Method for detecting cis-platinum resistance of bladder cancer based on exosome miRNA marker and application

Abstract

The invention relates to the technical field of drug resistance detection, in particular to a method for detecting cis-platinum drug resistance of bladder cancer based on an exosome miRNA marker and application, and the method comprises the following steps: (1) extracting an exosome from serum of a bladder cancer patient; (2) extracting total RNA (Ribonucleic Acid) from the exosome; (3) detecting the expression level of specific miRNA in the total RNA; (4) on the basis of the expression level of the miRNA, the drug resistance risk of a patient to cis-platinum is evaluated, and the prediction accuracy (AUC is increased from 0.72 to 0.89) of the cis-platinum drug resistance of bladder cancer is remarkably improved; compared with the prior art, the treatment effective rate is greatly improved (from 45% to 60%), meanwhile, the occurrence rate of serious adverse reactions is reduced (from 25% to 15%), and in a drug-resistant bladder cancer model, the comprehensive scheme shows that the tumor inhibition rate exceeds 70% and is far higher than that of a traditional method.

Description

Technical Field

[0001] The present invention relates to the technical field of drug resistance detection, and in particular to a method and application of detecting cisplatin resistance of bladder cancer based on exosome miRNA markers. Background Art

[0002] Bladder cancer is one of the common malignant tumors of the urinary system. A major challenge in its treatment is resistance to chemotherapeutic drugs, especially cisplatin. Traditional methods for detecting drug resistance mainly rely on tumor tissue biopsy, which has the disadvantages of being highly invasive and difficult to repeat. In recent years, the development of liquid biopsy technology has provided new ideas for cancer diagnosis and monitoring. Among them, biomarkers such as circulating tumor DNA (ctDNA) and circulating tumor cells (CTC) have shown certain potential in clinical applications. However, these methods still have limitations in detection sensitivity and specificity.

[0003] Recently, exosomal miRNA has attracted the attention of researchers as a new type of liquid biopsy marker. Exosomes are nanoscale membrane vesicles secreted by cells that can carry a variety of biomolecules, including miRNA. Compared with free miRNA, miRNA in exosomes is more stable and less susceptible to RNase degradation. In addition, the composition of exosomal miRNA can reflect the state of its source cells, providing new possibilities for cancer diagnosis and prognosis assessment.

[0004] However, existing methods for detecting exosomal miRNAs still have some limitations. First, the separation and purification process of exosomes is complex and difficult to standardize. Second, the specificity and sensitivity of a single miRNA as a marker are insufficient. Furthermore, although studies have explored the relationship between miRNA and tumor drug resistance, there is a lack of systematic multi-miRNA combination analysis and the construction of a predictive model. Finally, how to combine miRNA marker detection with targeted therapy strategies to achieve personalized precision treatment is still an urgent problem to be solved. Summary of the invention

[0005] The present invention aims to solve the above technical problems and proposes a comprehensive method for detecting cisplatin resistance in bladder cancer based on exosome miRNA markers and its application. This method not only innovatively integrates efficient exosome separation, multi-miRNA combination analysis and machine learning prediction models, but also further combines miRNA regulators with nano-delivery systems to develop targeted strategies to overcome drug resistance.

[0006] The object of the present invention is to provide a method for detecting cisplatin resistance in bladder cancer based on exosomal miRNA markers, comprising the following steps:

[0007] (1) Extracting exosomes from the serum of bladder cancer patients;

[0008] (2) extracting total RNA from the exosomes;

[0009] (3) detecting the expression level of specific miRNA in the total RNA;

[0010] (4) Based on the expression level of the miRNA, assess the patient's risk of cisplatin resistance.

[0011] Preferably, the method for extracting exosomes in step (1) comprises:

[0012] First, serum samples were centrifuged at 2000 g for 30 min at 4°C;

[0013] Secondly, the supernatant was filtered through a 0.22 μm filter membrane;

[0014] Then, ultracentrifugation was performed at 100,000 g at 4°C for 70 min;

[0015] Again, resuspend the pellet using PBS buffer;

[0016] Finally, density gradient centrifugation was performed using a 0.25M-2.5M sucrose gradient at 4°C and 210,000g for 18 h to collect the exosome layer with a density range of 1.13-1.19 g / mL.

[0017] Preferably, the method for extracting total RNA in step (2) uses miRNeasy Mini Kit, and the specific steps include:

[0018] (a) Add 700 μL QIAzol lysis reagent and incubate at room temperature for 5 minutes;

[0019] (b) Add 140 μL of chloroform, shake for 15 seconds, and let stand at room temperature for 2-3 minutes;

[0020] (c) Centrifuge at 12,000 g for 15 min at 4°C and remove the upper aqueous phase;

[0021] (d) Add 1.5 times the volume of anhydrous ethanol and transfer to the RNeasyMini column;

[0022] (e) centrifugation at 8,000 g for 15 seconds and discarding the filtrate;

[0023] (f) 700 μL RWT buffer and 500 μL RPE buffer were added sequentially, and centrifuged at 8,000 g for 15 s;

[0024] (g) Centrifuge at 14,000 g for 2 min to dry the membrane;

[0025] (h) Add 30-50 μL RNase-free water to elute RNA.

[0026] Preferably, the method for detecting miRNA expression level in step (3) is quantitative PCR, using TaqMan Advanced miRNA Assays, comprising the following steps:

[0027] (a) Reverse transcription: 37°C for 45 minutes, 65°C for 10 minutes;

[0028] (b) Adapter ligation: 16°C for 30 minutes;

[0029] (c) Reverse transcription: 42°C for 15 min, 85°C for 5 min;

[0030] (d) miR-Amp reaction: 95°C for 5 min, followed by 14 cycles (95°C, 3 s; 60°C, 30 s), and finally 99°C for 10 min;

[0031] (e) qPCR: 95°C for 20 seconds, followed by 40 cycles (95°C for 1 second; 60°C for 20 seconds).

[0032] Preferably, the specific miRNA is obtained by screening through the following steps:

[0033] (a) High-throughput screening was performed using a miRNA chip to select miRNAs with expression differences greater than 2-fold between the resistant group and the sensitive group and p < 0.05;

[0034] (b) qPCR was used to verify the screening results and the top 10 miRNAs with the most significant expression differences were selected;

[0035] (c) Overexpress or knockdown candidate miRNAs in bladder cancer cell lines, detect changes in the IC50 of cells to cisplatin, and select 3-5 miRNAs with the most significant effects as final markers.

[0036] Preferably, the method for assessing the risk of drug resistance in step (4) comprises:

[0037] (a) Logistic regression and random forest algorithm prediction models were established, with input variables including the expression levels of the screened miRNAs and clinical pathological characteristics, and output variables including resistance (PFS < 6 months) or sensitivity (PFS ≥ 6 months);

[0038] (b) ROC curve analysis was used to evaluate model performance and calculate sensitivity, specificity, and accuracy;

[0039] (c) Patients were divided into three groups: high risk (predicted probability > 0.7), intermediate risk (predicted probability 0.3-0.7), and low risk (predicted probability < 0.3).

[0040] A bladder cancer cisplatin resistance detection kit based on the method comprises:

[0041] Exosome extraction reagents, RNA extraction reagents, reverse transcription reagents, qPCR reagents and primer probes for specific miRNAs.

[0042] A method for formulating an individualized treatment plan for bladder cancer based on the method comprises the following steps:

[0043] (a) using the method to assess the risk of drug resistance in patients;

[0044] (b) Treatment options should be selected based on risk level: high-risk patients should consider combination therapy or alternative options, moderate-risk patients should be closely monitored and dose adjustments should be considered, and low-risk patients should use standard cisplatin regimens;

[0045] (c) Exosomal miRNA levels were retested after each course of treatment. When the miRNA change exceeded 50%, the risk of drug resistance was reassessed and the regimen was adjusted.

[0046] The strategy for overcoming drug resistance in bladder cancer based on the method includes:

[0047] (a) Designing regulators targeting miRNA;

[0048] (b) loading the miRNA regulator into exosomes to form a targeted delivery system;

[0049] (c) using the targeted delivery system in combination with cisplatin and / or immune checkpoint inhibitors.

[0050] Preferably, the preparation method of the targeted delivery system comprises:

[0051] (a) Loading miRNA antagonists or mimics into exosomes using electroporation: electroporation was performed at 400 V and 125 μF in a 4 mm electroporation cuvette, followed by incubation at 37 °C for 30 min;

[0052] (b) Click chemistry was used to link the targeting peptide on the surface of exosomes: exosomes were incubated with phospholipid azide (DBCO-PEG4-DSPE) at 37 °C for 2 h, and a targeting peptide with an alkyne group (CRGDK) was added and reacted at room temperature for 1 h, and then purified using a 100 kDa cutoff ultrafiltration tube;

[0053] (c) Preparation of a combined drug preparation: 1-10 parts by weight of miRNA regulator-loaded exosomes, 5-20 parts by weight of cisplatin liposomes, 2-15 parts by weight of PD-1 antibody, and physiological saline added to 100 parts by weight.

[0054] The core innovations and technical features of the present invention include:

[0055] 1. Efficient exosome separation: The method of combining ultracentrifugation with density gradient centrifugation significantly improved the purity and yield of exosomes. This improvement laid the foundation for subsequent miRNA analysis.

[0056] 2. Multi-miRNA combination analysis: Through high-throughput screening and functional validation, miR-21, miR-155 and miR-210 were identified as key markers. This multi-marker strategy significantly improved the accuracy of drug resistance prediction.

[0057] 3. Machine learning prediction model: By integrating miRNA expression data and clinical pathological characteristics, a prediction model based on logistic regression and random forest algorithm was constructed. This model can not only accurately predict the risk of drug resistance, but also dynamically adjust the treatment plan.

[0058] 4. Design of miRNA regulators: Specific antisense oligonucleotides (ASOs) are designed for key miRNAs (such as miR-21) to reverse drug resistance by regulating miRNA expression.

[0059] 5. Nano-delivery system: A multifunctional nano-carrier that integrates the delivery of miRNA regulators and targeted delivery of chemotherapeutic drugs has been developed. This system not only improves the targeting and bioavailability of drugs, but also achieves a synergistic therapeutic effect.

[0060] 6. Individualized treatment strategy: Based on the results of miRNA marker detection, an individualized treatment plan including dose adjustment and combination medication was developed to achieve true precision medicine.

[0061] From the perspective of molecular mechanism, the innovation and superiority of the present invention are mainly reflected in the following aspects:

[0062] First, the multi-miRNA combination analysis strategy takes into account the complexity and heterogeneity of tumor resistance. MiR-21, miR-155, and miR-210 are involved in regulating different resistance-related pathways, such as apoptosis inhibition, drug efflux, and DNA repair. By simultaneously detecting these miRNAs, the resistance status of tumor cells can be comprehensively evaluated.

[0063] Secondly, the design of miRNA regulators fully utilizes the RNA interference mechanism. Taking miR-21ASO as an example, its 2'-O-methyl modification and phosphorothioate backbone not only improve stability but also enhance binding affinity with target miRNA. This precise molecular design ensures the specificity and efficiency of miRNA regulation.

[0064] Furthermore, the design of the nano-delivery system reflects multiple synergistic effects. As a natural nano-carrier, exosomes have good biocompatibility and targeting. The CRGDK peptide modified by click chemistry further enhances tumor targeting. At the same time, the co-loading of miRNA regulators and cisplatin realizes the integration of "diagnosis-treatment" and effectively overcomes the treatment difficulties caused by tumor heterogeneity.

[0065] Finally, the formulation of individualized treatment strategies fully considers the dynamic evolution of tumors. By regularly monitoring the levels of exosomal miRNAs, treatment plans can be adjusted in a timely manner to effectively prevent the further development of drug resistance.

[0066] The beneficial effects of the present invention include but are not limited to:

[0067] 1. Significantly improved the prediction accuracy of cisplatin resistance in bladder cancer (AUC increased from 0.72 to 0.89).

[0068] 2. The treatment efficacy has been greatly improved (from 45% to 60%), while the incidence of serious adverse reactions has been reduced (from 25% to 15%).

[0069] 3. In the drug-resistant bladder cancer model, the comprehensive scheme of the present invention showed a tumor inhibition rate of more than 70%, which is much higher than the traditional method.

[0070] 4. The potential immunomodulatory effect was unexpectedly discovered, laying the foundation for future combined applications with immunotherapy.

[0071] 5. It has initially shown a broad-spectrum effect in overcoming resistance to multiple platinum drugs, expanding the potential scope of application.

[0072] In summary, this invention innovatively integrates multiple advanced technologies to not only solve the key problems of diagnosis and treatment of cisplatin resistance in bladder cancer, but also achieves precise regulation and multiple synergies at the molecular level. This comprehensive and systematic approach has opened up a new path for personalized treatment of bladder cancer, with important clinical translation value and broad application prospects. DETAILED DESCRIPTION

[0073] Example 1

[0074] This embodiment provides a method for detecting cisplatin resistance in bladder cancer based on exosome miRNA markers, the method comprising the following steps:

[0075] (1) Exosomes were extracted from the serum of bladder cancer patients. First, 10 mL of serum sample was centrifuged at 4°C and 2000 g for 30 minutes. Second, the supernatant was filtered through a 0.22 μm filter. Then, ultracentrifugation was performed at 4°C and 100,000 g for 70 minutes. Again, the precipitate was resuspended in 1 mL of PBS buffer (pH 7.4). Finally, density gradient centrifugation was performed using a 0.25 M-2.5 M sucrose gradient and centrifuged at 4°C and 210,000 g for 18 hours to collect the exosome layer with a density range of 1.13-1.19 g / mL.

[0076] (2) Extracting total RNA from the exosomes. This step is performed using the miRNeasyMini Kit, and the specific steps are as follows:

[0077] (a) Add 700 μL QIAzol lysis reagent and incubate at room temperature for 5 minutes;

[0078] (b) Add 140 μL of chloroform, shake for 15 seconds, and let stand at room temperature for 2 minutes;

[0079] (c) Centrifuge at 12,000 g for 15 min at 4°C and remove the upper aqueous phase;

[0080] (d) Add 1.5 times the volume of anhydrous ethanol and transfer to the RNeasyMini column;

[0081] (e) centrifugation at 8,000 g for 15 seconds and discarding the filtrate;

[0082] (f) 700 μL RWT buffer and 500 μL RPE buffer were added sequentially, and centrifuged at 8,000 g for 15 s;

[0083] (g) Centrifuge at 14,000 g for 2 min to dry the membrane;

[0084] (h) Add 30 μL RNase-free water to elute RNA.

[0085] (3) Detecting the expression level of specific miRNA in the total RNA. This step uses TaqMan Advanced miRNA Assays for quantitative PCR detection, including the following steps:

[0086] (a) Reverse transcription: 37°C for 45 minutes, 65°C for 10 minutes;

[0087] (b) Adapter ligation: 16°C for 30 minutes;

[0088] (c) Reverse transcription: 42°C for 15 min, 85°C for 5 min;

[0089] (d) miR-Amp reaction: 95°C for 5 min, followed by 14 cycles (95°C, 3 s; 60°C, 30 s), and finally 99°C for 10 min;

[0090] (e) qPCR: 95°C for 20 seconds, followed by 40 cycles (95°C for 1 second; 60°C for 20 seconds).

[0091] (4) Based on the expression level of the miRNA, the patient's risk of resistance to cisplatin was assessed. This step used a logistic regression model for prediction, and the input variables included the expression levels of miR-21, miR-155, and miR-210, as well as the patient's age and tumor stage. Patients with a predicted probability > 0.7 were judged to have a high risk of resistance.

[0092] Preferably, in this embodiment, the specific miRNA detected is screened by the following steps: First, high-throughput screening is performed using Agilent Human miRNA Microarray (8x60K v21.0), and miRNAs with expression differences greater than 2 times and p<0.05 between the resistant group and the sensitive group are selected. Secondly, qPCR is used to verify the screening results, and the top 10 miRNAs with the most significant expression differences are selected. Finally, candidate miRNAs are overexpressed or knocked down in T24 and 5637 bladder cancer cell lines, and the changes in the IC50 of cells to cisplatin are detected, and the three miRNAs with the most significant effects (miR-21, miR-155 and miR-210) are selected as the final markers. This multi-step screening strategy can effectively improve the specificity and sensitivity of the selected miRNA markers.

[0093] Example 2

[0094] This embodiment provides a kit for detecting cisplatin resistance in bladder cancer based on exosome miRNA markers, and the kit includes the following components:

[0095] (1) Exosome extraction reagents: including PBS buffer (pH 7.2), 0.25 M-2.5 M sucrose gradient solution, and 0.22 μm filter membrane;

[0096] (2) RNA extraction reagent: miRNeasyMini Kit (QIAGEN, catalog number 217004);

[0097] (3) Reverse transcription reagent: TaqMan Advanced miRNA cDNA Synthesis Kit (Thermo Fisher Scientific, catalog number A28007);

[0098] (4) qPCR reagents: TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific, catalog number 4444556);

[0099] (5) Primer probes for specific miRNAs: including TaqMan Advanced miRNA Assays (Thermo Fisher Scientific, catalog number A25576) for miR-21, miR-155 and miR-210.

[0100] The preparation method of the kit comprises the following steps:

[0101] First, the exosome extraction reagent, RNA extraction reagent, reverse transcription reagent, and qPCR reagent were packaged into separate sterile containers. Second, the primer probes for specific miRNAs were stored in PCR tubes in a freeze-dried form. Then, all components were stored at -20°C. Finally, a detailed operating manual was provided.

[0102] Preferably, in this embodiment, the kit also includes a software package for data analysis, which can automatically calculate the relative expression of miRNA according to the test results and provide the drug resistance risk assessment results. This integrated design can significantly improve the convenience and accuracy of the test.

[0103] Example 3

[0104] This embodiment provides a method for formulating an individualized treatment plan for bladder cancer based on exosomal miRNA markers, the method comprising the following steps:

[0105] (1) Assessing the patient's risk of drug resistance using the method described in Example 1;

[0106] (2) Choose treatment options based on risk level:

[0107] (a) For high-risk patients (predicted probability > 0.7), consider combined treatment: cisplatin (75 mg / m 2 , day 1) combined with gemcitabine (1000 mg / m 2 , Day 1, 8), each 3 weeks is a cycle;

[0108] (b) For patients with intermediate risk (predicted probability 0.3-0.7), standard cisplatin regimen (100 mg / m 2 , day 1), with each 3-week cycle and close monitoring;

[0109] (c) For low-risk patients (predicted probability < 0.3), an intensive cisplatin regimen (120 mg / m2 , Day 1), each 3 weeks is a cycle;

[0110] (3) After each course of treatment, the exosomal miRNA levels were retested. If the miRNA changed by more than 50%, the risk of drug resistance was reassessed and the regimen was adjusted.

[0111] Preferably, in this embodiment, for high-risk patients, immune checkpoint inhibitors (such as PD-1 antibody Pembrolizumab, 200 mg, once every 3 weeks) can also be considered. This combination strategy may produce a synergistic effect and more effectively overcome drug resistance. In addition, by dynamically monitoring miRNA levels, the treatment plan can be adjusted in time to achieve truly individualized precision treatment.

[0112] Example 4

[0113] This embodiment provides a strategy for overcoming drug resistance in bladder cancer based on exosomal miRNA markers, which includes the following steps:

[0114] (1) Design of a regulator targeting miRNA. In this example, an antisense oligonucleotide (ASO) targeting miR-21 was selected as a miRNA regulator, and its sequence was 5'-TCAACATCAGTCTGATAAGCTA-3', with a 2'-O-methyl modification and a phosphothioate backbone.

[0115] (2) Loading the miRNA regulator into exosomes to form a targeted delivery system. The specific steps are as follows:

[0116] (a) Loading miRNA antagonists into exosomes using electroporation: 100 μL of exosome suspension (protein concentration 1 mg / mL) was mixed with 50 pmol ASO in a 4 mm electroporation cup, electroporated at 400 V, 125 μF, and then incubated at 37 °C for 30 min.

[0117] (b) Click chemistry was used to link the targeting peptide on the surface of exosomes: the electroporated exosomes were incubated with 10 μM DBCO-PEG4-DSPE at 37 °C for 2 h, and then 20 μM CRGDK peptide with an alkynyl group was added and reacted at room temperature for 1 h. Finally, the exosomes were purified using a 100 kDa cutoff ultrafiltration tube.

[0118] (3) The targeted delivery system is used in combination with cisplatin and immune checkpoint inhibitors. The specific formula is as follows:

[0119] miR-21ASO loaded exosomes: 5 parts by weight;

[0120] Cisplatin liposomes: 10 parts by weight;

[0121] PD-1 antibody (Pembrolizumab): 5 parts by weight;

[0122] Physiological saline: q.s. to 100 parts by weight;

[0123] Dosage regimen: miRNA-regulated exosomes and cisplatin were injected intravenously once a week, and PD-1 antibody was injected intravenously once every 3 weeks for 4 consecutive cycles.

[0124] Preferably, in this embodiment, the cisplatin liposomes are prepared using HSPC / Chol / DSPE-PEG2000 (molar ratio 55:40:5), with a particle size of 100±20nm and an encapsulation rate of>90%. This nano-delivery system can improve the targeting and bioavailability of cisplatin. In addition, by combining miRNA regulators, chemotherapeutic drugs and immunotherapy drugs, it is expected to produce a synergistic anti-tumor effect and more effectively overcome drug resistance.

[0125] Through the above four embodiments, the present invention fully demonstrates the method and application of detecting cisplatin resistance in bladder cancer based on exosomal miRNA markers. From drug resistance detection, kit development, individualized treatment plan formulation to drug resistance overcoming strategy, a complete technical system has been formed. This multi-level and multi-angle research strategy not only helps to deeply understand the cisplatin resistance mechanism of bladder cancer, but also provides new diagnosis and treatment ideas for clinical practice, which is expected to significantly improve the prognosis of bladder cancer patients.

[0126] Comparative Example 1: Detection of free miRNA in serum by traditional RT-qPCR method

[0127] This comparative example aims to verify the superiority of exosomal miRNA as a marker compared to free miRNA. The method is basically the same as Example 1, but in step (1), total RNA is directly extracted from serum instead of isolating exosomes first. The specific steps are as follows:

[0128] (1) Total RNA was directly extracted from 1 mL of serum from bladder cancer patients using TRIzol LS reagent (Invitrogen, catalog number 10296028).

[0129] (2) The subsequent steps are the same as steps (2) to (4) of Example 1.

[0130] The results showed that the sensitivity and specificity of the method for directly detecting serum free miRNA were lower than those of exosomal miRNA. This may be because exosomes can protect miRNA from RNase degradation, and exosomal miRNA can better reflect the status of tumor cells. This result confirms the rationality and innovation of the present invention in selecting exosomal miRNA as a marker.

[0131] Comparative Example 2: Single miRNA marker prediction model

[0132] This comparative example is intended to verify the superiority of the multi-miRNA marker combination. The method is basically the same as Example 1, but in step (4), only miR-21 is used as a predictor variable to construct a logistic regression model.

[0133] The results showed that the prediction accuracy of the single miRNA marker model was significantly lower than that of the multi-miRNA combination model in Example 1 (AUC: 0.72 vs 0.89, p<0.001). This confirms the rationality of the multi-miRNA combination strategy adopted in the present invention and reflects its innovation in improving prediction accuracy.

[0134] Comparative Example 3: Conventional combined treatment regimen without miRNA regulator

[0135] This comparative example is intended to verify the role of miRNA regulators in overcoming drug resistance. The treatment plan is as follows:

[0136] Cisplatin liposomes: 15 parts by weight;

[0137] PD-1 antibody (Pembrolizumab): 5 parts by weight;

[0138] Physiological saline: q.s. to 100 parts by weight;

[0139] The administration schedule was the same as in Example 4.

[0140] The results showed that in the drug-resistant bladder cancer mouse model, the tumor inhibition rate of the regimen without miRNA regulator was 20% lower than that of the regimen of Example 4 (p<0.05). This confirms the innovation and effectiveness of the introduction of miRNA regulators in the present invention.

[0141] Comparative Example 4: Non-targeted exosome delivery system

[0142] This comparative example is intended to verify the necessity of modifying the exosome surface with a targeting peptide. The method is basically the same as Example 4, but the exosome surface modification step (b) is omitted in step (2).

[0143] The results showed that the tumor targeting of the non-targeted exosome delivery system was significantly reduced, and the drug enrichment at the tumor site was reduced by 40% (p<0.01). This confirms the innovation and superiority of the targeted modified exosomes used in the present invention.

[0144] Comparative Example 5: Conventional cisplatin preparations replace cisplatin liposomes

[0145] This comparative example is intended to verify the superiority of the nano-delivery system. The method is basically the same as Example 4, but the cisplatin liposomes are replaced with an equal dose of conventional cisplatin injection.

[0146] The results showed that the regimen using conventional cisplatin preparations was more toxic than the regimen of Example 4 (the incidence of bone marrow suppression increased by 30%, p<0.05), while the anti-tumor effect decreased by 15% (p<0.05). This confirms the rationality and innovation of the nano-delivery system used in the present invention.

[0147] Comparative Example 6: Fixed-dose regimen vs. individualized treatment regimen

[0148] This comparative example aims to verify the superiority of the personalized treatment plan based on miRNA markers. Compare the following two plans:

[0149] A. Fixed-dose regimen: All patients received standard dose cisplatin (100 mg / m 2 , once every 3 weeks).

[0150] B. Individualized plan: According to the method of Example 3, adjust the treatment plan according to the results of miRNA marker detection.

[0151] The results showed that compared with the fixed-dose regimen, the individualized regimen significantly improved the treatment efficacy (60% vs 45%, p<0.01) and reduced the incidence of serious adverse reactions (15% vs 25%, p<0.05). This fully confirms the innovation and clinical value of the individualized treatment strategy proposed in this invention.

[0152] Through these 6 comparative examples, the innovation and superiority of the present invention in terms of exosomal miRNA marker selection, multi-miRNA combination prediction model, miRNA regulator application, targeted delivery system design, nanoformulation development, and personalized treatment strategy are fully verified. These results not only confirm the core innovation of the present invention, but also provide strong support for its potential value in clinical applications. In particular, these comparative experiments reveal the synergy between the various technical features of the present invention, such as the combination of exosomal miRNA selection and multi-marker prediction model, the synergy of miRNA regulator and nanodelivery system, and the complementarity of personalized treatment strategy and precision medicine. This multi-level and multi-angle innovative combination makes the present invention have significant technical advantages and application prospects in the field of diagnosis and treatment of cisplatin resistance in bladder cancer.

[0153] Next, we will combine a method based on exosomal miRNA markers to detect cisplatin resistance in bladder cancer and its specific application scenarios, design multiple test experiments to evaluate the effectiveness of the scheme, and give detailed test results.

[0154] In order to comprehensively evaluate the effectiveness of the present invention, the following test experiments were designed:

[0155] 1. Exosome isolation purity and yield test

[0156] First, the purity and yield of the exosomes isolated in Examples 1-4 and Comparative Examples 1-6 were tested. The exosome concentration and particle size distribution were determined by nanoparticle tracking analysis (NTA), the protein content was determined by the BCA method, and the exosome purity (particle number / protein ratio) was calculated.

[0157] 2. miRNA extraction efficiency and quality assessment

[0158] Secondly, the extraction efficiency and quality of miRNA in each embodiment and comparative example were evaluated. The miRNA concentration was determined using the Qubit microRNA detection kit, and the RNA integrity (RIN value) was evaluated using the Agilent 2100 bioanalyzer.

[0159] 3. qPCR detection sensitivity and specificity analysis

[0160] Then, the sensitivity and specificity of the qPCR assay were evaluated. Standard curve analysis was performed using synthetic miRNAs of known concentrations, and the limits of detection (LOD) and quantification (LOQ) were calculated. At the same time, non-target sequences were used to evaluate specificity.

[0161] 4. Prediction Model Performance Evaluation

[0162] The performance of the prediction models of Example 1 and Comparative Example 2 was evaluated. The AUC value was calculated using ROC curve analysis, and the stability of the model was evaluated by cross-validation.

[0163] 5. In vitro cell experiments

[0164] In vitro cell experiments were performed to evaluate the effects of miRNA regulators. Cell viability was determined by CCK-8 assay, cell apoptosis rate was analyzed by flow cytometry, and the expression of drug resistance-related proteins was detected by Western blot.

[0165] 6. In vivo animal experiments

[0166] Finally, in vivo animal experiments were conducted to evaluate the therapeutic effects of Example 4 and Comparative Examples 3-5. A nude mouse subcutaneous transplant tumor model was established, the tumor volume and weight were measured, and the tumor inhibition rate was calculated. At the same time, hematological indicators were detected to evaluate toxicity.

[0167] The experimental results are as follows:

[0168] Table 1. Exosome isolation purity and yield test results

[0169]

[0170] Table 2. miRNA extraction efficiency and quality assessment results

[0171] sample miRNA concentration (ng / μL) RIN Value Example 1 25.3 9.2 Example 2 23.8 9 Example 3 24.5 9.1 Example 4 26.1 9.3 Comparative Example 1 10.2 6.5 Comparative Example 2 22.9 8.9 Comparative Example 3 23.6 9 Comparative Example 4 24.2 9.1 Comparative Example 5 25 9.2 Comparative Example 6 24.3 9.1

[0172] Table 3. qPCR detection sensitivity and specificity analysis results

[0173] miRNA LOD (copies / μL) LOQ (copies / μL) Specificity (non-target sequence interference rate) miR-21 10 50 <0.1％ miR-155 15 75 <0.1％ miR-210 12 60 <0.1％

[0174] Table 4. Prediction model performance evaluation results

[0175] Model AUC Sensitivity Specificity Accuracy Example 1 (Multi-miRNA Model) 0.89 0.85 0.87 0.86 Comparative Example 2 (Single miRNA Model) 0.72 0.68 0.7 0.69

[0176] Table 5. In vitro cell experiment results (T24 drug-resistant cell line)

[0177] deal with Cell viability inhibition rate (%) Apoptosis rate (%) P-gp expression (relative value) Comparison 0 5.2 1 Cisplatin 25.3 18.7 0.95 Example 4 68.9 45.6 0.42 Comparative Example 3 42.1 30.2 0.78 Comparative Example 4 55.3 38.4 0.6 Comparative Example 5 38.7 27.9 0.82

[0178] Table 6. In vivo animal experiment results (n=10 / group)

[0179]

[0180] Based on the above test results, the following conclusions can be drawn:

[0181] 1. Exosome separation: The exosomes separated by the method of the present invention (Examples 1-4) have higher purity and yield, which is better than the traditional method (Comparative Examples 2-6). This confirms the superiority of the present invention in exosome separation.

[0182] 2. miRNA extraction: miRNA extracted from exosomes (Examples 1-4 and Comparative Examples 2-6) has a higher concentration and integrity, which is significantly better than that extracted directly from serum (Comparative Example 1). This verifies the rationality of selecting exosome miRNA as a marker.

[0183] 3. qPCR detection: The qPCR method used in the present invention has high sensitivity and specificity, and can accurately detect low-concentration miRNA.

[0184] 4. Prediction model: The prediction performance of the multi-miRNA combination model (Example 1) is significantly better than that of the single miRNA model (Comparative Example 2), which confirms the innovation and effectiveness of the multi-marker strategy adopted in the present invention.

[0185] 5. In vitro experiments: The regimen containing miRNA regulators (Example 4) performed best in inhibiting the proliferation of drug-resistant cells, inducing apoptosis and reducing P-gp expression, confirming the key role of miRNA regulators in overcoming drug resistance.

[0186] 6. In vivo experiments: The comprehensive scheme of the present invention (Example 4) exhibited the best anti-tumor effect and the lowest toxicity, verifying its superiority in vivo.

[0187] Unexpected technical effects:

[0188] 1. Synergistic effect: The present invention combines miRNA marker detection, miRNA regulators and nano-delivery systems to produce unexpected synergistic effects. The tumor inhibition rate (72.3%) of Example 4 is much higher than the simple superposition of the effects of each single component. This may be because the miRNA regulator enhances the sensitivity of tumor cells to cisplatin, while the nano-delivery system increases the enrichment of the drug at the tumor site.

[0189] 2. Significantly reduced toxicity: Example 4 not only improves the anti-tumor effect, but also unexpectedly significantly reduces toxicity. The incidence of bone marrow suppression dropped from 40% in traditional cisplatin treatment to 20%, which may be due to the targeted delivery system reducing the impact of the drug on normal tissues.

[0190] 3. Reversal of drug resistance: In in vitro experiments, Example 4 not only inhibited the proliferation of drug-resistant cells, but also significantly reduced the expression of P-gp. This shows that the present invention can not only overcome existing drug resistance, but also prevent the further development of drug resistance, providing new possibilities for long-term treatment.

[0191] 4. Immunomodulatory effect: Although the present invention mainly targets chemotherapy resistance, in vivo experiments showed that the immune function of mice in group 4 of Example 4 was improved to a certain extent (data not shown). This suggests that this regimen may have potential immunomodulatory effects, laying the foundation for future combined applications with immunotherapy.

[0192] 5. Broad spectrum: Preliminary experiments show that the method of the present invention is not only effective against cisplatin resistance, but also has the potential to overcome resistance to other platinum drugs (such as carboplatin and oxaliplatin). This means that the present invention may have a wider application prospect.

[0193] In summary, this invention not only achieves the expected diagnosis and treatment of cisplatin resistance in bladder cancer by innovatively integrating multiple technologies, but also demonstrates unexpected technical effects in many aspects. These findings provide new ideas and methods for individualized precision treatment of bladder cancer and have important clinical translation value.

Claims

1. A method for detecting cisplatin resistance in bladder cancer based on exosomal miRNA markers, characterized in that , including the following steps: (1) Extracting exosomes from the serum of bladder cancer patients to detect cisplatin resistance in bladder cancer based on exosome miRNA markers and using them; (2) extracting total RNA from the exosomes; (3) detecting the expression level of specific miRNA in the total RNA; (4) Based on the expression level of the miRNA, assess the patient's risk of cisplatin resistance.

2. The method according to claim 1, characterized in that , the method for extracting exosomes in step (1) comprises: First, serum samples were centrifuged at 2000 g for 30 min at 4°C; Secondly, the supernatant was filtered through a 0.22 μm filter membrane; Then, ultracentrifugation was performed at 100,000 g at 4°C for 70 min; Again, resuspend the pellet using PBS buffer; Finally, density gradient centrifugation was performed using a 0.25M-2.5M sucrose gradient at 4°C and 210,000g for 18 h to collect the exosome layer with a density range of 1.13-1.19 g / mL.

3. The method according to claim 1, characterized in that The method for extracting total RNA in step (2) uses miRNeasyMini Kit, and the specific steps include: (a) Add 700 μL QIAzol lysis reagent and incubate at room temperature for 5 minutes; (b) Add 140 μL of chloroform, shake for 15 seconds, and let stand at room temperature for 2-3 minutes; (c) Centrifuge at 12,000 g for 15 min at 4°C and remove the upper aqueous phase; (d) Add 1.5 times the volume of anhydrous ethanol and transfer to the RNeasyMini column; (e) centrifugation at 8,000 g for 15 seconds and discarding the filtrate; (f) 700 μL RWT buffer and 500 μL RPE buffer were added sequentially, and centrifuged at 8,000 g for 15 s; (g) Centrifuge at 14,000 g for 2 min to dry the membrane; (h) Add 30-50 μL RNase-free water to elute RNA.

4. The method according to claim 1, characterized in that In step (3), the method for detecting miRNA expression level is quantitative PCR, using TaqMan Advanced miRNA Assays, including the following steps: (a) Reverse transcription: 37°C for 45 minutes, 65°C for 10 minutes; (b) Adapter ligation: 16°C for 30 minutes; (c) Reverse transcription: 42°C for 15 min, 85°C for 5 min; (d) miR-Amp reaction: 95°C for 5 min, followed by 14 cycles (95°C, 3 s; 60°C, 30 s), and finally 99°C for 10 min; (e) qPCR: 95°C for 20 seconds, followed by 40 cycles (95°C for 1 second; 60°C for 20 seconds).

5. The method according to claim 1, characterized in that , the specific miRNA is screened by the following steps: (a) High-throughput screening was performed using a miRNA chip to select miRNAs with expression differences greater than 2-fold between the resistant group and the sensitive group and p < 0.05; (b) qPCR was used to verify the screening results and the top 10 miRNAs with the most significant expression differences were selected; (c) Overexpress or knockdown candidate miRNAs in bladder cancer cell lines, detect changes in the IC50 of cells to cisplatin, and select 3-5 miRNAs with the most significant effects as final markers.

6. The method according to claim 1, characterized in that , the method for evaluating the risk of drug resistance in step (4) includes: (a) Logistic regression and random forest algorithm prediction models were established, with input variables including the expression levels of the screened miRNAs and clinical pathological characteristics, and output variables including resistance (PFS < 6 months) or sensitivity (PFS ≥ 6 months); (b) ROC curve analysis was used to evaluate model performance and calculate sensitivity, specificity, and accuracy; (c) Patients were divided into three groups: high risk (predicted probability > 0.7), intermediate risk (predicted probability 0.3-0.7), and low risk (predicted probability < 0.3).

7. A kit for detecting cisplatin resistance in bladder cancer based on the method according to any one of claims 1 to 6, characterized in that ,include: Exosome extraction reagents, RNA extraction reagents, reverse transcription reagents, qPCR reagents and primer probes for specific miRNAs.

8. A method for formulating an individualized treatment plan for bladder cancer based on the method according to any one of claims 1 to 6, characterized in that , including the following steps: (a) using the method to assess the risk of drug resistance in patients; (b) Treatment options should be selected based on risk level: high-risk patients should consider combination therapy or alternative options, moderate-risk patients should be closely monitored and dose adjustments should be considered, and low-risk patients should use standard cisplatin regimens; (c) Exosomal miRNA levels were retested after each course of treatment. When the miRNA change exceeded 50%, the risk of drug resistance was reassessed and the regimen was adjusted.

9. A strategy for overcoming drug resistance in bladder cancer based on the method according to any one of claims 1 to 6, characterized in that ,include: (a) Designing regulators targeting miRNA; (b) loading the miRNA regulator into exosomes to form a targeted delivery system; (c) using the targeted delivery system in combination with cisplatin and / or immune checkpoint inhibitors.

10. The strategy for overcoming drug resistance according to claim 9, characterized in that , the preparation method of the targeted delivery system comprises: (a) Loading miRNA antagonists or mimics into exosomes using electroporation: electroporation was performed at 400 V and 125 μF in a 4 mm electroporation cuvette, followed by incubation at 37 °C for 30 min; (b) Click chemistry was used to link the targeting peptide on the surface of exosomes: exosomes were incubated with phospholipid azide (DBCO-PEG4-DSPE) at 37 °C for 2 h, and a targeting peptide with an alkyne group (CRGDK) was added and reacted at room temperature for 1 h, and then purified using a 100 kDa cutoff ultrafiltration tube; (c) Preparation of a combined drug preparation: 1-10 parts by weight of miRNA regulator-loaded exosomes, 5-20 parts by weight of cisplatin liposomes, 2-15 parts by weight of PD-1 antibody, and physiological saline added to 100 parts by weight.

Images