Use of reagents for detecting reep proteins in the preparation of products for diagnosing prostate cancer

Abstract

This invention discloses the application of reagents for detecting REEP protein in the preparation of products for diagnosing prostate cancer, relating to the field of biomedical technology. Through proteomic analysis, ELISA assays, and immunohistochemical analysis of urinary proteins from prostate cancer patients, clear cell renal cell carcinoma patients, urothelial carcinoma patients, and healthy individuals, as well as paraffin-embedded tissue samples from prostate cancer patients and surrounding normal prostate tissue, urothelial carcinoma tissue, and clear cell renal cell carcinoma tissue, it was found that REEP6 and REEP5 proteins are related to the occurrence and development of prostate cancer and can be used to assist in the diagnosis of prostate cancer. Furthermore, in urinary testing, REEP6 and REEP5 have higher sensitivity compared to PSA, and the three proteins can complement each other, thus helping to improve the diagnostic rate and non-invasive screening capabilities. Research on the relationship between REEP6 and REEP5 proteins and prostate cancer facilitates non-invasive follow-up after treatment and guides medication, thereby improving survival rates.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of reagents for detecting REEP protein in the preparation of products for diagnosing prostate cancer. Background Technology

[0002] Prostate cancer is one of the most common malignant tumors in men, and its incidence has been gradually increasing in recent years. Early diagnosis of prostate cancer currently relies on biopsy combined with prostate-specific antigen (PSA) testing. However, diagnosis is difficult for some patients due to a lack of effective biomarkers. Therefore, seeking efficient biomarkers is crucial for early detection, early intervention, and prolonging the survival time of prostate cancer patients. Exploring non-invasive and efficient diagnostic methods is also essential.

[0003] Receptor expression-enhancing proteins (REEPs) belong to the DP1 / YOP1P family. REEPs act as membrane-forming adaptors in the endoplasmic reticulum (ER) to regulate expression or transport from the ER to the Golgi complex or plasma membrane. Based on their structural and sequence homology, at least six REEP family members (REEP1-6) have been identified, divided into two subfamilies (REEP1-4 and REEP5-6). REEP5 is involved in membrane transport and plays a crucial role in ER plasticity, receptor expression regulation, mitochondrial transport, and cellular stress responses. Its potential functions in various diseases make it a hot topic in biomedical research. REEP6 is a member of the receptor expression-enhancing protein (REEP) family, located on the short arm of chromosome 19, region 13.3. As a receptor accessory protein, REEP6 has a protein size of 20 kDa and may play a role in signal transduction pathways, influencing cell growth, differentiation, and apoptosis. Literature indicates that REEP6 is closely linked to the development of squamous cell carcinoma of the tongue, triple-negative breast cancer, lung cancer, colon cancer, certain hereditary eye diseases, and neurological disorders. Currently, there are no reports on the relationship between REEP5, REEP6, and the development of prostate cancer. Research into the relationship between REEP5, REEP6, and prostate cancer could provide a theoretical basis for prostate cancer screening and prognostic prediction. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and to provide the application of reagents for detecting REEP protein in the preparation of products for diagnosing prostate cancer.

[0005] The present invention achieves the above objectives through the following technical solutions: As a first aspect of the present invention, the use of reagents for detecting REEP protein in the preparation of products for diagnosing prostate cancer.

[0006] A further improvement is 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.

[0007] A further improvement is that the reagent is a binding agent 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.

[0008] A further improvement is that the product includes a protein chip or a protein detection kit.

[0009] A further improvement is that the protein chip includes a specific binding agent 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.

[0010] The present invention has the following beneficial effects: This invention has revealed that REEP5 and REEP6 proteins are highly expressed in prostate cancer, and their levels in urine are also higher than in the normal group. REEP5 and REEP6 proteins are related to the occurrence and development of prostate cancer, which is helpful for the diagnosis of prostate cancer and facilitates non-invasive detection and diagnosis of prostate cancer for patients. By studying the relationship between REEP5 and REEP6 proteins and prostate cancer, we aim to improve the diagnosis rate, survival rate and cure rate of patients. Attached Figure Description

[0011] Figure 1 The functional relationship curves of sample concentration and absorbance of REEP5 and REEP6 in urine ELISA provided by the present invention; Figure 2 The average concentrations of REEP5 and REEP6 in urine ELISA provided by this invention were the highest in the prostate cancer group (in the figure, the horizontal axis represents: 1 for benign prostatic hyperplasia group, 2 for urothelial carcinoma group, 3 for clear cell renal cell carcinoma group, and 4 for prostate cancer group). Figure 3 The comparison of mean REEP5 and REEP6 protein concentrations in the prostate cancer group (NA), benign prostatic hyperplasia group (NH), and clear cell renal cell carcinoma group (NK) provided by this invention; (Figure A shows REEP5 protein, and Figure B shows REEP6 protein). Figure 4ROC analysis of urinary REEP5 in patients with prostate cancer and benign prostatic hyperplasia provided by this invention; Figure 5 ROC analysis of urinary REEP6 in patients with prostate cancer and benign prostatic hyperplasia provided by this invention; Figure 6 The expression of REEP5 in prostate cancer tissue and normal tissue provided by this invention is shown in Figure A, which shows high expression of REEP5 in prostate cancer, and positive expression of REEP5 in normal prostate tissue. Figure 7 Survival curves for prostate cancer patients with high REEP5 expression provided by this invention; (In the figures, both Figure A and Figure B show that high REEP5 expression is not statistically significant with cancer survival time or progression-free survival time, P>0.05). Figure 8 The following figures illustrate the expression of REEP5 in clear cell carcinoma and urothelial carcinoma, as provided by this invention. (Figure A shows the positive expression of REEP5 in clear cell carcinoma (black arrow) and normal renal tubules (red arrow); Figure B is an enlarged version of Figure A, showing the positive expression of REEP5 in clear cell carcinoma; Figures C and D show the positive expression of REEP5 in two cases of urothelial carcinoma). Figure 9 The following figures illustrate the expression of REEP6 in prostate cancer tissues as provided by this invention: (Figures A and B show high REEP6 expression in one case of prostate cancer under different fields of view; Figures C and D show high REEP6 expression in one case of prostate cancer under different fields of view; Figures E and F show high REEP6 expression in another case of prostate cancer under different fields of view). Figure 10 The following figures illustrate the expression of REEP6 in normal tissue, clear cell renal cell carcinoma, and urothelial carcinoma, as provided by this invention. (In the figures, G shows negative REEP6 expression in normal prostate tissue; H shows low expression in this case of urothelial carcinoma, with arrows indicating staining of some remaining umbrella cells; I and J show negative REEP6 expression in the same case of clear cell renal cell carcinoma; red arrows in I show staining of surrounding normal renal tubules). Figure 11 The expression of REEP6 in prostate cancer tissue and normal tissue provided by this invention is shown in the figure. (In the figure, Figure AD shows that REEP6 is well expressed in prostate cancer tissue (black arrow), and low or no expression in normal tissue (red arrow). AB represents the same patient (Gleason score 3+4=7 points), and CD represents the same patient (Gleason score 5+5=10 points)). Figure 12Survival curves for prostate cancer patients with high REEP6 expression provided by the present invention; (Figure A shows that high REEP6 expression tends to result in longer cancer survival (P=0.067), and Figure B shows that high REEP6 expression results in longer progression-free survival (P=0.034)). Detailed Implementation

[0012] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection 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.

[0013] Unless otherwise specified, the experimental methods used in the following examples are conventional biochemical methods, and the experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0014] Receptor expression-enhancing protein (REEP) belongs to the DP1 / YOP1P family. Based on their structural and sequence homology, at least six members of the REEP family (REEP1-6) have been identified and divided into two subfamilies (REEP1-4 and REEP5-6).

[0015] Our team previously ruled out the association between REEP1-4 genes and prostate cancer through proteomics analysis, and determined the association between REEP5 and REEP6 genes 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.

[0016] 1. Urine protein analysis In October 2024, 20 ml of preoperative morning urine was collected from 10 patients with prostate cancer (referred to as the prostate cancer group), and the patients were pathologically confirmed to have prostate cancer. In addition, 20 ml of morning urine was collected from 5 healthy men as a control group. Proteomics analysis was performed using a t-test. The analysis process is as follows: (1) Protein extraction Urine samples were removed from -80°C, and Tris-HCl (pH=8.0) 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 then 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 mixture was shaken for 15 s and then incubated at room temperature for 5 min. The samples were then centrifuged at 12000 g for 15 min at room temperature, and the supernatant was discarded. An equal volume of methanol was added again, and the mixture was shaken for 15 s. The samples were then centrifuged at 12000 g for 15 min at room temperature, and the supernatant was discarded. The samples were reconstituted with 80 μl of lysis buffer (containing 1% SDC), and the protein concentration was determined using a BCA kit.

[0017] (2) Pancreatic enzyme digestion Take equal amounts of each extracted protein sample for enzymatic digestion, adjust the volume to be consistent with lysis buffer, add dithiothreitol (DTT) to a final concentration of 5 mM, reduce at 56 °C for 30 min, then add iodoacetamide (IAA) to a final concentration of 11 mM, incubate at room temperature in the dark for 15 min, add TEAB to dilute urea to ensure the concentration is below 2 M, add trypsin at a ratio of 1:50 (protease: protein, m / m), digest overnight, then add trypsin at a ratio of 1:100 (protease: protein, m / m), continue digestion for 4 h to obtain peptides.

[0018] (3) Liquid chromatography-mass spectrometry analysis The peptides obtained by trypsin hydrolysis were dissolved in mobile phase A of liquid chromatography and then separated using a NanoElute ultra-high performance liquid chromatography system. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was an acetonitrile-water solution containing 0.1% formic acid. The liquid phase gradient settings were: 0-14 min, 6-24% B; 14-16 min, 24-35% B; 16-18 min, 35-80% B; 18-20 min, 80% B, with the flow rate maintained at 500 nl / min. After separation by an ultra-high performance liquid chromatography (UHPLC) system, the peptides were injected into a Capillary ion source for ionization, and then data were acquired using a timsTOF Pro 2 mass spectrometer. The ion source voltage was set to 1.75 kV, and the peptide precursor ion and its secondary fragments were detected and analyzed using TOF. The data acquisition mode used was the data-independent parallel cumulative serial fragmentation (dia-PASEF) mode. The primary mass spectrometry scan range was set to 300-1500 m / z, and 20 PASEF mode acquisitions were performed after one primary mass spectrometry acquisition. The secondary mass spectrometry scan was performed in the range of 400-850 m / z, with each 7 m / z segment as a window.

[0019] Urine proteomics analysis, through protein extraction, pancreatic enzyme digestion, and liquid chromatography-mass spectrometry, revealed that... In the morning urine of 10 patients with prostate cancer, both REEP5 and REEP6 proteins were detected in all 10 cases. In the urine of 5 healthy men, REEP5 protein was detected in 4 cases, with a protein spectrum signal intensity significantly lower than that in the prostate cancer group. The concentration of REEP5 protein in urine was significantly higher than the normal value (P=0.00007), approximately 12.5 times that in the healthy men group (Table 1). In the healthy men group, REEP6 protein was detected in 2 cases, with a protein spectrum signal intensity significantly lower than that in the prostate cancer group. The concentration of REEP6 protein in urine in the prostate cancer group was significantly higher than the normal value (P=0.0017), approximately 17.3 times that in the healthy men group (Table 2).

[0020] Table 1. Comparison of urinary REEP5 protein levels between prostate cancer patients and healthy men. Note: C1 to C5 represent normal healthy males; P7_1 to P7_10 represent prostate cancer.

[0021] Table 2. Comparison of urinary REEP6 protein levels between prostate cancer patients and healthy men. Note: C1 to C5 represent normal healthy males; P7_1 to P7_10 represent prostate cancer.

[0022] For cross-sectional comparison, this study further collected morning urine from 16 patients with urothelial carcinoma (denoted as urothelial carcinoma group) and 15 patients with clear cell renal cell carcinoma (denoted as clear cell renal cell carcinoma group), and also collected 20 ml of urine from 5 healthy individuals (including 1 female) as a healthy control group. The urine proteinomics analysis was performed according to the same steps as above.

[0023] The results showed that REEP5 was detected in 3 out of 5 healthy controls with low intensities (192.463, 557.386, 166.925, different batches); in the urothelial carcinoma group, REEP5 was detected in 7 out of 16 cases (mean intensity 982.783), and the intensity of REEP5 in urothelial carcinoma was increased by 3.22 times compared with the healthy control group (P=0.042); in the clear cell renal cell carcinoma group, REEP5 was detected in 6 out of 15 cases (mean intensity 315.242), with no difference compared with the healthy control group (P>0.05). In the morning urine of 15 patients with clear cell renal cell carcinoma, only 2 cases showed detectable REEP6 protein proteomic signals, with intensities of 614.568 and 442.667, respectively, which was not statistically different from the healthy control group (P>0.05). In 16 patients with urothelial carcinoma, 3 cases showed detectable REEP6 protein proteomic signals, with intensities of 774.82, 2544.58 and 312.778, respectively, while 13 cases did not show detectable REEP6. The T-test showed no statistical significance compared with the healthy control group (P>0.05). The results further confirmed that REEP6 protein is relatively specific in the urine of prostate cancer and can be well used in urine tests to differentiate it from clear cell renal cell carcinoma and urothelial carcinoma; REEP5 has the highest intensity in prostate cancer but lacks specificity, and it is also slightly elevated in urothelial carcinoma.

[0024] 2. Urine ELISA test Preoperative morning urine samples were collected from 26 patients with prostate cancer (referred to as prostate cancer group) and 5 patients with benign prostatic hyperplasia (referred to as benign prostatic hyperplasia group) as controls, 20 ml from each patient. For cross-sectional comparison, morning urine samples were further collected from 5 patients with clear cell renal cell carcinoma (referred to as clear cell renal cell carcinoma group) and 5 patients with urothelial carcinoma (referred to as urothelial carcinoma group), 20 ml from each patient.

[0025] The concentrations of REEP5, REEP6, and PSA in urine were further detected using the ELISA method. The specific steps are as follows: (2.1) The collected urine samples were diluted appropriately according to the preliminary experimental results, and the standard was serially diluted. (2.2) Add 100 μL of the processed sample and standard to the corresponding microplate wells, cover with the plate membrane, and incubate at 37°C for 150 min. After incubation, discard the liquid in the wells and manually wash 5 times with the washing buffer injected into the wells to ensure that unbound substances are completely removed. After washing, add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to each well and incubate at 37°C for 150 min again. (2.3) After washing 5 times as in step (2.2) above, add 100 μL of freshly prepared substrate solution (TMB) to each well and incubate at 37°C in the dark for 150 min to develop the color. After a clear color gradient appears in the standard wells, add 50 μL of stop solution to each well, and the solution color will change from blue to yellow. Within 15 min after the reaction is terminated, use a microplate reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm (corrected wavelength 630 nm). (2.4) Calculate the actual concentration of the target substance in the urine sample based on the standard curve plotted by the concentration of the standard and the OD value, and perform corresponding factor correction on the results of the diluted sample.

[0026] When detecting REEP5 and REEP6 proteins in urine using ELISA, the sample concentration and absorbance follow a current functional relationship, R. 2 >0.99 indicates the detection is reliable ( Figure 1 ); The results are shown in Tables 3 and 4, and are as follows: Figures 2-3 As shown, the average concentration of REEP5 protein in the urine of the prostate cancer group (NA) was 0.338 ± 0.031 ng / ml, which was higher than the average concentration in the benign prostatic hyperplasia group (NH) (0.149 ± 0.017 ng / ml). The mean concentrations of REEP5 protein in urine were 0.104 ± 0.008 ng / ml in the urothelial carcinoma (NB) group and 0.163 ± 0.041 ng / ml in the clear cell renal cell carcinoma (NK) group, both of which were statistically significant (P < 0.0001). This indicates that the detection concentration of REEP5 protein in urine was highly specific in the prostate cancer (NA) group. The concentration of REEP5 protein was lowest in the urothelial carcinoma (NB) group, and it showed statistically significant differences compared to both the benign prostatic hyperplasia (NH) and clear cell renal cell carcinoma (NK) groups (P = 0.020 and P = 0.003, respectively). These results suggest that REEP5 protein is highly expressed and specific in the urine of the prostate cancer (NA) group. The average concentration of REEP6 protein in the urine of the prostate cancer group (NA) was 0.504±0.052 ng / ml, which was significantly higher than that in the benign prostatic hyperplasia group (NH) (0.284±0.018 ng / ml), the urothelial carcinoma group (NB) (0.246±0.022 ng / ml), and the clear cell renal cell carcinoma group (NK) (0.261±0.037 ng / ml) (all P<0.0001). This indicates that the detection concentration of REEP6 protein in the urine of the prostate cancer group (NA) is relatively specific. These results suggest that REEP6 protein is highly expressed and specific in the urine of the prostate cancer group (NA).

[0027] Table 3. Statistical analysis of REEP5 protein urinary ELISA results in the prostate cancer group, benign prostatic hyperplasia group, urothelial carcinoma group, and clear cell renal cell carcinoma group. Note: NB, urothelial carcinoma group; NH, benign prostatic hyperplasia group; NK, clear cell renal cell carcinoma group; NA, prostate cancer group; concentration unit is ng / ml.

[0028] Table 4. Statistical analysis of REEP6 protein urinary ELISA results in the prostate cancer group, benign prostatic hyperplasia group, urothelial carcinoma group, and clear cell renal cell carcinoma group. Note: NB, urothelial carcinoma group; NH, benign prostatic hyperplasia group; NK, clear cell renal cell carcinoma group; NA, prostate cancer group; concentration unit is ng / ml.

[0029] Furthermore, compared with benign prostatic hyperplasia, 10 out of 26 prostate cancer patients had urinary PSA concentrations below the threshold (1.794 pg / ml), with a sensitivity of 61.5% (16 / 26) and a high false negative rate (38.5%). Compared with PSA, urinary REEP detection had higher sensitivity (100.0% vs. 61.5%, P<0.05) and higher specificity.

[0030] In summary, REEP proteins (REEP5 and REEP6) are specific in urine ELISA detection, confirming that REEP proteins (REEP5 and REEP6) are highly expressed and specific in the urine of prostate cancer patients. Compared with benign prostatic hyperplasia, the concentration detection of REEP proteins (REEP5 and REEP6) is more advantageous. They can complement PSA and may even better replace PSA in urine detection.

[0031] 3. ROC detection and analysis Preoperative morning urine was collected from 26 patients with prostate cancer and 5 patients with benign prostatic hyperplasia, 20 ml from each patient, to verify the diagnostic efficacy. The concentrations of REEP5 and REEP6 proteins in the urine were used as detection variables to determine the diagnostic results. Analysis method: ROC of the indicators was analyzed using SPSS 26.0 software.

[0032] The results showed that, compared with the benign prostatic hyperplasia group, the cutoff concentration of REEP5 protein was 0.2275 ng / ml (approximately 0.23 ng / ml), the area under the ROC curve was 1, P < 0.001, and the specificity and sensitivity were both 100.0%. Figure 4 ); Compared with the prostate cancer hyperplasia group, the cutoff concentration of REEP6 protein was 0.3550 ng / ml (approximately 0.36 ng / ml), the area under the ROC curve was 1, P < 0.001, and the specificity and sensitivity were both 100.0%. Figure 5 ).

[0033] 4. Immunohistochemical analysis Prostate cancer paraffin-embedded tissue samples were collected from 91 patients diagnosed with prostate cancer between 2017 and 2024 (referred to as the prostate cancer group). Twenty adjacent normal prostate tissue samples were also collected (referred to as the normal control group). Additionally, paraffin-embedded tissue samples were collected from 10 cases of urothelial carcinoma (referred to as the urothelial carcinoma group) and 20 cases of clear cell renal cell carcinoma (referred to as the clear cell renal cell carcinoma group) for cross-sectional comparison to further understand REEP6 and REEP5 and facilitate differential diagnosis. AR immunohistochemistry of prostate cancer was also performed to explore the correlation and significance of REEP6 and REEP5 proteins and their expression, and to compare them with other clinical factors to comprehensively understand the significance of REEP6 and REEP5 protein expression.

[0034] The immunohistochemical analysis methods are as follows: The following antibodies were purchased: Abcam REEP6 (ab204341) antibody at a concentration of 1:400; Origene REEP5 (clone number: OTI4D2) antibody at a concentration of 1:200; and androgen receptor (AR, MXR023) and PSA (MAB-0146) antibodies were purchased from Maixin Biotechnology Co., Ltd.

[0035] Immunohistochemistry (formalin / PFA fixed paraffin-embedded sections) was performed using the EnVision one-step immunohistochemical method; 4µm thick sections were dewaxed to water, and EDTA was used for heat antigen retrieval, followed by DAB staining; statistical analysis was performed using SPSS 25.0 software and Fisher's method. ， The exact probability test method is used for analysis.

[0036] 4.1, REEP5 A positive result was defined as ≥10% cell staining. The positive rate for prostate cancer was 98.9% (90 / 91). In the prostate tissue of 20 normal controls, 18 cases showed ≥10% cell staining, with a positive rate of 90.0% and a negative rate of 10.0%. It can be seen that there was no statistically significant difference between the prostate cancer group and the normal control group (P>0.05). For REEP5, the sensitivity of the four-fold table in the prostate cancer group and the normal control group was 98.9%, and the specificity was 10.0%.

[0037] High expression was defined as intermediate to high positivity in cells with ≥60% staining. The high expression rate in prostate cancer was 93.4% (85 / 91). Figure 6 The comparison with clinicopathological factors is shown in Table 5. Univariate Kaplan-Meier survival function analysis (Log Rank) showed no statistical significance and no correlation with prognosis. Figure 7 ); The high expression rate in prostate tissue of the normal control group was 85.0%, and its staining intensity and density were lower than those of prostate cancer on average area (P<0.05); the high expression rate in urothelial carcinoma was 90.0% (9 / 10). Figure 8 The staining intensity was stronger in the urothelial carcinoma group than in the clear cell renal cell carcinoma group. All clear cell renal cell carcinoma groups showed high expression (100.0%, 20 / 20). Figure 8 There was no statistically significant difference between the prostate cancer group and other groups; immunohistochemical results showed that REEP5 lacked specificity between the prostate cancer group and the urothelial carcinoma group and the clear cell renal cell carcinoma group, and could not be used for differential diagnosis; however, the expression intensity of REEP5 in prostate cancer was higher than that in normal prostate tissue, which is helpful for the diagnosis of prostate cancer.

[0038] 4.2, REEP6 ≥10% of cells were considered positive for staining. The results showed that the positive rate of REEP6 in prostate cancer was 91.2% (83 / 91). Figure 9-11 The expression of REEP6 in prostate tissue of the normal control group was as follows: 18 cases did not express it, and 2 cases showed expression in the gland (10-30% of cells were stained), lacking diffuse expression. The positive rate was 10.0% and the negative rate was 90.0%. The four-fold table statistical analysis of prostate tissue of prostate cancer and normal control group showed that the sensitivity of REEP6 in prostate cancer was 91.2% and the specificity was 90.0%.

[0039] High expression was defined as intermediate to high positivity in cells with ≥60% staining, and comparisons with clinicopathological factors are shown in Table 5. This study showed that univariate Kaplan-Meier survival function analysis (Log Rank) indicated a correlation between high REEP6 expression and progression-free survival ((75.650±5.288) months vs. (41.255±4.068) months, P=0.034, see [reference needed]). Figure 12 REEP6 was correlated with cancer-specific survival time [(79.420±4.156) months vs. (51.705±5.417) months, P=0.067], and patients with high REEP6 expression had longer survival, suggesting that high expression has a better prognosis.

[0040] High REEP6 expression was positively correlated with PSA expression in prostate cancer tissue (r=0.219, P=0.037). In cases with negative / low REEP6 expression, 33.3% showed high REEP6 expression, and 26 out of 32 cases with low REEP6 expression (81.3%) showed positive PSA expression, suggesting that the two have a complementary effect to some extent.

[0041] In the cross-sectional comparison, REEP6 was expressed at low levels in the clear cell renal cell carcinoma group, while the expression rate of REEP6 in the prostate cancer group was significantly higher than that in the clear cell renal cell carcinoma group (91.2% vs. 25.0%, P<0.001). Figure 10 The expression of α was significantly different from that of urothelial carcinoma, with low expression in the urothelial carcinoma group (91.2% vs. 20.0%, P=0.016).

[0042] Immunohistochemical results showed that REEP6 exhibited a more significant difference in prostate cancer tissue compared to normal control prostate tissue, with a higher specificity (90.0%). REEP5, on the other hand, showed lower specificity in prostate cancer tissue, suggesting that REEP6 is more helpful in diagnosing prostate cancer than REEP5. REEP5 was more sensitive in prostate cancer, with a higher sensitivity (98.9%). Although REEP5 is less effective than REEP6 in the histological and pathological diagnosis of prostate cancer, both can assist in the examination. The combination of the high specificity of REEP6 and the high sensitivity of REEP5 can be used for screening and diagnosing prostate cancer tissue and urine. Furthermore, combining REEP6 with PSA testing can better complement each other, avoiding missed diagnoses and supplementing the deficiencies of existing examinations.

[0043] Table 5. Comparison of high expression of REEP6 and REEP5 in prostate cancer with clinicopathological factors. In addition, the detection of protein in urine for REEP5 and REEP6 can also be used for (1) health monitoring for prostate cancer; (2) risk monitoring for high-risk groups; (3) non-invasive re-examination and guidance on medication; and can also be used for drug resistance identification. During the treatment process, drug resistance can be detected early by self-testing for REEP5 and REEP6 proteins, which can guide the change of drugs or combination therapy strategies.

[0044] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. Application of reagents for detecting REEP protein in the preparation of products for diagnosing prostate cancer.

2. The application according to claim 1, characterized in that, The REEP protein is either 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 application according to claim 1, characterized in that, The reagent is a binding agent 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.

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

5. The application according to claim 4, characterized in that, The protein chip includes a specific binding agent 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.

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