Urine-based prostate cancer RNA diagnostic marker combination and application thereof in preparation of prostate cancer diagnostic kit

Abstract

The invention discloses a prostate cancer RNA (Ribonucleic Acid) detection marker combination based on urine and application of the prostate cancer RNA detection marker combination in preparation of a prostate cancer diagnostic kit. The RNA detection marker combination comprises a urine supernatant 12 gene combination and a urinary sediment 12 gene combination. The urine supernatant 12 gene combination and the urinary sediment 12 gene combination can be independently used according to the combination, and can also be combined for use. The detection marker combination further comprises primers and probes which respectively correspond to the urine supernatant 12 gene combination and the urinary sediment 12 gene combination. When the kit is used for detecting the prostatic cancer, morning urine or urine obtained after transrectal finger diagnosis does not need to be collected, only common random urine is needed, collection of samples is greatly facilitated, and the accessibility of detection is improved. The gene combination is more, the interference of a sample on a detection result is smaller, and the detection accuracy, sensitivity and specificity are higher.

Description

Technical Field

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

[0002] At present, 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 cause false positive results. Relying solely on PSA for prostate cancer screening will lead to over-puncture of patients with benign prostate diseases in the gray zone. 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 cancer, measures such as active surveillance can be selected, while clinically significant prostate cancer requires active treatment. However, due to the influence of the sampling site and tissue size of the 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 the patients with a 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, risk stratification of prostate cancer patients and identification of 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 into urine, so prostate cancer tumor markers can be detected through urine. The PCA3 (prostate cancer associated 3 Gene, abbreviated as PCA3, novel prostate cancer antigen 3) score is the first detection score based on urine tumor markers 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 the patients 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: Patent CN116377068A discloses a prostate cancer RNA biomarker NKX3-1 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.

[0006] 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.

[0007] Therefore, generally speaking, the existing urine-based prostate cancer detection methods have the following problems.

[0008] First, the requirements for test samples are relatively high, often requiring morning urine or urine after digital rectal examination, 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, making it difficult to collect qualified morning urine.

[0009] Second, the gene combinations used in the detection are few, and the samples have a large interference on the test results, affecting the accuracy of the detection.

[0010] 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.

[0011] Fourth, the 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, making it difficult to correctly reflect the occurrence and development of prostate cancer and unable to be truly used for clinical prostate cancer screening. Summary of the Invention

[0012] To solve the above problems, the present invention proposes a combination of prostate cancer RNA detection markers based on ordinary urine. The detection markers of the present invention can use ordinary random urine, without the need to use morning urine or urine after digital rectal examination, greatly facilitating 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.

[0013] The technical solution of the present invention is: a combination of RNA detection markers for prostate cancer based on urine, 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, 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 includes the 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.

[0014] Among them, 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.

[0015] Among them, the genes and corresponding primers in the urine supernatant 12-gene combination are shown in Table 1.

[0016] Table 1 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 Among them, the genes and corresponding primers of the urine sediment 12-gene group are shown in Table 2.

[0017] Table 2 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 Furthermore, the present invention also discloses an application of a combination of RNA detection markers for prostate cancer based on urine in the preparation of a prostate cancer diagnostic kit. The detection marker combination contains primers and probes corresponding to the urine supernatant 12-gene combination as shown in Table 3 below: Table 3 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 And, the detection marker combination further contains primers and probes corresponding to the urine sediment 12-gene combination as shown in Table 4 below: Table 4 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 The primers and probes corresponding to the urine supernatant 12-gene combination and the primers and probes 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.

[0018] Among them, the urine is random urine. Different from many prostate cancer detection reagents and methods in the prior art, the present invention does not have to use morning urine or urine after digital rectal examination, but only ordinary random urine is required.

[0019] The present invention respectively uses 12 RNA markers for urine supernatant and urine sediment, 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.

[0020] Through the above gene combination, the present invention can accurately distinguish prostate cancer from other prostate diseases, meeting the actual clinical needs.

[0021] 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.

[0022] The detection methods for the RNA markers of urine supernatant and urine sediment proposed by the present invention can be used alone or in combination.

[0023] Compared with the prior art, the present invention provides an RNA detection marker and method for prostate cancer based on ordinary urine, having the following beneficial effects: 1. 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 sample collection, improves the accessibility of detection and the possibility of popularization and application; 2. Respectively using 12 RNA markers for urine supernatant and urine sediment, there are more gene combinations, the interference of samples on the detection results is smaller, and the detection accuracy is improved; 3. By detecting a variety of RNA markers in urine supernatant and urine sediment, it can comprehensively reflect the occurrence and development of prostate cancer, providing an important basis for the early diagnosis and treatment of the disease; 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 popularization and application.

[0024] The detection marker combination of the present invention has high sensitivity, specificity, and detection accuracy. Description of the Drawings

[0025] Figure 1 It is the AUROC curve of the urine sediment sample in Example 2.

[0026] Figure 2 It is the AUROC curve of the urine supernatant sample in Example 3.

[0027] Figure 3 It is the comparison of the AUROC curves of the results of the RNA markers in the urine sediment of Example 5 with the detection results of PSA and PI-RADS.

[0028] Figure 4 It is the comparison of the AUROC curves of the results of the RNA markers in the urine supernatant of Example 5 with the detection results of PSA and PI-RADS.

[0029] Figure 5 It is the comparison of the AUROC curves 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 Embodiments

[0030] 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.

[0031] Example 1 Screening of Gene Combinations 1. Research Population Patients who visited the Cancer Hospital of Fudan University due to urinary system diseases in 2024 were selected as the research subjects. The inclusion criteria for the population in this study were: Inclusion Criteria: 1) Age ≥ 18 years old; 2) Urine sample volume ≥ 50 mL; 3) Meeting one of the following conditions: a. Patients with suspected prostate cancer indicated by elevated PSA, positive digital rectal examination, imaging, etc., and who are scheduled for prostate biopsy for a definite diagnosis; b. Patients with a definite diagnosis of prostate cancer by prostate biopsy, and who are scheduled for radical prostatectomy and / or endocrine therapy and / or radiotherapy; c. Patients with a clinical diagnosis of benign urinary system diseases, including: benign prostatic hyperplasia, prostatitis, urinary system stones, renal cysts, ureteral stricture, urethral stricture, hypospadias, urinary tract infection, glandular cystitis and other benign urinary system diseases; d. Patients with other tumors clinically or pathologically diagnosed as non-prostate cancer, including bladder cancer, renal cancer, ureteral cancer, renal pelvic cancer, etc.

[0032] Exclusion criteria: 1) Patients who have previously undergone transurethral resection or enucleation of the prostate, or prostate radiotherapy; 2) Patients who have previously received endocrine therapy or have taken 5α-reductase inhibitors within 6 months; 3) Any other conditions that the researcher deems unsuitable for participation in this trial.

[0033] The patients were from the outpatient or inpatient cases of the Department of Urology, Fudan University Shanghai Cancer Center. This study was approved by the Ethics Committee of our hospital, and all study subjects signed informed consent forms.

[0034] 2. Experimental methods 1) Urine collection Use a urine cup to collect approximately 50 - 120 mL of random urine from the study subjects.

[0035] 2) Pretreatment of urine 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.

[0036] 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).

[0037] 3) Enrichment of urine cfRNA (cell-free RNA, abbreviated as cfRNA, cell-free RNA) According to the ratio of urine: precipitant (16% PEG) (Polyethylene glycol, abbreviated as PEG, polyethylene glycol) ≈ 1:1, mix well and precipitate overnight at 4°C; Centrifuge the 50 mL centrifuge tube with the precipitate at 3000 g for 1 h at 4°C, carefully remove the supernatant, and keep the remaining precipitate for backup.

[0038] 4) Extraction of urine sediment and supernatant cfRNA Take the urine sediment and supernatant, add 1 mL of Trizol (Trizol reagent, total RNA extraction reagent) and mix well. Then add 200 μL of chloroform to the centrifuge tube, vortex to mix well, and centrifuge at 12000 rpm for 15 min to separate the layers; Gently aspirate the upper colorless and transparent liquid into a new centrifuge tube, add an equal volume of ethanol and mix well; Use the purification column method to extract RNA.

[0039] 5) RNA sequencing Enrich mRNA from total RNA using oligo (dT) magnetic beads. After fragmentation, synthesize the first-strand cDNA using random hexamer primers, and then synthesize the second-strand cDNA. After end repair, A-tailing, adapter ligation, fragment selection, amplification, and purification, the library is ready.

[0040] Perform high-throughput sequencing on the above library using an Illumina Novaseq 6000 sequencer. 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.

[0041] 6) Mining of biomarkers Divide the data at different levels (urinary sediment and urinary 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 distinguish prostate cancer from non-prostate cancer.

[0042] 7) Construction of a prostate cancer prediction model Table 5 12-gene combination in urinary supernatant

[0043] The primers and probes corresponding to the 12 genes in the urinary supernatant are shown in Table 6.

[0044] Table 6

[0045] Table 7 12-gene combination in urinary sediment

[0046] The primers and probes corresponding to the 12 genes in the urinary sediment are shown in Table 8.

[0047] Table 8 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 8) Detect new urine samples using the above-constructed prostate cancer prediction model.

[0048] Example 2 Detection using RNA biomarkers in urinary sediment 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). Analyze the samples using 12 RNA biomarkers in the urinary sediment. The analysis results are shown in Table 9.

[0049] Table 9 Analysis performance of 12-gene combination in urinary sediment

[0050] 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 .

[0051] For invasive prostate cancer (Gleason score ≥ 7), the detection accuracy was 83.72% (36 / 43).

[0052] Example 3 Using RNA markers in urine supernatant In this example, 102 random urine samples were analyzed, including 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). Twelve RNA markers in the urine supernatant were used to analyze the samples. The results are shown in Table 10.

[0053] Table 10 Analysis performance of 12-gene combination in urine supernatant

[0054] For these 102 samples, the sensitivity of the present invention was 80.77%, the specificity was 86.00%, and the AUROC reached 0.8746.

[0055] For invasive prostate cancer (Gleason score ≥ 7), the detection accuracy was 87.50% (35 / 40). The results are as Figure 2 .

[0056] Example 4 Combining RNA markers in urine sediment and urine supernatant 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), a total of 152 random urine samples were used. A combined detection method using urine sediment and urine supernatant RNA was used. A positive result was defined as positive for either the urine sediment or urine supernatant test. The results are shown in Table 11.

[0057] Table 11

[0058] For these 152 samples, the sensitivity of the present invention was 83.95%, the specificity was 80.28%, and the accuracy was 82.24%.

[0059] For invasive prostate cancer (Gleason score ≥ 7), the detection accuracy rate was 90.63% (58 / 64).

[0060] Example 5 Comparison of the RNA markers of the present invention with commonly used clinical indicators (PSA and PI-RADS) For prostate cancer, PI-RADS refers to the Prostate Imaging Reporting and Data System, which is a system for standardizing prostate magnetic resonance imaging (MRI) reports and aims to improve the accuracy and consistency of prostate cancer diagnosis. "PSA" and "MRI (i.e., the PI-RADS indicator)" are common biomarkers and imaging tools in prostate cancer diagnosis.

[0061] 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.

[0062] MRI is an imaging examination that can provide more intuitive tumor information, but there are certain diagnostic difficulties in actual applications, such as how to distinguish between benign and malignant lesions.

[0063] The PI-RADS score ranges from 1 to 5, among which, PI-RADS 1: Very low risk, and it is almost impossible to have clinically significant prostate cancer.

[0064] PI-RADS 2: Low risk, and it is unlikely to have clinically significant prostate cancer.

[0065] PI-RADS 3: Moderate risk, and clinically significant prostate cancer may or may not exist.

[0066] PI-RADS 4: High risk, and it is very likely to have clinically significant prostate cancer.

[0067] PI-RADS 5: Very high risk, and it is extremely likely to have clinically significant prostate cancer.

[0068] For the 152 samples in Example 4, their clinical data were collected simultaneously, PSA and PI-RADS results were obtained, and the results of the RNA markers in the urinary sediment constructed by the present invention were compared with the PSA and PI-RADS detection results as shown in Table 12 and Figure 3 .

[0069] Table 12

[0070] The results of the RNA markers in the urine supernatant constructed using the present invention, compared with the PSA and PI-RADS test results, are shown in Table 13 and Figure 4 。

[0071] Table 13

[0072] The results of the integrated analysis of the RNA markers in the urine sediment and urine supernatant constructed using the present invention, compared with the PSA and PI-RADS test results, are shown in Table 14 and Figure 5 。

[0073] Table 14

[0074] Note: If any one of the urine supernatant and urine sediment results is positive, it is judged as positive.

[0075] As can be seen from the comparison in Example 5: 1) The AUROC values of the RNA markers of the present invention are all the largest.

[0076] 2) The test results of the RNA markers of the present invention are better than the PI-RADS scoring system.

[0077] 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.

[0078] The present invention uses 12 RNA markers in the urine supernatant and urine sediment. Not only can the 12 RNA markers in the urine supernatant and urine sediment be used separately, but also the 12 RNA markers in the urine supernatant and urine sediment can be used in combination. There are more gene combinations, the interference of samples on the test 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.

[0079] The present invention does not require the collection of 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.

[0080] 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 prostate cancer RNA detection marker combination, characterized in that: The RNA detection marker combination includes a 12-gene combination of urine supernatant and a 12-gene combination of urine sediment; wherein the 12-gene combination of urine supernatant includes 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 12-gene combination of urine sediment includes 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, characterized in that: The urine supernatant 12-gene combination and the urine sediment 12-gene combination can be used alone or in combination.

3. The prostate cancer RNA detection marker combination according to claim 1, characterized in that: The combination also includes primers and probes corresponding to the urine supernatant 12 gene combination as described in the following table: 。 4. The prostate cancer RNA detection marker combination according to claim 1, characterized in that: The combination also includes primers and probes corresponding to the 12-gene combination of urine sediment as described in the following table: 。 5. Use of a urine-based prostate cancer RNA detection marker composition in the preparation of a prostate cancer diagnostic kit, characterized in that: The detection marker composition comprises primers and probes corresponding to the 12 gene combinations in urine supernatant as described in the following table: And, the detection marker composition further comprises primers and probes corresponding to the 12 gene combinations of urine sediment as described in the following table: The primer probes corresponding to the 12 gene combination in urine supernatant and the primer probes corresponding to the 12 gene combination in urine sediment can be used alone as a combination, or the two combinations can be used in combination.

6. Use of a urine-based prostate cancer RNA detection marker composition in the preparation of a prostate cancer diagnostic kit, characterized in that: The urine is random urine.

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