A set of plasma exosome microRNAs markers for gallbladder cancer and a detection method and application thereof

Abstract

The application relates to the field of molecular biology and medical detection technology, and specifically discloses a group of gallbladder cancer plasma exosome microRNA marker, a detection method and application thereof, the marker comprises at least one of miR-135b-5p, miR-224-5p, miR-451a, miR-148a-3p and miR-375-3p; the marker combination presents a characteristic expression profile (two rises and three falls) in gallbladder cancer patients; the application provides a non-diagnostic purpose detection method of the marker, the exosome separation and detection process is optimized, a joint diagnosis model is constructed, efficient and non-invasive identification of gallbladder cancer is realized, the AUC reaches 0.987, the sensitivity is 93.3%, and the specificity is 91.7%; the marker and the method can be used for preparing a gallbladder cancer related detection kit, and have important clinical application value.

Description

Technical Field

[0001] This application relates to the fields of molecular biology and medical detection technology, and more specifically, it relates to a group of microRNA markers of exosomal plasma from gallbladder cancer, as well as their detection methods and applications. Background Technology

[0002] Gallbladder cancer is a highly malignant tumor of the digestive system, ranking first among malignant gallbladder tumors and fifth among gastrointestinal tumors, with its incidence rate showing a continuous upward trend in recent years. my country is a high-incidence area for gallbladder cancer, and this disease seriously threatens the lives and health of the people. According to global cancer statistics, gallbladder cancer has an extremely high mortality rate after onset, with high age-standardized mortality rates in underdeveloped regions. Most patients are diagnosed at an advanced stage, resulting in a low surgical resection rate.

[0003] Gallbladder cancer often coexists with benign conditions such as gallstones and chronic cholecystitis, with chronic irritation from gallstones being a significant contributing factor. Early diagnosis of gallbladder cancer is extremely challenging due to the lack of specific clinical symptoms and signs. Currently, commonly used diagnostic methods include ultrasound, CT scans, and color Doppler flow imaging, but these methods have limited accuracy in diagnosing early-stage gallbladder cancer. Direct biopsy or bile extraction for cancer cells is invasive, may accelerate metastasis, and produces delayed results, limiting its widespread application. Traditional serological markers such as CA199 and CEA have low sensitivity and specificity, failing to meet clinical needs for early diagnosis, accurate differentiation, and monitoring of treatment efficacy.

[0004] Body fluid biopsy, as a non-invasive and convenient diagnostic method, was named one of the "Top 10 Breakthroughs of 2015" by MIT Technology Review and has received widespread attention and promotion in the field of tumor diagnosis in recent years. Body fluid biopsy diagnoses diseases by detecting biomarkers such as circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), circulating RNA, and exosomes in body fluids such as blood and urine. Exosomes are small, double-membrane vesicles with a diameter of 40-100 nm secreted by various cells, widely distributed in body fluids such as saliva, plasma, and breast milk. Their contents include various biomolecules such as proteins, mRNA, microRNA, and lipids. Compared with normal cells, tumor cells secrete a larger amount of exosomes, and the contents differ significantly. They are closely related to the occurrence, development, invasion, metastasis, and immune escape of tumors, making them an ideal source of tumor diagnostic biomarkers.

[0005] microRNAs are a class of small, non-coding RNAs, approximately 19-25 nt in length. They negatively regulate target gene expression by specifically binding to the 3' untranslated region (UTR) of target gene mRNA, playing a crucial role in tumorigenesis and development. Studies have shown that microRNAs can be encapsulated in exosomes and distributed throughout the body via fluid circulation. Their expression levels are stable and easily detected, making them a hot research topic in tumor diagnosis. However, current research on exosomal microRNAs as diagnostic biomarkers for gallbladder cancer is insufficient, lacking highly specific and sensitive biomarker combinations. Based on the above, this application proposes a set of plasma exosomal microRNA markers for gallbladder cancer, along with their detection methods and applications. Summary of the Invention

[0006] To address the challenges of gallbladder cancer, such as its insidious clinical manifestations, low early detection rate, and easy confusion with benign diseases like chronic cholecystitis, as well as the limited diagnostic efficacy of existing imaging and serum biomarkers and the lack of highly sensitive and specific non-invasive biomarkers, this application provides a set of plasma exosomal microRNA markers for gallbladder cancer, along with their detection methods and applications.

[0007] Firstly, this application provides a set of microRNA markers for exosomal plasma from gallbladder cancer, employing the following technical solution:

[0008] A group of plasma exosomal microRNA markers for gallbladder cancer, including at least one of miR-135b-5p, miR-224-5p, miR-451a, miR-148a-3p and miR-375-3p.

[0009] Preferably, the sequences of miR-135b-5p, miR-224-5p, miR-451a, miR-148a-3p, and miR-375-3p are as shown in SEQ ID No. 1 to SEQ ID No. 5, respectively:

[0010] miR-135b-5p: 5'-UAUGGCUUUUCAUUCCUAUGUGA-3' (SEQ ID No. 1);

[0011] miR-224-5p: 5'-UCAAGUCACUAGUGGUUCCGUUUAG-3' (SEQ ID No. 2);

[0012] miR-451a: 5'-AAACCGUUACCAUUACUGAGUU-3' (SEQ ID No. 3);

[0013] miR-148a-3p: 5'-UCAGUGCACUACAGAACUUUGU-3' (SEQ ID No. 4);

[0014] miR-375-3p: 5'-UUUGUUCGUUCGGCUCGCGUGA-3' (SEQ ID No. 5).

[0015] Compared with healthy individuals or patients with benign gallbladder disease, the expression levels of miR-135b-5p and miR-224-5p were significantly increased in the plasma exosomes of gallbladder cancer patients, while the expression levels of miR-451a, miR-148a-3p, and miR-375-3p were significantly decreased.

[0016] The miR-135b-5p and miR-224-5p mentioned above are believed to be involved in the proliferation, migration and drug resistance regulation of cancer cells, and their increased expression in gallbladder cancer may be related to their promoting effect on cancer cell growth.

[0017] The miR-451a, miR-148a-3p, and miR-375-3p mentioned above are tumor suppressor factors. Their expression levels were significantly reduced during the development of gallbladder cancer, indicating that they play an important role in inhibiting the proliferation and metastasis of cancer cells.

[0018] Secondly, this application provides a non-diagnostic detection method for exosomal microRNA markers in gallbladder cancer plasma, employing the following technical solution:

[0019] A non-diagnostic detection method for plasma exosomal microRNA markers in gallbladder cancer, comprising the following steps:

[0020] S1. Blood sample collection and preprocessing to obtain plasma samples;

[0021] S2. Plasma samples were separated and purified using a combination of differential centrifugation and ultrafiltration to obtain exosomes;

[0022] S3. Extraction and quality assessment of total exosome RNA;

[0023] S4. Total exosome RNA is reverse transcribed into cDNA template;

[0024] S5. Real-time quantitative PCR was used to detect the expression level of target microRNAs.

[0025] S6. Data standardization and performance analysis.

[0026] Preferably, the specific process of step S1 is as follows: collect 5-8 mL of fasting venous blood from the research subject, inject it into an EDTA anticoagulant tube, invert and mix 5-8 times to avoid blood coagulation, and obtain a blood sample; centrifuge the blood sample at 3-5℃, 2800-3200×g for 10-15 min within 2 hours after blood collection to separate and obtain a plasma sample.

[0027] Preferably, the specific process of step S2 is as follows: centrifuge the plasma sample at 3-5℃ at 400-600×g for 8-12 min to remove blood cells; centrifuge at 10000-14000×g for 25-35 min to remove cell debris; ultracentrifuge at 90000-110000×g for 80-100 min and collect the precipitate; resuspend the precipitate with PBS buffer, and then ultracentrifuge at 90000-110000×g for 60-80 min to collect the final precipitate to obtain exosomes.

[0028] Preferably, the volume ratio of the precipitate to the PBS buffer is 4-6:1.

[0029] Preferably, the specific process of step S3 is as follows: total RNA is extracted from exosomes using an exosome RNA extraction kit, and the operation is strictly performed according to the kit instructions; after extraction, the RNA purity (A260 / A280 ratio) is detected using a NanoDrop spectrophotometer, and the RNA integrity is detected using an Agilent Bioanalyzer (RIN value ≥ 7.0 is considered qualified), to obtain total exosome RNA.

[0030] Preferably, the specific process of step S4 is as follows: the total exosome RNA is prepared into a reaction system according to the instructions of the reverse transcription kit, the reaction system is mixed, and the reverse transcription reaction is carried out in a PCR instrument. The reaction program is as follows: incubation at 25℃ for 10 min (primer binding); incubation at 42℃ for 60 min (reverse transcription reaction); incubation at 70℃ for 15 min (reverse transcriptase inactivation); and cDNA template is obtained.

[0031] Preferably, in step S5, the real-time quantitative PCR uses endogenous U6 snRNA as an internal reference gene, and the expression level between samples is normalized by correcting the difference in Ct values ​​between the target microRNAs and the internal reference gene.

[0032] Preferably, the reaction system for real-time quantitative PCR in step S5 includes: 2×SYBR Green PCRMaster Mix, upstream and downstream primer mixture, cDNA template, and nuclease-free water;

[0033] Preferably, the reaction procedure for real-time quantitative PCR in step S5 is as follows:

[0034] Pre-denaturation: 94-95℃, 30-120s;

[0035] Cyclic amplification: 40-45 cycles, each cycle including: denaturation at 94-95℃ for 5-15 seconds, annealing / extension at 60-65℃ for 20-40 seconds;

[0036] After the cyclic amplification was completed, melting curve analysis was performed: first, the temperature was held at 94-95℃ for 10-20s, then at 55-65℃ for 30-90s, and finally the temperature was increased from 55-65℃ to 94-95℃ at a heating rate of 0.1-0.5℃ / s. Fluorescence signals were continuously collected during the heating process.

[0037] Preferably, the specific process of step S5 is as follows:

[0038] S5.1, upstream and downstream primer design: Real-time quantitative PCR was performed using the SYBR Green method. For each target microRNA, a specific upstream primer was designed, whose sequence was complementary to the antisense strand of the microRNA sequence (i.e., corresponding to its DNA coding strand, with the U base replaced by T). All target microRNAs used a universal downstream primer. The internal reference gene U6snRNA used conventional specific upstream and downstream primers. The GC content of all DNA primers was controlled between 45-55%, and Oligo 7 was used for evaluation to ensure no nonspecific binding, hairpin structures, and significant primer dimer tendency.

[0039] S5.2 Preparation of the reaction system: The total system volume is 20 μL, and the specific components are as follows:

[0040] 2×SYBR Green PCR Master Mix: 10μL;

[0041] Mixture of upstream and downstream primers (final concentration of each primer is 10 μM): 0.8 μL;

[0042] cDNA template (50 ng / μL): 2 μL;

[0043] Nuclease-free water: 7.2 μL;

[0044] S5.3 Mix the prepared reaction system and transfer it to a 96-well PCR plate. Set up 3 technical replicate wells for each sample and set up blank control wells (using nuclease-free water instead of cDNA template).

[0045] S5.4 Perform PCR amplification according to the following reaction procedure:

[0046] Pre-denaturation: 95℃, 30s;

[0047] Cyclic amplification: 40 cycles, each cycle consisting of: denaturation at 95℃ for 10 s, annealing / extension at 60℃ for 30 s;

[0048] After the cycle amplification was completed, melting curve analysis was performed: first, the temperature was held at 95℃ for 15 seconds, then at 60℃ for 60 seconds, and finally the temperature was increased from 60℃ to 95℃ at a rate of 0.3℃ / s. Fluorescence signals were continuously collected during the heating process to verify the specificity of the PCR product.

[0049] S5.5 After amplification, the Ct value of each well is obtained using the software of the real-time quantitative PCR instrument. Data with a Ct value variation coefficient greater than 5% in the technical replicate wells are removed, and the average value of the remaining data is taken as the final Ct value of the sample.

[0050] Preferably, in step S6, the 2^(-ΔΔCT) method is used for data standardization. The efficacy of the marker is analyzed by plotting the receiver operating characteristic curve, and the area under the curve, sensitivity, specificity and optimal cutoff value are calculated.

[0051] Preferably, the specific process of step S6 is as follows:

[0052] S6.1 Data Standardization: The relative expression levels of target microRNAs were calculated using the 2^(-ΔΔCT) method, with the specific formula as follows:

[0053] ΔCt = Ct value of target microRNAs - Ct value of internal reference gene U6 snRNA;

[0054] ΔCt = ΔCt value of the sample to be tested - ΔCt value of the calibration sample (the calibration sample is the average ΔCt value of plasma exosomes from healthy individuals).

[0055] Relative expression level = 2^(-ΔΔCT);

[0056] S6.2. Performance Analysis: Based on the relative expression levels of healthy individuals, patients with benign gallbladder disease, and patients with gallbladder cancer, SPSS statistical software was used to plot receiver operating characteristic (ROC) curves. The area under the ROC curve (AUC) was calculated, and the optimal cutoff value for each microRNA and combination marker was determined by the Youden index (sensitivity + specificity - 1). The sensitivity, specificity, positive predictive value, negative predictive value, and 95% confidence interval of each marker at the optimal cutoff value were calculated.

[0057] Thirdly, this application provides the application of gallbladder cancer plasma exosomal microRNAs markers in the preparation of gallbladder cancer-related reagent kits, which are used for the auxiliary diagnosis, early screening, efficacy evaluation, or early warning of recurrence and metastasis of gallbladder cancer.

[0058] Preferably, the gallbladder cancer-related kit contains at least one of the following: upstream and downstream primers for detecting plasma exosomal microRNA markers miR-135b-5p, miR-224-5p, miR-451a, miR-148a-3p and miR-375-3p; upstream and downstream primers for the internal reference gene U6 snRNA; RNA extraction reagent; reverse transcription reagent; and real-time quantitative PCR reaction reagent.

[0059] Compared with the prior art, this application has the following beneficial effects:

[0060] 1. This application is the first to screen and validate a combination of plasma exosomal microRNAs containing miR-135b-5p, miR-224-5p, miR-451a, miR-148a-3p, and miR-375-3p. These markers exhibit a characteristic expression pattern in gallbladder cancer patients (two significantly elevated and three significantly decreased), and participate in the development and progression of gallbladder cancer through either oncogenic or tumor-suppressive mechanisms. The differences in their expression are supported by clear biological functions. Compared with existing imaging tests and traditional serum markers, these markers can effectively distinguish gallbladder cancer-related samples from healthy individuals and samples related to benign gallbladder diseases.

[0061] 2. This application optimizes the exosome isolation protocol of "differential centrifugation + ultrafiltration purification," combining it with standardized procedures using commercial reagents to achieve efficient enrichment of exosomes and stable RNA extraction. In the real-time quantitative PCR step, U6 snRNA is used as an internal reference gene, and data standardization is performed using the 2^(-ΔΔCT) method. Technical replicate wells and blank controls are also included to effectively control experimental errors and ensure the accuracy and reproducibility of the detection results. This method has a clear operational procedure, optimized and reasonable conditions, requires no complex instruments or equipment, and is easy to apply on a large scale in laboratories.

[0062] 3. The microRNA markers in this application can not only be used for early risk screening and clinical auxiliary assessment of gallbladder cancer, but also provide a reference for efficacy evaluation by dynamically monitoring changes in expression levels before and after treatment. Furthermore, abnormal expression can precede clinical symptoms and imaging changes, providing a non-invasive monitoring method for early warning of recurrence and metastasis risks, covering the entire application process of gallbladder cancer from "risk screening to auxiliary assessment to efficacy monitoring to recurrence warning." The kit developed based on this marker integrates upstream and downstream primers, commercially available core reagents, and supporting consumables, offering convenient operation and controllable costs.

[0063] 4. This application utilizes the natural protective effect of exosomes on microRNAs, which solves the technical bottlenecks of traditional RNA biomarkers being easily degraded and having a short detection window. Moreover, sample collection can be completed with only routine venous blood collection, without the need for invasive tissue biopsy, which greatly reduces the threshold of detection trauma and patient compliance. It is more clinically practical and has greater promotional value than existing invasive or unstable biomarker detection. Detailed Implementation

[0064] The present application will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the present application and are not intended to limit the scope of the present application.

[0065] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0066] The RNA extraction reagent was purchased from Tiangen Biotech Co., Ltd., catalog number: DP419;

[0067] The reverse transcription reagent was purchased from Thermo Fisher Scientific, catalog number: K1691;

[0068] 2×SYBR Green PCR Master Mix was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., product code: 11201ES03.

[0069] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0070] Example 1: Screening and validation of exosomal microRNA markers in gallbladder cancer plasma

[0071] 1. Experimental materials

[0072] (1) Study subjects: 30 patients with gallbladder cancer (pathologically confirmed as gallbladder adenocarcinoma, TNM stage I-IV), 30 patients with benign gallbladder diseases (20 with chronic cholecystitis and 10 with gallstones) and 30 healthy individuals (without a history of gallbladder disease or other malignant tumors) were selected. There were no statistically significant differences in gender and age among the three groups (P>0.05). This experiment was approved by the hospital ethics committee, and all subjects signed informed consent forms.

[0073] (2) Primer sequence design: Based on the microRNA sequences shown in SEQ ID No. 1-5 and the internal reference gene U6 snRNA sequence, specific upstream primers and universal downstream primers were designed. Conventional specific upstream and downstream primers were used for the internal reference gene U6 snRNA. The primer sequences are shown in Table 1:

[0074] Table 1 Primer sequence listing

[0075]

[0076] 2. Experimental Methods

[0077] A non-diagnostic detection method for plasma exosomal microRNA markers in gallbladder cancer, comprising the following steps:

[0078] S1. Collect 5 mL of fasting venous blood from the study subjects and inject it into an EDTA anticoagulant tube. Invert and mix 5 times to avoid blood clotting to obtain a blood sample. Centrifuge the blood sample at 4°C and 3000×g for 15 min within 2 hours after blood collection to separate the plasma sample.

[0079] S2. Centrifuge the plasma sample at 500×g for 10 min at 4℃ to remove blood cells; centrifuge at 12000×g for 30 min to remove cell debris; ultracentrifuge at 100000×g for 90 min and collect the precipitate; resuspend the precipitate in PBS buffer at a volume ratio of 5:1, and ultracentrifuge again at 100000×g for 60 min to collect the final precipitate, which is the exosome.

[0080] S3. Total RNA was extracted from exosomes using an RNA extraction reagent, and the procedure was strictly performed according to the instructions of the RNA extraction reagent. After extraction, the RNA purity (A260 / A280 ratio) was detected using a NanoDrop spectrophotometer, and the RNA integrity was detected using an Agilent Bioanalyzer (RIN value ≥7.0 was considered acceptable).

[0081] S4. Prepare the reaction system with total exosome RNA according to the reverse transcription reagent instructions. Mix the reaction system well and place it in a PCR instrument for reverse transcription. The reaction program is as follows: incubate at 25℃ for 10 min (primer binding); incubate at 42℃ for 60 min (reverse transcription reaction); incubate at 70℃ for 15 min (reverse transcriptase inactivation); obtain cDNA template and adjust the concentration to 50 ng / μL.

[0082] S5.1, upstream and downstream primer design: Real-time quantitative PCR was performed using the SYBR Green method. For each target microRNA, a specific upstream primer was designed, whose sequence was complementary to the antisense strand of the microRNA sequence (i.e., corresponding to its DNA coding strand, with the U base replaced by T). All target microRNAs used a universal downstream primer. The internal reference gene U6snRNA used conventional specific upstream and downstream primers. The GC content of all DNA primers was controlled between 45-55%, and Oligo 7 was used for evaluation to ensure no nonspecific binding, hairpin structures, and significant primer dimer tendency, as shown in Table 1.

[0083] S5.2 Preparation of the reaction system: The total system volume is 20 μL, and the specific components are as follows:

[0084] 2×SYBR Green PCR Master Mix: 10μL;

[0085] Mixture of upstream and downstream primers (final concentration of each primer is 10 μM): 0.8 μL;

[0086] cDNA template (50 ng / μL): 2 μL;

[0087] Nuclease-free water: 7.2 μL;

[0088] S5.3 Mix the prepared reaction system and transfer it to a 96-well PCR plate. Set up 3 technical replicate wells for each sample and set up blank control wells (using nuclease-free water instead of cDNA template).

[0089] S5.4 Perform PCR amplification according to the following reaction procedure:

[0090] Pre-denaturation: 95℃, 30s;

[0091] Cyclic amplification: 40 cycles, each cycle consisting of: denaturation at 95℃ for 10 s, annealing / extension at 60℃ for 30 s;

[0092] After the cycle amplification was completed, melting curve analysis was performed: first, the temperature was held at 95℃ for 15 seconds, then at 60℃ for 60 seconds, and finally the temperature was increased from 60℃ to 95℃ at a rate of 0.3℃ / s. Fluorescence signals were continuously collected during the heating process to verify the specificity of the PCR product.

[0093] S5.5 After amplification, the Ct value of each well is obtained using the software of the real-time fluorescence quantitative PCR instrument. Data with a Ct value variation coefficient greater than 5% in the technical replicate wells are removed, and the average value of the remaining data is taken as the final Ct value of the sample.

[0094] S6.1 Data Standardization: The relative expression levels of target microRNAs were calculated using the 2^(-ΔΔCT) method, with the specific formula as follows:

[0095] ΔCt = Ct value of target microRNAs - Ct value of internal reference gene U6 snRNA;

[0096] ΔCt = ΔCt value of the sample to be tested - ΔCt value of the calibration sample (the calibration sample is the average ΔCt value of plasma exosomes from healthy individuals).

[0097] Relative expression level = 2^(-ΔΔCT);

[0098] S6.2. Performance Analysis: Based on the relative expression levels of healthy individuals, patients with benign gallbladder disease, and patients with gallbladder cancer, SPSS statistical software was used to plot receiver operating characteristic (ROC) curves. The area under the ROC curve (AUC) was calculated, and the optimal cutoff value for each microRNA and combination marker was determined by the Youden index (sensitivity + specificity - 1). The sensitivity, specificity, positive predictive value, negative predictive value, and 95% confidence interval of each marker at the optimal cutoff value were calculated.

[0099] 3. Experimental Results

[0100] (1) RNA quality identification results: The A260 / A280 ratio of total exosome RNA extracted from all samples was between 1.8 and 2.1, and the RIN value was ≥7.2, indicating that the RNA purity and integrity met the experimental requirements.

[0101] (2) Differences in microRNA expression levels: Compared with the healthy group and the benign gallbladder disease group, the relative expression levels of miR-135b-5p and miR-224-5p were significantly increased in the gallbladder cancer group, while the relative expression levels of miR-451a, miR-148a-3p and miR-375-3p were significantly decreased; there was no statistically significant difference in the expression levels of the above microRNAs between the healthy group and the benign gallbladder disease group. The specific data are shown in Table 2.

[0102] Table 2 Comparison of relative expression levels of plasma exosomal microRNAs in each group

[0103]

[0104] (3) Construction of the joint diagnostic model and results of diagnostic efficacy analysis: In order to further improve the diagnostic efficacy, the relative expression levels of the above 5 microRNAs were transformed by log2 and used as independent variables, and whether it was diagnosed as gallbladder cancer (1 for yes, 0 for no) was used as dependent variable. The Forward:LR method was used to perform binary logistic regression analysis, and finally the joint diagnostic model was constructed.

[0105] Its logistic regression equation is:

[0106] Logit(P)=1.85×[miR-135b-5p]+1.62×[miR-224-5p]-2.10×[miR-451a]-1.78×[miR-148a-3p]-2.30×[miR-375-3p]-2.50;

[0107] Where [miR-135b-5p], [miR-224-5p], [miR-451a], [miR-148a-3p], and [miR-375-3p] represent the expression values ​​of microRNAs after log2 transformation and standardization, and P represents the probability that the sample is predicted to be gallbladder cancer. Logit(P) = ln(P / (1-P)).

[0108] ROC curve analysis of single markers showed that the AUC of each marker in distinguishing between gallbladder cancer and healthy individuals with benign gallbladder disease was above 0.85, with miR-224-5p showing the highest AUC. Based on the combined diagnostic model, the Logit(P) value for each subject was calculated, and ROC curves were plotted using this new variable to assess diagnostic efficacy. The results showed that the AUC of the five-marker combination reached 0.987, significantly higher than any single microRNA marker (P<0.05). According to the principle of maximizing the Youden index (sensitivity + specificity - 1), the optimal cutoff value for this combined model was determined to be 0.48. At this cutoff value, the sensitivity for diagnosing gallbladder cancer was 93.3%, the specificity was 91.7%, the positive predictive value was 90.9%, and the negative predictive value was 94.0%. Specific data are shown in Table 3.

[0109] Table 3. Diagnostic efficacy analysis of single markers and combined markers

[0110]

[0111] Example 2: Preparation and Performance Verification of the Reagent Kit

[0112] 1. Preparation of the kit: A gallbladder cancer-related kit prepared from gallbladder cancer plasma exosomal microRNA markers, the specific composition of which is shown in Table 4:

[0113] Table 4. Specific components of the gallbladder cancer-related reagent kit

[0114]

[0115] 2. Reagent kit performance validation

[0116] (1) Validation subjects: 20 patients diagnosed with gallbladder cancer, 20 patients with benign gallbladder diseases, and 20 healthy individuals were selected. There were no statistically significant differences in gender and age among the three groups of subjects (P>0.05). This experiment was approved by the hospital ethics committee, and all subjects signed informed consent forms.

[0117] (2) Verification method: In accordance with the experimental method in Example 1, all samples were subjected to plasma exosome isolation, RNA extraction, reverse transcription and real-time quantitative PCR detection. The relative expression levels of the five target microRNAs were recorded and statistical analysis was performed using SPSS 26.0 software.

[0118] (3) Verification indicators and results:

[0119] Specificity verification: There was no significant difference in the relative expression levels of the five microRNAs between the healthy group and the benign gallbladder disease group (P>0.05); in the gallbladder cancer group, the expression of miR-135b-5p and miR-224-5p was significantly increased, while the expression of miR-451a, miR-148a-3p and miR-375-3p was significantly decreased, consistent with the results of Example 1, indicating that the kit can specifically distinguish between gallbladder cancer samples and non-cancer samples.

[0120] Sensitivity validation: Using the optimal cutoff value of 0.48 determined in Example 1 as the criterion, the kit's sensitivity for detecting gallbladder cancer was 90.0% (18 / 20), and the negative predictive value was 92.3% (12 / 13), indicating that its ability to detect gallbladder cancer was stable.

[0121] Repeatability validation: Five representative samples (3 cases of gallbladder cancer and 2 healthy individuals) were selected, and each sample was tested three times using the kit. The coefficient of variation (CV) of the Ct values ​​was calculated. The results showed that the CV of the Ct values ​​for all target microRNAs and internal reference genes was <3%, indicating that the kit had good repeatability.

[0122] Stability validation: The kit was stored at 4°C for 7 days and at -20°C for 6 months, respectively, and the relative expression level of the positive control was measured. The results showed that the fluctuation range of the relative expression level after storage at 4°C was <5%, and the fluctuation range after storage at -20°C was <3%, which met the stability requirements of clinical kits.

[0123] Limit of Detection (LOD) Validation: Positive controls were serially diluted (10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM), and the Ct values ​​were recorded using the kit. When the target microRNA concentration was ≥0.01 nM, the Ct values ​​were consistently detectable (Ct<38), indicating that the kit's LOD reaches 0.01 nM, meeting the requirements for trace RNA detection.

[0124] Example 3: Comparative analysis of the exosomal microRNA markers in gallbladder cancer plasma of this application with existing technologies.

[0125] To demonstrate the progressiveness of this application, we selected a publicly available combination of gallbladder cancer exosomal miRNA markers (miR-552-3p, miR-581, miR-4433a-3p, miR-496, miR-203b-3p) as a control. In the same cohort of 90 cases (30 gallbladder cancer cases, 30 chronic cholecystitis cases, and 30 healthy controls), the expression levels of the five miRNAs in this comparative document combination were detected using the same experimental method (Example 1), and diagnostic scores were calculated according to the published logistic regression equation (Logit(P) = 1.628 × miR-552-3p + 1.543 × miR-581 + 2.485 × miR-4433a-3p - 3.228 × miR-496 - 3.127 × miR-203b-3p - 2.254).

[0126] The results showed that, under the same conditions, the AUC of the comparative document combination was 0.898, the sensitivity was 83.3%, and the specificity was 86.7%. The novel combination provided in this application (AUC=0.987) is significantly superior to the existing technology combination in terms of diagnostic efficacy (AUC difference=0.089, Delong test P<0.05), and it also shows improvements in both sensitivity and specificity.

[0127] As demonstrated by the above embodiments, the specific plasma exosome microRNA combination provided in this application can effectively distinguish gallbladder cancer patients from healthy individuals and patients with benign gallbladder diseases, exhibiting excellent diagnostic capabilities. Based on the standardized detection method constructed in this application, the sensitivity and specificity of gallbladder cancer diagnosis are further improved. This method, based on non-invasive body fluid biopsy, achieves stable, early, and rapid detection of target microRNAs through optimized exosome isolation and detection procedures. It is simple to operate, has a short detection cycle, and possesses promising clinical application prospects and widespread application value.

[0128] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A group of plasma exosomal microRNA markers for gallbladder cancer, characterized in that, The markers consist of miR-135b-5p, miR-224-5p, miR-451a, miR-148a-3p, and miR-375-3p.

2. The gallbladder cancer plasma exosome microRNA marker according to claim 1, characterized in that, The sequences of miR-135b-5p, miR-224-5p, miR-451a, miR-148a-3p, and miR-375-3p are shown in SEQ ID No. 1 to SEQ ID No. 5, respectively: miR-135b-5p: 5'-UAUGGCUUUUCAUUCCUAUGUGA-3' (SEQ ID No. 1); miR-224-5p: 5'-UCAAGUCACUAGUGGUUCCGUUUAG-3' (SEQ ID No. 2); miR-451a: 5'-AAACCGUUACCAUUACUGAGUU-3' (SEQ ID No. 3); miR-148a-3p: 5'-UCAGUGCACUACAGAACUUUGU-3' (SEQ ID No. 4); miR-375-3p: 5'-UUUGUUCGUUCGGCUCGCGUGA-3' (SEQ ID No. 5).

3. The use of a reagent for detecting exosomal microRNAs markers in gallbladder cancer plasma according to any one of claims 1-2 in the preparation of a kit for the auxiliary diagnosis of gallbladder cancer.

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