Application of EF1G as a molecular marker in the preparation of a product for diagnosing benign prostatic hyperplasia

Abstract

The present invention provides the application of EF1G as a molecular marker in the preparation of a product for diagnosing benign prostatic hyperplasia. The present application discovers that EF1G, as an effective auxiliary diagnostic molecular marker for benign prostatic hyperplasia, the expression level of EF1G protein in BPH patients is positively correlated with clinical symptoms. The detection kit for EF1G can detect the expression level of EF1G in the serum of patients, thereby quickly diagnosing benign prostatic hyperplasia, with a sensitivity of more than 59.3% and a specificity of more than 77.8%.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of EF1G as a molecular marker in the preparation of products for diagnosing benign prostatic hyperplasia. Background Art

[0002] Benign prostatic hyperplasia (BPH) is one of the common diseases in elderly men, and its incidence increases with age. With the continuous intensification of the global population aging trend, the disease burden shows an increasing trend year by year. According to data from the World Health Organization, among men over 50 years old, more than half have varying degrees of prostatic hyperplasia, and about half of them will present clinical symptoms. These symptoms not only seriously affect the quality of life of patients, but may also trigger a series of complications, such as urethral obstruction, urinary tract infection, impaired renal function, etc., seriously affecting the health and life of patients. Currently, the clinical diagnosis of BPH mainly relies on the clinical symptoms of patients, imaging examinations, and the experience of clinicians. This method has certain subjectivity and limitations, especially in early diagnosis and differentiating BPH from other diseases such as prostate cancer.

[0003] Therefore, there is an urgent need to find new detection targets to improve the early diagnosis rate and accuracy of BPH, provide more timely and effective clinical decision-making schemes for patients, and thus maximize the improvement of their quality of life. Summary of the Invention

[0004] The object of the present invention is to provide the application of EF1G as a molecular marker in the preparation of products for diagnosing benign prostatic hyperplasia. Through experiments, the present invention has found that the expression of EF1G protein is increased in BPH patients and is positively correlated with clinical symptoms.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] In the first aspect of the present invention, there is provided the application of EF1G as a molecular marker in the preparation of products for diagnosing benign prostatic hyperplasia.

[0007] Furthermore, compared with healthy individuals, the expression level of EF1G in the serum of patients with benign prostatic hyperplasia is significantly increased.

[0008] Furthermore, the products for diagnosing prostatic hyperplasia include a detection kit for EF1G.

[0009] The detection kit for EF1G includes an ELISA detection kit for EF1G.

[0010] The detection kit for EF1G includes a real-time fluorescence quantitative PCR kit.

[0011] The detection kit for EF1G includes an immunohistochemistry detection kit.

[0012] In the second aspect of the present invention, there is provided the use of a detection reagent for EF1G in the preparation of a product for diagnosing benign prostatic hyperplasia.

[0013] The detection reagent for EF1G includes a reagent for immunohistochemistry of EF1G.

[0014] In the sensitivity and specificity of EF1G as a diagnostic marker for distinguishing healthy and BPH patients, the AUC of EF1G is 0.720, the sensitivity is 59.3%, and the specificity is 77.8%.

[0015] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0016] The present invention provides the use of EF1G as a molecular marker in the preparation of a product for diagnosing benign prostatic hyperplasia. In the research, through the serum proteomics sequencing of 50 normal and BPH patients, the expression of EF1G protein was analyzed. The results showed that compared with healthy people, the expression level of EF1G in the serum of BPH patients was significantly increased (P<0.01) ( Figure 1 A, B); through immunohistochemistry, it was found that the expression level of EF1G protein in BPH patients was positively correlated with clinical symptoms, and by plotting the receiver operating characteristic (ROC) curve of the EF1G protein expression level, the prostate volume (PV), and the international prostate symptom score (IPSS) in the serum proteomics sequencing of 50 normal and BPH patients, the sensitivity and specificity of EF1G as a diagnostic marker for distinguishing healthy and BPH patients were evaluated. The AUC of EF1G is 0.720, the sensitivity is 59.3%, and the specificity is 77.8% ( Figure 3 , Table 3). By detecting the expression level of EF1G, the disease severity of BPH patients can be more accurately evaluated, providing an important basis for clinical diagnosis and decision-making. Compared with the traditional diagnosis relying on the clinical symptoms of patients and the experience of clinicians, the detection of EF1G has high sensitivity and accuracy in the diagnosis of BPH, can detect the existence of early lesions of BPH, and reduces the risk of misdiagnosis and missed diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0018] Figure 1 It is the expression of EF1G protein. A: Heat map analysis of the expression levels of differentially expressed proteins (Fold Change > 2, p < 0.05) detected by serum proteomics in BPH patients (N = 30) and healthy individuals (N = 20). B: Volcano plot analysis of differentially expressed proteins detected by serum proteomics in BPH patients (N = 30) and healthy individuals (N = 20). C: Expression levels of EF1G protein in the sera of BPH patients and healthy individuals, **p < 0.01.

[0019] Figure 2 It is that the expression level of EF1G protein is positively correlated with clinical symptoms. A: Immunohistochemical staining of EF1G protein in prostate tissues of BPH patients with mild, moderate, and severe LUTS symptoms. B: Quantitative results of immunohistochemical staining of EF1G protein. C - F: Statistical analysis of age, prostate volume, quality of life score, and international prostate symptom score in healthy and BPH patients at different stages. *p < 0.05; **p < 0.01; ***p < 0.001.

[0020] Figure 3 It is the sensitivity and specificity of EF1G in differentiating healthy and BPH patients. A: PV, B: IPSS, C: EF1G. AUC: Area Under Curve, the area under the curve. Specific Embodiments

[0021] The present invention will be specifically described below in combination with specific embodiments and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the present invention, rather than limiting the present invention.

[0022] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood to have the meanings as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention pertains. In case of conflict, this specification shall prevail.

[0023] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchase or by existing methods.

[0024] The overall idea of the present invention is as follows:

[0025] EF1G (Elongation Factor 1 Gamma) belongs to one of the members of the EF1 family. The EF1 family is a class of important proteins that participate in the process of protein synthesis within cells. Its main function is to promote the binding between amino acids and transfer RNA (tRNA), enabling protein synthesis to proceed. In this process, EF1G plays an important catalytic role. In addition to its basic function in protein synthesis, EF1G also has other important biological functions in cell biology and physiology. For example, it may be involved in processes such as cell proliferation, differentiation, and cell signal transduction.

[0026] In some studies, EF1G has also been found to be associated with biological processes such as apoptosis and cell cycle regulation. Some studies have shown that the expression level of EF1G is abnormal in certain cancers and is closely related to the occurrence, development, and prognosis of tumors. For example, EF1G has been found to be overexpressed in various tumors such as prostate cancer, breast cancer, lung cancer, and colorectal cancer, and its overexpression may be related to mechanisms such as tumor cell proliferation, metastasis, and drug resistance. Currently, there is no research on EF1G in BPH.

[0027] The inventors of this application found that: compared with healthy people, the expression level of EF1G in the serum of BPH patients was significantly increased (P < 0.01), and the expression level of EF1G protein in BPH patients was positively correlated with clinical symptoms. The sensitivity and specificity of EF1G as a biomarker were evaluated by the ROS curve.

[0028] The following will combine examples and experimental data to elaborate in detail on the application of EF1G of this application as a molecular biomarker in the preparation of products for diagnosing benign prostatic hyperplasia.

[0029] Example 1: The expression level of EF1G protein as a detection biomarker increases in the serum of BPH patients

[0030] 1. Method

[0031] 1.1 Experimental reagents and instruments:

[0032] 1.1.1 Main reagents and consumables

[0033] Table 1

[0034]

[0035] 1.1.2 Main instruments

[0036] Table 2

[0037]

[0038] 1.2 Detection of the expression of EF1G in the serum of healthy people and BPH patients

[0039] A total of 50 serum samples from healthy individuals and BPH patients were collected clinically. The serum samples were subjected to protein extraction, followed by protein enrichment, quality control, peptide separation, and High pH RP separation. Finally, DDA (data dependent acquisition) library construction and DIA (data independent acquisition) quantitative detection were performed, and differential protein screening and analysis were carried out between different comparison groups based on the quantitative results. This study was approved by the Medical Ethics Committee of Zhongnan Hospital of Wuhan University (Ethical Approval Number: 2022173).

[0040] 1.3 Detection of EF1G expression in prostate tissues of BPH patients

[0041] Thirteen BPH patients were selected and divided into mild-LUTS (IPSS ≤ 7), moderate-LUTS (8 ≤ IPSS ≤ 19), and severe-LUTS (20 ≤ IPSS ≤ 35) according to the IPSS score. IHC staining of prostate tissues of BPH patients in different groups was performed with EF1G antibody. The specific method is as follows:

[0042] 1. Place the paraffin sections in an oven at 60 °C for 1 h for baking the sections.

[0043] 2. Then dewax the sections with 100% xylene three times, 5 min each time, until completely dewaxed.

[0044] 3. Place the sections in gradient alcohols of 100% × 2, 95%, and 70% for 5 min each for hydration, and finally wash them twice with double-distilled water, 5 min each time.

[0045] 4. Take a certain amount of citrate antigen retrieval working solution in a retrieval box. The amount of the retrieval solution must be sufficient to immerse the whole section. Place the retrieval box in a pressure cooker with an appropriate amount of tap water added. Preheat it over high heat until boiling. Place the dewaxed and hydrated tissue sections on a heat-resistant staining rack, and then slowly place the staining rack into the retrieval box. Cover the pot lid and continue heating until steam is emitted. Start timing. After 5 min, turn off the power of the pressure cooker, remove the valve and open the lid, and let it cool naturally at room temperature for 20 - 30 minutes. Then wash it twice with double-distilled water, 5 min each time.

[0046] 5. Drop 100 μL of 3% hydrogen peroxide solution on each section, place it in a wet box, incubate it in an oven at 37 °C for 20 min, and wash it 3 times with PBS, 5 min each time.

[0047] 6. Drop 100 μL of 10% goat serum on each section for blocking, place it in a wet box, and incubate it in an oven at 37 °C for 30 min.

[0048] 7. Discard the blocking solution, add 50 μL of EF1G primary antibody dilution (diluted 1:200) dropwise to each section, and incubate overnight at 4°C.

[0049] 8. Discard the primary antibody, wash the sections 3 times with PBS, 5 minutes each time.

[0050] 9. Add 100 μL of the corresponding secondary antibody dilution (1:1000) dropwise to each section, and incubate at 37°C for 30 minutes.

[0051] 10. Discard the secondary antibody, wash the sections 3 times with PBS, 5 minutes each time.

[0052] 11. Add 100 μL of freshly prepared DAB chromogenic solution dropwise to each section, and immediately rinse with running water to stop the reaction when the sections turn brownish-yellow.

[0053] 12. Then counterstain with hematoxylin for 1 minute, differentiate with 1% hydrochloric acid alcohol for 2 - 3 seconds, and then immediately rinse with tap water to blue back.

[0054] 13. Dehydrate the sections in gradient alcohols of 70%, 95%, and 100% for 3 minutes in sequence, place them in xylene solution twice, 5 minutes each time for clearing, air dry, and then add neutral balsam for mounting.

[0055] 14. Detect the staining intensity of EF1G by scanning microscope.

[0056] Receiver Operating Characteristic (ROC) curve plotting of serum EF1G expression levels, prostate volume (PV), and international prostate symptom score (IPSS) in healthy individuals and BPH patients

[0057] Plot the ROC curves based on the serum EF1G expression levels, PV, and IPSS of healthy individuals and BPH patients respectively, and determine the cut-off value using Graphpad Prism 8.0 software. The sensitivity calculation formula: TP / (TP + FN)×100%, where TP is the true positive in BPH patients and FN is the false negative in BPH patients; the specificity calculation formula: TN / (TN + FP)×100%, where TN is the true negative in healthy controls and FP is the false positive in healthy controls.

[0058] 2. Results

[0059] 2.1 Increased expression level of EF1G protein in the serum of BPH patients

[0060] By performing serum proteomic sequencing on 50 normal and BPH patients, the expression of EF1G protein was analyzed. The results showed that compared with healthy individuals, the expression level of EF1G in the serum of BPH patients was significantly increased (P<0.01) ( Figure 1 A, B).

[0061] 2.2 The expression level of EF1G protein in BPH patients was positively correlated with clinical symptoms

[0062] Prostate tissues of BPH patients with mild-LUTS (IPSS≤7), moderate-LUTS (8≤IPSS≤19), and severe-LUTS (20≤IPSS≤35) symptoms were selected according to the IPSS score for immunohistochemical staining. It was found that with the increase in the severity of symptoms, the expression level of EF1G gradually increased ( Figure 2 A, B). At the same time, statistical analysis was performed on the age, prostate volume, and quality of life score of the included patients according to the different disease state stages of BPH. It was found that with the progression of clinical stages, clinical indicators such as prostate volume (PV), quality of life score (QoL), and international prostate symptom score (IPSS) showed an upward trend ( Figure 2 C-E). The above results indicate that the expression of EF1G protein is increased in BPH patients and is positively correlated with clinical symptoms. EF1G may become a detection target for BPH.

[0063] Example 2: Sensitivity and specificity of EF1G in differentiating healthy and BPH patients

[0064] By plotting the receiver operating characteristic (ROC) curve for the expression level of EF1G protein, prostate volume (PV), and international prostate symptom score (IPSS) in the serum proteomic sequencing of 50 normal and BPH patients in Example 1, the sensitivity and specificity of EF1G as a diagnostic marker in differentiating healthy and BPH patients were evaluated.

[0065] Table 3 - Statistical analysis of the ROC curve.

[0066]

[0067] Note: AUC: Area Under Curve, the area under the curve; SE: Standard Error, standard error; CI: Confidence interval, confidence interval.

[0068] From Figure 3 and the results in Table 3, it can be seen that the AUC of EF1G is 0.720, the sensitivity is 59.3%, and the specificity is 77.8%.

[0069] In summary, it can be seen that by detecting the expression level of EF1G, the disease severity of BPH patients can be more accurately evaluated, providing an important basis for clinical diagnosis and decision-making. Compared with the traditional diagnosis relying on the clinical symptoms of patients and the experience of clinicians, the detection of EF1G has higher sensitivity and accuracy in the diagnosis of BPH, can detect the existence of early lesions of BPH, and reduces the risk of misdiagnosis and missed diagnosis. This diagnostic method helps doctors detect the changes in the patient's condition early, and the reliable diagnostic results are crucial for the early diagnosis and clinical decision-making of patients, can effectively reduce the incidence of BPH-related complications, and improve the quality of life of patients. The detection of EF1G can be performed through the patient's serum protein, without the need for prostate tissue, with simple operation, low cost, and reduced harm to patients. As a new detection target for BPH, EF1G has certain clinical application prospects. By developing a diagnostic kit or detection method targeting EF1G, an effective tool can be provided for the early screening and disease monitoring of BPH.

[0070] Finally, it should also be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, such that a process, method, article or device comprising a series of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article or device.

[0071] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0072] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. The application of EF1G as a molecular marker in the preparation of a product for diagnosing benign prostatic hyperplasia. Compared with healthy individuals, the expression level of EF1G in the serum of patients with benign prostatic hyperplasia is significantly increased.

2. The application according to claim 1, wherein The product for diagnosing prostatic hyperplasia includes a detection kit for EF1G.

3. The application according to claim 2, wherein The detection kit for EF1G includes an ELISA detection kit for EF1G.

4. The application according to claim 2, characterized in that, The detection kit for EF1G includes a real-time fluorescence quantitative PCR kit.

5. The application according to claim 2, wherein The detection kit for EF1G includes an immunohistochemical detection kit. Use of the detection reagent for EF1G in the preparation of a product for diagnosing benign prostatic hyperplasia, characterized in that, Compared with healthy individuals, the expression level of EF1G in the serum of patients with benign prostatic hyperplasia is significantly increased. Among the sensitivity and specificity of using EF1G as a diagnostic marker to distinguish healthy and BPH patients, the AUC of EF1G is 0.720, the sensitivity is 59.3%, and the specificity is 77.8%.

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