Application of reagent for detecting REEP protein in preparation of product for diagnosing prostatic cancer

Abstract

The invention discloses application of a reagent for detecting REEP protein in preparation of a product for diagnosing prostatic cancer, and relates to the technical field of biological medicine. Proteome analysis on urine proteins of a prostate cancer patient and a normal male and immunohistochemical analysis on archive wax blocks and surrounding normal prostate tissues of the prostate cancer patient find that REEP5 and REEP6 proteins are highly expressed in the prostate cancer tissues, and the content of REEP5 and REEP6 proteins in urine is higher than that of the normal group; the REEP5 protein and the REEP6 protein have a certain relationship with occurrence and development of the prostate cancer, are helpful for diagnosis of the prostate cancer and are also helpful for detection and diagnosis of noninvasive urine prostate cancer, and through research on the relationship between the REEP5 protein and the REEP6 protein and the prostate cancer, the diagnosis rate of a patient is increased, early discovery and early treatment are realized, postoperative noninvasive review, medication guidance and self-test are also facilitated, and the prostate cancer diagnosis method is suitable for clinical application. The survival rate and the cure rate are improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of a reagent for detecting REEP protein in the preparation of a product for diagnosing prostate cancer. Background Art

[0002] Prostate cancer is one of the common malignant tumors in men, and its incidence has been gradually increasing in recent years. The early diagnosis of prostate cancer currently relies on puncture biopsy, combined with prostate-specific antigen. For some patients, the diagnosis is difficult due to the lack of effective markers. To achieve the goals of early detection, early intervention, and prolonging the survival time of prostate cancer patients, it is very meaningful to seek highly efficient biomarkers, and it is also necessary to seek non-invasive and highly efficient diagnostic methods.

[0003] Receptor expression-enhancing protein (REEP) belongs to the DP1 / YOP1P family. REEP acts as a membrane-forming adaptor protein in the endoplasmic reticulum (ER) to regulate the expression or transport from the ER to the Golgi complex or the plasma membrane. According to its structure and sequence homology, at least six REEP family members (REEP1-6) have been identified and divided into two subfamilies (REEP1-4 and REEP5-6). REEP5 is involved in membrane trafficking and plays a key role in aspects such as endoplasmic reticulum shaping, receptor expression regulation, mitochondrial transport, and cellular stress response. Its potential functions in various diseases have made it one of the hotspots in biomedical research. REEP6 is a member of the receptor expression-enhancing protein (REEPs) family and is a gene located in the short arm of chromosome 19 at region 13.3. As a receptor accessory protein with a protein size of 20 kDa, REEP6 may play a role in signal transduction pathways and affect cell growth, differentiation, and apoptosis. According to the literature, REEP6 is also closely related to the occurrence of tongue squamous cell carcinoma, triple-negative breast cancer, lung cancer, colon cancer, certain hereditary eye diseases, and nervous system diseases. Currently, there is no report on the relationship between REEP5, REEP6 and the occurrence and development of prostate cancer. Through the study of the relationship between REEP5, REEP6 and prostate cancer, a theoretical basis can be provided for prostate cancer screening and prognosis prediction. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide the application of a reagent for detecting REEP protein in the preparation of a product for diagnosing prostate cancer.

[0005] The present invention realizes the above purpose through the following technical solutions:

[0006] As a first aspect of the present invention, the use of a reagent for detecting REEP protein in the preparation of a product for diagnosing prostate cancer.

[0007] A further improvement lies in that the REEP protein is REEP5 or REEP6, the amino acid sequence of the REEP5 protein is as shown in SEQ ID No.1, and the amino acid sequence of the REEP6 protein is as shown in SEQ ID No.2.

[0008] A further improvement lies in that the reagent is a binder that specifically binds to the REEP protein, an oligonucleotide probe that specifically recognizes the gene encoding the REEP protein, or a primer pair that specifically amplifies the gene encoding the REEP protein.

[0009] A further improvement lies in that the product includes a protein chip or a protein detection kit.

[0010] A further improvement lies in that the protein chip includes a specific binder for the REEP protein, and the protein detection kit includes reagents, chips, probes, colloidal gold, or test strips for detecting the expression level of the REEP protein.

[0011] The present invention has the following beneficial effects:

[0012] Through the research of the present invention, it is found that the REEP5 and REEP6 proteins are highly expressed in prostate cancer and their content in urine is also higher than that in the normal group. The REEP5 and REEP6 proteins are related to the occurrence and development of prostate cancer, which is helpful for the diagnosis of prostate cancer and also convenient for patients to perform non-invasive detection and diagnosis of prostate cancer. By studying the relationship between the REEP5 and REEP6 proteins and prostate cancer, the diagnostic rate of patients can be improved, and the survival rate and cure rate can be increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This shows the expression of REEP5 provided by the present invention in prostate cancer tissues and normal tissues. In the figure, Figures A - D show that REEP5 is highly expressed in prostate cancer tissues (black arrows), mainly expressed in the cytoplasm, and is lowly expressed in normal tissues (red arrows). A - D are the same patient (Gleason score 4 + 3 = 7).

[0014] Figure 2 This shows the expression of REEP6 provided by the present invention in prostate cancer tissues and normal tissues. In the figure, Figures A - D show that REEP6 is highly expressed in prostate cancer tissues (black arrows), and is lowly expressed or not expressed in normal tissues (red arrows). A - B are the same patient (Gleason score 3 + 4 = 7), and C - D are the same patient (Gleason score 5 + 5 = 10). DETAILED DESCRIPTION OF THE INVENTION

[0015] The following further describes the present application in conjunction with the accompanying drawings. It is necessary to point out here that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the protection scope of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0016] In the experimental methods in the following embodiments, unless otherwise specified, they are all conventional biochemical methods. The test materials used in the following embodiments are all obtained from conventional biochemical reagent stores unless otherwise specified.

[0017] Receptor expression-enhancing protein (REEP) belongs to the DP1 / YOP1P family. According to its structure and sequence homology, at least six REEP family members (REEP1-6) have been identified and divided into two subfamilies (REEP1-4 and REEP5-6).

[0018] Our team previously excluded the correlation between REEP1-4 genes and prostate cancer through proteomic analysis and determined the correlation between REEP5 gene, REEP6 gene and prostate cancer. The amino acid sequence of the protein encoded by the REEP5 gene is shown in SEQ ID No.1, and the GeneBank accession number is Q00765. The amino acid sequence of the REEP6 protein encoded by the REEP6 gene is shown in SEQ ID No.2, and the GeneBank accession number is Q96HR9.

[0019] 1. Urine proteome analysis

[0020] In October 2024, 20 ml of preoperative morning urine from 10 patients clinically diagnosed with prostate cancer was collected. At the same time, 20 ml of urine from 5 normal men was collected as a control group for analysis. The proteomics was analyzed using the T-test. The analysis process is as follows:

[0021] (1) Protein extraction

[0022] The urine samples were taken out from -80°C. Tris-HCl with a pH of 8.0 was added to all samples to a final concentration of 50 mM, and centrifuged at 1000 g for 5 min. The supernatant was taken, centrifuged at 17000 g for 10 min, and the supernatant was taken and an equal volume of methanol and 1 / 4 volume of chloroform were added. After shaking for 15 s, it was left at room temperature for 5 min, centrifuged at 12000 g at room temperature for 15 min, and then the supernatant was discarded. Then, an equal volume of methanol was added, shaken for 15 sec, and centrifuged at 12000 g at room temperature for 15 min, and then the supernatant was discarded. 80 μl of lysis buffer (containing 1% SDC) was added for reconstitution, and the protein concentration was measured using a BCA kit.

[0023] (2) Trypsin digestion

[0024] Equal amounts of proteins from each sample were digested with trypsin. The volume was adjusted to be the same with the lysis buffer, and then dithiothreitol (DTT) was added to a final concentration of 5 mM, followed by reduction at 56 °C for 30 min. After that, iodoacetamide (IAA) was added to a final concentration of 11 mM, and the mixture was incubated in the dark at room temperature for 15 min. TEAB was added to dilute urea to ensure the concentration was below 2 M. Trypsin was added at a ratio of 1:50 (protease: protein, m / m) and digested overnight. Then trypsin was added at a ratio of 1:100 (protease: protein, m / m) and digestion continued for 4 h.

[0025] (3) Liquid chromatography - mass spectrometry analysis

[0026] The peptides obtained by trypsin digestion were dissolved in mobile phase A of liquid chromatography and separated using a NanoElute ultra - high - performance liquid system. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was an acetonitrile - aqueous solution containing 0.1% formic acid. The liquid - phase gradient was set as follows: 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 was maintained at 500 nl / min. After separation by the ultra - high - performance liquid system, the peptides were injected into a Capillary ion source for ionization and then into a timsTOF Pro 2 mass spectrometer for data acquisition. The ion source voltage was set at 1.75 kV, and both the peptide precursor ions and their fragment ions were detected and analyzed using TOF. The data acquisition mode used data - independent parallel accumulation serial fragmentation (dia - PASEF) mode. The first - order mass spectrometry scan range was set at 300 - 1500 m / z. After one first - order mass spectrometry acquisition, 20 PASEF mode acquisitions were performed. The second - order mass spectrometry scan interval was 400 - 850, with a window of every 7 m / z.

[0027] Using urine proteome analysis, through protein extraction, trypsin digestion, and liquid chromatography - mass spectrometry analysis, it was found that the concentration of REEP5 in urine was significantly higher than the normal value (P = 0.00007), 12.5 times that of the normal group, as shown in Table 1. The concentration of REEP6 in urine was significantly higher than the normal value (P = 0.0017), 17.3 times that of the normal group, as shown in Table 2.

[0028] Table 1. Comparison of REEP5 protein content in urine of prostate cancer patients and normal group

[0029]

[0030] Note: C1 to C5 are the normal male (without prostate cancer) group; P7_1 to P7_10 are the prostate cancer group.

[0031] Table 2. Comparison of REEP6 protein content in urine of prostate cancer patients and normal group

[0032]

[0033]

[0034] Note: C1 to C5 are the normal male (without prostate cancer) group; P7_1 to P7_10 are the prostate cancer group.

[0035] 2. Immunohistochemical analysis

[0036] During the period from May 2022 to November 2024, 20 archival paraffin blocks of patients clinically diagnosed with prostate cancer were collected, and 10 cases of surrounding normal prostate tissues were collected. Antibodies against REEP6 (ab204341) from abcam company were purchased, and the antibody concentration was diluted at 1:400. Antibodies against REEP5 (clone number: OTI4D2) from Origene company were purchased, and the antibody concentration was diluted at 1:200. By immunohistochemistry (formalin / PFA-fixed paraffin-embedded sections) method, using EnVision one-step method, 4 μm thick sections were dewaxed to water, and heat antigen repair was carried out with EDTA, and DAB was used for color development. Using the statistical software SPSS 25.0, Fisher ， 's exact probability test method was used for analysis.

[0037] The immunohistochemical staining results showed that the expression of REEP5 in 20 cases of prostate adenocarcinoma was higher than that in normal tissues (P<0.05). REEP5 was mainly localized in the cytoplasm, showing brownish-yellow( Figure 1 ), and partial staining of the cell membrane was accompanied. The expression intensity of REEP5 in normal tissues was lower than that in the cancer group( Figure 1 ). The immunohistochemical staining results showed that the expression of REEP6 in 20 cases of prostate adenocarcinoma was higher than that in normal tissues (P<0.05). REEP6 was localized in the cytoplasm and cell membrane, showing brownish-yellow( Figure 2 ), and the expression of REEP6 in normal tissues was low( Figure 2 ).

[0038] The above-described embodiments only express several implementation manners of the present invention, and the description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the present invention patent. It should be noted that for those of ordinary skill in the art, without departing from the 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. Application of a reagent for detecting REEP protein in the preparation of a product for diagnosing prostate cancer.

2. The use according to claim 1, characterized in that: The REEP protein is REEP5 or REEP6, the amino acid sequence of the REEP5 protein is shown in SEQ ID No.1, and the amino acid sequence of the REEP6 protein is shown in SEQ ID No.

2.

3. The use according to claim 1, characterized in that: The reagent is a binding agent that specifically binds to REEP protein, an oligonucleotide probe that specifically recognizes a gene encoding REEP protein, or a primer pair that specifically amplifies a gene encoding REEP protein.

4. The use according to claim 1, characterized in that: The products include protein chips or protein detection kits.

5. The use according to claim 4, characterized in that: The protein chip comprises a specific binding agent for REEP protein, and the protein detection kit comprises a reagent, a chip, a probe, colloidal gold or a test paper for detecting the expression level of REEP protein.

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