Bladder cancer diagnostic kit based on multiple urine exosome lncRNAs

Abstract

The invention provides a bladder cancer diagnostic kit based on a plurality of urine exosome lncRNAs (long non-coding ribonucleic acids). Experimental results show that the expression levels of the four lncRNAs (MALAT1, SCARNA10, LINC00963 and LINC01578) in the urine exosomes of bladder cancer patients are obviously higher than those of a healthy control group and a benign disease group. The lncRNA diagnosis combination shows excellent diagnosis efficiency (the sensitivity is 75.59%, the specificity is 84.37%, and AUC is equal to 0.862); after the kit is further combined with an existing bladder cancer marker NMP22, the diagnosis efficiency is remarkably improved (the sensitivity is 79.53%, the specificity is 87.50%, and AUC is equal to 0.900). Besides, the lncRNA combination has outstanding advantages on differential diagnosis of pathological high-grade bladder cancer, AUC reaches 0.917, and the limitation of accurate classification of bladder cancer in existing liquid biopsy is broken through.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, relates to the diagnosis and detection of bladder cancer, and particularly relates to a diagnostic kit for bladder cancer based on multiple urinary exosomal lncRNAs. Background Art

[0002] Bladder cancer (BLCA), as a highly prevalent malignant tumor in the urinary system, has seen a continuous increase in its incidence and mortality rate in recent years, posing a serious threat to global public health. Data from the International Agency for Research on Cancer of the World Health Organization shows that in 2022, the global incidence of bladder cancer ranked 9th among malignant tumors, and the mortality rate ranked 15th. According to the depth of tumor invasion, BLCA can be divided into non-muscle-invasive (NMIBC, accounting for 75%) and muscle-invasive (MIBC, accounting for 25%). Among them, the 5-year survival rate of NMIBC patients reaches 90%, while the 5-year survival rates of MIBC and metastatic cases drop sharply to 50% and 5% respectively, highlighting the clinical importance of early recurrence monitoring and accurate prognosis assessment of bladder cancer.

[0003] Currently, the diagnosis of bladder cancer mainly relies on cystoscopy and pathological biopsy. However, its high cost and invasive operation are prone to sampling bias and missed detection of deep lesions, making it difficult to meet the needs of large-scale screening. Auxiliary imaging methods are mostly limited to tumor staging assessment due to radiation risks and economic burdens, and there is a significant lag in the identification of recurrence and metastasis. Although molecular markers such as bladder tumor antigen (BTA) and nuclear matrix protein 22 (NMP22), as well as urine exfoliative cytology detection, are used for clinical auxiliary diagnosis, their sensitivity and specificity are insufficient, especially in the diagnosis of high-grade bladder cancer. The lack of effective diagnostic markers has led to the fact that most cases are diagnosed at the middle and late stages, and the tumor may have invaded the muscle layer, missing the best treatment window. Therefore, developing new diagnostic markers to improve the early detection rate of bladder cancer has become an urgent need.

[0004] Exosomes are lipid bilayer vesicles with a diameter of about 30 - 150 nm secreted by cells and are widely present in various body fluids such as blood and urine. Exosomes carry biomolecules such as proteins, nucleic acids (such as DNA, mRNA, miRNA, lncRNA, circRNA), and lipids, can stably exist in body fluids, and reflect the biological characteristics of their source cells. In the tumor microenvironment, exosomes, as key mediators of cell - cell communication, participate in the occurrence, development, and metastasis of tumors by transmitting bioactive molecules. Bladder cancer cells can secrete exosomes into urine, and the long non - coding RNA (lncRNA) in exosomes shows high stability due to being encapsulated by a rigid biological membrane and can become potential novel biomarkers. Given that previous studies have mostly been limited to single indicators with limited diagnostic efficacy, the present invention aims to establish a multi - indicator combined detection model of exosomal lncRNA to improve the sensitivity and specificity of bladder cancer diagnosis. Summary of the Invention

[0005] Based on the above problems, the present invention aims to provide a diagnostic biomarker for bladder cancer, and also aims to provide a new use of the combination of MALAT1, SCARNA10, LINC00963, and LINC01578 in lncRNA, that is, its application in the preparation of a diagnostic kit for bladder cancer.

[0006] Through high - throughput sequencing technology, the present invention identified a series of significantly up - regulated lncRNAs in urine exosomes of bladder cancer patients and healthy individuals, including MALAT1, SCARNA10, LINC00963, and LINC01578; the combined use of these lncRNAs can effectively distinguish bladder cancer patients from healthy individuals and bladder cancer patients from patients with benign urinary system diseases. Further research shows that in combination with the existing bladder cancer biomarker nuclear matrix protein (NMP22), this combination can significantly improve the differential diagnostic efficacy of bladder cancer, especially showing excellent efficacy in the diagnosis of high - grade bladder cancer. Therefore, the present invention provides a liquid biopsy biomarker with high sensitivity and high specificity for the clinical management and differential diagnosis of bladder cancer patients.

[0007] Based on this, the technical solutions claimed by the present invention are as follows:

[0008] In the first aspect of the present invention, there is provided the application of the lncRNA combination of MALAT1, SCARNA10, LINC00963, and LINC01578 in urine exosomes as a diagnostic biomarker for bladder cancer.

[0009] In the second aspect of the present invention, there is provided the application of this lncRNA combination in urine exosomes in the preparation of a diagnostic reagent or a diagnostic kit for bladder cancer.

[0010] In a third aspect of the present invention, there is provided an application of the above-mentioned urinary exosome lncRNA combination in combination with NMP22 protein in the preparation of a diagnostic reagent or diagnostic kit for bladder cancer.

[0011] Preferably, the diagnostic reagent is a reagent for detecting the contents of MALAT1, SCARNA10, LINC00963 and LINC01578 in urinary exosomes;

[0012] The diagnostic kit may separately contain reagents for detecting the contents of MALAT1, SCARNA10, LINC00963 and LINC01578, or may also simultaneously contain reagents for detecting the content of NMP22 protein in a urine sample.

[0013] More preferably, the reagents for detecting the contents of MALAT1, SCARNA10, LINC00963 and LINC01578 in urinary exosomes are selected from PCR primers with detection specificity for the four lncRNAs, and the sequences are as follows:

[0014]

[0015] In the present invention, urinary exosomes are used as samples to detect the expression levels of four lncRNAs. Compared with the need for a large amount of samples and difficult extraction when directly extracting free RNA from urine, collecting exosomes is equivalent to concentrating urine, improving the extraction efficiency, and significantly reducing the required urine volume.

[0016] In the examples of the present invention, exosomes are extracted from urine samples by differential centrifugation, and the steps are as follows:

[0017] (1) After the urine sample is allowed to stand to remove the precipitate, it is centrifuged at 600×g for 20 minutes, and the supernatant is taken after removing the dead cell precipitate layer;

[0018] (2) The supernatant in step (1) is centrifuged at 2000×g for 30 minutes, and the supernatant is taken after removing the cell fragments;

[0019] (3) The supernatant in step (2) is centrifuged successively under the following conditions: 10000×g, 60 min, 100000×g, 120 min, and the extracellular vesicle precipitate in the precipitate layer is taken as urinary exosomes.

[0020] In a fourth aspect of the present invention, there is provided a diagnostic kit for bladder cancer, which kit contains reagents for detecting the contents of MALAT1, SCARNA10, LINC00963 and LINC01578 in a urine sample. In terms of specific composition, it consists of a reverse transcription system, a primer system and an amplification system, and the primer system includes the above-mentioned PCR primers.

[0021] Preferably, the kit may further contain a reagent for detecting the expression level of NMP22 protein in urine, enabling the simultaneous detection of MALAT1, SCARNA10, LINC00963, LINC01578, and NMP22.

[0022] In the fifth aspect of the present invention, a combination of bladder cancer diagnostic kits is provided, which is composed of the diagnostic kit described in the fourth aspect and an NMP22 detection kit. The NMP22 detection kit is a finished product and can be directly used in combination.

[0023] In the sixth aspect of the present invention, a method for diagnosing bladder cancer using the above diagnostic kit is provided as follows:

[0024] A. Differentially centrifuge the urine sample to be tested to obtain urinary exosomes, and extract total RNA therefrom.

[0025] B. Reverse transcribe the total RNA in step A to obtain cDNA.

[0026] C. Use qRT-PCR technology to quantitatively detect the copy numbers of MALAT1, SCARNA10, LINC00963, and LINC0157. The primers used in the detection process are as shown in the above SEQ ID NO.1 - 10; simultaneously detect the NMP22 protein in urine.

[0027] Through bioinformatics analysis, the expression levels of four lncRNAs (MALAT1, SCARNA10, LINC00963, and LINC01578) in urinary exosomes of bladder cancer patients are significantly higher than those in the healthy control group and the benign disease group. The lncRNA diagnostic combination exhibits excellent diagnostic efficacy (sensitivity: 75.59%; specificity: 84.37%; AUC = 0.862).

[0028] After further combining with NMP22, the diagnostic efficacy is significantly improved (sensitivity: 79.53%; specificity: 87.50%; AUC = 0.900). In addition, the lncRNA combination has outstanding advantages in the differential diagnosis of high-grade bladder cancer, with an AUC of 0.917, breaking through the limitations of existing liquid biopsies for the precise grading of bladder cancer.

[0029] In addition, the present invention first detected lncRNA SCARNA10 and LINC01578 in urinary exosomes, and there has been no relevant report so far.

[0030] The beneficial guarantees and effects of the present invention are as follows:

[0031] First, in terms of technology, the detection of the four lncRNAs is essentially a quantitative PCR detection of urine genome, which is characterized by simple operation, high sensitivity, good specificity, high repeatability, etc. Nowadays, it has been increasingly applied to clinical testing technologies.

[0032] The basic detection method adopted in the present invention is qRT-PCR. This technology has been proven to be a highly sensitive and accurate detection method in modern experimental diagnostics. The test technology has been very mature, and the standard curve quantification method in this technology is used, which can accurately quantify specific nucleic acid molecules in various samples.

[0033] Secondly, as a non-invasive and easily collectable sample type, urine samples have high patient compliance. When conducting the detection of the present invention, only 8 mL of urine sample is required, and random urine can be taken. It has strong clinical operability and is not restricted by the collection time, providing an efficient and convenient new strategy for the early screening and stratified diagnosis and treatment of bladder cancer.

[0034] Thirdly, the expressions of the indexes MALAT1, SCARNA10, LINC00963, and LINC01578 involved in the present invention are significantly up-regulated in the urine exosomes of bladder cancer patients, and the differences are statistically significant (P<0.05), and their clinical reference value and credibility are relatively high. Combined with the existing bladder cancer marker nuclear matrix protein (NMP22), this combination can significantly improve the differential diagnostic efficacy of bladder cancer, especially showing excellent efficacy in the diagnosis of high-grade bladder cancer, which is helpful for the accurate grading of bladder cancer.

[0035] Therefore, taking urine exosome lncRNA as the research object, compared with the existing bladder cancer diagnostic methods, this model not only has excellent diagnostic efficacy, but also can realize the differential diagnosis of high-grade bladder cancer, providing a new reference plan for the rapid diagnosis of bladder cancer. Description of the Drawings

[0036] Figure 1 Shows a schematic diagram of the preparation process of urine exosomes;

[0037] Figure 2 Shows the detection of urine exosomes. a~c respectively show the detection results of transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot;

[0038] Figure 3 Shows the differential expression heat map of long non-coding RNA (lncRNA);

[0039] Figure 4 Shows the differential expression volcano map of long non-coding RNA (lncRNA);

[0040] Figure 5 Shows the relative expression levels of lncRNAs in urinary exosomes: a - d respectively show the expression differences of MALAT1, SCARNA10, LINC00963, and LINC01578 in healthy individuals (Normal), benign lesions (Benign lesion), and bladder cancer (BLCA); e The freeze - thaw cycle experiment shows the Ct values of exosomes of MALAT1, SCARNA10, LINC00963, and LINC01578.

[0041] Figure 6 Shows the correlation between urinary exosome lncRNAs and clinical parameters: a, The expression difference of MALAT1 in tumors with a volume less than 3 cm and ≥ 3 cm; b, The expression difference of SCARNA10 in low - grade and high - grade bladder cancer; c, The expression difference of SCARNA10 in tumors with a volume less than 3 cm and ≥ 3 cm; d, The expression difference of LINC00963 in low - grade and high - grade bladder cancer; e, The expression difference of LINC01578 in low - grade and high - grade bladder cancer.

[0042] Figure 7 Shows the diagnostic efficacy of urinary exosome lncRNAs and NMP22 for bladder cancer: a - e respectively show the diagnostic efficacy of MALAT1, SCARNA10, LINC00963, LINC01578, and NMP22 alone as diagnostic markers; f shows the diagnostic efficacy of the combination of four lncRNAs as a diagnostic marker; g shows the diagnostic efficacy of the combination of four lncRNAs and NMP.

[0043] Figure 8 Shows the diagnostic efficacy of urinary exosome lncRNAs and NMP22 for high - grade bladder cancer: a - e respectively show the diagnostic efficacy of MALAT1, SCARNA10, LINC00963, LINC01578, and NMP22 alone as diagnostic markers for high - grade bladder cancer; f shows the diagnostic efficacy of the combination of four lncRNAs as a diagnostic marker for high - grade bladder cancer; g shows the diagnostic efficacy of the combination of four lncRNAs and NMP for high - grade bladder cancer. Detailed implementation manners

[0044] The following examples and experimental examples further illustrate the present invention and should not be construed as limiting the present invention. The examples do not include a detailed description of traditional methods, such methods being well - known to those of ordinary skill in the art and having been described in many publications.

[0045] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to persons skilled in the art. In addition, any methods and materials similar or equivalent to those described may be applied to the present invention, and the preferred methods and materials described in the specific embodiments are for illustrative purposes only.

[0046] I. Urine exosome extraction

[0047] Exosomes were extracted from urine samples from 3 healthy subjects and 4 patients clinically and pathologically diagnosed with high-grade bladder cancer. Differential ultracentrifugation was used to extract urine exosomes, and the experimental procedure is shown in Figure 1 as follows:

[0048] (1) After the urine sample was allowed to stand to remove the precipitate, it was centrifuged at 600×g for 20 minutes, and the supernatant was taken after removing the dead cell pellet layer;

[0049] (2) The supernatant in step (1) was centrifuged at 2000×g for 30 minutes, and the supernatant was taken after removing the cell fragments;

[0050] (3) The supernatant in step (2) was centrifuged successively under the following conditions: 10000×g, 60 min, 100000×g, 120 min, and the extracellular vesicle precipitate in the pellet layer was taken as urine exosomes.

[0051] To ensure the accuracy of subsequent experimental results, in accordance with the ISEV2023 standard, see Figure 2 and transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot techniques were used to strictly identify the extracted exosomes. Through these methods, the presence, quantity, and integrity of exosomes were evaluated to verify the effect of exosome isolation and purification.

[0052] II. Screening of differentially expressed lncRNAs

[0053] High-throughput sequencing was performed by Shanghai Kangcheng Biotech Co., Ltd., and the specific process was as follows: The concentration and purity were measured by NanoDrop2000. After taking at least 1 μg of total RNA for oligo dT enrichment (rRNA removal) treatment, the library was constructed according to the standard process, and long-chain RNA sequencing was performed using the Illumina Novaseq 6000 system (Illumina, San Diego, CA, USA), and differential transcripts were screened according to the thresholds of fold change>1.5 and P≤0.05.

[0054] Figure 3 and Figure 4The differential expression heatmap and differential expression volcano plot of long non-coding RNA (lncRNA) are shown respectively. After screening, four differentially expressed lncRNAs (MALAT1, SCARNA10, LINC00963 and LINC01578) were finally identified, among which SCARNA10 and LINC01578 were detected in exosomes for the first time.

[0055] III. qRT-PCR detection of four lncRNAs

[0056] Urine samples were collected from healthy individuals (Nomal), patients with clinically diagnosed benign urinary system diseases (Benignlesion) and patients with bladder cancer (BLCA). Total RNA was extracted from the exosomes after extraction, and detected by qRT-PCR. First, MALAT1, SCARNA10, LINC00963 and LINC01578 were used as diagnostic markers separately to compare the expression levels of lncRNAs in the three groups of samples. The results of the differential analysis of the expression levels are shown in Figure 5 ; After freeze-thaw cycles, the ct values of MALAT1, SCARNA10, LINC00963 and LINC01578 in exosomes were detected to prove the good stability of the expression levels of the four lncRNAs derived from exosomes.

[0057] During the qRT-PCR detection process, the primer sequences of 18S, MALAT1, LINC00963, LINC01578 and SCARNA10 are shown in Table 1 below:

[0058] Table 1 Summary of primer sequences

[0059]

[0060] The results are as Figure 5 shown. The expression levels of the four lncRNAs (MALAT1, SCARNA10, LINC00963 and LINC01578) in the urine exosomes of bladder cancer patients were significantly higher than those in the healthy control group and the benign disease group.

[0061] IV. Clinical correlation analysis

[0062] Analyze the correlations between the expression levels of MALAT1, SCARNA10, LINC00963, and LINC01578 in urinary exosomes and clinical features (including age, gender, stage, grade, number of tumors, tumor size, lymph node metastasis, recurrence, FPSA / TPSA, and urine cytology). The results show that high expression of MALAT1 is related to tumor diameter, and high expression of SCARNA10 is related to high pathological grade and tumor diameter. In patients with high-grade histological bladder cancer, LINC00963 and LINC01578 also showed higher expression levels in urinary exosomes ( Figure 6 ).

[0063] Further combined with Figure 7 and Figure 8 , by analyzing the ROC curve, it was found that the diagnostic efficacy based on the above four lncRNAs in urinary exosomes was better than the existing clinical index nuclear matrix protein (NMP22), and the combination of the four lncRNAs and NMP22 had significant diagnostic value for BLCA and high-grade BLCA: The expression levels of the four lncRNAs (MALAT1, SCARNA10, LINC00963, and LINC01578) in urinary exosomes of bladder cancer patients were significantly higher than those in the healthy control group and the benign disease group. The lncRNA diagnostic combination showed excellent diagnostic efficacy (sensitivity: 75.59%; specificity: 84.37%; AUC = 0.862). After further combining with NMP22, the diagnostic efficacy was significantly improved (sensitivity: 79.53%; specificity: 87.50%; AUC = 0.900). In addition, the lncRNA combination had outstanding advantages in the differential diagnosis of high-grade bladder cancer, with an AUC of 0.917, breaking through the limitations of existing liquid biopsies for the accurate grading of bladder cancer. Therefore, this urinary exosome lncRNA combination can be used as an excellent biomarker for the diagnosis of bladder cancer and high-grade bladder cancer.

[0064] In summary, the present invention has developed a novel lncRNA combined diagnostic model for liquid biopsy of bladder cancer, which has not been reported before and provides a new reference scheme for the rapid diagnosis of bladder cancer. Taking urinary exosome lncRNAs as the research object, compared with the existing diagnostic methods for bladder cancer, this model not only has excellent diagnostic efficacy but also can achieve the differential diagnosis of high-grade bladder cancer. At the same time, only 8 mL of urine sample is required, and random urine can be taken. It has strong clinical operability and is not limited by the collection time, providing an efficient and convenient new strategy for the early screening and stratified diagnosis and treatment of bladder cancer.

[0065] The parts not described in the present invention are the same as those in the prior art or can be implemented by using the prior art. The applicant declares that the present invention uses the above embodiments to illustrate the detailed method of the present invention, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvement to the present invention, the equivalent substitution of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc. all fall within the protection scope and the disclosure scope of the present invention.

Claims

1. The use of a combination of urine exosome lncRNA and NMP22 protein in the preparation of a bladder cancer diagnostic reagent or diagnostic kit, characterized in that: The urinary exosomal lncRNA combination consists of MALAT1, SCARNA10, LINC00963 and LINC01578.

2. The use according to claim 1, characterized in that: The diagnostic reagent is a reagent for detecting the content of MALAT1, SCARNA10, LINC00963 and LINC01578 in urine exosomes; the diagnostic kit contains reagents for detecting the content of NMP22 protein in urine samples and the content of MALAT1, SCARNA10, LINC00963 and LINC01578 in urine exosomes.

3. The use according to claim 2, characterized in that: The reagents for detecting the contents of MALAT1, SCARNA10, LINC00963 and LINC01578 in urine exosomes are selected from PCR primers with detection specificity for the four lncRNAs, and the sequences are shown in SEQ ID NO.1~8.

4. The use according to claim 1, characterized in that: Exosomes were extracted from urine samples by differential centrifugation as follows: (1) After the urine sample is allowed to stand to remove the precipitate, it is centrifuged at 600 × g for 20 minutes to remove the dead cell precipitate and collect the supernatant; (2) The supernatant in step (1) was centrifuged at 2000 × g for 30 minutes, and the cell fragments were removed and the supernatant was collected; (3) The supernatant in step (2) was centrifuged under the following conditions: 10,000 × g, 60 min, 100,000 × g, 120 min, and the extracellular vesicle precipitate in the precipitate layer was taken as urine exosomes.

5. A bladder cancer diagnostic kit, characterized in that: The kit contains reagents for detecting the levels of MALAT1, SCARNA10, LINC00963 and LINC01578 in urine samples.

6. The bladder cancer diagnostic kit according to claim 5, characterized in that The diagnostic kit is composed of a reverse transcription system, a primer system and an amplification system, wherein the primer system includes PCR primers as shown in SEQ ID NOs. 1 to 8.

7. The bladder cancer diagnostic kit according to claim 5, characterized in that The invention also comprises a reagent for detecting the expression amount of NMP22 protein in urine.

8. A bladder cancer diagnostic kit combination, characterized in that: The method is composed of a combination of the diagnostic kit according to claim 5 or 6 and the NMP22 detection kit.

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