Protein marker composition for diagnosing prostate cancer and application thereof

Abstract

The invention provides a protein marker composition for diagnosing prostatic cancer and application of the protein marker composition, and relates to the technical field of clinical medicine. RPL34, RPL37A, RPL9, RPL38, UQCR10, SLC4A1, MGST1, ATP5F1E, TMEM109, POLR1C, RAN, CYB5A, HAS1 and RPL31 are adopted as relatively remarkable differential urine proteins of the prostate cancer, 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 prostate 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 particularly relates to a protein marker composition for diagnosing prostate cancer and its application. Background Art

[0002] Prostate cancer is one of the most common malignant tumors in the urogenital male reproductive system. The survival time of prostate cancer patients is closely related to the stage of malignant tumors at the time of clinical diagnosis. Therefore, "early screening, early diagnosis, and early treatment" of high-risk groups of prostate cancer is an effective means to improve the overall survival rate of prostate cancer patients in China.

[0003] At present, in the early stage, prostate cancer is prone to have some symptoms that overlap with those of prostate hyperplasia, such as common symptoms like urgency, frequency, dysuria, difficulty in urination, or thinning of the urinary stream, dribbling after urination, etc., which have no specific differences from prostate hyperplasia. Therefore, many prostate cancer patients have no specific symptoms in the early stage clinically, and the methods of diagnosis often rely on tumor markers, imaging examinations, and prostate puncture. The overall examination is relatively cumbersome and costly, or the examination requires blood sampling and puncture, which is somewhat harmful. On this basis, early-stage patients usually do not want to undergo further examinations, which easily leads to delays in the condition and affects subsequent treatment. On this basis, it is objectively important to develop a new convenient and low-cost diagnostic method.

[0004] Urine is not regulated by a homeostatic mechanism. It is a metabolite of the body, enriched with various changes in the body, can be obtained in large quantities in a non-invasive manner, and is an ideal source of disease markers, having great clinical application prospects in the early diagnosis of diseases. Based on this, it is necessary to develop urine proteins as markers for the early diagnosis of prostate cancer. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the present invention provides a protein marker composition for diagnosing prostate cancer and its application. By determining the differential urine proteins between the healthy group and the disease group and using these differential proteins as markers, prostate cancer can be preliminarily diagnosed non-invasively and quickly, providing a new direction for clinical diagnosis.

[0006] To achieve the above objectives, the present invention is realized through the following technical solutions:

[0007] A protein marker composition for diagnosing prostate cancer, wherein the protein marker composition is any one or more of: RPL34, RPL37A, RPL9, RPL38, UQCR10, SLC4A1, MGST1, ATP5F1E, TMEM109, POLR1C, RAN, CYB5A, HAS1, RPL31.

[0008] Preferably, the protein marker composition further contains any one or more of CAPS, ATP5F1B, RPL11, RPS28, MT-CO2, PRPS1, MGST2, ACOT11, ATP5F1A, CAT, ATIC, SH3BGRL2, FAM98A, HADHB, BCAS1, TUBB4B, RPL12, PLEKHA6, PDLIM1, SLC25A5, HSPB1, RPL30, SYNGR2, RPL29, NANS, RPSA2, DNPEP, SFN, RPL13, ACADSB, UBE2L3, UQCRC1, HADH, CCT4, CCT7, RPL32, CTNNA1, EIF3I.

[0009] Preferably, the protein marker composition is urinary protein.

[0010] Preferably, compared with healthy controls, an increase in the expression level of the protein marker composition indicates that the subject has a risk of prostate cancer.

[0011] Use of an identification reagent for a protein marker composition for diagnosing prostate cancer in the preparation of a product for diagnosing prostate cancer, wherein the identification reagent is a reagent for determining the content of any one or more of RPL34, RPL37A, RPL9, RPL38, UQCR10, SLC4A1, MGST1, ATP5F1E, TMEM109, POLR1C, RAN, CYB5A, HAS1, RPL31 in urine.

[0012] Preferably, the identification reagent also determines the content of any one or more of CAPS, ATP5F1B, RPL11, RPS28, MT-CO2, PRPS1, MGST2, ACOT11, ATP5F1A, CAT, ATIC, SH3BGRL2, FAM98A, HADHB, BCAS1, TUBB4B, RPL12, PLEKHA6, PDLIM1, SLC25A5, HSPB1, RPL30, SYNGR2, RPL29, NANS, RPSA2, DNPEP, SFN, RPL13, ACADSB, UBE2L3, UQCRC1, HADH, CCT4, CCT7, RPL32, CTNNA1, EIF3I in urine.

[0013] Design a product for diagnosing and identifying prostate cancer using one or more of the above identification reagents, and the product can be a test strip, kit, chip, antigen-antibody conjugate, probe or test strip.

[0014] The present invention provides a protein marker composition for diagnosing prostate cancer and its applications. Compared with the prior art, the advantages are as follows:

[0015] Through experimental research, the present invention finds that compared with the urine of the healthy group, the contents of 52 typical proteins in the urine of prostate cancer patients are significantly increased. Additionally, among these significantly increased proteins, the contents of 14 proteins, namely RPL34, RPL37A, RPL9, RPL38, UQCR10, SLC4A1, MGST1, ATP5F1E, TMEM109, POLR1C, RAN, CYB5A, HAS1, and RPL31, are particularly significantly increased. Through the comprehensive changes of these proteins, it is possible to further accurately determine whether a patient has prostate cancer. Designing detection reagents or related products for these proteins can preliminarily screen the risk of prostate cancer in patients non-invasively, showing good application prospects for actual clinical detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a bar chart showing the distribution differences of protein intensity values in different samples in the embodiments of the present invention;

[0017] Figure 2 It is a heat map of differential proteins in different samples in the embodiments of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0019] Example 1:

[0020] Obtaining differential proteins:

[0021] 1. Obtaining samples:

[0022] Recruit 10 prostate cancer patients and 6 healthy male volunteers, collect urine, and record them as the healthy group (C1 - C6) and the patient group (P7 - 1 to P7 - 10) respectively, and store them at -80 °C for later use (it was found that the urine of sample C2 was insufficient during the detection process, so the following experiments do not refer to the detection results of C2);

[0023] 2. Protein extraction:

[0024] Take out each group of samples from -80°C, add pH 8.0 Tris-HCl to all samples to a final concentration of 50 mM, centrifuge at 1000 g for 5 min, take the supernatant, centrifuge at 17000 g for 10 min, take the supernatant and add an equal volume of methanol and 1 / 4 volume of chloroform. After shaking for 15 s, let it stand at room temperature for 5 min. Centrifuge at 12000 g at room temperature for 15 min, then discard the supernatant. Add an equal volume of methanol again, shake for 15 sec, centrifuge at 12000 g at room temperature for 15 min, and then discard the supernatant. Add 80 μl of lysis buffer (1% SDC, 100 mM Tris-HCl, pH 7.6) for resuspension, and use the BCA kit to measure the protein concentration.

[0025] 3. Trypsin digestion:

[0026] Take equal amounts of the proteins extracted from the above samples for digestion. Adjust the volume to the same with lysis buffer (4% SDS, 100 mM Tris-HCl, pH 7.6), then add dithiothreitol (DTT) to a final concentration of 5 mM, and reduce at 56°C for 30 min. Then add iodoacetamide (IAA) to a final concentration of 11 mM, and incubate in the dark at room temperature for 15 min. Add TEAB to dilute urea to ensure the concentration is less than 2 M. Add trypsin at a ratio of 1:50 (protease:protein, m / m), and digest overnight. Then add trypsin at a ratio of 1:100 (protease:protein, m / m) and continue to digest for 4 h.

[0027] 4. Liquid chromatography-mass spectrometry analysis:

[0028] The peptides are dissolved in mobile phase A of liquid chromatography and separated using a NanoElute ultra-high performance liquid system. Mobile phase A is an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B is an acetonitrile-aqueous solution containing 0.1% formic acid. Liquid phase gradient setting: 0 - 14 min, 6% - 24% B; 14 - 16 min, 24% - 35% B; 16 - 18 min, 35% - 80% B; 18 - 20 min, 80% B, and the flow rate is maintained at 500 nl / min. After the peptides are separated by the ultra-high performance liquid system, they are injected into the Capillary ion source for ionization and then into the timsTOF Pro 2 mass spectrometer for data acquisition. The ion source voltage is set to 1.75 kV, and both the peptide parent ions and their secondary fragments are detected and analyzed using TOF. The data acquisition mode uses the data-independent parallel accumulation serial fragmentation (dia-PASEF) mode. The primary mass spectrometry scanning range is set to 300 - 1500 m / z. After one primary mass spectrometry acquisition, 20 PASEF mode acquisitions are performed. The secondary mass spectrometry scanning interval is 400 - 850, and each window is 7 m / z.

[0029] 5. Cluster analysis based on protein function enrichment:

[0030] (1) Protein annotation was performed by means of Gene Ontology analysis, protein domain annotation of the identified proteins based on the Pfam database and the corresponding PfamScan tool, subcellular structure prediction analysis of the identified proteins using the PSORTb software, COG / KOG annotation, Reactome annotation, WikiPathways pathway annotation, HallMark signature gene set annotation, and annotation of the transcription factor information corresponding to the proteins using the databases TRRUST and GTRD.

[0031] (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.

[0032] (3) Based on the differential proteins in different groups, functional enrichment clustering analysis was used to study the potential connections and differences in their specific functions (GO, KEGG pathway, protein domain, Reactome, WikiPathways). First, collect the functional classification information and the corresponding enrichment P value of all protein groups, and then screen out the functional classifications that are significantly enriched (P value < 0.05) in at least one protein group. The obtained P value data matrix was first logarithmically transformed with base -Log10, and one - sided clustering analysis was performed on the transformed data set using the hierarchical clustering (Euclidean distance, average linkage clustering) method; in order to explore the distribution and differences in protein intensity values between different samples, the protein intensity values of each sample were extracted and presented in the form of a distribution histogram ( Figure 1 ). Since there are certain differences in the number of protein expressions and the expression level distribution of the samples, the protein intensity values in the samples can be divided into different intervals, calculate the number of proteins expressed in samples in different expression intervals, and draw a stacked histogram for display. The horizontal axis is the sample name, the vertical axis is the percentage of sample proteins, and different colors in the figure represent different ranges of intensity values. Horizontal comparison can compare the distribution of intensity values in each sample.

[0033] (4) Draw an expression heat map ( Figure 2 ) of the union of differential proteins in all healthy and patient groups (with quantitative values in at least 2 / 3 of the total samples) to show the relative expression levels of multiple differential proteins in different samples and present the clustering relationship of the relative expression levels of differential proteins. Each row represents a differential protein, and each column represents a sample. Red represents high expression, blue represents low expression, and gray indicates non - quantifiable in the corresponding sample.

[0034] (5) Screen out the up-regulated proteins among all the differential proteins, perform mass spectrometry detection, calculate and statistically analyze the ratio of the protein spectrum signal intensities between the patient group and the healthy group of the up-regulated proteins in each group. Those with a numerical value ≥ 7 are considered significantly up-regulated. A total of 52 proteins, namely RPL34, RPL37A, RPL9, RPL38, UQCR10, SLC4A1, MGST1, ATP5F1E, TMEM109, POLR1C, RAN, CYB5A, HAS1, RPL31, CAPS, ATP5F1B, RPL11, RPS28, MT-CO2, PRPS1, MGST2, ACOT11, ATP5F1A, CAT, ATIC, SH3BGRL2, FAM98A, HADHB, BCAS1, TUBB4B, RPL12, PLEKHA6, PDLIM1, SLC25A5, HSPB1, RPL30, SYNGR2, RPL29, NANS, RPSA2, DNPEP, SFN, RPL13, ACADSB, UBE2L3, UQCRC1, HADH, CCT4, CCT7, RPL32, CTNNA1, EIF3I, were screened out. The ratio data of the protein spectrum signal intensities between the patient group and the healthy group of each protein are shown in the following table:

[0035]

[0036]

[0037] Among them, the four proteins RPL34, RPL37A, RPL9, and RPL38 were not detected in the healthy group. When calculating, the healthy group was recorded as 0.01.

[0038] As can be seen from the above table, the P / C ratios of the 14 proteins RPL34, RPL37A, RPL9, RPL38, UQCR10, SLC4A1, MGST1, ATP5F1E, TMEM109, POLR1C, RAN, CYB5A, HAS1, and RPL31 are all greater than 10, indicating the highest up-regulation significance in this group.

[0039] That is, there are significant differences in these up-regulated proteins between the healthy group and the patient group, which can provide a new detection direction for the early detection of prostate cancer disease in the follow-up.

[0040] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A protein marker composition for diagnosing prostate cancer, characterized in that: The protein marker composition is any one or more of RPL34, RPL37A, RPL9, RPL38, UQCR10, SLC4A1, MGST1, ATP5F1E, TMEM109, POLR1C, RAN, CYB5A, HAS1, and RPL31.

2. The protein marker composition according to claim 1, characterized in that: The protein marker composition also contains any one or more of CAPS, ATP5F1B, RPL11, RPS28, MT-CO2, PRPS1, MGST2, ACOT11, ATP5F1A, CAT, ATIC, SH3BGRL2, FAM98A, HADHB, BCAS1, TUBB4B, RPL12, PLEKHA6, PDLIM1, SLC25A5, HSPB1, RPL30, SYNGR2, RPL29, NANS, RPSA2, DNPEP, SFN, RPL13, ACADSB, UBE2L3, UQCRC1, HADH, CCT4, CCT7, RPL32, CTNNA1, and EIF3I.

3. The protein marker composition according to any one of claims 1-2, characterized in that: The protein marker composition is urine protein.

4. The protein marker composition according to any one of claims 1-2, characterized in that: Compared to healthy controls, an increased expression level of the protein marker composition indicates that the subject has a risk of developing prostate cancer.

5. Use of an identification reagent of a protein marker composition for diagnosing prostate cancer in the preparation of a product for diagnosing prostate cancer, characterized in that: The identification reagent is a reagent for identifying the content of any one or more proteins of RPL34, RPL37A, RPL9, RPL38, UQCR10, SLC4A1, MGST1, ATP5F1E, TMEM109, POLR1C, RAN, CYB5A, HAS1, and RPL31 in urine.

6. The use according to claim 5, characterized in that: The identification reagent also identifies the content of any one or more of the proteins CAPS, ATP5F1B, RPL11, RPS28, MT-CO2, PRPS1, MGST2, ACOT11, ATP5F1A, CAT, ATIC, SH3BGRL2, FAM98A, HADHB, BCAS1, TUBB4B, RPL12, PLEKHA6, PDLIM1, SLC25A5, HSPB1, RPL30, SYNGR2, RPL29, NANS, RPSA2, DNPEP, SFN, RPL13, ACADSB, UBE2L3, UQCRC1, HADH, CCT4, CCT7, RPL32, CTNNA1, and EIF3I in urine.

7. A product for diagnosing prostate cancer, characterized in that: The product contains the identification reagent described in any one of claims 5-6.

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

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