Urine miRNA markers, diagnostic reagents and kits for bladder cancer diagnosis

Abstract

The present invention discloses a urine miRNA marker, a diagnostic reagent and a kit for diagnosing bladder cancer, wherein the urine miRNA marker is selected from a combination of one or more of hsa-miR-143-3p, hsa-miR-183-5p, hsa-miR-21-3p, hsa-miR-219a-5p, hsa-miR-1-3p and hsa-miR-18a-3p. The present invention has higher population specificity and is more reliable than other miRNA molecular markers.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular diagnosis, and in particular to a urine miRNA marker, a diagnostic reagent and a kit for diagnosing bladder cancer. Background Art

[0002] Bladder cancer (BC) is a malignant tumor originating from the urothelium of the bladder. It is one of the most common malignant tumors of the urinary system and ranks first in the incidence of urogenital tumors in my country. The incidence of bladder cancer worldwide: The incidence rate ranks 11th among all malignant tumors. There are regional, racial and gender differences in the incidence of bladder cancer. It can be seen in all age groups, with a high incidence age of 50 to 70 years old. The incidence rate gradually increases with age.

[0003] BC has the characteristics of high survival rate if detected early, and high recurrence rate requiring frequent and long-term monitoring. Although treatment methods such as surgery, radiotherapy, and chemotherapy are available, the prognosis and 5-year survival rate are extremely poor due to late diagnosis and rapid metastasis. Although treatment methods such as surgery, radiotherapy, and chemotherapy have significantly improved the quality of life of bladder cancer patients, early diagnosis is the key to improving survival rates.

[0004] Currently, cystoscopy and biopsy are considered the gold standard for BC screening and diagnosis, but they are expensive and may cause complications such as urogenital infection, urethral and bladder bleeding, urethral injury and urethral stricture, and have low detection sensitivity for low-grade tumors. Urine cytology of urine or bladder washing specimens is one of the main methods for bladder cancer diagnosis and postoperative follow-up. Although this detection method is non-invasive, it has extremely low sensitivity in the diagnosis of early BC. Many urine-based markers such as nuclear matrix protein 22 (NMP22), bladder tumor antigen (BTA) and cytokeratin have been developed over the past few decades, but none of them is an ideal marker that can be used for large-scale cancer screening. Therefore, there is an urgent need to develop screening markers that can be used for early stage BC diagnosis and monitoring with high sensitivity and specificity.

[0005] Multiple studies have shown that urine contains many different types of RNA, including mRNA, microRNA (miRNA) and other non-coding protein (nc) RNA. Urine is a particularly attractive source of biomarkers for urological cancer screening because it is easier to detect than blood and can capture the disease at an earlier stage.

[0006] miRNA is a short non-coding RNA with a length of 18-25 nucleotides. It can prevent protein expression by decomposing specific target mRNA or inhibiting its translation. It participates in regulating life activities such as individual development, cell apoptosis, proliferation and differentiation, and plays a role similar to oncogenes or tumor suppressor genes in the occurrence and development of tumors. The expression profile of miRNA has obvious tissue specificity and has specific expression patterns in different tumors. These characteristics make miRNA a new biological marker and therapeutic target for tumor diagnosis.

[0007] Although existing studies have reported some miRNA markers for early diagnosis of urinary system cancer, these markers have more or less detection limitations. The urine miRNA biomarkers and biomarker combination markers that can ultimately be used for bladder cancer screening have not yet been finalized. There is an urgent need to develop new urine miRNA markers suitable for bladder cancer diagnosis. Summary of the invention

[0008] The purpose of the present invention is to provide a urine miRNA marker, diagnostic reagent and kit for bladder cancer diagnosis, which has higher population specificity than other miRNA markers reported internationally; the disclosed miRNA diagnostic markers are all proposed for the first time and are more reliable than other miRNA molecular markers, providing a new optional approach for bladder cancer diagnosis.

[0009] The technical solution adopted by the present invention to solve its technical problem is:

[0010] A urine miRNA marker for diagnosing bladder cancer, wherein the urine miRNA marker is selected from a combination of one or more of hsa-miR-143-3p, hsa-miR-183-5p, hsa-miR-21-3p, hsa-miR-219a-5p, hsa-miR-1-3p, and hsa-miR-18a-3p.

[0011] Preferably, the urine miRNA marker is a combination of hsa-miR-143-3p, hsa-miR-183-5p, hsa-miR-21-3p, hsa-miR-219a-5p, hsa-miR-1-3p and hsa-miR-18a-3p.

[0012] A bladder cancer diagnostic reagent, comprising the urine miRNA marker according to claim 1.

[0013] A kit for diagnosing bladder cancer, comprising the urine miRNA marker of claim 1 as a standard, and corresponding primers for detecting the urine miRNA marker of claim 1.

[0014] The beneficial effect of the present invention is that the present invention identifies 6 specific diagnostic markers and specific diagnostic marker combinations suitable for bladder cancer in Chinese people. The above-mentioned miRNA diagnostic markers are proposed for the first time and are more reliable than other miRNA molecular markers.

[0015] Compared with the prior art, the present invention has the following positive and beneficial effects:

[0016] (1) The present invention first discovered that hsa-miR-143-3p, hsa-miR-183-5p, hsa-miR-21-3p, hsa-miR-219a-5p, hsa-miR-1-3p, and hsa-miR-18a-3p can be used as new bladder cancer markers, and the above markers have important value in the early detection and diagnosis of urinary system tumors;

[0017] (2) The miRNA marker detection of the present invention is simple to operate, has strong feasibility, high sensitivity and specificity, and has clinical diagnosis and guidance significance.

[0018] (3) The present invention can detect miRNA expression through urine, thereby achieving the purpose of non-invasiveness. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Heat map of expression levels of 16 differentially expressed (P < 0.05) miRNA diagnostic markers for bladder cancer. The expression levels of miRNAs (copies / mL) are presented in log2 scale and normalized to zero mean. The color of the points indicates the concentration. Hierarchical clustering was performed based on Euclidean distance for both dimensions (miRNA and sample). For the horizontal dimension, color is used to indicate case-control subjects.

[0020] Figure 2 The AUC graph of the bladder cancer miRNA diagnostic marker combination in each cohort; Figure 2 -A is the AUC graph of the combination of 16 bladder cancer miRNA diagnostic markers in each cohort, and 2-B is the AUC graph of the combination of 6 bladder cancer miRNA diagnostic markers in each cohort.

[0021] Figure 3 Box plots of the control and cancer prediction values ​​of the combination of six bladder cancer miRNA diagnostic markers. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is further described in detail below through specific embodiments.

[0023] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following embodiments, unless otherwise specified, are all conventional methods in the art.

[0024] The detection method for bladder cancer includes the following steps: a) determining hsa-miR-143-3p, hsa-miR-183-5p, hsa-miR-21-3p, hsa-miR-219a-5p, hsa-miR-1-3p, and hsa-miR-18a-3p in a test sample and a reference sample of human origin; b) comparing the expression levels of hsa-miR-143-3p, hsa-miR-183-5p, hsa-miR-21-3p, hsa-miR-219a-5p, hsa-miR-1-3p, and hsa-miR-18a-3p in the test sample and the reference sample; wherein a significant change in the expression level of any one of the above miRNAs in the test sample compared with the expression level of the corresponding miRNA in the reference sample indicates that the individual may have bladder cancer.

[0025] Specifically, when the expression level of any one miRNA in the detected bladder cancer is significantly different from the expression level of the miRNA in the reference sample, the sample provider may have bladder cancer. In a preferred embodiment, when the expression level of any 16 miRNAs in the detected urine sample is significantly different from the expression level of the miRNA in the reference sample, the sample provider may have bladder cancer. In a more preferred embodiment, when the expression level of 6 miRNAs in the detected urine sample is significantly different from the expression level of the miRNA in the reference sample, the sample provider may have bladder cancer.

[0026] In the present invention, the reference sample is a urine sample from a healthy person.

[0027] On the other hand, the present invention also provides a method for detecting bladder cancer microRNA. Specifically, the method includes reverse transcription and real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). More specifically, in the real-time fluorescence quantitative polymerase chain reaction, the primer combination used is a reverse transcription primer and PCR primer combination for hsa-miR-143-3p, hsa-miR-183-5p, hsa-miR-21-3p, hsa-miR-219a-5p, hsa-miR-1-3p, and hsa-miR-18a-3p detection.

[0028] The primer sequences of the present invention can be appropriately adjusted and modified according to the sequences of the relevant miRNAs, and these modified primer sequences can still be used to detect the miRNA markers. The present invention also includes these equivalent technical solutions.

[0029] The present invention uses the RT-qPCR method, which has more reliable clinical feasibility and practicality compared to chips based on hundreds of molecules, and is low-cost and easier to promote. In addition, the detection method of the above miRNA is not limited to the RT-qPCR method provided by the present invention, and other methods such as sequencing, microarray, Northern blot, bioluminescence and probe method can also be used to detect the miRNA markers.

[0030] The applicant has found in the research that any one miRNA marker can be used to diagnose bladder cancer, and any combination of 2 to 5 miRNA markers can be used to diagnose bladder cancer. Using the above markers, bladder cancer can be reliably identified.

[0031] The present invention discloses bladder cancer diagnostic markers including hsa-miR-143-3p, hsa-miR-183-5p, hsa-miR-21-3p, hsa-miR-219a-5p, hsa-miR-1-3p, and hsa-miR-18a-3p; hsa-miR-143-3p, hsa-miR-183-5p, hsa-miR-21-3p, hsa-miR-219a-5p, hsa-miR-1-3p, and hsa-miR-18a-3p are composed of multiple nucleic acid molecules, and each nucleic acid molecule encodes at least one miRNA sequence. The specific sequence information is shown in Table 1 below.

[0032] All miRNA sequences disclosed in the present invention have been stored in the miRBase database ( http: / / www.mirbase.org / ).

[0033] Table 1

[0034]

[0035] The present invention discloses a bladder cancer diagnostic marker combination, comprising a plurality of nucleic acid molecules encoding hsa-miR-143-3p, hsa-miR-183-5p, hsa-miR-21-3p, hsa-miR-219a-5p, hsa-miR-1-3p, and hsa-miR-18a-3p. The expression of any one or any two or any three or any four or any five or any six nucleic acid molecules encoding hsa-miR-143-3p, hsa-miR-183-5p, hsa-miR-21-3p, hsa-miR-219a-5p, hsa-miR-1-3p, and hsa-miR-18a-3p in the urine of patients diagnosed with bladder cancer is significantly changed compared with the expression in the control. In the optimal embodiment for bladder cancer diagnosis, the multiple nucleic acid molecules include a combination of six nucleic acid molecules: hsa-miR-143-3p, hsa-miR-183-5p, hsa-miR-21-3p, hsa-miR-219a-5p, hsa-miR-1-3p, and hsa-miR-18a-3p.

[0036] Example 1: Validation cohort validation of 16 miRNA combinations for bladder cancer diagnosis

[0037] 1. Requirements, collection and extraction of urine samples for the bladder cancer diagnostic marker panel development cohort

[0038] Two groups of samples were used in the study of bladder cancer diagnostic markers, namely the case group and the control group. The case group contained 29 bladder cancer samples and the control group was 88 healthy human urine samples. Biomarkers and biomarker combinations for detecting early bladder cancer were discovered and verified. Bladder cancer cases and healthy samples in the research and development cohort were all from the Second Affiliated Hospital of Zhejiang University School of Medicine.

[0039] 2. Reverse transcription-real-time fluorescence PCR operation process and results

[0040] Extraction was performed according to the Zymo serum plasma cfDNA rapid extraction kit operating procedures; after extraction, reverse transcription was performed using the MIRXE reverse transcription kit operating procedures, and after reverse transcription, qPCR amplification was performed for quality inspection.

[0041] After the quality inspection, the samples were amplified by qPCR to detect the expression of 312 miRNAs. There was no obvious grading in the expression of all 312 miRNAs in cancer and healthy controls. Further research found a single miRNA biomarker for bladder cancer detection. After correction, it was found that the p-value of 16 miRNAs was less than 0.05, of which 13 were upregulated and 3 were downregulated in bladder cancer subjects. These 16 miRNAs were extracted from the R&D cohort to draw a heatmap, and significant differences in miRNA expression between cancer and control subjects were observed, such as Figure 1 shown.

[0042] 3: Validation cohort validation of the above 16 miRNAs

[0043] The case-control cohort used the leave-one-out cross-validation method to detect these 16 urine miRNA biomarkers. The validation cohort samples were 117 samples, and the diagnostic efficacy AUC = 0.732. Figure 2 -A.

[0044] Example 2: Validation of the optimal 6 miRNA combinations for bladder cancer diagnosis by validation cohort The urine sample requirements, collection and extraction, reverse transcription-real-time fluorescence PCR operation process and results of the research and development cohort are the same as those described in Example 1. Based on the 16 miRNAs screened previously, the miRNA combination was optimized using algorithms such as Lasso, Stepwise, and exhaustive methods, and the optimal 6 miRNA combinations were finally determined. The cross-validation (Leave-one-out Cross-validation) method was used to validate the 6 urine biomarker combinations (optimal combination) including hsa-miR-143-3p, hsa-miR-183-5p, hsa-miR-21-3p, hsa-miR-219a-5p, hsa-miR-1-3p, and hsa-miR-18a-3p. The validation cohort samples consisted of 117 samples, and the diagnostic efficacy of the 6 miRNA combination in the validation cohort was AUC = 0.857, as shown in Figure 2. Figure 2 -B. The results of the model interpretation are shown in Figure 3 As shown, it can be seen that the model output probability of cancer patients is significantly higher than that of control subjects, indicating that the model can better predict and classify cancer patients and control subjects.

[0045] Based on the above comparison, the combination of 6 miRNAs was confirmed as the optimal combination for diagnosing bladder cancer.

[0046] The present invention establishes a complete workflow for discovering and validating urine miRNA biomarker combinations, and successfully determines biomarkers and biomarker combinations for detecting bladder cancer.

[0047] The above-described embodiment is only a preferred solution of the present invention and does not limit the present invention in any form. There are other variations and modifications without exceeding the technical solution described in the claims. SEQUENCE LISTING <110> Mirui (Hangzhou) Biotechnology Co., Ltd.; Mirui Laboratory Pte Ltd <120> Urine miRNA markers, diagnostic reagents and kits for bladder cancer diagnosis <130> 2021.11 <160> 16 <170> PatentIn version 3.3 <210> 1 <211> twenty one <212> RNA <213> Artificial sequence <400> 1 ugagaugaag cacuguagcu c 21 <210> 2 <211> twenty two <212> RNA <213> Artificial sequence <400> 2 uauggcacug guagaauuca cu 22 <210> 3 <211> twenty one <212> RNA <213> Artificial sequence <400> 3 caacaccagu cgaugggcug u 21 <210> 4 <211> twenty one <212> RNA <213> Artificial sequence <400> 4 ugauugucca aacgcaauuc u 21 <210> 5 <211> twenty two <212> RNA <213> Artificial sequence <400> 5 uggaauguaa agaaguaugu au 22 <210> 6 <211> twenty three <212> RNA <213> Artificial sequence <400> 6 acugcccuaa gugcuccuuc ugg 23 <210> 7 <211> twenty two <212> RNA <213> Artificial sequence <400> 7 cuuaucagau uguauuguaa uu 22 <210> 8 <211> twenty three <212> RNA <213> Artificial sequence <400> 8 caaagugcug uucgugcagg uag 23 <210> 9 <211> twenty one <212> RNA <213> Artificial sequence <400> 9 uacaguacug ugauaacuga a 21 <210> 10 <211> twenty two <212> RNA <213> Artificial sequence <400> 10 uagcagcaca ucaugguuua ca 22 <210> 11 <211> twenty three <212> RNA <213> Artificial sequence <400> 11 caacggaauc ccaaaagcag cug 23 <210> 12 <211> twenty two <212> RNA <213> Artificial sequence <400> 12 cauugcacuu gucucggucu ga 22 <210> 13 <211> 20 <212> RNA <213> Artificial sequence <400> 13 ucacgcggag agauggcuuu 20 <210> 14 <211> twenty three <212> RNA <213> Artificial sequence <400> 14 uagugcaaua uugcuuauag ggu 23 <210> 15 <211> twenty two <212> RNA <213> Artificial sequence <400> 15 uccuucauuc caccggaguc ug 22 <210> 16 <211> twenty three <212> RNA <213> Artificial sequence <400> 16 aaugacacga ucacuucccgu uga 23

Claims

1. Use of urine miRNA markers and / or their detection reagents in the preparation of a kit for diagnosing bladder cancer, It is characterized in that The urine miRNA marker is a combination of hsa-miR-143-3p, hsa-miR-183-5p, hsa-miR-21-3p, hsa-miR-219a-5p, hsa-miR-1-3p and hsa-miR-18a-3p.

2. A bladder cancer diagnostic reagent, It is characterized in that The bladder cancer diagnostic reagent comprises the corresponding primers of the urine miRNA marker according to claim 1.

3. A kit for diagnosing bladder cancer, It is characterized in that The bladder cancer diagnosis kit comprises the urine miRNA marker of claim 1 as a standard, and corresponding primers for detecting the urine miRNA marker of claim 1.

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