Biomarker for diagnosing bladder cancer and application thereof

Abstract

The invention provides a biomarker for diagnosing bladder cancer and application of the biomarker, and by analyzing single-cell transcriptome data of normal bladder epithelial cells and bladder cancer cells, ZDHHC20 is found to be continuously increased in the process of transforming from the bladder epithelial cells to vicious cells, and is remarkably and highly expressed in human bladder cancer tissues. Functional studies show that overexpression of ZDHHC20 can significantly activate PI3K / AKT, Wnt / beta-catenin, cell cycle and other cancer-promoting signal channels, so that normal bladder epithelial cells obtain malignant growth ability independent of adherence. The results show that ZDHHC20 is an important molecule for driving the vicious transformation of bladder epithelial cells. The invention discloses a key effect of the ZDHHC20 in bladder cancer generation, and shows that the ZDHHC20 can be used as a bladder cancer diagnosis and treatment target.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to biomarkers for diagnosing bladder cancer and their applications. Background Technology

[0002] The development and progression of bladder cancer is a complex process involving multiple factors. On the one hand, its occurrence is closely related to epidemiological factors such as smoking and occupational exposure; on the other hand, it also involves multi-level biological regulation, including the combined effects of gene mutations, abnormal gene expression, and various mechanisms such as cellular behavior, immune responses, and the tumor microenvironment. Although related research has deepened in recent years, the molecular events revealed so far only reflect a part of the bladder cancer regulatory network. Existing studies have shown that there are complex and extensive cross-regulatory relationships among multiple signaling pathways, and the common upstream regulators of these pathways have not yet been fully elucidated. It is worth noting that many genes reported to be associated with bladder cancer, while promoting tumor progression, are often insufficient to drive tumorigenesis on their own. In addition, there are significant differences in the mutation or expression patterns of these genes among different patients, suggesting that they may not be stable and core regulatory factors in tumorigenesis. Therefore, current early screening and intervention strategies based on single genes still face certain limitations in clinical application. Systematically identifying and analyzing key molecular events that drive bladder cancer is of great significance for establishing effective early warning indicators and precision prevention strategies.

[0003] The zinc finger DHHC palmitoyltransferase family (ZDHHC) is a class of enzymes that catalyze palmitoylation of proteins. Their mechanism of action involves transferring the palmitic acid group from palmitoyl-CoA to a cysteine ​​residue on the target protein, thereby regulating protein localization, stability, and function. Early research on this family of proteins focused primarily on metabolic diseases, subsequently expanding to areas such as neurological disorders and immune regulation. In recent years, increasing research has focused on the role of the ZDHHC family in tumorigenesis and development. Summary of the Invention

[0004] To address the shortcomings and deficiencies of existing technologies, this invention provides a biomarker for diagnosing bladder cancer and its application. It demonstrates that ZDHHC20 overexpression can activate multiple pro-cancer signaling pathways and induce malignant transformation of human bladder epithelial cells without other exogenous stimuli. ZDHHC20 may play a key driving role in the development of bladder cancer.

[0005] The technical solution adopted in this invention is: a biomarker for diagnosing bladder cancer, wherein the biomarker is the enzyme ZDHHC20, which catalyzes the modification of protein palmitoylation.

[0006] Application of a biomarker ZDHHC20 detection reagent in the preparation of reagents for detecting and diagnosing bladder cancer.

[0007] The biomarker ZDHHC20 is a marker for the early diagnosis of bladder cancer.

[0008] Application of ZDHHC20 as a target in the preparation of drugs for treating bladder cancer.

[0009] Furthermore, the drug used to treat bladder cancer is a ZDHHC20 overexpression inhibitor.

[0010] Furthermore, the bladder cancer treatment drug is a pharmaceutically acceptable vector containing inhibition of ZDHHC20 gene overexpression.

[0011] The beneficial effects of this invention are as follows: This invention provides a biomarker for diagnosing bladder cancer and its application. Analysis of single-cell transcriptome data from normal bladder epithelial cells and bladder cancer cells revealed that ZDHHC20 is continuously elevated during the malignant transformation of bladder epithelial cells and is significantly overexpressed in human bladder cancer tissue. Functional studies showed that ZDHHC20 overexpression can significantly activate pro-cancer signaling pathways such as PI3K / AKT, Wnt / β-catenin, and cell cycle, enabling normal bladder epithelial cells to acquire adherent-independent malignant growth ability. These results indicate that ZDHHC20 is an important molecule driving the malignant transformation of bladder epithelial cells. This invention reveals the crucial role of ZDHHC20 in the development of bladder cancer, suggesting that ZDHHC20 can serve as a target for the diagnosis and treatment of bladder cancer. Attached Figure Description

[0012] Figure 1 : To uncover key molecules that drive malignant transformation of bladder epithelial cells. Figure 1 In section A: Single-cell sequencing data from normal and bladder cancer tissues (20:20) were processed, and cells that highly expressed the BC oncogene and the gene maintaining NBU stability were defined as the NBU / BC population, and Monocle time simulation analysis was performed on them. Figure 1 BD in the data: Pathway enrichment of genes with significant changes in BC compared to NBU ( Figure 1 (B in the original text) Observe the changes in gene transcription levels in the most significantly enriched pathway as malignant transformation occurs. Figure 1 (C in the text), while also showing changes in other genes and labeling classic oncogenes ( Figure 1 (D in the middle). Figure 1 E: histochemical staining was used to detect the expression of ZDHHC20 in clinical bladder cancer samples. Figure 1F: Proteomic sequencing results of bladder epithelial cells before and after ZDHHC20 overexpression in UROtsa (3:3), control group transfected with empty plasmid (i.e., Vector). n, number of samples; # P <0.0001.

[0013] Figure 2 ZDHHC20 overexpression enables bladder epithelial cells to acquire growth-independent capabilities. Figure 2 AB in the text: bladder epithelial cells UROtsa ( Figure 2 (A) or SV-HUC-1 ( Figure 2 B) Results of soft agar colony formation experiment after ZDHHC20 overexpression. Compared to the Medium group transfected with Vector P<0.05 Compared to transfecting Vector with EGF-induced group P<0.05 #Compared to the Medium group that overexpressed ZDHHC20 P<0.05 . Detailed Implementation

[0014] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative effort are all within the scope of protection of the present invention.

[0015] Experimental methods:

[0016] Analysis methods for single-cell sequencing: We used the Seurat, Harmony, and Monocle R packages to perform integrated and pseudo-time series analysis on single-cell transcriptome data from bladder cancer. First, we read the 10× Genomics expression matrix of the samples, including bladder cancer samples and adjacent normal tissue samples, and constructed a Seurat object using CreateSeuratObject, with the selection criteria set to min.cells = 3 and min.features = 200. Then, we merged the samples and calculated the proportions of mitochondrial genes (percent.mt) and erythrocyte-related genes (percent.rbc); cells with percent.mt < 5 and percent.rbc < 1 were retained for subsequent analysis. Next, we performed data standardization, hypervariable gene identification, data scaling, and principal component analysis (PCA). We used the Harmony method to correct for batch effects, using the sample origin (orig.ident) as the correction variable. Based on the Harmony dimensionality reduction results, we performed cluster analysis using FindNeighbors and FindClusters, and visualized the results using UMAP. In the initial ensemble analysis, the neighbor graph was constructed using the first 15 dimensions, the clustering resolution was set to 0.1, and the UMAP dimensionality reduction used the first 16 dimensions.

[0017] During the cell type annotation phase, FeaturePlot was used to determine the expression patterns of epithelial cell markers (such as EPCAM, KRT7, KRT20, MUC1, ERBB2, UPK1A / UPK1B / UPK2 / UPK3A), immune cell markers (such as CD3D, CD3E, CD4, CD8A, CD19, MS4A1, CD68, CD14), and matrix / endothelial cell markers (such as FAP, ACTA2, PECAM1, CDH5). The main cell populations were annotated as tumor / epithelial cells, T cells, B cells, fibroblasts, macrophages, and endothelial cells. Subsequently, cells annotated as "Tumor mixed with normal epithelial" were extracted for secondary analysis. This subpopulation underwent normalization, hypervariable gene screening, scaling, PCA, Harmony batch correction, clustering, and UMAP dimensionality reduction. In this step, the neighbor graph construction also uses the first 15 dimensions, increasing the clustering resolution to 0.5. UMAP uses the first 16 dimensions to obtain higher resolution epithelial cell heterogeneity features. Further, a subset of tumor / adjacent normal tissue epithelial cells is extracted based on sample grouping for downstream analysis.

[0018] In the differential expression analysis, RNA was set as the default analysis matrix, and layer information was merged using JoinLayers when necessary. Subsequently, FindAllMarkers was used to identify marker genes between different clusters or groups, with parameters set to only.pos = TRUE, min.pct = 0.25, and logfc.threshold = 0.25. Genes with corrected p_val_adj < 0.05 and |avg_log2FC| > 0.5 were further selected as significantly differentially expressed genes.

[0019] The pseudo-temporal analysis was performed using Monocle. First, the selected Seurat epithelial cell objects were converted into Monocle CellDataSet objects. An expression matrix was constructed using the raw RNA counting matrix, with cell annotation information used as phenoData and gene annotation information as featureData. `lowerDetectionLimit = 0.5` and `expressionFamily = negbinomial.size()` were set. Normalization and dispersion estimation were then performed using `estimateSizeFactors` and `estimateDispersions`. Expressed genes were identified using `detectGenes(min_expr = 0.1)`, retaining genes expressed in at least 10 cells. Next, hypervariable genes identified by Seurat were used as ordering genes, and pseudo-temporal ordering was performed using `setOrderingFilter`. Dimensionality reduction was performed using the DDRTree method, and cell developmental trajectories and pseudo-temporal sequences were constructed using `orderCells`. The trajectory results were visualized according to pseudo-time, cell state, and sample grouping.

[0020] To identify key genes related to cell evolution, the DifferentialGeneTest was used to screen pseudo-time-related genes. The model was set to ~sm.ns (Pseudotime), and the top 100 genes were selected by q-value to generate pseudo-time heatmaps. Furthermore, BEAM (branch expression analysis modeling) was used to analyze branch-related genes at trajectory branch points (branch_point = 1), screening for branch-dependent expression genes and generating branch heatmaps to analyze transcriptional dynamics in different evolutionary branches.

[0021] Immunohistochemistry First, tissue specimens were fixed in 4% paraformaldehyde at 4°C for 24–48 hours. After fixation, the specimens were rinsed with running water for approximately 20 minutes to remove residual fixative, and then dehydrated sequentially with different concentrations of ethanol (30%, 50%, and 70% for 30 minutes each, followed by overnight incubation in 70% ethanol at 4°C). Dehydration was then continued in 80%, 85%, 95%, 100% I, and 100% II ethanol, each step lasting approximately 1 hour. After dehydration, the specimens were cleared with xylene, followed by paraffin embedding. The embedded tissue blocks were then cut into 4–5 cm sections. μ The sections were cut to a thickness of m and spread in a 42°C water bath. The sections were then attached to a detachable glass slide for later use.

[0022] Before immunohistochemical staining, sections were heated in a 65°C oven for 2–3 hours. Dewaxing was then performed (twice with xylene for 10 minutes each), followed by a brief treatment with a xylene and ethanol mixture before a gradient ethanol rehydration process (100%, 95%, 90%, 80%, 70%, 50%), each step lasting approximately 90 seconds. After rehydration, sections were thoroughly rinsed with distilled water. Antigen retrieval was then performed: sections were placed in pH 6.0, 0.01 M citrate buffer and retrieval was performed using microwave heating. The solution was heated to boiling and then continuously heated at medium-low power for 7 minutes per cycle, for a total of 4 cycles, followed by natural cooling to room temperature.

[0023] After cooling, the sections were washed with 1×TBS buffer and the tissue areas were delineated using a histochemical pen. Then, 3% H2O2 was added for approximately 30 minutes to block endogenous peroxidase activity. After washing again with TBS, the sections were incubated with 5% BSA in a humidified chamber at room temperature for 30 minutes to block non-specific binding sites. Primary antibody solution was then added to cover the tissue areas, and the sections were incubated overnight at 4°C (12–16 hours). A blank control (TBS instead of primary antibody) and an IgG negative control were provided to assess staining specificity.

[0024] The next day, after sectioning and rewarming, the sections were washed with TBS and incubated with the corresponding biotinylated secondary antibody at 37°C for 1 hour. Washing with TBS was then repeated, followed by incubation with the SABC complex at 37°C for 1 hour. After thorough washing, DAB chromogenic solution was added for the colorimetric reaction. The color development was observed under a microscope in real time, and the reaction was terminated with distilled water.

[0025] After staining, hematoxylin was used for counterstaining. After staining for 1–2 minutes, the sections were rinsed with running water to make the cell nuclei blue. The sections were then dehydrated again with graded ethanol and cleared with xylene. Finally, they were mounted with neutral resin and observed and imaged under a microscope after drying.

[0026] Construction of Urotsa or SV-HUC-1 cells overexpressing ZDHHC20 To construct UROtsa or SV-HUC-1 cells stably overexpressing ZDHHC20, the human ZDHHC20 coding sequence was cloned into a lentiviral expression vector to construct a ZDHHC20 overexpression plasmid; an empty vector was used as a negative control. The recombinant expression vector and packaging plasmid were co-transfected into 293T cells for viral packaging. Viral supernatants were collected at 48 and 72 hours post-transfection. After centrifugation to remove cell debris, the cells were subjected to 0.45 μL of thiocyanate solution. μ m-filter membrane filtration for later use.

[0027] UROtsa or SV-HUC-1 cells were seeded in culture plates. When the cells reached approximately 30%–50% confluence, they were infected with culture medium containing ZDHHC20 overexpressing lentivirus or an empty control virus. An appropriate amount of polybrene was added as needed to improve infection efficiency. After 24 hours of infection, the medium was replaced with fresh complete medium, and the cells were cultured for another 48–72 hours. Drug screening was then performed, maintaining the selection until all uninfected cells in the control group died. The surviving cells were retained and further cultured to obtain stable ZDHHC20 overexpressing UROtsa or SV-HUC-1 cell lines and their corresponding negative control cell lines. After construction, total protein was extracted from each group of cells, and the protein expression level of ZDHHC20 was detected by Western blot to verify the successful construction of the overexpression cell lines. The validated cells were used for subsequent functional experiments.

[0028] Soft agar colony formation experiment To assess the anchorage-independent growth capacity of cells, a soft agar colony formation assay was used. This assay indirectly reflects the survival, continuous proliferation, and malignant transformation potential of cells by observing their ability to form colonies in a semi-solid culture system. The main reagents used in the experiment included agar, BME medium, fetal bovine serum (FBS), antibiotics, glutamine, and 7.5% NaHCO3. The basal culture medium used in the experiment was prepared by mixing BME, FBS, PBS, antibiotics, glutamine, and NaHCO3 in a ratio of 4:1:1:0.1:0.1:0.1; the agar stock solution concentration was 1.25%.

[0029] First, prepare the agar solution. Dissolve agar powder in double-distilled water to prepare a 1.25% agar solution, autoclave, and keep warm for later use. If using pre-prepared and refrigerated agar solution, heat it until fully melted before use and maintain it in a constant temperature water bath to prevent premature solidification during subsequent gel preparation. Meanwhile, preheat the prepared culture medium to approximately 45°C. When laying the lower gel, mix the preheated culture medium and agar solution at a volume ratio of 3:2, add 3 mL to each well of a six-well plate, ensuring even coverage of the bottom of the wells, and allow to stand at room temperature until completely solidified. Next, prepare a single-cell suspension. Collect cells and thoroughly disperse them by pipetting to ensure they remain in a single-cell state as much as possible. Then count the cells and calculate the required seeding volume. Seed each well with 1 × 10⁶ cells. 4 Cells. When preparing the upper gel, first mix the culture medium and agar solution according to the specified volume, with each well containing 736 cells. μ L medium and 264 μ The upper gel is prepared by adding the appropriate volume of cell suspension, mixing rapidly, and then spreading it onto the surface of the solidified lower gel, with a total volume of 1 mL per well. To reduce premature solidification of the upper gel during the operation, the six-well plate can be preheated appropriately, and the mixture should be kept at a suitable temperature before adding cells. After the upper gel solidifies at room temperature, the culture plate is sealed and incubated in a 37°C, 5% CO2 incubator for 7–15 days.

[0030] After culture, colony formation was observed and photographed under a microscope. Each experimental group had three replicates, with five fields of view randomly selected from each well for recording. For subsequent statistical analysis, the average number of colonies in each well (five images) was first calculated, followed by the average of the three replicates, to obtain the average colony formation number N. The final result was converted to N × 190.8 to represent the number of anchorage-independent colonies formed after 10,000 cells were seeded. Statistical analysis and graphing were performed using GraphPad Prism software.

[0031] Western blot Western blot technology is used to detect the expression level of target proteins in cells or tissues. First, cultured cells are collected, and lysis buffer is added for protein lysis. Subsequently, sonication is used to further break down nucleic acids and cell structures, allowing for the full release of proteins. The lysed sample is then centrifuged at high speed to remove cell debris, and the supernatant is collected as the total protein sample. The sample concentration is determined using protein quantification methods, and an appropriate amount of buffer is added to bring the volume to a final level. Protein loading buffer is also added, and the sample is then heated at 100°C for approximately 5 minutes to denature the protein.

[0032] After protein sample processing, separation was performed using SDS-PAGE electrophoresis. Separating and stacking gels of appropriate concentrations were prepared according to the target protein molecular weight. The processed protein samples, along with molecular weight markers, were added to the gel wells. In the electrophoresis buffer system, a higher voltage was first applied to concentrate the proteins in the stacking gel, and then electrophoresis continued in the separating gel to ensure thorough separation of proteins of different molecular weights.

[0033] After electrophoresis, the proteins in the gel are transferred to a PVDF membrane. Before transfer, the PVDF membrane needs to be activated with methanol and equilibrated in transfer buffer. Then, the transfer clamp is assembled in the following order: sponge—filter paper—gel—PVDF membrane—filter paper—sponge. Transfer is performed under constant voltage conditions, allowing the proteins to migrate from the gel to the membrane surface.

[0034] After transfer, the membrane is blocked, typically using a protein-containing blocking buffer incubated at room temperature for a period to reduce non-specific binding. Then, a primary antibody targeting the target protein is added, and the membrane is incubated overnight at 4°C to allow for complete antibody binding to the target protein on the membrane. After incubation, the membrane is repeatedly washed with buffer to remove unbound primary antibody, and then an enzyme-labeled secondary antibody corresponding to the primary antibody is added for further incubation. After secondary antibody incubation, the membrane is washed thoroughly again, followed by the addition of a chemiluminescent substrate for development. Protein band signals are recorded in an imaging system to analyze the expression level of the target protein.

[0035] Experimental results (1) ZDHHC20 may be a key molecule driving the malignant transformation of bladder epithelial cells.

[0036] This invention defines a bladder cancer (BC) cell / normal bladder epithelial cell (NBU) population (Figure 1, A) by analyzing single-cell sequencing data from normal and bladder cancer tissues. Pseudo-temporal analysis shows that NBUs are located at the differentiation initiation stage, while BCs are located at the mid-terminus, consistent with the general pattern of NBUs gradually transforming into malignant cells (Figure 1, A). Further analysis revealed that differentially expressed genes between BCs and NBUs were most significantly enriched in post-translational regulatory pathways; compared with all differentially expressed genes, ZDHHC20 It is the only gene that steadily increases with the malignant transformation of NBU, even exceeding... EGFR , MYC , CCND1 Classic oncogenes ( Figure 1 ZDHHC20 expression was significantly elevated in human bladder cancer tissue (5 times higher than normal). Figure 1 (E in the text).

[0037] Proteomic analysis showed that overexpression of ZDHHC20 in bladder epithelial cells led to upregulation of pro-cancer signaling pathways such as PI3K / AKT, Wnt / β-catenin, and cell cycle, suggesting that ZDHHC20 may be a common upstream molecule in these pathways and plays a crucial role in bladder cancer development. Figure 1 (F in the text).

[0038] This invention discovered that bladder epithelial cells overexpressing ZDHHC20 acquired adhesion-independent malignant growth ability, with a malignancy level even exceeding that of EGF-induced cells. Figure 2 (AB in the text). These results suggest that ZDHHC20 overexpression endows bladder epithelial cells with the ability to proliferate malignantly and evade growth control, which is a key feature of their malignant transformation.

[0039] (2) High expression of ZDHHC20 alone can directly lead to malignant transformation of bladder epithelial cells.

[0040] This invention discovered that bladder epithelial cells overexpressing ZDHHC20 acquired adhesion-independent malignant growth ability, with a malignancy level even exceeding that of EGF-induced cells. Figure 2 (AB in the text). These results suggest that ZDHHC20 overexpression endows bladder epithelial cells with the ability to proliferate malignantly and evade growth control, which is a key feature of their malignant transformation.

[0041] The specific embodiments described in this invention are merely illustrative of the spirit of the invention. Those skilled in the art to which this invention pertains can make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of the invention or exceeding its defined scope. Although the invention has been detailed and described in the accompanying drawings and foregoing description, such descriptions are considered illustrative or exemplary rather than restrictive. It should be understood that changes and modifications can be made by those skilled in the art within the scope of the following claims.

Claims

1. The application of a biomarker detection reagent in the preparation of reagents for detecting and diagnosing bladder cancer, characterized in that, The biomarker is ZDHHC20, an enzyme that catalyzes the palmitoylation modification of proteins.

2. The application according to claim 1, characterized in that, The enzyme ZDHHC20 is a biomarker for early diagnosis of bladder cancer.

3. Application of ZDHHC20 as a target in the preparation of drugs for treating bladder cancer.

4. The application according to claim 3, characterized in that, The drug mentioned for treating bladder cancer is a ZDHHC20 overexpression inhibitor.

5. The application according to claim 4, characterized in that, The aforementioned drug for treating bladder cancer is a pharmaceutically acceptable vector containing inhibition of ZDHHC20 gene overexpression.

Images