Application of RRBP1 as target in preparation of pharmaceutical composition for treating bladder cancer

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of RRBP1 as a target spot in preparation of a pharmaceutical composition for treating bladder cancer. The pharmaceutical composition for treating bladder cancer is a targeted inhibitor of RRBP1. The inhibitor is shRNA (short hairpin ribonucleic acid). Experimental results show that the RRBP1 is a potential therapeutic target for bladder cancer progression, and development of drugs for inhibiting the expression level of the RRBP1 is expected to provide a new strategy for bladder cancer treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of RRBP1 as a target in preparing a pharmaceutical composition for treating bladder cancer. Background Art

[0002] Bladder cancer, a highly prevalent malignant tumor of the urinary system, has attracted considerable attention for its aggressive nature and poor prognosis. According to the 2023 Global Cancer Statistics, bladder cancer ranks 8th in incidence among all malignant tumors, with a higher incidence rate in men, ranking 4th in incidence and 8th in mortality. Pathologically, bladder cancer is divided into two main types: non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC). Currently, surgical resection is the primary treatment option for early-stage cases, but patients with advanced disease face challenges with traditional adjuvant therapies, which have limited efficacy and sustained efficacy. Recent studies have demonstrated that tumor immune escape mechanisms mediated by the PD-1 / PD-L1 pathway play a key role in the progression of bladder cancer. Immune checkpoint inhibitors targeting this pathway have ushered in a new era in bladder cancer treatment. PD-1 / PD-L1 inhibitors are increasingly prominent in the treatment of advanced-stage bladder cancer, significantly improving patient prognosis and survival. However, clinical practice has found that PD-1 / PD-L1 immunotherapy still faces bottlenecks such as low treatment responsiveness, unclear resistance mechanisms, and difficulty in screening beneficiary populations. It is urgent to further elucidate the key regulatory factors and molecular mechanisms of responsiveness to PD-1 / PD-L1 immunotherapy for bladder cancer, explore potential therapeutic targets, and further improve the sensitivity and efficacy of treatment through combined treatment strategies.

[0003] RRBP1 (ribosome-binding protein 1) plays a crucial role in the development, progression, and prognosis of various tumors. Studies have shown that RRBP1 is highly expressed in a variety of malignant tumors, including colorectal cancer, lung cancer, ovarian cancer, oral squamous cell carcinoma, bladder cancer, endometrial cancer, osteosarcoma, and cervical cancer. Its high expression is closely associated with tumor malignancy, invasiveness, metastatic potential, and poor patient prognosis. The mechanisms of action of RRBP1 vary across different cancer types. For example, in oral squamous cell carcinoma (OSCC), RRBP1 induces cisplatin resistance in tumor cells by regulating the expression of YAP1, a key effector molecule in the Hippo pathway. In prostate cancer, RRBP1 expression is regulated by m6A methylation, and its mRNA stability depends on the action of the methyltransferase METTL3. High RRBP1 expression is significantly associated with enhanced tumor invasiveness and poor prognosis. Furthermore, RRBP1 overexpression promotes tumor metastasis. For example, in osteosarcoma, high RRBP1 expression is closely associated with an increased risk of distant metastasis. However, the specific molecular mechanism of how RRBP1 promotes the occurrence and development of bladder cancer and immune escape has not yet been fully elucidated. Summary of the Invention

[0004] In response to the deficiencies of the existing technology, the present invention aims to provide a new target for the treatment of bladder cancer and further explore the specific regulatory mechanism of RRBP1 on the occurrence and development of bladder cancer and immune escape.

[0005] The technical solution provided by the present invention is: use of RRBP1 as a target in preparing a pharmaceutical composition for treating bladder cancer.

[0006] Preferably, knockdown of RRBP1 can inhibit bladder cancer cell proliferation, invasion and migration.

[0007] Preferably, the pharmaceutical composition for treating bladder cancer is a targeted inhibitor of RRBP1.

[0008] Preferably, the targeted inhibitor of RRBP1 is shRNA.

[0009] Preferably, the nucleotide sequence of the shRNA is shown in SEQ ID NO. 1, 2, 3 or 4 in the sequence listing.

[0010] The present invention also provides a pharmaceutical composition for treating bladder cancer, wherein the pharmaceutical composition comprises a targeted inhibitor of RRBP1.

[0011] Preferably, the pharmaceutical composition comprises anti-PD-L1.

[0012] The experimental results of the present invention found that RRBP1 has a positive regulatory effect on the proliferation and invasion ability of bladder cancer cells. After knocking down RRBP1 in bladder cancer cells, the proliferation rate and invasion ability of bladder cancer cells were significantly reduced. Animal experiments further verified the above results. In addition, RRBP1 can enhance the efficacy of anti-PD-L1 immunotherapy by reshaping the immune microenvironment of bladder cancer. The research results of the present invention suggest that RRBP1 is a potential therapeutic target for the progression of bladder cancer. The development of drugs that inhibit RRBP1 expression levels is expected to provide a new strategy for the treatment of bladder cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Figure 1 shows the expression level of RRBP1 in bladder cancer tissues and its correlation with the prognosis of bladder cancer. Figure 1 shows the expression level of RRBP1 in bladder cancer tissues analyzed in the TCGA bladder cancer dataset. Figure 1 shows the expression level of RRBP1 in bladder cancer tissues detected by immunohistochemistry. Figure 1 shows the correlation analysis between RRBP1 and the prognosis of bladder cancer patients. Figure 2After constructing RRBP1 knockdown stable expression lines in T24 and MB49 cells using lentiviral vectors, the following were performed: a. RT-qPCR was used to detect the mRNA expression level of RRBP1 in T24 and MB49 cells; b. Western blotting was used to detect the protein expression level of RRBP1 in T24 and MB49 cells; Figure 3 The proliferation rate, invasion and migration abilities of bladder cancer cells were significantly decreased after knockdown of RRBP1, among which: a. Cell clone assay for the proliferation rate of T24 bladder cancer cells; b. CCK-8 assay for the proliferation rate of T24 bladder cancer cells; c. Cell clone assay for the proliferation rate of MB49 bladder cancer cells; d. CCK-8 assay for the proliferation rate of MB49 bladder cancer cells; e. Transwell assay for the migration and invasion abilities of T24 bladder cancer cells; f. Transwell assay for the migration and invasion abilities of MB49 bladder cancer cells; Figure 4 The results are in vivo experiments validating the effect of RRBP1 on tumor growth, including: a. a BALB / C-nude mouse model used to test the effect of RRBP1 on mouse tumor growth; b. a C57BL / 6J mouse model used to test the effect of RRBP1 on mouse tumor growth; Figure 5 The results are as follows: ac. are the correlation between RRBP1 expression and immune cell infiltration in the TCGA bladder cancer cohort; de. are the correlation between RRBP1 and CD8+ T cell infiltration in bladder cancer tissues detected by immunohistochemistry; f. are the differentially expressed genes in tumor tissues of shNC and shRRBP1 mice; g. are the results of GSEA enrichment analysis of differentially expressed genes; hi. are the effects of knocking down RRBP1 combined with anti-PD-L1 antibodies on mouse tumor growth using a C57BL / 6J mouse model. DETAILED DESCRIPTION

[0014] To facilitate understanding of the present invention, the present invention will be described in more detail below with reference to the accompanying drawings and specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. On the contrary, the purpose of providing these embodiments is to make the understanding of the present invention more thorough and comprehensive.

[0015] 1. Experimental methods: 1. Main materials Four lentiviral knockdown vectors were constructed by Shanghai GeneGene.

[0016] 2. Cell culture The immortalized human bladder cancer cell lines used in this study, including T24 and the immortalized mouse bladder cancer cell line MB49, were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences. All cells were cultured under strict sterile conditions and verified by mycoplasma testing and STR analysis. To thaw cells, cells were removed from the liquid nitrogen tank and quickly transferred to a 37°C water bath, where they were gently shaken to rapidly thaw. After thawing, the cells were quickly transferred to a UV-sterilized laminar flow hood. The cell suspension was transferred to a 15 mL centrifuge tube, and the appropriate amount of complete medium was added. After mixing, the suspension was centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cell pellet was then resuspended in fresh complete medium and transferred to a cell culture flask. The cells were cultured in a 37°C, 5% CO2 incubator. The next day, cell growth was observed microscopically, and medium changes or subcultures were performed as needed.

[0017] 3. Lentiviral vector construction and transfection Bladder cancer cells were transfected with lentiviral vectors to construct stable knockdown of RRBP1 in T24 and MB49 bladder cancer cells (Hu-shRRBP1#1, Hu-shRRBP1#2, MS-shRRBP1#1, Ms-shRRBP1#2). The specific sequences are as follows: Hu-shRRBP1#1: 5'-GTGAAGCATCTCGAAGAGATT-3' (SEQ ID NO. 1 in the Sequence Listing), Hu-shRRBP1#2: 5'-GCCAAGAAGAAGTCTGGTTCA-3' (SEQ ID NO. 2 in the Sequence Listing), MS-shRRBP1#1: GACAGAUACAGUUGCUAAUTT (SEQ ID NO. 3 in the Sequence Listing), and Ms-shRRBP1#2: GCACCUGCAAAGAAGAAGUTT (SEQ ID NO. 4 in the Sequence Listing). All RRBP1 knockdown lentiviruses used in the experiments were constructed by Shanghai GeneGene.

[0018] The specific steps for lentiviral transfection of cells are as follows: (1) Prepare 2 ml of cell suspension with a density of 3-5 × 104 cells / ml using complete culture medium. Take 500 μl of cell suspension and inoculate it into a 24-well culture plate. Incubate at 37°C for 16-24 hours until the cell confluence reaches 20-30%. (2) Take the virus out of the refrigerator and slowly thaw it on ice. Dilute the virus with complete culture medium according to the experimental requirements; (3) Based on the cell MOI and virus titer, calculate the formula: virus volume = (MOI × number of cells) / virus titer. Aspirate the supernatant from the well, add the corresponding volume of virus and the corresponding infection enhancement solution, mix well, and continue to culture; (4) Culture at 37°C for 16 hours, then replace with complete culture medium and continue culturing. Observe cell morphology during infection. If changes occur, change the medium 8 hours in advance to maintain normal cell growth. (5) The medium can be changed according to the growth status of the cells to maintain cell activity; (6) About 72 hours after infection, when the fluorescence expression is high, observe under a microscope. The group with an infection efficiency of about 80% and good cell growth is passaged and cultured; (7) When the cells grow well, add puromycin to the cell culture medium and continue culturing; (8) Regularly observe the growth status and morphological changes of cells under a microscope. Untransfected cells gradually die because they cannot tolerate puromycin, while transfected cells can survive and continue to grow.

[0019] 4. RT-qPCR Experiment After establishing stable RRBP1 knockdown cell lines in T24 and MB49 cells, RRBP1 mRNA expression levels were assessed by RT-qPCR. According to the experimental plan, cells were seeded in 24-well plates in advance (using 24-well plates as an example). One-fifth volume of chloroform (approximately 100 μL) was added to the EP tube containing the lysate. The tube was shaken vigorously to mix thoroughly. After ensuring no stratification, the tube was placed on ice for 10 minutes. After this, the tube was centrifuged at 12,000 rpm for 15 minutes at 4°C. Separation of the EP tube will occur at this point, with proteins and phenolic substances located in the inorganic phase (the white film in the lower and middle layers) and RNA in the top aqueous phase. Carefully aspirate the colorless, transparent liquid from the top layer and transfer it to a new, enzyme-inactivated EP tube. An equal volume of isopropanol was added to the tube, mixed by inversion, and the tube was placed on ice for 10 minutes. The tube was then centrifuged at 12,000 rpm for 20 minutes at 4°C. Discard the supernatant, add 1 mL of 75% ethanol to wash the precipitate, and centrifuge at 12,000 rpm for 5 minutes at 4°C. Repeat this step once. After discarding the supernatant, open the EP tube and place it in a clean bench to dry for 15-30 minutes until the residual ethanol is completely evaporated. Add 20-30 μL of RNase-free water to the precipitate, mix well, and store at -80°C or proceed to the next concentration determination. RT-qPCR reactions were performed using the KODSYBR qPCR kit (TOYOBO Co., Ltd, Osaka, Japan) on a real-time fluorescence quantitative PCR instrument (LightCycler 480Ⅱ, Mannheim, Germany). The primers for each gene were synthesized at Suzhou Jikai Company.

[0020] 5. Western blotting After establishing stable knockdown cell lines for RRBP1 in T24 and MB49 cells, an appropriate amount of cell lysis buffer (containing protease inhibitors) was added based on the cell load in the wells. After lysis, cells were collected into EP tubes, sonicated, and centrifuged at 12,000 rpm for 5 minutes. Protein concentration was determined by the BCA assay, followed by denaturation by heating in a 95°C metal bath for 5 minutes. 35 μg of protein sample was loaded per well and separated by 10% SDS-PAGE. The membranes were transferred to PVDF membranes (Millipore, USA). After blocking with 5% BSA for 1 hour, the membranes were incubated with primary antibodies (RRBP1, PCNA, Ub, K48, K63, and GAPDH) overnight at 4°C. The following day, the membranes were washed three times with PBST and incubated with secondary antibody working solution for 1 hour at room temperature. Bound proteins were detected by ECL chemiluminescence.

[0021] 6. CCK8 experiment After establishing stable RRBP1 knockdown cell lines in T24 and MB49 cells, bladder cancer cells in the logarithmic growth phase were trypsinized, centrifuged, and resuspended. Cells were counted, and 2,000 cells were plated per well in a 96-well plate. 100 μl of culture medium was added to each well. A zero well (culture medium only) was also set up, and five replicates were set up for each group. Growth was observed at 24, 48, 72, 96, and 120 hours. At each time point, 10 μL of CCK8 reagent was added to each well, and the cells were incubated for an additional 3-4 hours. After incubation, the cells were thoroughly shaken and dissolved, and the absorbance of each well at 450 nm was measured using a microplate reader. Cell growth curves were plotted based on the results, and growth trends between the experimental and control groups were compared.

[0022] 7. Cell clone formation experiment After constructing RRBP1 stable knockdown cell lines in T24 and MB49 cells, bladder cancer cells in the logarithmic growth phase were obtained, digested with trypsin, centrifuged and resuspended, and then the cells were counted. The cells were seeded into 6-well plates at a density of 500 cells per well and cultured for 7-10 days. The culture medium was replaced every 3 days, and the growth status of the cells was observed. After the culture was completed, the cells were fixed with 4% paraformaldehyde for 15-30 minutes and then washed three times with PBS. Then, crystal violet stain was added for 10-20 minutes and then washed three times with PBS. Finally, the clone formation was observed and photographed using a microscope, and the number of clones formed was calculated.

[0023] 8. Transwell experiment After constructing RRBP1 stable knockdown cell lines in T24 and MB49 cells, the migration and invasion abilities of bladder cancer cells were tested. The specific steps are as follows: (1) Digest the cells, resuspend them in serum-free medium, and adjust the cell density to 1-5×10 5 cells / mL; (2) Add 200 μL of cell suspension to the upper chamber of the Transwell and 500-600 μL of culture medium containing 10% FBS to the lower chamber. Incubate in a 37°C, 5% CO2 incubator for 12-24 hours. (3) Gently wipe away the cells that have not migrated / invaded in the upper chamber with a cotton swab. Fix with 4% paraformaldehyde for 15-30 minutes and wash 1-2 times with PBS. Stain with 0.1% crystal stain for 20-30 minutes and gently rinse with PBS to remove excess dye. (4) Randomly select five fields of view (×100 or ×200) under a microscope, take photos, and count the number of migrated / invaded cells. Use ImageJ or other software for quantitative analysis.

[0024] 9. Construction of mouse tumor model (1) T24 or MB49 bladder cancer cells in the logarithmic growth phase were seeded in a 10 cm culture dish. After the cell confluence exceeded 80%, the cells were digested with trypsin and collected by centrifugation. The cell pellet was resuspended in a 1:1 mixture of PBS and Matrigel matrix gel and the cell density was adjusted to 5×10 6 / mL; (2) When inoculating tumor cells, draw 300 μL of cell suspension with a 1 mL syringe and slowly inject it into the subcutaneous tissue of the left groin to form a skin mound, ensuring that no liquid overflows during the injection process; (3) About 3 days after tumor cell inoculation, visible bulges began to appear on the tumor. Mice were randomly divided into a control group and an experimental group. The control group mice were injected with normal saline intraperitoneally, while the experimental group mice were injected with 10 mg / kg SB431542 or 8 mg / kg ENOblock drugs intraperitoneally. The injections were performed every other day. During the injection, attention was paid to withdrawing the blood vessels to avoid injection into the peritoneal cavity. The reaction of the mice was observed after the injection. (4) Use a vernier caliper to measure the length and width of the tumor and record the data every 2 days. Calculate the tumor volume using the following formula: tumor volume (mm³) = (length × width) / 2. Draw a tumor growth curve based on the measurement results. (5) After the drug treatment, the mice were killed by cervical dislocation, and the tumor tissues were removed, measured and weighed, and photographed and recorded. (6) The isolated tumor tissue was divided into two parts, one part was used for tissue protein extraction, and the other part was fixed in formalin and made into wax blocks for subsequent mouse immunohistochemistry experiments.

[0025] 10. Statistical analysis Data processing and statistical analysis were primarily performed using GraphPad Prism 8.0 software. Intergroup comparisons were performed using the independent sample t-test or one-way analysis of variance (ANOVA). If the data did not conform to a normal distribution, the Wilcoxon rank-sum test or the Kruskal-Wallis test was used for intergroup comparisons. All experimental data are presented as mean ± standard deviation (SD), and each experiment was repeated at least three times. A P value of less than 0.05 was considered statistically significant.

[0026] 2. Experimental Results 1. RRBP1 is highly expressed in bladder cancer tissues and is closely related to prognosis By analyzing the TCGA bladder cancer dataset, it was found that the mRNA level of RRBP1 was significantly upregulated in bladder cancer tissues compared with normal tissues, such as Figure 1 As shown in Figure a. Immunohistochemistry experiments confirmed that the expression level of RRBP1 protein was also significantly upregulated in bladder cancer tissues, as shown in Figure 4. Figure 1 As shown in b and c. Survival analysis showed that in the TCGA bladder cancer dataset, high expression of RRBP1 was closely associated with poor prognosis of bladder cancer patients, as shown in Figure 1 As shown in middle d, it suggests that RRBP1 can be used as a marker related to the prognosis of bladder cancer.

[0027] 2. RRBP1 knockdown significantly inhibits bladder cancer cell proliferation, invasion, and migration Lentivirus was used to construct T24 and MB49 bladder cancer cell lines with stable knockdown of RRBP1. RT-qPCR and western blotting results showed that the expression level of RRBP1 in the shRRBP1 group was significantly downregulated compared with the shNC group (control group). Figure 2 As shown in a and b. CCK-8 assay, cell cloning assay and Transwell assay found that knocking down RRBP1 significantly inhibited the proliferation, migration and invasion of bladder cancer cells. Figure 3 At the same time, by constructing BALB / C-nude mouse models and C57BL / 6J mouse models, it was found that knocking down RRBP1 could significantly inhibit tumor growth in mice, as shown in Figure 5. Figure 4 As shown in a and b.

[0028] 3. RRBP1 knockdown reshapes the immune microenvironment and enhances the efficacy of anti-PD-L1 immunotherapy RRBP1 was found to be associated with anti-tumor CD8 in the TCGA bladder cancer cohort. + The infiltration levels of T cells and helper T cells were negatively correlated, but correlated with the tumor-promoting M2 macrophages, e.g. Figure 5Immunohistochemical experiments also confirmed that RRBP1 expression in bladder cancer tissues was associated with CD8 + T cell infiltration levels were significantly negatively correlated, such as Figure 5 As shown in d and e. RNA-seq of mouse tumor tissues revealed that knockdown of RRBP1 activated multiple immune-related pathways in the tumor microenvironment, such as Figure 5 In addition, knockdown of RRBP1 combined with anti-PD-L1 immunotherapy can significantly inhibit the growth of tumors in mice, as shown in f and g. Figure 5 As shown in h and i.

Claims

1. Use of RRBP1 as a target in the preparation of a pharmaceutical composition for the treatment of bladder cancer.

2. The use according to claim 1, characterized in that: Knockdown of RRBP1 can inhibit the proliferation, invasion and migration of bladder cancer cells.

3. The use according to claim 1, characterized in that: The pharmaceutical composition for treating bladder cancer is a targeted inhibitor of RRBP1.

4. A targeted inhibitor of RRBP1, characterized in that: The inhibitor is shRNA.

5. The targeted inhibitor of RRBP1 according to claim 4, characterized in that: The nucleotide sequence of shRNA is shown in any one of SEQ ID NO. 1-4 in the sequence listing.

6. A pharmaceutical composition for treating bladder cancer, characterized in that: The pharmaceutical composition comprises the targeted inhibitor of RRBP1 according to claim 4 or 5.

7. The pharmaceutical composition for treating bladder cancer according to claim 6, characterized in that: The pharmaceutical composition contains anti-PD-L1.

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