteins (Fig. 5H). These findings strongly suggest an essential role for the K321 site in facilitating binding between LINC00525 and YAP.

LINC00525 regulates bladder cancer tumor growth through YAP

To validate the phenotype of the LINC00525/YAP pathway in vivo, we subcutaneously injected T24/87 cells treated as indicated into nude mice to investigate the impact of the LINC00525/YAP axis on BLCA growth. As anticipated, knockdown of LINC00525 significantly decelerated BLCA growth. Importantly, GA-017-mediated activation of YAP reversed the effect of LINC00525 knockdown on tumor progression (Fig. 6A and B). Conversely, overexpression of LINC00525 enhanced tumor growth, and this effect was attenuated upon inhibition of YAP (Fig. 6E and F). Furthermore, in bladder tumors, we also observed a positive regulatory relationship between LINC00525/YAP. Knockdown of LINC00525 suppressed the expression levels of YAP and pYAPS127; however, these effects were reversed by GA-017 treatment (Fig. 6C and D). Overexpression of LINC00525 activated the YAP pathway. Subsequent administration of YAP inhibitor weakened the regulation exerted by LINC00525 on YAP-related proteins (Fig. 6G and H). Collectively, these results demonstrate the promoting effect of LINC00525 on bladder tumor growth and the feasibility and effectiveness of YAP inhibitors as a therapeutic approach against LINC00525-high bladder tumorigenesis.

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Discussion

LncRNA refers to RNA molecules longer than 200 nt that do not encode proteins but function as regulators of gene expression at multiple levels in the form of RNA. In recent years, the emerging role of lncRNAs in disease pathogenesis has been gradually unveiled, as they participate in diverse cellular processes including chromatin modification, substance metabolism, cell proliferation and apoptosis. Mechanistically, lncRNAs primarily exert their regulatory effects on gene expression through transcriptional and post-transcriptional modifications, as well as epigenetic alterations. Fang et al. [5] demonstrated that LINC00525 induces repression of P21 expression by modulating chromatin structure changes. Meng et al. [6] reported that LINC00525 binds to miR-338-3p to enhance HIF-1α-mediated glycolysis in colorectal cancer cells. However, the functional significance of LINC00525 in BLCA remains elusive. Our findings reveal upregulation of LINC00525 in BLCA compared to normal tissue and its correlation with tumor development. Notably, we demonstrate that the regulation of LINC00525 in bladder cancer is mediated through YAP.

The YAP pathway is hyperactivated in numerous cancer types and associated with the acquisition of cancer stem cell properties, enhanced proliferation, drug resistance, and metastasis [14, 15]. Nuclear accumulation of YAP is crucial for its transcriptional regulation of target genes and tumor progression. In this context, TEAD family transcription factors play a pivotal role as nuclear chaperones that modulate transcriptional activation. Meanwhile, LATS1 kinase, a central component of the Hippo pathway, assumes a critical function in regulating YAP phosphorylation and thereby influencing its cytoplasmic localization. Consequently, targeting the Hippo-YAP pathway holds promise for novel approaches in cancer therapy. Moreover, c-Abl can directly phosphorylate YAP at residue Y357 in a kinase-dependent manner to stabilize it [16]. ARID1A-containing SWI/SNF complexes inhibit YAP’s transcriptional activity by disrupting its interaction with TEAD [17]. Additionally, non-coding RNAs have been implicated in modulating YAP expression within the Hippo-YAP pathway. Specifically, lncRNAs have been discovered to interact with key components of this pathway at various levels. For example, lncSNHG9 interacts with LATS1’s C-terminal domain to facilitate liquid-liquid phase separation and consequently impede LATS1-mediated YAP phosphorylation [18]. Studies have previously demonstrated that lncMALAT1 can enhance the radioresistance of CRC by promoting the transcriptional coactivation of YAP and regulating DNA damage repair [19]. However, the essential noncoding RNAs involved in YAP signaling, particularly those that directly interact with YAP, remain largely uncharacterized. Consequently, their roles in cancer progression, including BLCA, remain poorly understood. In this study, it was discovered that LINC00525 binds to the CC domain of YAP and modulates the interaction between YAP and LATS1, thereby inhibiting phosphorylation at S127 of YAP in BLCA cells and facilitating its nuclear translocation. Moreover, LINC00525 interacts with the K321 site of YAP to impede ubiquitination and degradation mediated by this site. Through these two pathways, LINC00525 promotes the nuclear accumulation of YAP, enhances the interaction between YAP and TEAD1, thereby activating YAP’s transcriptional activity. Functional analyses and mouse xenograft experiments underscored a significant increase in BLCA cell proliferation and invasion upon overexpression of LINC00525. Importantly, depletion of YAP significantly counteracted the stimulatory impact exerted by LINC00525 on BLCA tumor advancement both in vitro and in vivo. Our findings shed light on a pivotal mechanism through which LINC00525 promotes BLCA progression: direct binding to YAP inhibits its phosphorylation and subsequent ubiquitin-mediated degradation. Overexpressed LINC00525 facilitates nuclear localization of YAP, thereby stimulating transcriptional activity towards target genes. This cascade contributes to a more aggressive phenotype within the context of BLCA. This lncRNA’s role in regulating stability may provide novel insights for cancer treatment. The identification of the LINC00525/YAP axis may serve as a reference point for exploring novel treatments for BLCA.

However, this study has several limitations. Regarding the collected pathological samples, we employed a simplified classification of NMIBC and MIBC to investigate the role of LINC00525 in BLCA, without conducting deeper analyses stratified by T-stage. Analysis of TCGA database revealed elevated LINC00525 expression in stage II, III, and IV BLCA tissues compared to normal tissues (Figures S5A). Although an upward trend was observed in stage I samples, statistical significance was not achieved due to the limited sample size (n = 4), potentially attributable to the TCGA database’s predominant inclusion of intermediate-to-advanced stage cases. Notably, no significant expression differences were detected among stage II, III, and IV tumors. Based on these findings, we propose the following hypotheses: (1) LINC00525 may be upregulated during malignant transformation from non-muscle-invasive to muscle-invasive tumors (Stage I to II), maintaining stable expression during subsequent progression; (2) Molecular heterogeneity across stages (e.g., genomic variations, microenvironmental disparities) may diminish detection sensitivity for expression differences. Future studies should expand early-stage sample sizes and incorporate single-cell sequencing or spatial transcriptomics to validate these hypotheses, thereby advancing the potential of LINC00525 as an early diagnostic and stratification biomarker.

Additionally, TCGA-based survival analysis revealed no significant prognostic value of LINC00525 expression in bladder cancer patients, potentially confounded by insufficient sample size (n(High) = 20, n(Low/Medium) = 59) and inherent limitations of retrospective study design (Figures S5B). Prospective cohort studies or functional experiments are required to clarify its mechanistic role. Despite the lack of prognostic or advanced-stage differentiation significance in TCGA data, the observed overexpression of LINC00525 in tumor tissues and its interaction with the YAP signaling axis retain biological relevance. These findings warrant further investigation into its functional contributions to tumorigenesis.

Conclusion

Collectively, our studies unveil a functional association between LINC00525 and YAP signaling. LINC00525 directly interacts with YAP, thereby inhibiting phosphorylation at the S127 site and preventing ubiquitination degradation mediated by the K321 site. This interaction facilitates nuclear localization of YAP, subsequently activating its signaling pathway. Based on our findings, we propose that the LINC00525/YAP axis plays a pivotal role in BLCA progression. These findings provide a potential novel avenue for therapeutic intervention in BLCA.

Data availability

No datasets were generated or analysed during the current study.

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