A urine-based RNA diagnostic marker combination for prostate cancer and its application in the preparation of a diagnostic kit for prostate cancer

Abstract

The present invention discloses a combination of RNA detection markers for prostate cancer based on urine and its application in the preparation of a diagnostic kit for prostate cancer. The RNA detection marker combination includes a combination of 12 genes in urine supernatant and a combination of 12 genes in urine sediment. The combination of 12 genes in urine supernatant and the combination of 12 genes in urine sediment can be used separately according to the combination, or the two combinations can be used jointly. The detection marker combination also includes primers and probes corresponding to the combination of 12 genes in urine supernatant and the combination of 12 genes in urine sediment respectively. When used for detecting prostate cancer, the present invention does not require the collection of early morning urine or urine after digital rectal examination, and only ordinary random urine is needed, which greatly facilitates the collection of samples and improves the accessibility of detection. The gene combination of the present invention is relatively large, the interference of samples on the detection results is small, and the accuracy, sensitivity and specificity of detection are relatively high.

Description

Technical Field

[0001] The present invention relates to the field of early diagnosis of prostate cancer, and specifically relates to a combination of RNA detection markers for prostate cancer based on ordinary urine and its application. Background Art

[0002] Currently, the diagnosis of prostate cancer mainly relies on serum PSA (prostate specific antigen, abbreviated as PSA) detection and prostate biopsy after evaluation by digital rectal examination (abbreviated as DRE). PSA is the first tumor biomarker approved by the US Food and Drug Administration (FDA). A serum PSA level > 4 ng / ml is considered elevated, and when the serum PSA > 10 ng / ml, it indicates that the blood-epithelial cell barrier of the prostate system is broken, and the probability of the patient having prostate cancer increases. However, the specificity and sensitivity of serum PSA detection are both less than satisfactory. Especially when the serum PSA detection is in the gray zone (4 - 10 ng / ml), only 20% - 50% of the patients have a positive prostate biopsy result. Some benign diseases, such as prostate hyperplasia, prostatitis, urinary tract infections, etc., can also lead to false positive results. Relying solely on PSA for prostate cancer screening will cause patients with benign prostate diseases in the gray zone to be over-punctured. This will not only cause pain to the patients, but may also trigger a series of adverse consequences that seriously affect the quality of life, such as bleeding, urinary retention, permanent urinary incontinence, and sexual dysfunction.

[0003] Prostate cancer is a focal lesion, and there is a large area of histologically normal tissue around the tumor, so there is a possibility of missed diagnosis by biopsy. In addition, most prostate cancers are indolent cancers with slow disease progression and a long course. For non-clinically significant prostate cancers, measures such as active surveillance can be chosen, while clinically significant prostate cancers require active treatment. However, due to the influence of the biopsy site and tissue size of the needle biopsy, there is a lack of consistency between the biopsy and the Gleason (Gleason grading, the Gleason scoring system for prostate cancer) score of radical prostatectomy. According to Cheng Wanli, Pang Cheng, Song Xinda, etc., Analysis of risk factors for Gleason score upgrading in postoperative pathology of low-risk prostate cancer ([J]. Chinese Journal of Geriatrics, 2020, 39(9): 1059-1062. DOI: 10.3760 / cma.j.issn.0254-9026.2020.09.016), 50% of patients with a needle biopsy Gleason score of 6 had an upgrade in postoperative pathology. The inconsistency between the biopsy Gleason score and the Gleason score of radical prostatectomy is one of the important problems in the clinical treatment of prostate cancer. Therefore, stratifying prostate cancer patients by risk and identifying clinically significant prostate cancer are crucial for prostate cancer management.

[0004] Previous studies have shown that prostate cancer cells can release tumor markers into prostatic fluid and then enter the urine, so prostate cancer tumor markers can be detected through urine. The PCA3 (prostate cancer associated 3 Gene, abbreviated as PCA3, a novel prostate cancer antigen 3) score is the first urine tumor marker-based test score approved by the US FDA. The concentrations of PCA3 and PSA mRNA (Messenger RNA, abbreviated as mRNA, messenger ribonucleic acid) in urine after digital rectal examination are quantitatively detected, and the ratio of the two is the PCA3 score. In addition, the ExoDx Prostate IntelliScore product of Bio-Techne Corporation is based on RNA in urine exosomes to predict whether patients with PSA in the gray zone are at risk of prostate cancer and whether they have high-grade and more aggressive prostate cancer. Therefore, urine-based auxiliary diagnosis of prostate cancer is feasible.

[0005] Regarding the solution to the problem of early diagnosis of prostate cancer, there are already some invention patents. For example:

[0006] Patent CN116377068A discloses an RNA biomarker NKX3-1 for prostate cancer in urine. By comparing the urine of prostate cancer patients and healthy individuals, this patent found that NKX3-1 is highly expressed in prostate cancer patients and lowly expressed in healthy individuals. Therefore, prostate cancer can be detected solely through urine samples. However, this patent can only distinguish between prostate cancer and healthy individuals, and it is still affected by other urinary system diseases.

[0007] Patent CN118562932A discloses a urine RNA preservation solution, a urine RNA extraction kit, and the application of the gene combination of PCA3, HOXC6, and DLX1 in the early screening of prostate cancer. It provides a urine RNA preservation solution, a urine RNA extraction kit, and the application of the gene combination of PCA3, HOXC6, and DLX1 in the early screening of prostate cancer, which has relatively high detection accuracy and specificity, simple test operation, low cost, and can use morning urine samples instead of DRE urine samples for detection, reducing the pain of patient sampling and having good patient compliance. However, this patent still requires the collection of morning urine and cannot use ordinary random urine.

[0008] Therefore, generally speaking, the existing urine-based prostate cancer detection methods have the following problems.

[0009] First, the requirements for test samples are relatively high. Often, morning urine or urine after digital rectal examination is needed, which greatly limits the practicality of user screening and reduces the accuracy of sampling. Some elderly patients have the habit of getting up at night, and it is very difficult to collect qualified morning urine.

[0010] Second, the gene combinations used in detection are few, and the interference of samples on the test results is large, affecting the accuracy of detection.

[0011] Third, the detection accuracy is not ideal enough, with insufficient sensitivity and specificity, often not reaching 80%, and the accuracy also needs to be further improved.

[0012] Fourth, detection is carried out only through a single biomarker, and the detection information is limited. Although it can be used to distinguish the expression between prostate cancer patients and healthy individuals, it is often affected by other urinary diseases and is difficult to correctly reflect the occurrence and development of prostate cancer, and cannot be truly used for clinical prostate cancer screening. Summary of the Invention

[0013] To solve the above problems, the present invention proposes a combination of RNA detection markers for prostate cancer based on ordinary urine. The detection markers of the present invention can use ordinary random urine, without the need for morning urine or urine after digital rectal examination, which greatly facilitates the collection of samples. Moreover, the markers used in the present invention are numerous and scientifically screened, and the detection sensitivity, specificity, and accuracy are all very high.

[0014] The technical solution of the present invention is: a urine-based prostate cancer RNA detection marker combination, and the RNA detection marker combination includes a urine supernatant 12-gene combination and a urine sediment 12-gene combination; wherein, the urine supernatant 12-gene combination includes the AOX1 gene, the C2orf72 gene, the CYP3A5 gene, the FA2H gene, the LOC105370113 gene, the TNFRSF19 gene, the MIPEP gene, the MX2 gene, the NSG1 gene, the PCA3 gene, the PCOTH gene, and the S100A4 gene; the urine sediment 12-gene combination includes the AGPAT4 gene, the CD24 gene, the CRABP2 gene, the CTSK gene, the FDFT1 gene, the GDPD3 gene, the KAT2B gene, the MFSD13A gene, the PLS3 gene, the SDC3 gene, the TLE2 gene, and the UCA1 gene.

[0015] Wherein, the urine supernatant 12-gene combination and the urine sediment 12-gene combination can be used alone according to the combination, or the two combinations can be used in combination.

[0016] Wherein, the genes in the urine supernatant 12-gene combination and the corresponding primers are shown in Table 1.

[0017] Table 1

[0018] Gene identification Forward primer Probe Reverse primer AOX1 CTCGTGGTCCAGACCAATATAAA CCACGGAGTTGCACATTGCTTTGT TCTCCCAGACCCTTAGATGAA C2orf72 AGAAGCTCAGCTCAGGAAGA CTGAGGAGGAGCTGCCACTAACAG CATCACAGTCTCCATTGGGAAATA CYP3A5 TTTGCCCAATAAGGCACCAC CAAGGTACTCCATCTGTACCACGGCA CACCACCATTGACCCTTTGG FA2H GACCCATTACTACCTGCACTTT CACGTCAAGCACCACTTTGCACAT GCTGATACCAAATCCTGACTTCT LOC105370113 ACCTGGGTGCTGATGAAAG TCTGTTTCCCAGAATGAGCTGGCA TCCTGACAAACTGGCAATACA TNFRSF19 GGACTGCTTGCCAGGATTT ATGGAGTGTGTGCCTTGTGGAGAC CACAGTGCGGTTCGTAAGG MIPEP CTGGCTGTTGTTCATGAATCTG TCAGCGAGCAGACAAACCACATCA GCCTCCACGGATAGTGAAAT MX2 AGTCTTCGGTTTCCTCCTTTAC CTTGGAAACCAGCAAACGTCTCGC CTGCAAGGAGTCACCATTCT NSG1 GTCGTCTTCCTGGTTGTCTAC TCCTCAAGAACACCCAGTGCATCC TAGTAGCTCTCCAAGCCTTCT PCA3 ACAGAGATCCCTGGGAGAAA CCGCCATCTTGGGTCATCGATGAG TTCTAATGTCCTTCCCTCACAAG PCOTH ATCTCCTTTCCAGCCTCTTATTC TTGGGACCAGGCTTTCCAATAGGG TGGGCCCAACTGGTTTAC S100A4 CGAGGTGGACTTCCAAGAGT TGTCTTCCTGTCCTGCATCGCCA TCATTTCTTCCTGGGCTGCT

[0019] Wherein, the genes in the urine sediment 12-gene combination and the corresponding primers are shown in Table 2.

[0020] Table 2

[0021] Gene identification Forward primer Probe Reverse primer AGPAT4 CATTCAGCTCTTCACTCTCCTC TCTGGCCCATTAACAAGCAGCTCT AGCTGGCTTGAGATGCAATA CD24 TCGTGGTCTCACTCTCTCTT AGAGACTCAGGCCAAGAAACGTCT CGCCATTTGGATTGGGTTTAG CRABP2 CCTGTAAGAGCCTGGTGAAA CGTGGACCAGAGAACTGACCAACG CGTCATGGTCAGGATCAGTT CTSK GGGACAGGAAGAGAGTTGTATG CAACAGGCAAGGCAGCTAAATGCA CCTCTTCAGGGCTTTCTCATT FDFT1 CCAACTCTATGGGCCTGTTT TGGAAGACCAGCAAGGAGGAAGAGA CATACCTGCTCCAAACCTCTT GDPD3 GAAGAGCTCATCCGTGAGATAG ATGAAATCACCATCTGGGCCTCGG GTTGGCAGCCTTGCATTT KAT2B CTCGTCTTTGACCCGAAACA TGGCCGTGTTATTGGTGGTATCTGT GTGAATCCTTGAGATGGGAACA MFSD13A AACTTCCTGTGGTTCGTGAG AGGTCTTCCACTGCCACTTCAACA CAGGAAGAGAGGGAAGAAGTTG PLS3 TGAAGCCTTGGCTGCTTTA TCCGAGATGGTGAGACTTTGGAGGA TCCAAATGAAAGTTTGCCCATC SDC3 TCAGCTGCCTCAGAAGAGTA AGGTGCTCGTAGCTGTGATTGTGG GATGAGCAGTGTGACCAAGAA TLE2 CATGACTTCAGCTCCCAGATT TGGAGAGTAGCAACGTGGAGATCCT GAGGTGCAGCTGGTATTTCT UCA1 AAATCGGATCTCCTCGGCTT TGCCCGATCGCCTCAGAAGCCC TATGGCTGGGAATCCTCCAC

[0022] Furthermore, the present invention also discloses an application of a urine-based prostate cancer RNA detection marker composition in the preparation of a prostate cancer diagnostic kit, and the detection marker composition contains primers and probes corresponding to the urine supernatant 12-gene combination as shown in Table 3 below:

[0023] Table 3

[0024] Gene identification Forward primer Probe Reverse primer AOX1 CTCGTGGTCCAGACCAATATAAA CCACGGAGTTGCACATTGCTTTGT TCTCCCAGACCCTTAGATGAA C2orf72 AGAAGCTCAGCTCAGGAAGA CTGAGGAGGAGCTGCCACTAACAG CATCACAGTCTCCATTGGGAAATA CYP3A5 TTTGCCCAATAAGGCACCAC CAAGGTACTCCATCTGTACCACGGCA CACCACCATTGACCCTTTGG FA2H GACCCATTACTACCTGCACTTT CACGTCAAGCACCACTTTGCACAT GCTGATACCAAATCCTGACTTCT LOC105370113 ACCTGGGTGCTGATGAAAG TCTGTTTCCCAGAATGAGCTGGCA TCCTGACAAACTGGCAATACA TNFRSF19 GGACTGCTTGCCAGGATTT ATGGAGTGTGTGCCTTGTGGAGAC CACAGTGCGGTTCGTAAGG MIPEP CTGGCTGTTGTTCATGAATCTG TCAGCGAGCAGACAAACCACATCA GCCTCCACGGATAGTGAAAT MX2 AGTCTTCGGTTTCCTCCTTTAC CTTGGAAACCAGCAAACGTCTCGC CTGCAAGGAGTCACCATTCT NSG1 GTCGTCTTCCTGGTTGTCTAC TCCTCAAGAACACCCAGTGCATCC TAGTAGCTCTCCAAGCCTTCT PCA3 ACAGAGATCCCTGGGAGAAA CCGCCATCTTGGGTCATCGATGAG TTCTAATGTCCTTCCCTCACAAG PCOTH ATCTCCTTTCCAGCCTCTTATTC TTGGGACCAGGCTTTCCAATAGGG TGGGCCCAACTGGTTTAC S100A4 CGAGGTGGACTTCCAAGAGT TGTCTTCCTGTCCTGCATCGCCA TCATTTCTTCCTGGGCTGCT

[0025] And, the detection marker composition also contains primers and probes corresponding to the urine sediment 12-gene combination as shown in Table 4 below:

[0026] Table 4

[0027] Gene identification Forward primer Probe Reverse primer AGPAT4 CATTCAGCTCTTCACTCTCCTC TCTGGCCCATTAACAAGCAGCTCT AGCTGGCTTGAGATGCAATA CD24 TCGTGGTCTCACTCTCTCTT AGAGACTCAGGCCAAGAAACGTCT CGCCATTTGGATTGGGTTTAG CRABP2 CCTGTAAGAGCCTGGTGAAA CGTGGACCAGAGAACTGACCAACG CGTCATGGTCAGGATCAGTT CTSK GGGACAGGAAGAGAGTTGTATG CAACAGGCAAGGCAGCTAAATGCA CCTCTTCAGGGCTTTCTCATT FDFT1 CCAACTCTATGGGCCTGTTT TGGAAGACCAGCAAGGAGGAAGAGA CATACCTGCTCCAAACCTCTT GDPD3 GAAGAGCTCATCCGTGAGATAG ATGAAATCACCATCTGGGCCTCGG GTTGGCAGCCTTGCATTT KAT2B CTCGTCTTTGACCCGAAACA TGGCCGTGTTATTGGTGGTATCTGT GTGAATCCTTGAGATGGGAACA MFSD13A AACTTCCTGTGGTTCGTGAG AGGTCTTCCACTGCCACTTCAACA CAGGAAGAGAGGGAAGAAGTTG PLS3 TGAAGCCTTGGCTGCTTTA TCCGAGATGGTGAGACTTTGGAGGA TCCAAATGAAAGTTTGCCCATC SDC3 TCAGCTGCCTCAGAAGAGTA AGGTGCTCGTAGCTGTGATTGTGG GATGAGCAGTGTGACCAAGAA TLE2 CATGACTTCAGCTCCCAGATT TGGAGAGTAGCAACGTGGAGATCCT GAGGTGCAGCTGGTATTTCT UCA1 AAATCGGATCTCCTCGGCTT TGCCCGATCGCCTCAGAAGCCC TATGGCTGGGAATCCTCCAC

[0028] The primer-probe corresponding to the 12-gene combination in the urine supernatant and the primer-probe corresponding to the 12-gene combination in the urine sediment can be used separately according to the combination, or the two combinations can be used jointly.

[0029] Among them, the urine is random urine. Different from many existing prostate cancer detection reagents and methods that must use morning urine or urine after digital rectal examination, the present invention only requires ordinary random urine.

[0030] The present invention adopts 12 RNA markers for urine supernatant and urine sediment respectively, including AGPAT4 gene, AOX1 gene, C2orf72 gene, CD24 gene, CRABP2 gene, CTSK gene, CYP3A5 gene, FA2H gene, FDFT1 gene, GDPD3 gene, KAT2B gene, LOC105370113 gene, MFSD13A gene, MIPEP gene, MX2 gene, NSG1 gene, PCA3 gene, PCOTH gene, PLS3 gene, S100A4 gene, SDC3 gene, TLE2 gene, TNFRSF19 gene, UCA1 gene.

[0031] Through the above gene combination, the present invention can accurately distinguish prostate cancer from other prostate diseases, meeting the actual clinical needs.

[0032] The sequences of the above gene markers are publicly available in biological databases. For example, by entering the number in the Nuleotide (RefSeq) database of the NCBI (National Center for Biotechnology Information) website, the sequences can be obtained. For example, the sequence information of AOX1 can be obtained in the following form: https: / / www.ncbi.nlm.nih.gov / nuccore / NM_001159.4.

[0033] The detection methods for the RNA markers of urine supernatant and urine sediment proposed by the present invention can be used separately or jointly.

[0034] Compared with the prior art, the present invention provides a prostate cancer RNA detection marker and method based on ordinary urine, having the following beneficial effects:

[0035] 1. The present invention does not require collecting morning urine or urine after digital rectal examination, only ordinary random urine is needed, which greatly facilitates the sample collection, improves the accessibility of detection and the possibility of popularization and application;

[0036] 2. Twelve RNA markers were used for urine supernatant and urine sediment respectively. With a relatively large number of gene combinations and less interference from samples on the test results, the accuracy of the test was improved.

[0037] 3. By detecting multiple RNA markers in urine supernatant and urine sediment, the occurrence and development of prostate cancer can be comprehensively reflected, providing an important basis for the early diagnosis and treatment of the disease.

[0038] 4. The detection method is simple to operate, without complex sample pretreatment and experimental conditions. The urine supernatant sample or urine sediment sample can be used alone or in combination, which is conducive to clinical promotion and application.

[0039] The detection marker combination of the present invention has high sensitivity, specificity and detection accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is the AUROC graph of the urine sediment sample in Example 2.

[0041] Figure 2 It is the AUROC graph of the urine supernatant sample in Example 3.

[0042] Figure 3 It is the comparison of the AUROC graph of the results of the RNA markers in the urine sediment in Example 5 with the detection results of PSA and PI-RADS.

[0043] Figure 4 It is the comparison of the AUROC graph of the results of the RNA markers in the urine supernatant in Example 5 with the detection results of PSA and PI-RADS.

[0044] Figure 5 It is the comparison of the AUROC graph of the results of the RNA markers integrating urine sediment and urine supernatant in Example 5 with the detection results of PSA and PI-RADS. DETAILED DESCRIPTION OF THE INVENTION

[0045] The technical solutions of the present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited to these embodiments.

[0046] Example 1 Screening of Gene Combinations

[0047] 1. Research Population

[0048] Patients who visited Fudan University Shanghai Cancer Center due to urinary system diseases in 2024 were selected as the research objects. The inclusion criteria for the population in this study were:

[0049] Inclusion Criteria:

[0050] 1) Age ≥ 18 years old;

[0051] 2) Urine sample volume ≥ 50 mL;

[0052] 3) Meet one of the following conditions:

[0053] a. Patients suspected of having prostate cancer based on elevated PSA, positive digital rectal examination, imaging, etc., and who are scheduled for prostate biopsy for a definite diagnosis;

[0054] b. Patients with a definite diagnosis of prostate cancer by prostate biopsy, and who are scheduled for radical prostatectomy, and / or endocrine therapy, or radiotherapy;

[0055] c. Patients clinically diagnosed with benign urinary system diseases, including: benign prostatic hyperplasia, prostatitis, urinary tract stones, renal cysts, ureteral stricture, urethral stricture, hypospadias, urinary tract infection, glandular cystitis, and other benign urinary system diseases;

[0056] d. Patients with other tumors clinically or pathologically diagnosed as non-prostate cancer, including bladder cancer, renal cancer, ureteral cancer, renal pelvic cancer, etc.

[0057] Exclusion criteria:

[0058] 1) Patients who have previously undergone transurethral prostate resection or enucleation, or prostate radiotherapy;

[0059] 2) Patients who have previously received endocrine therapy, or have taken 5α-reductase inhibitors within 6 months;

[0060] 3) Any other situations that the researcher deems inappropriate for the patient to participate in this trial.

[0061] The patients are from the outpatient or inpatient cases of the Department of Urology, Fudan University Shanghai Cancer Center. This study has been approved by the Ethics Committee of our hospital, and all research subjects have signed the informed consent form.

[0062] 2. Experimental methods

[0063] 1) Collection of urine

[0064] Collect approximately 50 - 120 mL of random urine from the research subjects using a urine cup.

[0065] 2) Pretreatment of urine

[0066] Place the freshly collected urine in a 50 mL centrifuge tube, centrifuge at 1500 g for 15 min to obtain urine sediment (for backup) and supernatant.

[0067] Transfer the supernatant to a new 50 mL centrifuge tube, then centrifuge at 3000 g for 15 min, discard the bottom, and place the secondary supernatant in a new 50 mL centrifuge tube for backup (15 - 20 mL / tube).

[0068] 3) Enrichment of urine cfRNA (cell-free RNA, abbreviated as cfRNA)

[0069] According to the ratio of urine: precipitant (16% PEG) (Polyethylene glycol, abbreviated as PEG) ≈ 1:1, after thorough mixing, precipitate overnight at 4°C;

[0070] Centrifuge the precipitated 50 mL centrifuge tube at 3000 g for 1 h at 4°C, carefully remove the supernatant, and reserve the remaining precipitate.

[0071] 4) Extraction of urinary sediment and supernatant cfRNA

[0072] Take the urinary sediment and supernatant, add 1 mL of Trizol (Trizol reagent, total RNA extraction reagent), mix well, add 200 μL of chloroform to the centrifuge tube, vortex to mix thoroughly, and centrifuge at 12000 rpm for 15 min to separate the layers;

[0073] Gently aspirate the upper colorless and transparent liquid into a new centrifuge tube, add an equal volume of ethanol, and mix well;

[0074] Use the purification column method to extract RNA.

[0075] 5) RNA sequencing

[0076] Enrich mRNA from total RNA using oligo (dT) magnetic beads composed of thymine. After fragmentation, synthesize the first-strand cDNA using random hexamer primers, and then perform the synthesis of the second-strand cDNA. After end repair, A-tailing, adapter ligation, fragment selection, amplification, and purification, the library is ready.

[0077] Use the Illumina Novaseq 6000 sequencer to perform high-throughput sequencing on the above library. The sequencing data volume is 6 gb (gb, a gene length unit, representing 1000 mb), and use the fastq program to analyze the high-throughput sequencing data.

[0078] 6) Mining of biomarkers

[0079] Divide the data at different levels (urinary sediment and urine supernatant cfRNA) into two groups, namely the prostate cancer group and the control group. Based on the relative expression levels of each biomarker in the prostate cancer group and the control group, use statistical methods to mine RNA biomarkers that can be used to distinguish prostate cancer from non-prostate cancer.

[0080] 7) Construction of a prostate cancer prediction model

[0081] Table 5 12-gene combination in urine supernatant

[0082]

[0083] The primers and probes corresponding to the 12 genes in the urine supernatant are shown in Table 6.

[0084] Table 6

[0085]

[0086] Table 7 Urinary sediment 12-gene combination

[0087]

[0088] The primers and probes corresponding to the 12 genes in the urinary sediment are shown in Table 8.

[0089] Table 8

[0090] Gene identification Forward primer Probe Reverse primer AGPAT4 NO.37 CATTCAGCTCTTCACTCTCCTC NO.38 TCTGGCCCATTAACAAGCAGCTCT NO.39 AGCTGGCTTGAGATGCAATA CD24 NO.40 TCGTGGTCTCACTCTCTCTT NO.41 AGAGACTCAGGCCAAGAAACGTCT NO.42 CGCCATTTGGATTGGGTTTAG CRABP2 NO.43 CCTGTAAGAGCCTGGTGAAA NO.44 CGTGGACCAGAGAACTGACCAACG NO.45 CGTCATGGTCAGGATCAGTT CTSK NO.46 GGGACAGGAAGAGAGTTGTATG NO.47 CAACAGGCAAGGCAGCTAAATGCA NO.48 CCTCTTCAGGGCTTTCTCATT FDFT1 NO.49 CCAACTCTATGGGCCTGTTT NO.50 TGGAAGACCAGCAAGGAGGAAGAGA NO.51 CATACCTGCTCCAAACCTCTT GDPD3 NO.52 GAAGAGCTCATCCGTGAGATAG NO.53 ATGAAATCACCATCTGGGCCTCGG NO.54 GTTGGCAGCCTTGCATTT KAT2B NO.55 CTCGTCTTTGACCCGAAACA NO.56 TGGCCGTGTTATTGGTGGTATCTGT NO.57 GTGAATCCTTGAGATGGGAACA MFSD13A NO.58 AACTTCCTGTGGTTCGTGAG NO.59 AGGTCTTCCACTGCCACTTCAACA NO.60 CAGGAAGAGAGGGAAGAAGTTG PLS3 NO.61 TGAAGCCTTGGCTGCTTTA NO.62 TCCGAGATGGTGAGACTTTGGAGGA NO.63 TCCAAATGAAAGTTTGCCCATC SDC3 NO.64 TCAGCTGCCTCAGAAGAGTA NO.65 AGGTGCTCGTAGCTGTGATTGTGG NO.66 GATGAGCAGTGTGACCAAGAA TLE2 NO.67 CATGACTTCAGCTCCCAGATT NO.68 TGGAGAGTAGCAACGTGGAGATCCT NO.69 GAGGTGCAGCTGGTATTTCT UCA1 NO.70 AAATCGGATCTCCTCGGCTT NO.71 TGCCCGATCGCCTCAGAAGCCC NO.72 TATGGCTGGGAATCCTCCAC

[0091] 8) Use the above-constructed prostate cancer prediction model to detect new urine samples.

[0092] Example 2 Detection using RNA markers in urinary sediment

[0093] In this example, 108 random urine samples were analyzed, including 54 prostate cancer patients (43 of whom had a Gleason score ≥7) and 54 patients with other urinary system diseases but without prostate cancer (controls). Twelve RNA markers in the urinary sediment were used to analyze the samples. The analysis results are shown in Table 9.

[0094] Table 9 Analysis performance of urinary sediment 12-gene combination

[0095]

[0096] For these 108 samples, the sensitivity of the present invention was 77.78%, the specificity was 81.48%, and the AUROC (Area Under Receiver Operating Characteristic Curve, abbreviated as AUROC, the area under the receiver operating characteristic curve) reached 0.8563. The results are as Figure 1 .

[0097] For invasive prostate cancer (Gleason score ≥7), the detection accuracy was 83.72% (36 / 43).

[0098] Example 3 Using RNA markers in urine supernatant

[0099] In this example, 52 prostate cancer patients (40 of whom had a Gleason score ≥7) and 50 patients with other urinary system diseases but without prostate cancer (controls) were analyzed, for a total of 102 randomly selected urine samples. Twelve RNA markers in the urine supernatant were used to analyze the samples. The results are shown in Table 10.

[0100] Table 10 Analysis performance of the 12-gene combination in urine supernatant

[0101]

[0102] For these 102 samples, the sensitivity of the present invention was 80.77%, the specificity was 86.00%, and the AUROC reached 0.8746.

[0103] For invasive prostate cancer (Gleason score ≥7), the detection accuracy was 87.50% (35 / 40). The results are as Figure 2 .

[0104] Example 4 Combined use of RNA markers in urine sediment and urine supernatant

[0105] Eighty-one prostate cancer patients (64 of whom had a Gleason score ≥7) and 71 patients with other urinary system diseases but without prostate cancer (controls) were included, for a total of 152 randomly selected urine samples. A combined detection method using both urine sediment and urine supernatant RNA was used. A positive result was defined as positive if either the urine sediment or the urine supernatant test was positive. The results are shown in Table 11.

[0106] Table 11

[0107]

[0108] For these 152 samples, the sensitivity of the present invention was 83.95%, the specificity was 80.28%, and the accuracy was 82.24%.

[0109] For invasive prostate cancer (Gleason score ≥7), the detection accuracy was 90.63% (58 / 64).

[0110] Example 5 Comparison of the RNA markers of the present invention with commonly used clinical indicators (PSA and PI-RADS)

[0111] Prostate Cancer PI-RADS refers to the Prostate Imaging Reporting and Data System, a system used to standardize prostate magnetic resonance imaging (MRI) reports, aiming to improve the accuracy and consistency of prostate cancer diagnosis. "PSA" and "MRI (i.e., PI-RADS indicators)" are common biomarkers and imaging tools in prostate cancer diagnosis.

[0112] PSA is an important indicator for prostate cancer screening. When its level is abnormally elevated, it indicates a possible risk of prostate cancer and further examinations are needed for confirmation.

[0113] MRI is an imaging examination that can provide more intuitive tumor information, but there are certain diagnostic difficulties in practical applications, such as how to distinguish between benign and malignant lesions.

[0114] The PI-RADS score ranges from 1 to 5 points, among which,

[0115] PI-RADS 1: Very low risk, almost impossible to have clinically significant prostate cancer.

[0116] PI-RADS 2: Low risk, less likely to have clinically significant prostate cancer.

[0117] PI-RADS 3: Moderate risk, may or may not have clinically significant prostate cancer.

[0118] PI-RADS 4: High risk, very likely to have clinically significant prostate cancer.

[0119] PI-RADS 5: Very high risk, extremely likely to have clinically significant prostate cancer.

[0120] For the 152 samples in Example 4, their clinical data were collected simultaneously, and PSA and PI-RADS results were obtained. The results of the RNA markers in urinary sediment constructed by the present invention were compared with the PSA and PI-RADS test results in Table 12 and Figure 3 .

[0121] Table 12

[0122]

[0123] The results of the RNA markers in urinary supernatant constructed by the present invention were compared with the PSA and PI-RADS test results in Table 13 and Figure 4 .

[0124] Table 13

[0125]

[0126] The results of the integrated analysis of the RNA markers in urine sediment and urine supernatant constructed by the present invention were compared with the results of PSA and PI-RADS tests. See Table 14 and Figure 5 。

[0127] Table 14

[0128]

[0129] Note: If any of the results of urine supernatant and urine sediment is positive, it is judged as positive.

[0130] As can be seen from the comparison in Example 5:

[0131] 1) The AUROC values of the RNA markers of the present invention are all the largest.

[0132] 2) The detection results of the RNA markers of the present invention are superior to the PI-RADS scoring system.

[0133] 3) The PSA test has a relatively high sensitivity and can detect most prostate cancer patients. However, the specificity of the PSA test is relatively low and is easily affected by various factors, such as benign prostatic hyperplasia, prostatitis, etc. This may lead to false positive results. While maintaining a high sensitivity, the specificity of the RNA markers of the present invention is also 80%. The overall accuracy is the highest.

[0134] The present invention uses 12 RNA markers in urine supernatant and urine sediment. Not only can the 12 RNA markers in urine supernatant and urine sediment be used separately, but also the 12 RNA markers in urine supernatant and urine sediment can be used in combination. There are more gene combinations, the interference of samples on the detection results is small, the sensitivity is high, it can correctly distinguish prostate cancer from other urinary system diseases, and there is a good balance between sensitivity and specificity, and the detection accuracy is high.

[0135] The present invention does not require the collection of early morning urine or urine after digital rectal examination, only ordinary random urine is needed, which greatly facilitates the collection of samples and improves the accessibility of detection and the possibility of popularization and application.

[0136] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the inventive concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A urine-based RNA detection marker combination for prostate cancer, characterized in that, The RNA detection marker combination is a urine supernatant 12-gene combination and a urine sediment 12-gene combination; among them, the urine supernatant 12-gene combination is a combination of AOX1 gene, C2orf72 gene, CYP3A5 gene, FA2H gene, LOC105370113 gene, TNFRSF19 gene, MIPEP gene, MX2 gene, NSG1 gene, PCA3 gene, PCOTH gene, and S100A4 gene; the urine sediment 12-gene combination is a combination of AGPAT4 gene, CD24 gene, CRABP2 gene, CTSK gene, FDFT1 gene, GDPD3 gene, KAT2B gene, MFSD13A gene, PLS3 gene, SDC3 gene, TLE2 gene, and UCA1 gene.

2. The prostate cancer RNA detection marker combination according to claim 1, wherein The urine supernatant 12-gene combination and the urine sediment 12-gene combination can be used alone according to the combination, or the two combinations can be used in combination.

3. A reagent combination for detecting the prostate cancer RNA detection marker combination according to claim 1, characterized in that, The reagent combination contains primers and probes corresponding to the urine supernatant 12-gene combination described in the following table:

4. The reagent combination according to claim 3, characterized in that, The reagent combination also contains primers and probes corresponding to the urine sediment 12-gene combination described in the following table:

5. Use of a detection reagent combination in the preparation of a prostate cancer diagnostic kit, characterized in that, The detection reagent combination is used to detect the prostate cancer RNA detection marker combination described in claim 1, and the detection reagent combination contains primers and probes corresponding to the urine supernatant 12-gene combination described in the following table: And, the detection reagent combination also contains primers and probes corresponding to the urine sediment 12-gene combination described in the following table: The primer-probe corresponding to the urine supernatant 12-gene combination and the primer-probe corresponding to the urine sediment 12-gene combination can be used alone according to the combination, or the two combinations can be used in combination.

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