Urine protein composition for diagnosing urothelial carcinoma and application thereof

Abstract

The invention provides a urine protein composition for diagnosing urothelial carcinoma and application of the urine protein composition, and relates to the technical field of clinical medicine. The urine protein composition is at least one of SUSD3, IGLC7, IQCA1, FCN3, C4BPB, BPGM, SERPIND1, UBR4, FCGR1A, RNASE3, PCDHGA6, TCEAL2 and F13B, and the urine protein composition is at least one of the following components. According to the kit, the defects in the prior art are overcome, the differential urine proteins of a healthy group and a disease group are determined, and the differential proteins are adopted as markers, so that the urinary tract epithelial cancer can be noninvasively, rapidly and preliminarily diagnosed, and a new direction is provided for clinical diagnosis.

Description

Technical Field

[0001] The present invention relates to the technical field of clinical medicine, and in particular to a urine protein composition for diagnosing urothelial carcinoma and an application thereof. Background Art

[0002] Urothelial carcinoma (UC) is a common malignant tumor of the urinary system. It originates from malignant tumors of the urothelial cells of the urinary tract and includes renal pelvic, ureteral, bladder, and urethral cancers. The prognosis of UC is influenced by multiple factors, including tumor stage, grade, patient age, and physical condition. Early-stage UC has a relatively good prognosis with aggressive treatment, whereas advanced UC has a poorer prognosis, with a low 5-year survival rate. Upper tract UC is particularly characterized by rapid disease progression, a high recurrence rate, and a poor prognosis.

[0003] Based on the above-mentioned treatment characteristics of urothelial carcinoma, early detection and early treatment can effectively prolong the patient's survival period, so the early diagnosis of urothelial carcinoma is crucial. The existing diagnostic methods for urothelial carcinoma usually include urine examination, ultrasound examination, CT examination, magnetic resonance imaging, intravenous urography, cystoscopy, pathological examination, etc. Except for urine examination, the costs are relatively high, and some examinations are more painful. Patients often avoid these examinations when they are uncertain. The existing urine examination is usually based on the discovery of hematuria in routine urine examination to remind patients, and to look for cancer cells by examining the exfoliated cells in the urine. However, the overall sensitivity of the examination is low, and most of the examinations cannot be self-tested by patients with test strips, so the existing examinations often cannot achieve more accurate and convenient identification of patients in clinical practice.

[0004] Urine is not regulated by a steady-state mechanism. It is a metabolic product of the body and is enriched with various changes in the body. It can be obtained in large quantities in a non-invasive way. It is an ideal source of disease markers and has significant clinical application prospects in the early diagnosis of diseases. Based on this, urine protein can be used as a marker for diagnosis and non-invasive self-testing of the development of urothelial carcinoma. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, the present invention provides a urine protein composition for diagnosing urothelial carcinoma and its application. By determining the differential urine proteins between the healthy group and the disease group and using these differential proteins as markers, a non-invasive and rapid preliminary diagnosis of urothelial carcinoma can be made, providing a new direction for clinical diagnosis.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions: A urine protein composition for diagnosing urothelial carcinoma, wherein the urine protein composition is at least one of SUSD3, IGLC7, IQCA1, FCN3, C4BPB, BPGM, SERPIND1, UBR4, FCGR1A, RNASE3, PCDHGA6, TCEAL2, and F13B.

[0007] Preferably, the urine protein marker composition further contains: any one or more of IGKV1D-43, C8G, ZFYVE19, IGHG3, SRP9, AFDN, FETUB, GPLD1, SNU13, MINDY3, SERPINC1, IGLV1-40, MTPN, SERPINA10, SH3GLB2, ATP5MG, AP3B1, PRPS1, UBL4A, NRDC, BDH1, EFHD2, ABCB6, FBLL1, GATD3, IGHV5-51, SMG8, NCF1B, PLCB3, NSFL1C, IGHV3-49, PSMB3, SLC13A2, IGHV1-45, IGHV3-35, RBM12, IGHV1-69, and ST3GAL2.

[0008] Preferably, compared with healthy controls, an increased expression level of the urine protein marker composition indicates that the subject has a risk of developing urothelial cancer.

[0009] The invention relates to the use of an identification reagent for a protein marker composition for diagnosing urothelial carcinoma in the preparation of a product for diagnosing urothelial carcinoma, wherein the identification reagent is a reagent for identifying the content of any one or more proteins including SUSD3, IGLC7, IQCA1, FCN3, C4BPB, BPGM, SERPIND1, UBR4, FCGR1A, RNASE3, PCDHGA6, TCEAL2, and F13B in urine.

[0010] Preferably, the identification reagent also identifies the content of any one or more proteins of IGKV1D-43, C8G, ZFYVE19, IGHG3, SRP9, AFDN, FETUB, GPLD1, SNU13, MINDY3, SERPINC1, IGLV1-40, MTPN, SERPINA10, SH3GLB2, ATP5MG, AP3B1, PRPS1, UBL4A, NRDC, BDH1, EFHD2, ABCB6, FBLL1, GATD3, IGHV5-51, SMG8, NCF1B, PLCB3, NSFL1C, IGHV3-49, PSMB3, SLC13A2, IGHV1-45, IGHV3-35, RBM12, IGHV1-69, and ST3GAL2 in urine.

[0011] Preferably, the product contains any of the above-mentioned identification reagents.

[0012] Preferably, the product is a test paper, a test kit, a chip, an antigen-antibody conjugate, a probe or a test strip.

[0013] The present invention provides a urine protein composition for diagnosing urothelial carcinoma and its application, which has the following advantages over the prior art: Through experimental research, the present invention found that compared with the urine of the healthy group, the levels of 51 typical proteins in the urine of patients with urothelial carcinoma were significantly increased. In addition, among these significantly increased proteins, the levels of 13 proteins, including SUSD3, IGLC7, IQCA1, FCN3, C4BPB, BPGM, SERPIND1, UBR4, FCGR1A, RNASE3, PCDHGA6, TCEAL2, and F13B, increased particularly significantly. The comprehensive changes in these proteins can further accurately determine whether the patient has urothelial carcinoma. Designing detection reagents or related products for related proteins based on this phenomenon can preliminarily screen patients for the risk of urothelial carcinoma in a non-invasive manner, and has good application prospects for actual clinical testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a heat map of the union of differentially expressed proteins in the healthy group and the patient group in the embodiment of the present invention. DETAILED DESCRIPTION

[0015] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0016] Example 1: Acquisition of differentially expressed proteins: 1. Sample acquisition: Preoperative urine samples from 16 patients with urothelial carcinoma (NB 1-16) were collected between June and July 2024. The patients included 13 males and 3 females, ranging in age from 42 to 86 years (median age 67.5 years, mean age 66.3 years). Ten cases arose in the bladder, and 5 in the renal pelvis / ureter. Pathological diagnosis revealed one low-grade urothelial carcinoma, one predominantly low-grade urothelial carcinoma (containing >5% high-grade components), and the remainder high-grade urothelial carcinomas (including one with squamous differentiation and one with glandular differentiation). Five urine samples from healthy adults (four males and one female) were collected as a control group (CTRL 1-5) and stored at -80°C.

[0017] 2. Protein extraction: Each group of samples was removed from -80°C, and pH 8.0 Tris-HCl was added to all samples to a final concentration of 50 mM. The samples were centrifuged at 1000 g for 5 min, and the supernatant was collected. The samples were centrifuged at 17000 g for 10 min, and an equal volume of methanol and 1 / 4 volume of chloroform were added to the supernatant. The samples were shaken for 15 s and allowed to stand at room temperature for 5 min. The supernatant was discarded, and an equal volume of methanol was added. The samples were shaken for 15 sec and centrifuged at 12000 g for 15 min at room temperature. The supernatant was discarded, and 80 μL of lysis buffer (1% SDC, 100 mM Tris-HCl, pH 7.6) was added for resolubilization. The protein concentration was determined using a BCA kit.

[0018] 3. Enzymatic hydrolysis with pancreatic enzymes: Equal amounts of protein from each sample were digested with lysis buffer (4% SDS, 100mM Tris-HCl, pH 7.6). Dithiothreitol (DTT) was then added to a final concentration of 5 mM, and the cells were reduced at 56°C for 30 min. Iodoacetamide (IAA) was then added to a final concentration of 11 mM, and the cells were incubated at room temperature in the dark for 15 min. Urea was diluted with TEAB to ensure a concentration below 2 M. Trypsin was then added at a 1:50 ratio (protease:protein, m / m) and digestion was allowed to proceed overnight. Trypsin was then added at a 1:100 ratio (protease:protein, m / m) and digestion continued for 4 h.

[0019] 4. Liquid chromatography-mass spectrometry analysis Peptides were dissolved in liquid chromatography mobile phase A and separated using a NanoElute ultra-high performance liquid chromatography system. Mobile phase A consisted of 0.1% formic acid and 2% acetonitrile in water; mobile phase B consisted of acetonitrile in water containing 0.1% formic acid. The gradient was set as follows: 6% to 24% B (0–14 min); 24% to 35% B (14–16 min); 35% to 80% B (16–18 min); and 80% B (18–20 min), with a flow rate maintained at 500 nL / min. Following ultra-high performance liquid chromatography separation, peptides were injected into a Capillary ion source for ionization and subsequently acquired on a timsTOF Pro mass spectrometer. The source voltage was set to 1.75 kV, and peptide precursor ions and their secondary fragments were detected and analyzed using the TOF. The data acquisition mode used was data-independent parallel accumulation serial fragmentation (dia-PASEF) mode. The primary mass spectrometry scan range was set to 300-1500 m / z. After one primary mass spectrum was acquired, 20 PASEF mode acquisitions were performed. The secondary mass spectrometry scan range was 400-850, with every 7 m / z as a window.

[0020] 5. Cluster analysis based on protein function enrichment: (1) Gene Ontology analysis was used to annotate the identified proteins using the Pfam database and the corresponding PfamScan tool. The subcellular structure prediction analysis, COG / KOG annotation, Reactome annotation, WikiPathways pathway annotation, and HallMark signature gene set annotation were performed using the PSORTb software. The protein annotation was performed by annotating the transcription factor information corresponding to the protein using the databases TRRUST and GTRD. (2) Protein function enrichment was performed using GO enrichment analysis, KEGG pathway enrichment analysis, protein domain enrichment analysis, Reactome pathway enrichment analysis, and WikiPathways pathway enrichment analysis.

[0021] (3) Cluster analysis of differentially expressed proteins (functional enrichment) based on different groups was used to study their potential connections and differences in specific functions (GO, KEGG pathways, protein domains, Reactome, WikiPathways). First, the functional classification information and corresponding enrichment P values ​​of all protein groups were collected, and then the functional classifications that were significantly enriched (P value < 0.05) in at least one protein group were screened. The P value data matrix obtained by screening was first logarithmically transformed with -Log10 as the base, and the hierarchical clustering (Euclidean distance, average linkage clustering) method was used to perform unilateral cluster analysis on the transformed data set; in order to explore the distribution and differences of protein intensity values ​​between different samples, the protein intensity values ​​of each sample were extracted.

[0022] Draw an expression heat map of the union of all differentially expressed proteins in the healthy group and the patient group (at least 2 / 3 of the total samples have quantitative values) ( Figure 1 ), used to display the relative expression levels of multiple differentially expressed proteins in different samples, presenting the clustering relationship of the relative expression of differentially expressed proteins. Each row represents a differentially expressed protein, and each column represents a sample. Red represents high expression, blue represents low expression, and gray indicates that the corresponding sample cannot be quantified.

[0023] The upregulated proteins were screened out from all the differentially expressed proteins and detected by mass spectrometry. The protein spectrum signal intensity ratios of the upregulated proteins in the patient group and the healthy group were calculated and statistically analyzed. The value ≥4 was considered to be significantly upregulated. A total of 51 differentially expressed proteins were screened out: SUSD3, IGLC7, IQCA1, FCN3, C4BPB, BPGM, SERPIND1, UBR4, FCGR1A, RNASE3, PCDHGA6, TCEAL2, F13B, IGKV1D-43, C8G, ZFYVE19, IGHG3, SRP9, AFDN, FETUB, GPLD1, SNU13, MINDY3, SER PINC1, IGLV1-40, MTPN, SERPINA10, SH3GLB2, ATP5MG, AP3B1, PRPS1, UBL4A, NRDC, BDH1, EFHD2, ABCB6, FBLL1, GATD3, IGHV5-51, SMG8, NCF1B, PLCB3, NSFL1C, IGHV3-49, PSMB3, SLC13A2, IGHV1-45, IGHV3-35, RBM12, IGHV1-69, ST3GAL2; and the protein spectrum signal intensity ratio data of the patient group and the healthy group of each protein are shown in the following table: As can be seen from the above table, the NB / CTRL ratios of 13 proteins, including SUSD3, IGLC7, IQCA1, FCN3, C4BPB, BPGM, SERPIND1, UBR4, FCGR1A, RNASE3, PCDHGA6, TCEAL2, and F13B, are all greater than 8, indicating the highest upregulation significance in this group.

[0024] That is, there are significant differences in these upregulated proteins between the healthy group and the patient group, which can provide a new detection direction for subsequent detection of urothelial carcinoma.

[0025] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A urine protein composition for diagnosing urothelial carcinoma, characterized in that: The urine protein composition is at least one of SUSD3, IGLC7, IQCA1, FCN3, C4BPB, BPGM, SERPIND1, UBR4, FCGR1A, RNASE3, PCDHGA6, TCEAL2, and F13B.

2. The protein marker composition according to claim 1, characterized in that: The urine protein marker composition further contains: any one or more of IGKV1D-43, C8G, ZFYVE19, IGHG3, SRP9, AFDN, FETUB, GPLD1, SNU13, MINDY3, SERPINC1, IGLV1-40, MTPN, SERPINA10, SH3GLB2, ATP5MG, AP3B1, PRPS1, UBL4A, NRDC, BDH1, EFHD2, ABCB6, FBLL1, GATD3, IGHV5-51, SMG8, NCF1B, PLCB3, NSFL1C, IGHV3-49, PSMB3, SLC13A2, IGHV1-45, IGHV3-35, RBM12, IGHV1-69, and ST3GAL2.

3. The urine protein marker composition according to any one of claims 1-2, characterized in that: Compared to healthy controls, an increased expression level of the urine protein marker composition indicates that the subject is at risk of developing urothelial cancer.

4. Use of an identification reagent for a protein marker composition for diagnosing urothelial carcinoma in the preparation of a product for diagnosing urothelial carcinoma, characterized in that: The identification reagent is a reagent for identifying the content of any one or more proteins of SUSD3, IGLC7, IQCA1, FCN3, C4BPB, BPGM, SERPIND1, UBR4, FCGR1A, RNASE3, PCDHGA6, TCEAL2, and F13B in urine.

5. The use according to claim 4, characterized in that: The identification reagent also identifies the content of any one or more proteins of IGKV1D-43, C8G, ZFYVE19, IGHG3, SRP9, AFDN, FETUB, GPLD1, SNU13, MINDY3, SERPINC1, IGLV1-40, MTPN, SERPINA10, SH3GLB2, ATP5MG, AP3B1, PRPS1, UBL4A, NRDC, BDH1, EFHD2, ABCB6, FBLL1, GATD3, IGHV5-51, SMG8, NCF1B, PLCB3, NSFL1C, IGHV3-49, PSMB3, SLC13A2, IGHV1-45, IGHV3-35, RBM12, IGHV1-69, and ST3GAL2 in urine.

6. A product for diagnosing urothelial carcinoma, characterized in that: The product contains the identification reagent according to any one of claims 4-5.

7. The product according to claim 6, characterized in that: The product is a test paper, a test kit, a chip, an antigen-antibody conjugate, a probe or a test strip.

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