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

Abstract

The present invention discloses a urine miRNA marker, a diagnostic reagent and a kit for diagnosing prostate cancer, wherein the urine miRNA marker is selected from a combination of one or more of hsa-miR-375, hsa-miR-520d-5p, hsa-miR-199b-5p, hsa-miR-518e-5p, hsa-miR-31-3p and hsa-miR-4306. 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 prostate cancer. Background Art

[0002] According to data from the National Cancer Center, prostate cancer has become the most common tumor in the male urinary system since 2008. The incidence rate in 2014 reached 9.8 / 100,000, ranking sixth in the incidence of male malignant tumors; the mortality rate reached 4.22 / 100,000, and prostate cancer (PCa) is one of the top ten leading causes of cancer-related deaths in Chinese men. Moreover, prostate cancer in my country has been increasing year by year. It is worth noting that there is a large difference in the incidence of prostate cancer between urban and rural areas in my country, especially in large cities. Worldwide, the incidence of prostate cancer ranks second in the United States and third in Europe.

[0003] Although prostate cancer is a disease with a high incidence, little is known about its causes. Established risk factors are limited to age, family history of malignant tumors, and certain gene mutations (for example, BRCA1 and BRCA2) and conditions (Lynch syndrome). At the same time, its incidence is also somewhat related to race, with black men having the highest incidence among different races. In addition, the more established risk factors also include lifestyle and environmental factors, such as smoking, overweight and nutritional factors, which may increase the risk of advanced prostate cancer in general.

[0004] PCa is a slow-growing tumor, and up to 40% of men diagnosed with it have no clinical symptoms at all. However, if PCa is diagnosed at an early stage (i.e., localized), the patient's five-year survival rate is greater than 99%, but if it is diagnosed at an advanced, high-grade metastatic stage, the patient's survival rate is only about 30%. Therefore, early diagnostic PCa screening has the potential to radically reduce the healthcare burden of this cancer.

[0005] Prostate-specific antigen (PSA) is a 34kDa, single-chain glycoprotein composed of 237 amino acids, which is almost exclusively produced by prostate epithelial cells. In 2018, the U.S. Preventive Medicine Task Force issued a statement that PSA screening is not recommended for people over 70 years old. For people aged 55-69, whether to conduct regular PSA screening depends on individual circumstances. For a long time, PSA-based prostate cancer screening has been widely used, but the false positive rate of PSA results is high, which can easily lead to overdiagnosis and overtreatment.

[0006] Prostate biopsy is the gold standard test for screening and diagnosing prostate cancer. However, the main limitations of transrectal or transperineal prostate biopsy are false negatives, missed diagnosis of high-risk prostate cancer, and overdiagnosis. How to improve the positive rate of puncture while avoiding overdiagnosis is a huge challenge in the early diagnosis of prostate cancer. Therefore, it is urgent to develop safer, more efficient, and more specific methods for detecting prostate cancer and / or high-risk prostate cancer.

[0007] 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.

[0008] 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. Studies have shown that miRNA can exist in free form in urine. Recently, many studies have shown that miRNA in patient urine can be used as a biomarker for the diagnosis of early urinary system cancer.

[0009] Although existing studies have reported some miRNA markers for the 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 prostate cancer screening have not yet been finalized. There is an urgent need to develop new urine miRNA markers suitable for the diagnosis of prostate cancer. Summary of the invention

[0010] The purpose of the present invention is to provide a urine miRNA marker, diagnostic reagent and kit for prostate 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 prostate cancer diagnosis.

[0011] The technical solution adopted by the present invention to solve its technical problem is:

[0012] A urine miRNA marker for diagnosing prostate cancer, wherein the urine miRNA marker is selected from one of the following schemes:

[0013] ① Any one of hsa-miR-375, hsa-miR-520d-5p, hsa-miR-199b-5p, hsa-miR-518e-5p, hsa-miR-31-3p, hsa-miR-4306;

[0014] ② A combination of two or more of hsa-miR-375, hsa-miR-520d-5p, hsa-miR-199b-5p, hsa-miR-518e-5p, hsa-miR-31-3p, and hsa-miR-4306;

[0015] ③A combination of hsa-miR-375 and any one or more of hsa-miR-520d-5p, hsa-miR-199b-5p, hsa-miR-518e-5p, hsa-miR-31-3p, and hsa-miR-4306.

[0016] As the most preferred combination, the urine miRNA markers are a combination of hsa-miR-375, hsa-miR-520d-5p, hsa-miR-199b-5p, hsa-miR-518e-5p, hsa-miR-31-3p and hsa-miR-4306.

[0017] A prostate cancer diagnostic reagent, comprising the urine miRNA marker according to claim 1.

[0018] A prostate cancer diagnosis kit, comprising the urine miRNA marker of claim 1 as a standard, and corresponding primers for detecting the urine miRNA marker of claim 1.

[0019] The beneficial effect of the present invention is that the present invention clarifies hsa-miR-375 alone and two specific diagnostic marker combinations of 6 types of prostate cancer suitable for the Chinese population, including hsa-miR-375. The above miRNA diagnostic markers are all proposed for the first time and are more reliable than other miRNA molecular markers.

[0020] Compared with the prior art, the present invention has the following positive and beneficial effects:

[0021] (1) The present invention first discovered that hsa-miR-375, hsa-miR-520d-5p, hsa-miR-199b-5p, hsa-miR-518e-5p, hsa-miR-31-3p, and hsa-miR-4306 can be used as a new prostate cancer marker combination, and the above marker combination has important value in the early detection and diagnosis of prostate cancer;

[0022] (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.

[0023] (3) The present invention can detect miRNA expression through urine, thereby achieving the purpose of non-invasiveness. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 10 differentially expressed (P < 0.05) prostate cancer miRNA diagnostic markers expression level heat map. The expression level of miRNA (copy number / ml) is presented in log2 scale and is standardized relative to the zero mean. The color of the point represents the concentration. Hierarchical clustering is performed for two dimensions (miRNA and sample) based on Euclidean distance. For the horizontal dimension, the subject of the case-control is represented by color.

[0025] Figure 2 The AUC graph of the prostate cancer miRNA diagnostic marker combination in each cohort; Figure 2 -A is the AUC graph of the combination of 10 prostate cancer miRNA diagnostic markers in each cohort. Figure 2 -B is the AUC graph of the combination of 6 prostate cancer miRNA diagnostic markers in each cohort.

[0026] Figure 3 Box plot of the control and cancer predictive values ​​of the combination of six prostate cancer miRNA diagnostic markers. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described in detail below through specific embodiments.

[0028] 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.

[0029] The detection method for prostate cancer includes the following steps: a) determining the expression levels of hsa-miR-375, hsa-miR-520d-5p, hsa-miR-199b-5p, hsa-miR-518e-5p, hsa-miR-31-3p, and hsa-miR-4306 in a test sample and a reference sample of human origin; b) comparing the expression levels of hsa-miR-375, hsa-miR-520d-5p, hsa-miR-199b-5p, hsa-miR-518e-5p, hsa-miR-31-3p, and hsa-miR-4306 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 prostate cancer.

[0030] Specifically, when the expression level of any one miRNA in the detected prostate cancer is significantly different from the expression level of the miRNA in the reference sample, the sample provider may have prostate cancer. In a preferred embodiment, when the expression level of any 10 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 prostate 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 prostate cancer.

[0031] In the present invention, the reference sample is a urine sample from a healthy person.

[0032] On the other hand, the present invention provides a method for detecting prostate 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 a PCR primer combination for hsa-miR-375, hsa-miR-520d-5p, hsa-miR-199b-5p, hsa-miR-518e-5p, hsa-miR-31-3p, and hsa-miR-4306 detection.

[0033] 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.

[0034] 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.

[0035] The applicant has found in the research that any one of the miRNA markers can be used to diagnose prostate cancer, and any combination of 2 to 5 miRNA markers can also be used to diagnose prostate cancer. Using the above markers, prostate cancer can be reliably identified.

[0036] The present invention discloses a single diagnostic marker of hsa-miR-375; and a combination of diagnostic markers for prostate cancer including hsa-miR-375, hsa-miR-520d-5p, hsa-miR-199b-5p, hsa-miR-518e-5p, hsa-miR-31-3p and hsa-miR-4306, wherein hsa-miR-375, hsa-miR-520d-5p, hsa-miR-199b-5p, hsa-miR-518e-5p, hsa-miR-31-3p and hsa-miR-4306 are composed of multiple nucleic acid molecules, each of which encodes at least one miRNA sequence. The specific sequence information is shown in Table 1 below.

[0037] All miRNA sequences disclosed in the present invention have been stored in the miRBase database ( http: / / www.mirbase.org / ).

[0038] Table 1

[0039]

[0040]

[0041] The present invention discloses two prostate cancer diagnostic markers, including a single hsa-miR-375 nucleic acid molecule; and multiple nucleic acid molecules of hsa-miR-375, hsa-miR-520d-5p, hsa-miR-199b-5p, hsa-miR-518e-5p, hsa-miR-31-3p and hsa-miR-4306. 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-375, hsa-miR-520d-5p, hsa-miR-199b-5p, hsa-miR-518e-5p, hsa-miR-31-3p and hsa-miR-4306 in the urine of patients diagnosed with prostate cancer is significantly changed compared with the expression in the control. In the optimal embodiment for prostate cancer diagnosis, the multiple nucleic acid molecules include a combination of six nucleic acid molecules: hsa-miR-375, hsa-miR-520d-5p, hsa-miR-199b-5p, hsa-miR-518e-5p, hsa-miR-31-3p and hsa-miR-4306.

[0042] Example 1: Validation cohort validation of 10 miRNA combinations for prostate cancer diagnosis

[0043] 1. Requirements, collection and extraction of urine samples for the prostate cancer diagnostic marker panel development cohort

[0044] Two groups of samples were used in the study of prostate cancer diagnostic markers, namely the case group and the control group. The case group contained 83 prostate cancer samples and the control group contained 88 healthy human urine samples. Biomarkers and biomarker combinations for detecting early prostate cancer were discovered and verified. Both prostate cancer cases and healthy samples in the research and development cohort came from the Second Affiliated Hospital of Zhejiang University School of Medicine.

[0045] 2. Reverse transcription-real-time fluorescence PCR operation process and results

[0046] 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. After reverse transcription, qPCR amplification was performed for quality inspection.

[0047] 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 prostate detection. After correction, it was found that the p-value of 10 miRNAs was less than 0.05, of which 5 were upregulated and 5 were downregulated in prostate cancer subjects. These 10 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.

[0048] 3. Validation cohort validation of the above 10 miRNAs

[0049] The case-control cohort used the leave-one-out cross-validation method to detect these 10 urinary miRNA biomarkers. The validation cohort sample consisted of 171 samples, and the diagnostic efficacy AUC = 0.738, such as Figure 2 -A.

[0050] Example 2: Validation of the optimal 6 miRNA combinations for prostate cancer diagnosis by validation cohort The requirements for urine samples of the research and development cohort, collection and extraction, reverse transcription-real-time fluorescence PCR operation process and results are the same as those described in Example 1. Based on the 10 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) of hsa-miR-375, hsa-miR-520d-5p, hsa-miR-199b-5p, hsa-miR-518e-5p, hsa-miR-31-3p and hsa-miR-4306. The validation cohort samples consisted of 171 samples, and the diagnostic efficacy of the 6 miRNA combination in the validation cohort was AUC=0.75, 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.

[0051] Based on the above comparison, a combination of 6 miRNAs was confirmed as the optimal combination for diagnosing prostate cancer.

[0052] The present invention establishes a complete workflow for discovering and validating urine miRNA biomarker combinations, and successfully determines biomarkers and biomarker combinations for detecting prostate cancer.

[0053] 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-375, hsa-miR-520d-5p, hsa-miR-199b-5p, hsa-miR-518e-5p, hsa-miR-31-3p and hsa-miR-4306 in the urine of patients diagnosed with prostate cancer is significantly changed compared to the expression in the control group.

[0054] In the best embodiment, the multiple nucleic acid molecules include a combination of six nucleic acid molecules: hsa-miR-375, hsa-miR-520d-5p, hsa-miR-199b-5p, hsa-miR-518e-5p, hsa-miR-31-3p and hsa-miR-4306.

[0055] 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 prostate cancer diagnosis <130> 2021.12 <160> 10 <170> PatentIn version 3.3 <210> 1 <211> twenty three <212> RNA <213> Artificial sequence <400> 1 gcgacgagcc ccucgcacaa acc 23 <210> 2 <211> 20 <212> RNA <213> Artificial sequence <400> 2 cuacaaaggg aagcccuuuc 20 <210> 3 <211> twenty three <212> RNA <213> Artificial sequence <400> 3 cccaguguuu agacuaucug uuc 23 <210> 4 <211> twenty two <212> RNA <213> Artificial sequence <400> 4 cucuagaggg aagcgcuuuc ug 22 <210> 5 <211> twenty two <212> RNA <213> Artificial sequence <400> 5 ugcuaugcca acauauugcc au 22 <210> 6 <211> 17 <212> RNA <213> Artificial sequence <400> 6 uggagagaaa ggcagua 17 <210> 7 <211> twenty two <212> RNA <213> Artificial sequence <400> 7 uauggcacug guagaauuca cu 22 <210> 8 <211> twenty three <212> RNA <213> Artificial sequence <400> 8 ucuuugguua ucuagcugua uga 23 <210> 9 <211> twenty two <212> RNA <213> Artificial sequence <400> 9 uccagcauca gugauuuugu ug 22 <210> 10 <211> twenty two <212> RNA <213> Artificial sequence <400> 10 aacaauaucc uggugcugag ug 22

Claims

1. Use of urine miRNA markers and / or detection reagents thereof in the preparation of a prostate cancer diagnosis kit, characterized in that: The urine miRNA markers are a combination of hsa-miR-375, hsa-miR-520d-5p, hsa-miR-199b-5p, hsa-miR-518e-5p, hsa-miR-31-3p and hsa-miR-4306.

2. A prostate cancer diagnostic reagent, characterized in that: The prostate cancer diagnostic reagent comprises corresponding primers for detecting the urine miRNA marker according to claim 1.

3. A kit for diagnosing prostate cancer, characterized in that: The prostate 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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