Application of HBG protein in preparation of product for auxiliary diagnosis of prostate cancer

Abstract

The invention discloses application of HBG protein in preparation of a product for auxiliary diagnosis of prostate cancer, and relates to the technical field of biological medicine. The application way is as follows: the reagent for specifically detecting HBG protein expression in a biological sample is used for preparing a product for auxiliary diagnosis of prostate cancer. Proteome analysis on urine proteins of a prostate cancer patient and a normal male shows that the concentration of the HBG1 protein in the urine of the prostate cancer patient is obviously higher than that of the normal male group, and immunohistochemical analysis on archive wax blocks and paracancerous normal prostate tissues of the prostate cancer patient shows that the HBG1 protein can be applied to the prostate cancer patient. The HBG1 and HBG2 proteins are strongly positive in prostate cancer tissues and are low-expressed or non-expressed in normal prostate tissues, which indicates that the HBG1 and HBG2 proteins have a certain relationship with the occurrence and development of the prostate cancer, can be used for assisting the diagnosis of the prostate cancer and the diagnosis of difficult cases, and can also be used for the review after the treatment of the prostate cancer, so as to improve the diagnosis rate and the survival rate of patients.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of HBG protein in the preparation of products for assisting in the diagnosis of prostate cancer. Background Art

[0002] Prostate cancer (PCa) is a common malignant tumor in men. Early diagnosis of prostate cancer currently relies on needle biopsy combined with prostate-specific antigen (PSA). However, diagnosis is difficult for some patients due to the lack of effective biomarkers. To achieve early detection, early intervention, and prolong survival for prostate cancer patients, it is essential to develop efficient biomarkers for non-invasive and effective diagnosis.

[0003] HBG1 (Hemoglobin subunit gamma-1) is a protein-coding gene. A key paralog of this gene is HBG2. The gamma-globin genes (HBG1 and HBG2) are commonly expressed in the fetal liver, spleen, and bone marrow. During the embryonic period, fetal hemoglobin (HbF), primarily composed of α-globin and γ-globin, is responsible for oxygen transport and is often replaced by adult hemoglobin (HbA) at birth. After birth, the gamma-globin genes (HBG1 / 2) are silenced and replaced by β-globin. HBG1 / 2 expression is regulated by multiple transcription factors. The gamma chains of the two subunits differ at residue 136, with glycine present in the G-gamma product (HBG2) and alanine in the A-gamma product (HBG1). The former predominates at birth. In some cases of β-thalassemia and related disorders, gamma chain production persists into adulthood.

[0004] Abnormal expression of HBG1 is closely associated with tumor development and progression. Studies have shown that HBG1 is reactivated in various tumors, including liver cancer, breast cancer, and colorectal cancer, through epigenetic regulation (such as DNA hypomethylation and altered histone modifications) and transcription factor network imbalance (such as BCL11A / KLF1 inactivation), thereby affecting tumor oxidative stress, microenvironment remodeling, and drug resistance. Clinical data analysis shows that high HBG1 expression is significantly associated with tumor progression and poor prognosis. In the diagnostic field, HBG1, as a biopsy marker, combined with circulating tumor DNA testing, can improve the detection rate of early lung cancer. In addition, the coordinated regulation of HBG1 with miRNAs (such as miR-26b), genetic variations (such as promoter region deletions), and other molecules (GATA1, HDA C) provides new avenues for multi-target combination therapy. HBG2 is a γ-2-globin subunit. HBG2-related diseases include cyanosis and transient neonatal and fetal hemoglobinemia. HBG2 primarily binds to oxygen to exert its biological functions. At present, there are no reports on the relationship between HBG1 protein, HBG2 protein and the occurrence and development of prostate cancer. By studying the relationship between HBG1 protein, HBG2 protein and prostate cancer, a theoretical basis can be provided for prostate cancer screening and prognosis prediction. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide the use of HBG protein in the preparation of products for assisting in the diagnosis of prostate cancer.

[0006] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0007] The present invention provides an application of HBG protein in preparing a product for assisting in the diagnosis of prostate cancer. The application approach is: using a reagent for specifically detecting HBG protein expression in a biological sample to prepare a product for assisting in the diagnosis of prostate cancer.

[0008] As a further optimized solution of the present invention, the HBG protein is HBG1 protein or HBG2 protein, the amino acid sequence of the HBG1 protein is shown in SEQ ID No. 1, and its GeneBank accession number is P69891, and the amino acid sequence of the HBG2 protein is shown in SEQ ID No. 2, and its GeneBank accession number is P69892.

[0009] As a further optimized solution of the present invention, the biological sample is urine, blood or prostate tissue or cells.

[0010] As a further optimized solution of the present invention, the detection method is any one of enzyme-linked immunosorbent assay, protein immunoblotting, immunohistochemistry or antigen-antibody binding detection.

[0011] As a further optimized solution of the present invention, the reagent is a binding agent that specifically binds to HBG protein, an oligonucleotide probe that specifically recognizes the gene encoding HBG protein, or a primer pair that specifically amplifies the gene encoding HBG protein.

[0012] A further improvement is that the binding agent is a monoclonal antibody or a polyclonal antibody.

[0013] A further improvement is that the product is any one of a detection kit, a detection test paper, colloidal gold, a color developing card, a detection scale, a chip or a probe.

[0014] The present invention has the following beneficial effects:

[0015] The urine proteome analysis of the present invention found that the content of HBG1 protein in the urine of prostate cancer patients was higher than that in the normal male group. The immunohistochemical analysis showed that HBG1 and HBG2 proteins were strongly positively expressed in prostate cancer tissue and were lowly expressed or not expressed in normal prostate tissue. The present invention indicates that HBG1 and HBG2 proteins have a certain relationship with the occurrence and development of prostate cancer, has the application potential to assist in the diagnosis of prostate cancer and the follow-up of prostate cancer after treatment, can provide guidance for the treatment of prostate cancer, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The present invention provides the expression of HBG1 protein in prostate cancer tissue and normal prostate tissue. In the figure, Figures AC are from the same prostate cancer patient; Figures AB show positive expression of HBG1 protein in prostate cancer tissue (black arrows), and Figures A and C show low expression in normal prostate tissue adjacent to the cancer (red arrows); Figure D shows positive expression of HBG1 protein in prostate cancer tissue from another prostate cancer patient.

[0017] Figure 2 The present invention provides the expression of HBG2 protein in prostate cancer tissue and normal prostate tissue. In the figure, Figures AB are from the same prostate cancer patient, showing positive expression of HBG2 protein; Figures CD are from another prostate cancer patient, showing positive expression of HBG2 protein; Figure E is from a third prostate cancer patient, showing positive expression of HBG2 protein; Figure F shows no expression or low expression of HBG2 protein in normal prostate tissue. DETAILED DESCRIPTION

[0018] The present application is described in further detail below in conjunction with the accompanying drawings. It is necessary to point out that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technicians in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0019] The experimental methods in the following examples are conventional biochemical methods unless otherwise specified, and the experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0020] 1. Urine proteome analysis

[0021] In October 2024, 20 ml of morning urine was collected from 10 patients clinically diagnosed with prostate cancer before surgery as the experimental group. 20 ml of urine was collected from 5 normal men as the control group. Proteomics was analyzed using the T test. The analysis process was as follows:

[0022] (1) Protein extraction

[0023] Urine samples were taken out 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 1000g for 5 min, and the supernatant was collected. The samples were centrifuged at 17000g 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 then placed at room temperature for 5 min. The samples were centrifuged at 12000g for 15 min at room temperature, and the supernatant was discarded. An equal volume of methanol was added, shaken for 15 s, and the samples were centrifuged at 12000g for 15 min at room temperature. The supernatant was discarded, and 80 μl of lysis buffer (containing 1% SDC) was added for resolubilization. The protein concentration was determined using a BCA kit.

[0024] (2) Enzymatic hydrolysis with pancreatic enzymes

[0025] An equal amount of protein from each sample was enzymatically digested. The volume was adjusted to the same value with lysis buffer, and dithiothreitol (DTT) was added to a final concentration of 5 mM. The cells were reduced at 56°C for 30 minutes. 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 minutes. TEAB was added to dilute urea to ensure that the concentration was below 2 M. Trypsin was added at a ratio of 1:50 (protease: protein, m / m) and the cells were digested overnight. Trypsin was then added at a ratio of 1:100 (protease: protein, m / m) and the digestion continued for 4 hours.

[0026] (3) Liquid chromatography-mass spectrometry analysis

[0027] Peptides obtained by trypsin digestion 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-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). The flow rate was maintained at 500 nl / min. Following ultra-high performance liquid chromatography separation, the peptides were injected into the Capillary ion source for ionization and then acquired on a timsTOF Pro 2 mass spectrometer. The source voltage was set to 1.75 kV. The 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.

[0028] Using urine proteome analysis, through a series of analyses including protein extraction, trypsin hydrolysis, and liquid chromatography-mass spectrometry analysis, it was found that, as shown in Table 1, the content of HBG1 in the urine of prostate cancer patients was higher than that of the normal group. The content in the prostate cancer group was about 53 times that of the normal group, which was statistically significant (P≈0.004).

[0029] Table 1. Comparison of HBG1 protein levels in urine between prostate cancer and normal male groups

[0030]

[0031]

[0032] Note: C1 to C5 are normal male groups; P1 to P10 are prostate cancer groups.

[0033] As shown in Table 2, HBG2 was present in the urine of two prostate cancer patients (2 / 10), but was not detected in the normal male group, lacking statistical significance (P>0.05). The detection of HBG2 in urine has certain limitations.

[0034] Table 2. Comparison of HBG2 protein levels in urine between prostate cancer group and normal male group

[0035]

[0036] Note: C1 to C5 are normal male groups; P1 to P10 are prostate cancer groups.

[0037] 2. Immunohistochemical Analysis

[0038] 2.1 Immunohistochemical analysis of HBG1 protein

[0039] Archival paraffin blocks from 30 prostate cancer patients and 20 adjacent normal prostate tissues were collected from May 2017 to November 2024. HBG1 monoclonal antibody (clone number OTI35F4) purchased from Origene was diluted to a titer of 1:50 and immunohistochemistry was performed using the EnVision one-step method. 4 μm thick sections were dewaxed to water, heat antigen retrieval was performed with EDTA, and DAB staining was performed. Statistical analysis was performed using SPSS 25.0 software and Fisher's ， s exact probability test method was used for analysis.

[0040] The results of immunohistochemical staining for HBG1 protein are as follows Figure 1 As shown, Figure 1 Figures AC and D are from the same patient with prostate cancer, and another patient with prostate cancer. HBG1 protein is strongly positive in prostate cancer tissue ( Figure 1 Black arrows in the middle AB figure), HBG1 protein was lowly expressed in normal prostate tissue adjacent to the cancer ( Figure 1 The results of immunohistochemical analysis showed that the expression intensity of HBG1 protein in normal prostate tissue adjacent to the cancer was lower than that in prostate cancer tissue.

[0041] 2.2 Immunohistochemical analysis of HBG2 protein

[0042] The HBG2 polyclonal antibody (clone number TA388061) purchased from Origene was 17 kDa in size and diluted at 1:400. The immunohistochemistry EnVision one-step method was used. 4 μm thick sections were dewaxed to water, and heat antigen retrieval was performed with EDTA, and DAB was used for color development. Statistical analysis was performed using SPSS 25.0 software and a four-cell chi-square test. The immunohistochemical analysis results showed that the expression of HBG2 protein in prostate cancer patients was higher than that in normal tissues (P < 0.05), with a certain positive rate. In normal prostate tissue, HBG2 protein was not expressed or was expressed at a low level. HBG2 protein was brownish yellow in the cytoplasm of prostate cancer cells ( Figure 2 ).

[0043] The above-described embodiments merely illustrate several implementations of the present invention. 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 a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. Application of HBG protein in the preparation of a product for assisting in the diagnosis of prostate cancer, characterized in that: The application approach is to use a reagent for specifically detecting HBG protein expression in a biological sample to prepare a product for assisting in the diagnosis of prostate cancer.

2. The use according to claim 1, characterized in that The HBG protein is HBG1 protein or HBG2 protein. The amino acid sequence of the HBG1 protein is shown in SEQ ID No. 1, and the amino acid sequence of the HBG2 protein is shown in SEQ ID No.

2.

3. The use according to claim 1, characterized in that The biological sample is urine, blood, or prostate tissue or cells.

4. The use according to claim 1, characterized in that The detection method is any one of enzyme-linked immunosorbent assay, protein immunoblotting, immunohistochemistry or antigen-antibody binding detection.

5. The use according to claim 1, characterized in that The reagent is a binding agent that specifically binds to the HBG protein, and the binding agent is a monoclonal antibody or a polyclonal antibody.

6. The use according to claim 1, characterized in that The reagent is an oligonucleotide probe that specifically recognizes the gene encoding the HBG protein or a primer pair that specifically amplifies the gene encoding the HBG protein.

7. The use according to claim 1, characterized in that The product is any one of a detection kit, a detection test paper, colloidal gold, a color developing card, a detection scale, a chip or a probe.

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