WO2023105295A2

WO2023105295A2 - Urine mirna marker for renal cancer diagnosis, diagnostic reagent and kit

Info

Publication number: WO2023105295A2
Application number: PCT/IB2022/020080
Authority: WO
Inventors: 陈阳; 蒋贝尔; 郭辉; 杜晴晴; 张盼; 张楠; 程赫; 邹瑞阳; 刘昊
Original assignee: 觅瑞（杭州）生物科技有限公司; 觅瑞实验室私人有限公司
Priority date: 2021-12-09
Filing date: 2022-12-09
Publication date: 2023-06-15
Also published as: WO2023105295A3; CN114182021A

Abstract

Disclosed in the present invention are a urine miRNA marker for renal cancer diagnosis, a kit and a diagnostic reagent. The urine miRNA marker is selected from one or more of the following miRNAs: hsa-miR-671-3p, hsa-miR-518e-5p, hsa-miR-153-3p, hsa-miR-21-3p, hsa-miR-331-5p, hsa-miR-885-5p, hsa-miR-4306, hsa-miR-I-3p. Provided in the present invention are urine miRNA markers of various combinations. The test operation is simple and convenient, the feasibility is high, the sensitivity and the specificity are high, and there is significance in clinical diagnosis and guidance. In addition, by using the present solution, the miRNA expression quantity can be detected using urine, such that the non-invasive purpose is achieved.

Description

Description Urinary miRNA markers, diagnostic reagents and kits for kidney cancer diagnosis Technical Field The present invention relates to the technical field of molecular diagnosis, in particular to a urinary miRNA markers, diagnostic reagents and kits for kidney cancer diagnosis. Background Art Renal cell carcinoma (RCC, referred to as renal cell carcinoma) is a malignant tumor originating from renal tubular epithelium, accounting for 80-90% of renal malignant tumors - the incidence of renal cell carcinoma is second only to prostate cancer and bladder cancer, accounting for Urinary system tumors ranked third. The most common histopathological type of RCC is clear cell carcinoma, which accounts for 75-80% of all RCC, and about 90,000 deaths worldwide each year. The incidence of RCC has gradually increased over the past decades. Although RCC's mortality rate remains high, it is a salvageable disease until metastases occur. Clinically, it has been shown that RCC is often resistant to chemotherapy and radiotherapy, and tumor resection remains the only definitive treatment. After curative nephrectomy, nearly 55% of RCC patients survive. However, detection of cancer cells in early stages is challenging due to the lack of early symptoms, inability to correctly differentiate between benign and malignant masses by imaging or needle biopsy, and therefore, the development of innovative methods for early RCC detection is required. non-invasive method. Recently, many molecular markers such as carbonic anhydrase IX (CAIX), vascular endothelial growth factor (vascular endothelial growth factor, VEGF), hypoxia-inducible factor (hypoxia-inducible factor, HIF), KI67, P53, P2L PTEN (phosphatase and tensin homolog), E-Cadherin, osteopontin and CD44, CXCR4 and other cell cycle and proliferation markers. None of these potential RCC biomarkers has significantly improved the predictive accuracy of current prognostic systems, most have not been validated on a large scale, and the use of the aforementioned markers is not recommended in routine clinical practice. So far, no highly effective biomarkers for early diagnosis of RCC have been identified, resulting in poor late detection and treatment of the disease. Therefore, identification of RCC biomarkers that can improve early diagnosis and prognosis of RCC patients is an important focus of cancer therapy. miRNA is a short non-coding RNA with a length of 18-25 nucleotides, which can prevent protein expression by decomposing specific target mRNA or inhibiting its translation, and participate in the regulation of individual development, cell apoptosis, proliferation and differentiation and other life activities. In the occurrence and development of tumors, they play a role similar to oncogenes or tumor suppressor genes. The expression profile of miRNA has obvious tissue specificity, and has specific expression patterns in different tumors. These characteristics make miRNAs likely to become new biological markers and therapeutic targets for tumor diagnosis. Although many studies have evaluated miRNA expression in RCC patient tissue and serum, the urinary miRNA biomarkers and biomarker combination markers that can be used for kidney cancer screening have not yet been determined. For example: Patent US10457994B2 discloses a A combination of miRNA markers that can predict the metastasis and prognosis of clear cell renal cell carcinoma, miR-10b, miR-139-5p, miR-130b and miR-199b-5p; miR-199b-5p and miR-130b are in the metastasis miR-10b and miR-139-5p were downregulated in metastatic RCC. The literature microRNA-210-3p depletion by CRISPR/Cas9 promoted tumorigenesis through revival of TWIST 1 in renal cell carcinoma ( Hirofumi Yoshino, et. Oncotarget, 2017,8(13):20881-20894.) discloses that in clear cell renal cell carcinoma The five miRNA markers that were up-regulated in the CD were: miR-885-5p> miR-1274, miR-210-3p, miR-224 and miR-1290. In summary, in view of the fact that there is no clear conclusion in the prior art, which single miRNA marker or miRNA marker Combination of biomarkers can be used to accurately and effectively predict or diagnose renal cell carcinoma, and the sample collection route of miRNA markers is from urine, plasma, or tissue. There are different methods and no conclusions; therefore, those skilled in the art urgently need A urine miRNA marker combination suitable for the diagnosis of kidney cancer and having high population specificity was developed. SUMMARY OF THE INVENTION The technical problem to be solved by the present invention is to provide a urinary miRNA marker, diagnostic reagent and kit for kidney cancer diagnosis, which has higher population specificity than other miRNA markers reported internationally; The disclosed miRNA diagnostic markers and their combinations are proposed for the first time, which are more reliable than other miRNA molecular markers, and provide a new alternative way for the diagnosis of kidney cancer. In order to solve the above technical problems, the technical solution provided by the present invention is as follows: The present invention provides a urine miRNA marker for kidney cancer diagnosis, which is selected from any one or more of the following 8 miRNAs: hsa-miR-671 -3p, hsa-miR-518e-5p, hsa-miR-153-3p, hsa-miR-21-3p, hsa-miR-331-5p, hsa-miR-885-5p>hsa-miR-4306>hsa -miR-1-3p _o In some embodiments, the urine miRNA marker can be selected from any one or more of the following 7 miRNAs: hsa-miR-671-3p^hsa-miR-518e- 5p^hsa-miR-153-3p>hsa-miR-21-3p^hsa-miR-331-5p^hsa-miR-885-5p>hsa-miR-1-3p _o In some embodiments, the The urine miRNA markers can be selected from any one or more of the following 7 miRNAs: hsa-miR-671-3px hsa-miR-518e-5p^ hsa-miR-153-3p> hsa-miR-21- 3p^hsa-miR-331-5p^hsa-miR-885-5p>hsa-miR-4306o In some embodiments, the urine miRNA marker can be selected from any one or more of the following 6 miRNAs One: hsa-miR-671-3p, hsa-miR-518e-5p > hsa-miR-153-3p, hsa-miR-21-3p^ hsa-miR-331-5p > hsa-miR-885-5p ₀ In the present invention, the above is selected from hsa-miR-671-3p>hsa-miR-518e-5p hsa-miR-153-3p>hsa-miR-21-3p>hsa-miR-331-5p>hsa-miR- At least 2 of the 6 miRNAs 885-5p constitute urine miRNA markers whose AUC value is higher than that of a single miRNA (such as hsa-miR-518e-5p or hsa-miR13p), including but not limited to the following The combination can be used as a preferred embodiment:

1) Preferably, the urine miRNA marker can be selected from at least 2 of the following 5 miRNAs: hsa-miR-671-3p>hsa-miR-518e-5p, hsa-miR-153-3p>hsa-miR -21-3p> hsa-miR-331-5p _o hsa-miR-671-3p> hsa-miR-518e-5p, hsa-miR-153-3p^ hsa-miR-21-3p^ hsa-miR-885 -5p ₀ hsa-miR-671-3p>hsa-miR-518e-5p, hsa-miR-153-3p>hsa-miR-331-5p>hsa-miR-885-5p _o hsa-miR-671-3p > hsa-miR-518e-5p, hsa-miR-21-3p > hsa-miR-331-5p > hsa-miR-885-5p ₀ hsa-miR-671-3p > hsa-miR-153-3p > hsa -miR-21-3p>hsa-miR-331-5p, hsa-miR-885-5p ₀ hsa-miR-518e-5p>hsa-miR-153-3p>hsa-miR-21-3p>hsa-miR -331-5p> hsa-miR-885-5p ₀

2) Preferably, the urine miRNA marker can be selected from at least 2 of the following 4 miRNAs: hsa-miR-671-3p>hsa-miR-518e-5p, hsa-hsa-miR-21-3p _o hsa -miR-671-3p>hsa-miR-518e-5p, hsa-hsa-miR-331-5p _o hsa-miR-671-3p>hsa-miR-518e-5p, hsa-hsa-miR-885-5p _o hsa-miR-671-3p>hsa-miR-518e-5p, hsa-hsa-miR-331-5p _o hsa-miR-671-3p>hsa-miR-518e-5p, hsa-

hsa-miR-885-5p o Instructions hsa-miR-671-3p > hsa-miR-518e-5p > hsa-miR-331-5p > hsa-miR-885-5p _o hsa-miR-671-3p > hsa-miR-153-3p > hsa -miR-21-3p > hsa-miR-331-5p _o hsa-miR-671-3p > hsa-miR-153-3p > hsa-miR-21-3p > hsa-miR-885-5p ₀ hsa-miR -671-3p>hsa-miR-153-3p>hsa-miR-331-5p>hsa-miR-885-5p _o hsa-miR-671-3p>hsa-miR-21-3p>hsa-miR-331 -5p> hsa-miR-885-5p ₀ hsa-miR-518e-5p, hsa-miR-153-3p>hsa-miR-21-3p> hsa-miR-331-5p _o hsa-miR-518e-5p , hsa-miR-153-3p > hsa-miR-21-3p > hsa-miR-885-5p _o hsa-miR-518e-5p, hsa-miR-153-3p > hsa-miR-331-5p > hsa -miR-885-5p _o hsa-miR-518e-5p, hsa-miR-21-3p > hsa-miR-331-5p > hsa-miR-885-5p _o hsa-miR-153-3p > hsa-miR -21-3p> hsa-miR-331-5p^ hsa-miR-885-5p _o

3) Preferably, the urine miRNA marker can be selected from at least 2 of the following 3 miRNAs: hsa-miR-671-3p> hsa-miR-518e-5p, hsa-miR-153-3p _o hsa-miR -671-3p> hsa-miR-518e-5p, hsa-miR-21-3p _o hsa-miR-671-3p> hsa-miR-518e-5p, hsa-miR-331-5p _o hsa-miR-671 -3p> hsa-miR-518e-5p, hsa-miR-885-5p _o hsa-miR-671-3p>hsa-miR-153-3p> hsa-miR-21-3p _o hsa-miR-671-3p >hsa-miR-153-3p> hsa-miR-331-5p _o hsa-miR-671-3p>hsa-miR-153-3p> hsa-miR-885-5p _o hsa-miR-671-3p> hsa -miR-21-3p > hsa-miR-331-5p _o hsa-miR-671-3p > hsa-miR-21-3p > hsa-miR-885-5p _o hsa-miR-518e-5p, hsa-miR -153-3p>hsa-miR-21-3p _o hsa-miR-518e-5p, hsa-miR-153-3p>hsa-miR-331-5p _o hsa-miR-518e-5p, hsa-miR-153 -3p>hsa-miR-885-5p _o hsa-miR-518e-5p, hsa-miR-21-3p>hsa-miR-331-5p _o hsa-miR-518e-5p, hsa-miR-21-3p ^ hsa-miR-885-5p _o hsa-miR-153-3p > hsa-miR-21-3p > hsa-miR-331-5p _o hsa-miR-153-3p > hsa-miR-21-3p > hsa -miR-885-5p _o hsa-miR-518e-5p > hsa-miR-331-5p > hsa-miR-885-5p _o hsa-miR-153-3p > hsa-miR-331-5p > hsa-miR -885-5p _o hsa-miR-21-3p > hsa-miR-331-5p > hsa-miR-885-5p _o

4) Preferably, the urine miRNA marker can be selected from the following two miRNAs: hsa-miR-671-3p>hsa-miR-518e-5p. hsa-miR-671-3p> hsa-miR-153-3p _o hsa-miR-671-3p> hsa-miR-21-3p _o hsa-miR-671-3p> hsa-miR-885-5p _o hsa- miR-518e-5p > hsa-miR-21-3p _o hsa-miR-518e-5p > hsa-miR-331-5p ₀ hsa-miR-518e-5p > hsa-miR-885-5p ₀ hsa-miR- 153-3p> hsa-miR-21-3p _o Instructions hsa-miR-153-3p> hsa-miR-331-5p _o hsa-miR-21-3p> hsa-miR-331-5p _o hsa-miR-21-3p> hsa-miR-885-5p _o

5) Preferably, the urine miRNA marker can be selected from the following 1 miRNA: hsa-miR hsa-miR in some

The characteristics are: when the urine miRNA marker can be selected from hsa-miR-671-3p>hsa-miR-518e-5p>hsa-miR-153-3p>hsa-miR-21-3p>hsa-miR-331- 5p> hsa-miR-885-5p^ hsa-miR-4306^ hsa-miR-1-3p When at least 2 of these 8 miRNAs, or when the urine miRNA marker can be selected from hsa-miR-671- 3p^ hsa-miR-518e-5p hsa-miR-153-3p, hsa-miR-21-3p > hsa-miR-331-5p > hsa-miR-885-5p > hsa-miR-4306 these 7 miRNAs When at least 2 of them, or when the urine miRNA markers can be selected from hsa-miR-671-3p^ hsa-miR-518e-5p hsa-miR-153-3p^ hsa-miR-21-3p> hsa- miR-331-5p>hsa-miR-885-5p>hsa-miR-1-3p at least 2 of these 7 miRNAs, or when the urine miRNA markers can be selected from hsa-miR-671-3p^ hsa-miR-518e-5p hsa-miR-153-3p^hsa-miR-21-3p>hsa-miR-331-5p>hsa-miR-885-5p At least 2 of these 6 miRNAs, wherein One miRNA must be hsa-miR-518e-5p □ In some preferred embodiments, the present invention also provides a urine miRNA marker containing a specific miRNA, which is characterized in that: When the urine miRNA marker can be selected from hsa-miR-671-3p^ hsa-miR-518e-5p hsa-miR-153-3p, hsa-miR-21-3p>hsa-miR-331-5p>hsa-miR-885-5p^hsa-miR -4306>hsa-miR-1-3p when at least 2 of these 8 miRNAs, or when the urine miRNA markers can be selected from hsa-miR-671-3p^hsa-miR-518e-5p hsa-miR- 153-3p^ hsa-miR-21-3p>hsa-miR-331-5p>hsa-miR-885-5p> hsa-miR-l-3p at least 2 of these 7 miRNAs, or one of them Each miRNA must be hsa-miR- _1-3p . In the present invention, the above is selected from hsa-miR-671-3p>hsa-miR-518e-5p hsa-miR-153-3p>hsa-miR-21-3p>hsa-miR-331-5p>hsa-miR-885-5p> hsa-miR-4306, hsa-miR-l-3p at least 2 of the 8 miRNAs, and contain specific miRNAs (such as hsa-miR-518e -5p or hsa-miR-1-3p) _? Urinary miRNA markers, whose AUC value is not less than 0.681, including but not limited to the following combinations, can be used as a preferred embodiment:

1) Preferably, the urine miRNA marker can be selected from at least 2 of the following 8 miRNAs: hsa-miR-671-3p>hsa-miR-518e-5p hsa-miR-153-3p>hsa-miR- 21-3p^hsa-miR-331-5p>hsa-miR-885-5p>hsa-miR-4306^hsa-miR-l- _3p0

2) Preferably, the urine miRNA marker can be selected from at least 2 of the following 7 miRNAs: hsa-miR-671-3p>hsa-miR-518e-5p hsa-miR-153-3p^hsa-miR- 21-3p^hsa-miR-331-5p^hsa-miR-885-5p>hsa-miR-4306; Instructions hsa-miR-671-3p>hsa-miR-518e-5p hsa-miR-153-3p, hsa-miR-21-3p^hsa-miR-331-5p, hsa-miR-885-5p>hsa- miR-1-3p; hsa-miR-518e-5p hsa-miR-153-3p, hsa-miR-21-3p, hsa-miR-331-5p^hsa-miR-885-5p^hsa-miR-4306 , hsa-miR-l-3p;

3) Preferably, the urine miRNA marker can be selected from at least 2 of the following 6 miRNAs: hsa-miR-671-3p>hsa-miR-518e-5p, hsa-miR-153-3p^hsa-miR -21-3p^hsa-miR-331-5p, hsa-miR-4306; hsa-miR-671-3p, hsa-miR-518e-5p, hsa-miR-153-3p^hsa-miR-21-3p >hsa-miR-331-5p^hsa-miR-l-3p; hsa-miR-671-3p>hsa-miR-518e-5p, hsa-miR-153-3p^hsa-miR-21-3p^hsa -miR-331-5p hsa-miR-885-5p; hsa-miR-671-3p, hsa-miR-518e-5p, hsa-miR-21-3p, hsa-miR-331-5p, hsa-miR -4306, hsa-miR-l-3p; hsa-miR-671-3p> hsa-miR-518e-5p, hsa-miR-21-3p, hsa-miR-331-5p, hsa-miR-885-5p , hsa-miR-4306; hsa-miR-671-3p> hsa-miR-518e-5p, hsa-miR-21-3p, hsa-miR-331-5p, hsa-miR-885-5p, hsa-miR -l-3p; hsa-miR-518e-5p, hsa-miR-153-3p, hsa-miR-21-3p, hsa-miR-331-5p, hsa-miR-4306, hsa-miR-l-3p ; hsa-miR-518e-5p, hsa-miR-153-3p > hsa-miR-21-3p, hsa-miR-331-5p, hsa-miR-885-5p, hsa-miR-4306; hsa-miR -518e-5p, hsa-miR-153-3p, hsa-miR-21-3p, hsa-miR-885-5p, hsa-miR-4306, hsa-miR-l-3p; hsa-miR-518e-5p , hsa-miR-153-3p, hsa-miR-21-3p, hsa-miR-331-5p, hsa-miR-885-5p, hsa-miR-l-3p; hsa-miR-518e-5p, hsa - miR-21-3p, hsa-miR-331-5p, hsa-miR-885-5p, hsa-miR-4306, hsa-miR-1-3p;

4) Preferably, the urine miRNA marker can be selected from at least 2 of the following 5 miRNAs: hsa-miR-671-3p>hsa-miR-518e-5p, hsa-miR-153-3p>hsa-miR -21-3p>hsa-miR-331-5p;hsa-miR-671-3p>hsa-miR-518e-5p,hsa-miR-153-3p>hsa-miR-21-3p>hsa-miR-885-5p;hsa-miR-671-3p>hsa-miR-518e-5p,hsa-miR-21-3p>hsa-miR-331-5p>hsa-miR-4306;hsa-miR-671-3p>hsa -miR-518e-5p, hsa-miR-21-3p>hsa-miR-331-5p^hsa-miR-l-3p;hsa-miR-671-3p>hsa-miR-518e-5p, hsa-miR -21-3p^hsa-miR-331-5p^hsa-miR-885-5p; hsa-miR-518e-5p, hsa-miR-153-3p>hsa-miR-21-3p>hsa-miR-331 -5p^hsa-miR-4306; hsa-miR-518e-5p, hsa-miR-153-3p>hsa-miR-21-3p>hsa-miR-331-5p>hsa-miR-l-3p; hsa -miR-518e-5p, hsa-miR-153-3p> hsa-miR-21-3p^ hsa-miR-331-5p^ hsa-miR-885-5p; hsa-miR-518e-5p, hsa-miR -21-3p>hsa-miR-331-5p> hsa-miR-4306 hsa-miR-l-3p; hsa-miR-518e-5p, hsa-miR-21-3p>hsa-miR-331-5p>hsa-miR-885-5p>hsa-miR-4306; hsa-miR-518e-5p, hsa-miR-21-3p>hsa-miR-331-5p>hsa-miR-885-5p>hsa-miR- l-3p; manual

5) Preferably, the urine miRNA marker can be selected from at least 2 of the following 4 miRNAs: hsa-miR-671-3p>hsa-miR-518e-5p>hsa-miR-21-3p>hsa-miR -331-5p; hsa-miR-518e-5p, hsa-miR-153-3p>hsa-miR-21-3p>hsa-miR-331-5p; hsa-miR-518e-5p, hsa-miR-153 -3p>hsa-miR-21-3p>hsa-miR-885-5p; hsa-miR-518e-5p, hsa-miR-21-3p>hsa-miR-331-5p>hsa-miR-4306; hsa -miR-518e-5p, hsa-miR-21-3p>hsa-miR-331-5p>hsa-miR-l-3p; hsa-miR-518e-5p, hsa-miR-21-3p>hsa-miR -331-5p>hsa-miR-885-5p;

6) Preferably, the urine miRNA marker can be selected from at least 2 of the following 3 miRNAs: hsa-miR-518e-5p > hsa-miR-21-3p >hsa-miR-331-5p; the following types Urine miRNA markers consisting of 3 to 8 miRNAs with an AUC value of 30.681 have a common feature that they all include at least hsa-miR-518e-5p, hsa-miR-21-3p, and hsa-miR-331-5p: In some preferred embodiments, the urine miRNA marker may be composed of the following 8 miRNAs: hsa-miR-671-3p, hsa-miR-518e-5p, hsa-miR-153-3p, hsa- miR-21-3p, hsa-miR-331-5p, hsa-miR-885-5p > hsa-miR-4306 > hsa-miR- _1-3p. In some preferred embodiments, the urine miRNA The marker can be composed of the following seven miRNAs: hsa-miR-671-3p, hsa-miR-518e-5p, hsa-miR-153-3p, hsa-miR-21-3p, hsa-miR-331-5p, hsa-miR-885-5p>hsa-miR-4306 o In some preferred embodiments, the urine miRNA marker may be composed of the following seven miRNAs: hsa-miR-671-3p, hsa-miR- 518e-5p, hsa-miR-153-3p, hsa-miR-21-3p, hsa-miR-331-5p, hsa-miR-885-5p>hsa-miR-1-3p _o In some preferred embodiments Among them, the urine miRNA marker can be composed of the following 6 miRNAs: hsa-miR-671-3p, hsa-miR-518e-5p, hsa-miR-153-3p, hsa-miR-21-3p, hsa-miR-331-5p, hsa-miR-885-5p _o In some more preferred embodiments, the urine miRNA markers may be composed of the following six miRNAs: hsa-miR-671-3p>hsa -miR-518e-5p > hsa-miR-153-3p^ hsa-miR-21-3p^ hsa-miR-331-5p ₀ In some more preferred embodiments, the urine miRNA marker can be obtained by The following 4 miRNAs consist of: hsa-miR-518e-5p>hsa-miR-21-3p>hsa-miR-331-5p>hsa-miR- _1-3p . In some more preferred embodiments, the Urine miRNA markers may consist of the following 4 miRNAs: hsa-miR-518e-5p > hsa-miR-153-3p > hsa-miR-21-3p > hsa-miR-331-5p _o In some more preferred In an embodiment, the urine miRNA marker may be composed of the following three miRNAs: hsa-miR-518e-5p > hsa-miR-21-3p > hsa-miR-331-5p _o In addition, the present invention also provides A diagnostic reagent for kidney cancer is provided, including a reagent for specifically detecting one or more urinary miRNA markers as described above. In addition, the present invention also provides a diagnostic kit for kidney cancer, including using one or more urinary miRNA markers as described above as a standard, and detecting various urinary miRNA markers as described above corresponding primers. In addition, the present invention also provides an application of various urinary miRNA markers as mentioned above, mainly for the preparation of products for early diagnosis and/or recurrence monitoring. The products include but are not limited to devices (such as oligonucleotide probes or their integration, chip substrates or high-throughput miRNA detection chips on detection substrates, and microfluidic detection chips), kits and equipment. Instructions In some preferred embodiments, the prepared product for early diagnosis and/or recurrence monitoring includes one or more of the following functions:

1) Identification or auxiliary identification of renal cell carcinoma;

2) Diagnosis or auxiliary diagnosis of primary renal cancer;

3) Diagnosis or auxiliary diagnosis of recurrent renal cell carcinoma. In addition, the present invention also provides a method for early diagnosis and/or recurrence monitoring of kidney cancer using one or more urine miRNA markers as described above, including the following steps: Step 1, detecting the the presence of miRNA in the urine sample; step 2, measuring the expression level of at least one miRNA in the miRNA markers in the urine sample; step 3, using the score based on the previously measured miRNA expression level to compare with the reference, by comparing Whether there is a significant difference between the subject sample and the reference sample is used for early diagnosis and/or recurrence monitoring of the possibility of the subject suffering from kidney cancer. Also provided herein is a method of determining whether a subject has kidney cancer or determining the risk of kidney cancer in a subject, the method comprising detecting at least one miRNA in a urine sample obtained from the subject The expression level of wherein at least one miRNA is selected from hsa-miR-671-3p, hsa-miR-518e-5p, hsa-miR-153-3p, hsa-miR-21-3p, hsa-miR-331-5p , hsa-miR-885-5p, hsa-miR-4306, hsa-miR-l-3p; and wherein compared with the control, the differential expression level of miRNA indicates that the subject has kidney cancer or is at risk of suffering from kidney cancer . In some embodiments, as described in the method, the at least one miRNA comprises a combination of urinary miRNA markers as previously described. In some embodiments, wherein the expression level of the miRNA is up-regulated or down-regulated in a subject with or at risk of developing kidney cancer compared to a control. In some embodiments, the control is selected from subjects without cancer, subjects without kidney cancer, subjects without kidney cancer or subjects without risk of kidney cancer. In some embodiments, the methods provided herein also include performing clinical procedures and/or measuring additional biomarkers in biological samples (ie, urine, tissue blood, plasma, serum samples). Other biomarkers may include but are not limited to carbonic anhydrase IX (CAIX), vascular endothelial growth factor (vascular endothelial growth factor, VEGF), hypoxia-inducible factor (hypoxia-inducible factor, HIF), KI67, P53, P21 , PTEN (phosphatase and tensin homolog, phosphatase and thionin homolog), E-Cadherin, osteopontin and CD44, CXCR4 and other cell cycle and proliferation markers. Clinical procedures may include biopsy, fine needle aspiration, and diagnostic imaging tests including, but not limited to, ultrasound, computed tomography (CT), magnetic resonance imaging (MRI), or positron emission tomography (PET) imaging. In some embodiments, the present disclosure provides methods of treating a subject determined to have kidney cancer using the methods disclosed herein. Subjects determined to have kidney cancer will be referred to a licensed physician and, where determined appropriate, treated with an appropriate anticancer compound, surgery, targeted therapy, immunotherapy, or radiation therapy, or a combination of these treat. Compared with the prior art, the beneficial effects of the present invention are as follows:

1. The present invention discovers for the first time multiple miRNAs that can detect kidney cancer, such as hsa-miR-518e-5p or hsa-miR-1-3p, which have not been reported in existing literature or patents and can be used to detect kidney cancer.

2. The present invention initially finds 16 miRNA markers that can be used to detect kidney cancer from the urine samples of cancer and healthy control subjects. After further research, it is creatively discovered: When the urine miRNA markers are selected from the following 8 When one or more of these miRNAs were detected, kidney cancer could be significantly differently detected (p<0.05): hsa-miR-671-3p>hsa-miR-518e-5p>hsa-miR-153-3p>hsa- miR-21-3pAhsa-miR-331-5p^hsa-miR-885-5p>hsa-miR-4306>hsa-miR-l-3po

3. The present invention also unexpectedly finds that: as long as it is selected from hsa-miR-671-3p>hsa-miR-518e-5p>hsa-miR-153-3p^ Instructions hsa-miR-21-3p, hsa-miR-331-5p, hsa-miR-885-5p at least 2 of the 6 miRNAs, the urinary miRNA markers, the AUC value will be higher than The AUC value of a single miRNA (such as hsa-miR-518e-5p, etc.); however, when the urine miRNA marker is composed of hsa-miR-518e-5p and hsa-miR-153-3p, its AUC value is 0.561, instead Lower than the AUC value of single hsa-miR-518e-5p.

4. Among 8 miRNAs (hsa-miR-671-3p, hsa-miR-518e-5p > hsa-miR-153-3p, hsa-miR-21-3p > hsa-miR-331-5p > hsa- miR-885-5p>hsa-miR-4306 and hsa-miR-l-3p) based on the research of urine miRNA marker AUC=0.681, the present invention accidentally found a urine miRNA marker with AUCN0.681 through optimization and screening Liquid miRNA markers, the common features of which are: the urine miRNA markers are selected from hsa-miR-671-3p, hsa-miR-518e-5p, hsa-miR-153-3p>hsa-miR-21-3p >hsa-miR-331-5p>hsa-miR-885-5p> hsa-miR-4306, hsa-miR-l-3p at least 2 of these 8 miRNAs, and must contain 1 specific miRNA (hsa- miR-518e-5p or hsa-miR-l-3p), especially hsa-miR-518e-5p plays an up-regulation role. Therefore, the present invention tries to hsa-miR-671-3p, hsa-miR-518e-5p>hsa-miR-153-3p, hsa-miR-21-3p, hsa-miR-331-5p>hsa-miR- 885-5p is used as a new combination of kidney cancer markers, and the collection and verification of clinical samples are carried out. The results show that the combination of markers has important value in the early detection and diagnosis of kidney cancer. In conclusion, the present invention provides various combinations of urinary miRNA markers, which are easy to operate, highly feasible, high in sensitivity and specificity, and have clinical diagnostic and guiding significance; moreover, the present invention can detect miRNA expression through urine , so as to achieve the purpose of non-invasive. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a heat map of the expression levels of 8 (P<0.05) differentially expressed renal cancer miRNA diagnostic markers. miRNA expression levels (copy number/ml) are presented on a log2 scale and normalized to the zero mean. The color of the dots indicates the concentration. Hierarchical clustering was performed for two dimensions (miRNA and sample) based on Euclidean distance. For the horizontal dimension, color is used to represent case-control subjects. Figure 2 is the AUC chart of renal cancer miRNA diagnostic marker combinations in each cohort; among them, Figure 2-A is the AUC chart of 8 renal cancer miRNA diagnostic marker combinations in each cohort, and Figure 2-B is 6 renal cancer miRNAs AUC plots of diagnostic marker combinations in each cohort. Figure 3 is a line box plot of the control and cancer predictive values of the six kidney cancer miRNA diagnostic marker combinations. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The technical solution of the present invention will be further specifically described through specific examples below. In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or commonly used in the field. The methods in the following examples, unless otherwise specified, are conventional methods in the art. Definitions of Terms The term "miRNA" refers to microRNAs, ie small non-coding RNA molecules, which contain about 22 nucleotides in some embodiments, and exist in plants, animals and some viruses. miRNAs are known to have functions in RNA silencing and post-transcriptional regulation of gene expression. These highly conserved RNAs regulate gene expression by binding to the 3'-untranslated region (3'-UTR) of specific mRNAs. For example, each miRNA is thought to regulate multiple genes, and since hundreds of miRNA genes are predicted to exist in higher eukaryotes. miRNAs tend to be transcribed from several different loci in the genome. These genes encode long RNAs with hairpin structures, which are processed by a series of RNases manual

III enzymes (including Drosha and Dicer) form miRNA duplexes, typically about 22 nucleotides long, with a 2 nucleotide overhang on the 3' terminus when processed. The term "regulation" refers to the process by which a cell increases or decreases the amount of a cellular component such as RNA or protein in response to an external variable. An increase in a cellular component is referred to as upregulation, while a decrease in a cellular component is referred to as downregulation. The term "dysregulation" or "dysregulation" as used herein means up-regulation or down-regulation. An example of downregulation is a reduction in the number of receptors in a cell for a molecule such as a hormone or neurotransmitter, which reduces the sensitivity of the cell to that molecule. This phenomenon is an example of a locally acting negative feedback mechanism. An example of upregulation is the increased number of cytochrome P450 enzymes in hepatocytes when xenobiotic molecules such as dioxins are administered, causing greater degradation of these molecules. Upregulation and downregulation can also occur in response to toxins or hormones. An example of upregulation in pregnancy is a hormone that causes cells in the uterus to become more sensitive to oxytocin. The term "differential expression" refers to the measurement of a cellular component in comparison to a control or another sample, thereby determining a difference in, for example, concentration, presence or intensity of said cellular component. The results of such comparisons may be given in absolute values, ie, the component is present in the sample but not in the control, or in relative values, ie, the expression or concentration of the component is increased or decreased compared to the control. In this case, the term "increase" is synonymous and interchangeable with the term "up-regulation", and the term "decrease" is synonymous and interchangeable with the term "down-regulation". The term "miRNA combination" relates to combinations of miRNAs of the invention. The amount of miRNA can be determined in a sample from a subject by techniques well known in the art. Depending on the nature of the sample, the amount can be determined by PCR-based techniques for quantifying the amount of polynucleotides or by other methods such as mass spectrometry or sequencing, among others. The term "probe" refers to a single-stranded oligonucleotide commonly used to detect target RNA and/or RNA sequences complementary to the sequence of the probe. probe and nucleotide sequence single-stranded nucleic acid that allows nucleotide pairing due to complementarity between the probe and target sequence

(DNA or RNA) hybridization. The length of the probe depends on the intended use as well as the desired specificity of the probe. Usually, the length of the probe is 20 to 500 (ie 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180 , 190, 200, 300, 400, 500) nucleotides, preferably 20 to 100, more preferably 20 to 50 nucleotides. For the detection of microRNAs, the probes are 12 to 30 nucleotides. The probes are used in various experimental settings, such as but not limited to Southern and Northern blotting, real-time PCR and in situ hybridization (In Situ Hybridization, ISH), and microarray experiments. Probes may be unlabeled, directly labeled or indirectly labeled, for example with biotin to which the streptavidin complex can subsequently bind. The label may be a molecule detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical or other physical means. For example, suitable labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (eg, commonly used in ELISAs), biotin, digoxigenin, or haptens and others that are or can be made detectable. entity. Labels can be introduced anywhere in the nucleic acid, eg 3', 5' or internally. The term "probe" also covers nucleic acids whose backbone composition differs, such as, but not limited to, peptide nucleic acid (PNA), locked nucleic acid (LNA), diol nucleic acid (GNA) and threose nucleic acid (TNA). The terms "index" and ""Mark" is used interchangeably herein and refers to a sign or signal of a disorder or to monitor a disorder. Such "disorder" refers to the biological state of a cell, tissue or organ, or to the state of health and/or disease of an individual. An indicator may be the presence or absence of molecules including, but not limited to, peptides, proteins, and nucleic acids, or may be a change in the expression level or pattern of such molecules in a cell, or in a tissue, organ, or individual. An indicator may be the occurrence, development or presence of a disease in an individual or a sign of the further progression of such a disease. An indicator can also be a sign of the risk of developing a disease in an individual. The term "fraction" refers to an integer or number, which may be determined mathematically, for example, by using computational models known in the art (which may include, but not limited to, SMV as an example), and is determined using many numbers It is calculated by any one of bibliographic equations and/or algorithms to achieve the purpose of statistical classification. Such a score is used to enumerate an outcome over a range of possible outcomes. The relevance and statistical significance of such a score depends on the size and quality of the underlying data set used to establish the range of results. For example, a blind sample can be input into an algorithm, and a score can be calculated based on the information provided by analyzing the blind sample. This enables the scoring of the blind sample to be generated. Based on this score, decisions can be made, such as how likely it is that the patient generating the blind sample has cancer or does not have cancer. The end values of the ranges may be defined logically based on the data provided or arbitrarily defined at the request of the experimenter. In both cases, the range needs to be defined before testing blind samples. The term "expression level" refers to the amount of a gene product present in vivo or in a sample at a particular point in time. The expression level can be measured/quantified/detected eg by the protein or mRNA expressed by the gene. Expression levels can be quantified, for example, by using the total amount of the same type of gene product (total protein or mRNA) to normalize the amount of the gene product of interest present in the sample, or to determine the amount of the gene product of interest/defined sample size (weight, volume, etc.). Expression levels can be measured or detected by any method known in the art, such as methods for the direct detection and quantification of the gene product of interest (e.g., mass spectrometry), or typically by a combination of a gene product of interest and a gene specific for the gene product of interest. A method for the indirect detection and measurement of a gene product of interest that works in combination with one or more different molecular or detection devices (eg, primers, probes, antibodies, protein scaffolds). It is also known to the skilled person to determine the level of gene copies, which also includes determining the absence or presence of one or more fragments (for example, by nucleic acid probes or primers, such as quantitative PCR, multiplex ligation-dependent probe amplification PCR) o The detection method for kidney cancer includes the following steps: a) Determination of hsa-miR-671-3p, hsa-miR-518e-5p, hsa-miR-153-3p, hsa -miR-21-3p, hsa-miR-331-5p, hsa-miR-885-5p; b) compare hsa-miR-671-3p, hsa-miR-518e-5p, hsa-miR-518e-5p, The expression level of hsa-miR-153-3p, hsa-miR-21-3p, hsa-miR-331-5p, hsa-miR-885-5p; wherein, the expression level of any one of the above miRNAs in the detected sample A significant change compared to the expression level of the corresponding miRNA in the reference sample indicates that the individual may have kidney cancer. Specifically, when the expression level of any miRNA in the detected kidney cancer is significantly different from that in the reference sample, the sample provider may have kidney cancer. In a preferred embodiment, when the expression levels of any 8 miRNAs in the tested urine sample are significantly different from the expression levels of miRNAs in the reference sample, the sample provider may suffer from renal cancer. In a more preferred embodiment, when the expression levels of the six miRNAs in the tested urine sample are significantly different from those in the reference sample, the sample provider may suffer from kidney cancer. In the present invention, the reference samples come from healthy human urine samples. On the other hand, the present invention also provides a method for detecting microRNA in renal cancer. Specifically, the method includes reverse transcription and real-time quantitative polymerase chain reaction (RT-qPCR). More specifically, in the real-time fluorescent quantitative polymerase chain reaction, the primer combination used is hsa-miR-671-3p) hsa-miR-518e-5p) hsa-miR-153-3p, hsa-miR-21- 3p, hsa-miR-33 l-5p, hsa-miR-885-5p detection of reverse transcription primers and PCR primer combinations. Appropriate adjustments and modifications can be made to the primer sequences of the present invention according to the sequences of related miRNAs, and these modified primer sequences can still be used to detect the miRNA markers. The present invention also includes these equivalent technical solutions. Compared with chips based on hundreds of molecules, the present invention uses RT-qPCR method, which has more reliable clinical feasibility and The instruction manual is practical, and the cost is low, and it is easier to popularize. In addition, the above-mentioned miRNA detection method is not limited to the RT-qPCR method provided by the present invention, other methods such as sequencing, microarray, northern blotting, bioluminescence and probe methods can also be used to detect the miRNA markers. The applicant found in the research that any 1 miRNA marker combination that can be used for the diagnosis of renal cancer, and any 2-5 miRNA marker combinations that can be used for the diagnosis of renal cancer. Using the above markers, renal cancer can be reliably diagnosed Identification. From the 16 miRNA markers that can detect kidney cancer, the present invention found that 8 miRNA markers can detect kidney cancer with significant difference (p<0.05): hsa-miR-671-3p> hsa -miR-518e-5p, hsa-miR-153-3p>hsa-miR-21-3p, hsa-miR-331-5p, hsa-miR-885-5p, hsa-miR-4306, hsa-miR-l -3p; and, after further optimization and screening, a combination of the above-mentioned 8 miRNA markers with an AUC value higher than hsa-miR-671-3p, hsa-miR-518e-5p, hsa-miR-153- Kidney cancer marker combination including 3p, hsa-miR-21-3p, hsa-miR-331-5p, hsa-miR-885-5p, hsa-miR-671-3p, hsa-miR-518e-5p , hsa-miR-153-3p, hsa-miR-21-3p, hsa-miR-331-5p, hsa-miR-885-5p 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. All miRNA sequences disclosed in the present invention have been stored in the miRBase database (http://www.mirbase.org/) o Table 1. The sequence list of 8 miRNAs

The invention discloses a kidney cancer diagnostic marker combination, comprising hsa-miR-671-3p>hsa-miR-518e-5p>hsa-miR-153-3p>hsa-miR-21-3p, hsa-miR Multiple nucleic acid molecules of -331-5p>hsa-miR-885-5p. Encoding hsa-miR-671-3p, hsa-miR-518e-5p > hsa-miR-153-3p, hsa-miR-21-3p, hsa-miR-331-5p, hsa-miR-885-5p The expression of any one or any two or any three or any four or any five or any six nucleic acid molecules in the urine of a patient diagnosed with kidney cancer is significantly changed compared to the expression in a control. In an optimal embodiment for the diagnosis of kidney cancer, the above multiple nucleic acid molecules include hsa-miR-671-3p>hsa-miR-518e-5p, hsa-miR-153-3p>hsa-miR-21-3p>hsa -6 nucleic acid molecule combinations of miR-331-5p>hsa-miR-885-5p. The miRNAs and methods disclosed herein are useful for making a diagnosis of kidney cancer. Thus, as a result of a determination based on the methods provided herein, a subject who has been diagnosed with kidney cancer using the methods described herein may receive necessary and relevant drugs (e.g., chemotherapeutic agents) as a result of said diagnosis treatment or be arranged for necessary treatment options, such as radiation therapy, surgical resection, targeted therapy, immunotherapy, etc. Accordingly, the methods disclosed herein allow for the treatment of subjects diagnosed with renal cancer with compounds and compositions known in the art to be effective in treating renal cancer. Thus, in one embodiment, a method as disclosed herein results in a subject being diagnosed with kidney cancer, wherein the subject is then administered a treatment for kidney cancer as known in the art. As herein The disclosed method thus enables the treatment of kidney cancer. The invention described illustratively herein may be suitably implemented without any one or more elements, one or more limitations not specifically disclosed herein. Therefore , for example, the terms "comprising", "including", "containing", etc. This specification should be read broadly and without limitation. Furthermore, the terms and expressions used herein have been used as terms of description rather than limitation, and there is no intention in the use of these terms and expressions to exclude any equivalents to the features shown and described or parts thereof, but It should be appreciated that various modifications are possible within the scope of the claimed invention. Therefore, it should be understood that although the invention has been explicitly disclosed by way of preferred embodiments and optional features, modifications and variations of the invention embodied therein disclosed herein may be adopted by those skilled in the art, And such modifications and variations are considered to fall within the scope of the present invention. Example 1: Validation cohort Validation of 8 miRNA combinations for kidney cancer diagnosis 1. Urine sample requirements for the kidney cancer diagnostic marker combination research and development cohort. Two groups of samples were used in the collection and extraction of kidney cancer diagnostic markers research, respectively It is a case combination control group, the case group contains 69 cases of kidney cancer samples, and the control group is 88 cases of healthy human urine samples, discovering and validating biomarkers and biomarker combinations for detecting early kidney cancer. Kidney cancer cases and healthy samples in the R&D cohort came to the Second Affiliated Hospital of Zhejiang University School of Medicine. 2. The operation process and results of reverse transcription-real-time fluorescent PCR were extracted according to the operation steps of the Zymo Serum Plasma cfDNA Rapid Extraction Kit. After the extraction was completed, reverse transcription was performed using the operation steps of the MIRXE reverse transcription kit After completion, qPCR amplification was performed for quality inspection. After the quality inspection was completed, the samples were amplified by qPCR to detect the expression levels of 312 miRNAs. The expression levels of all 312 miRNAs in cancer and healthy controls were not significantly graded. 16 miRNA markers that can be used to detect kidney cancer were initially found, and further The study found 8 miRNA biomarkers with significant differences in the detection of kidney cancer. After correction, the P values of 8 miRNAs were found to be less than 0.05; among them, 3 were up-regulated and 5 were down-regulated in kidney cancer subjects. Up-regulated miRNAs include: hsa-miR-518e-5p > hsa-miR-153-3p 153 and hsa-miR-21-3p; Down-regulated miRNAs include: hsa-miR-671-3p, hsa-miR-4306, hsa -miR-331-5p, hsa-miR-l-3p and hsa-miR-885-5p _o extracted these 8 miRNAs in the research and development team to draw Heatmap heat map, observed that there were differences between cancer and control subjects Significant differences in miRNA expression levels, as shown in Figure 1. III. Validation cohort Validation of the above 8 miRNA case-control cohorts The 8 urine miRNA biomarkers were detected by the method of Leave-one-out Cross-validation. There were 157 samples in the verification cohort, and the diagnostic performance was AUC=0.681, as shown in Figure 2-A. Example 2: Validation cohort Validation of the optimal 6 miRNA combination R&D cohort for kidney cancer diagnosis. The urine sample requirements, collection and extraction, reverse transcription-real-time fluorescent PCR operation process and results are the same as those described in Example 1, based on For the 8 miRNAs screened out above, the algorithms of Lasso, Stepwise and exhaustive method were used to optimize the miRNA combinations, and finally the optimal 6 miRNA combinations were determined, and the cross-validation (Leave-one-out Cross-validation) method was used Verify hsa-miR-671-3p, hsa-miR-518e-5p, hsa-miR-153-3p, hsa-miR-21-3p, hsa-miR-331-5p > hsa-miR-885-5p included 6 urine biomarker panels. There were 157 samples in the validation cohort, and the diagnostic efficacy of the 6 miRNA combinations in the validation cohort was AUC=0.703, as shown in 2-B. The results of model interpretation are shown in Figure 3. 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. Based on the above comparison, it is confirmed that the combination of 6 miRNAs is one of the preferred combinations for the diagnosis of renal cancer. Instructions Example 3: Optimization of miRNA combinations In this example, the method of cross-validation (Leave-one-out Cross-validation) was used to detect the AUC value of the multivariate miRNA combination, and the method was the same as in Examples 1 and 2. AUC values were calculated for the multi-miRNA panel in the validation cohort for the 8 miRNAs used in the clinical trial (hsa-miR-671-3p > hsa-miR-518e-5p > hsa-miR-153-3p > hsa-miR-21-3p , hsa-miR-331-5p, hsa-miR-885-5p> hsa-miR-4306, hsa-miR-1-3p), choose one miRNA as a non-variable, and then use the non-variable miRNA as a benchmark , to detect the average AUC value of a multivariate combination of 1-, 2-, 3-, 4-, 5-, 6-, 7- or 8-miRNA, and analyze the difference in AUC value. The results are shown in Table 2. The average AUC from training and testing was calculated for each miRNA from the results of cross-validation experiments involving 2 to 8 miRNAs (Table 2). When used alone, the 8 miRNAs we identified were able to identify subjects with or at risk of developing kidney cancer. Combining two or more of these miRNAs further increased AUC values. It can be seen from Table 2 that when three or more miRNAs are combined, the AUC values of most multivariate miRNA combinations can reach a higher level. Table 2. Average AUC values for miRNAs alone or combinations of up to 8 mimas

2. Combinations of miRNAs used for the diagnosis of kidney cancer In order to further verify the utility of miRNA combinations for the diagnosis of renal cancer, the exemplary combinations of miRNAs listed in Table 2 above were analyzed. The mean AUC, mean sensitivity, mean specificity, mean positive predictive value (PPV) and mean negative predictive value (NPV) of the validation cohort are shown in Table 3, respectively. Exemplary combinations comprising 1-8 miRNA signatures can be used to predictively classify cancer patients and control subjects with mean AUCs ranging from 0.553 to 0.681. Table 3. Mean AUC, Sensitivity, Specificity, Positive Predictive Value, and Negative Predictive Value for 1 to 8 Feature Combinations

As an example, these miRNAs showed good performance in both the test cohort and the validation cohort. One or more of these miRNAs are then selected and combined with other miRNAs. The example combination in which hsa-miR-518e-5p was used as a fixed miRNA significantly increased AUC after further adding the number of miRNAs (other miRNAs listed in Table 2) (Table 4). However, after adding more than 6 miRNAs, AUC began to decrease slightly. Exemplary combinations with hsa-miR-1-3p and hsa-miR-671-3p are shown in Tables 5 and 6, respectively. As the number of combined miRNAs increases, AUC gradually increases, but AUC begins to decrease when more than 6 miRNAs are combined. Taking hsa-miR-518e-5p as an example, compare a single hsa-miR-518e-5p, and add one, two, three, four, five or six from any of the remaining 7 miRNAs with For the multivariate miRNA combination composed of hsa-miR-518e-5p, the average AUC value was detected, and the difference in AUC value was analyzed. The results are shown in Table 4. Table 4. Average AUC, Sensitivity, Specificity, Positive Predictive Value and Negative Predictive Value of hsa-miR-518e-5p and related parameters selected from the above miRNA feature combinations

Taking hsa-miR-l-3p as an example, compare a single hsa-miR-l-3p, and add one, two, three, four, five or six from any of the remaining 7 miRNAs with For the multivariate miRNA combination composed of hsa-miR-l-3p, the average AUC value was detected, and the difference in AUC value was analyzed. The results are shown in Table 5. Table 5. The average AUC, sensitivity, specificity, positive predictive value and negative predictive value of hsa-miR-1-3p and the relevant parameters of the combination of miRNA features selected from the difference analysis

Taking hsa-miR-671-3p as an example, compare a single hsa-miR-671-3p, and any selection from the remaining 7 miRNAs The multivariate miRNA combinations composed of hsa-miR-671-3p and hsa-miR-671-3p were described after one, three, four, five or six, and the difference in AUC value was analyzed. The results are shown in Table 6.

6. Difference analysis average AUC, sensitivity, specificity, positive predictive value and negative predictive value of hsa-miR-671-3p and related parameters selected from the combination of the above miRNA features

In order to better illustrate that various miRNA combinations provided by the present invention have good AUC, the present invention lists several miRNA combinations with AUCN0.681 as shown in Table 7, and the common features are No.1 to No.32 hsa-miR-518e-5p is present in all miRNA combinations shown, but it does not mean that the range of miRNA combinations to be protected in the present invention is limited to Table 7. Table 7. Exemplary miRNA combinations and their respective AUCs

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The present invention establishes a complete workflow for discovering and validating urine miRNA biomarker combinations, and successfully determines biomarkers and biomarker combinations for detecting kidney cancer. It should be noted that the urine miRNA marker combination provided by the present invention is obtained based on statistics, screening, and analysis of clinical test data of human urine samples, so it is suitable for high population-specific detection and has a significant difference in AUC Expressed miRNA combinations; but it does not mean that such miRNA combinations can be generally used in the detection of human-derived plasma or tissue samples. The embodiment described above is only a preferred solution of the present invention, and does not limit the present invention in any form, and there are other variations and modifications on the premise of not exceeding the technical solution described in the claims.

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SEQUENCE LISTING

<H0> Miray (Hangzhou) Biotechnology Co., Ltd.; Miray Laboratory Private Limited

<120> Urinary miRNA markers, diagnostic reagents and kits for the diagnosis of kidney cancer

<130> 2021.12

<160> 8

<170> Patent version 3.3

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Claims

claims

1. a kidney cancer diagnosis urine miRNA marker, is characterized in that, is selected from one or more in following miRNA: hsa-miR-518e-5p, hsa-miR-671-3p, hsa-miR-153 -3p, hsa-miR-21-3p, hsa-miR-331-5p, hsa-miR-885-5p>hsa-miR-4306>hsa-miR-1-3p _o

2. kidney cancer diagnosis according to claim 1 uses urine miRNA marker, it is characterized in that described miRNA marker is selected from one or more in following miRNA: hsa-miR-518e-5p^hsa-miR -671-3p>hsa-miR-153-3p>hsa-miR-21-3p>hsa-miR-331-5p>hsa-miR-885-5p^ hsa-miR-l-3p _o

3. kidney cancer diagnosis according to claim 1 uses urine miRNA marker, is characterized in that, described miRNA marker is selected from one or more in following miRNA: hsa-miR-518e-5p^hsa-miR -671-3p>hsa-miR-153-3p>hsa-miR-21-3p>hsa-miR-331-5p>hsa-miR-885-5p, hsa-miR-4306o

4. kidney cancer diagnosis according to claim 1 uses urine miRNA marker, it is characterized in that described miRNA marker is selected from one or more in following miRNA: hsa-miR-518e-5p^hsa-miR -671-3p>hsa-miR-153-3p>hsa-miR-21-3p>hsa-miR-331-5p>hsa-miR-885-5p _o

5. The urine miRNA marker for kidney cancer diagnosis according to any one of claims 1 to 4, characterized in that, when there are at least two miRNA markers, one of the miRNA must be hsa-miR -518e-5p _o

6. The urine miRNA marker for kidney cancer diagnosis according to any of claims 1 to 4, characterized in that, when there are at least two miRNA markers, one of the miRNA must be hsa-miR -l-3p _o

7. The urine miRNA marker for kidney cancer diagnosis according to claim 1, wherein the miRNA marker comprises any one or more of the following combinations: hsa-miR-671-3p>hsa- miR-518e-5p>hsa-miR-153-3p>hsa-miR-21-3p>hsa-miR-331-5p>hsa-miR-885-5p>hsa-miR-4306, hsa-miR-l- 3p; or consist of hsa-miR-671-3p>hsa-miR-518e-5p>hsa-miR-153-3p>hsa-miR-21-3p>hsa-miR-331-5p>hsa-miR-885 -5p>hsa-miR-4306; or consisting of hsa-miR-671-3p>hsa-miR-518e-5p>hsa-miR-153-3p>hsa-miR-21-3p>hsa-miR-331- 5p>hsa-miR-885-5p>hsa-miR-l-3p; or composed of hsa-miR-518e-5p^hsa-miR-153-3p^hsa-miR-21-3p^hsa-miR-331 -5p>hsa-miR-885-5p^hsa-miR-4306, hsa-miR-l-3p; or by hsa-miR-671-3p>hsa-miR-518e-5p>hsa-miR-153- 3p>hsa-miR-21-3p>hsa-miR-331-5p>hsa-miR-4306; or composed of hsa-miR-671-3p>hsa-miR-518e-5p>hsa-miR-153-3p >hsa-miR-21-3p>hsa-miR-331-5p>hsa-miR-l-3p; or composed of hsa-miR-671-3p>hsa-miR-518e-5p>hsa-miR-153- 3p>hsa-miR-21-3p>hsa-miR-331-5p> The claim consists of hsa-miR-885-5p; or consists of hsa-miR-671-3p>hsa-miR-518e-5p>hsa-miR-21-3p>hsa-miR-331-5p>hsa-miR- 4306>hsa-miR-l-3p; or composed of hsa-miR-671-3p>hsa-miR-518e-5p>hsa-miR-21-3p>hsa-miR-331-5p>hsa-miR-885 -5p>hsa-miR-4306; or composed of hsa-miR-671-3p>hsa-miR-518e-5p>hsa-miR-21-3p^hsa-miR-331-5p>hsa-miR-885- 5p>hsa-miR-l-3p; or composed of hsa-miR-518e-5p>hsa-miR-153-3p>hsa-miR-21-3p>hsa-miR-331-5p>hsa-miR-4306 >hsa-miR-l-3p composition; or hsa-miR-518e-5p>hsa-miR-153-3p>hsa-miR-21-3p, hsa-miR-331-5p>hsa-miR-885-5p >hsa-miR-4306 composition; or hsa-miR-518e-5p>hsa-miR-153-3p>hsa-miR-21-3p>hsa-miR-331-5p>hsa-miR-885-5p>hsa -miR-l-3p; or by hsa-miR-518e-5p^hsa-miR-21-3p>hsa-miR-331-5p>hsa-miR-885-5p>hsa-miR-4306, hsa- Composed of miR-1-3p; or composed of hsa-miR-671-3p>hsa-miR-518e-5p>hsa-miR-153-3p>hsa-miR-21-3p>hsa-miR-331-5p; or consist of hsa-miR-671-3p>hsa-miR-518e-5p>hsa-miR-153-3p^ hsa-miR-21-3p>hsa-miR-885-5p; or consist of hsa-miR-671 -3p>hsa-miR-518e-5p>hsa-miR-21-3p^hsa-miR-331-5p^hsa-miR-4306; or consisting of hsa-miR-671-3p>hsa-miR-518e- 5p>hsa-miR-21-3p>hsa-miR-331-5p, hsa-miR-l-3p; or composed of hsa-miR-671-3p>hsa-miR-518e-5p>hsa-miR-21 -3p>hsa-miR-331-5p>hsa-miR-885-5p; or composed of hsa-miR-518e-5p>hsa-miR-153-3p>hsa-miR-21-3p, hsa-miR- 331-5p, hsa-miR-4306; or composed of hsa-miR-518e-5p>hsa-miR-153-3p>hsa-miR-21-3p>hsa-miR-331-5p>hsa-miR-l -3p; or by hsa-miR-518e-5p>hsa-miR-153-3p>hsa-miR-21-3p, hsa-miR-331-5p>hsa-miR-885-5p; or by hsa -miR-518e-5p consisting of hsa-miR-21-3p, hsa-miR-331-5p, hsa-miR-4306, hsa-miR-l-3p; or consisting of hsa-miR-518e-5p>hsa-miR -21-3p> composed of hsa-miR-331-5p, hsa-miR-885-5p, hsa-miR-4306; or The claim consists of hsa-miR-518e-5p, hsa-miR-21-3p, hsa-miR-331-5p, hsa-miR-885-5p, hsa-miR-1-3p; or consists of hsa-miR -671-3p, hsa-miR-518e-5p>hsa-miR-21-3p, hsa-miR-331-5p; or composed of hsa-miR-518e-5p>hsa-miR-153-3p, hsa- Composed of miR-21-3p, hsa-miR-331-5p; or composed of hsa-miR-518e-5p>hsa-miR-153-3p, hsa-miR-21-3p, hsa-miR-885-5p; Or consist of hsa-miR-518e-5p>hsa-miR-21-3p, hsa-miR-331-5p, hsa-miR-4306; or consist of hsa-miR-518e-5p>hsa-miR-21-3p , hsa-miR-331-5p, hsa-miR-1-3p; or by hsa-miR-518e-5p>hsa-miR-21-3p, hsa-miR-331-5p, hsa-miR-885- 5p; or composed of hsa-miR-518e-5p, hsa-miR-21-3p, hsa-miR-331-5p.

8. A diagnostic reagent for kidney cancer, characterized in that it includes a reagent for specifically detecting the urine miRNA marker described in any one of claims 1 to 7

9. A diagnostic kit for kidney cancer, characterized in that it comprises the urine miRNA marker described in any one of claims 1 to 7 as a standard product, and detects the miRNA marker described in any one of claims 1 to 7. Corresponding primers for the urine miRNA markers described above.

10. An application of the urine miRNA marker according to any one of claims 1 to 7, characterized in that, it is used to prepare products for early diagnosis and/or recurrence monitoring; preferably, it includes the following one or Several functions:

1) Identification or auxiliary identification of renal cell carcinoma;

2) Diagnosis or auxiliary diagnosis of primary renal cancer;

3) Diagnosis or auxiliary diagnosis of recurrent renal cell carcinoma.

11. A method for early diagnosis and/or recurrence monitoring of kidney cancer using the urine miRNA marker according to any one of claims 1 to 7, characterized in that it comprises the following steps: Step 1, detecting the The presence of miRNA in the urine sample obtained by the test subject; Step 2, measuring the expression level of at least one miRNA in the miRNA markers in the urine sample; Step 3, using a score based on the expression level of the previously measured miRNA, to Early diagnosis and/or recurrence monitors the likelihood that the subject has kidney cancer.