Application of reagent for detecting LARS1 protein in preparation of product for diagnosing prostatic cancer

Abstract

The invention discloses application of a reagent for detecting LARS1 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 shows that the content of the LARS1 protein in the urine of the prostate cancer is 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 content of the LARS1 protein in the urine of the prostate cancer patient is higher than that of the normal male group. The LARS1 protein is strongly positively expressed in prostate cancer tissues, the coloring intensity of the LARS1 protein in normal prostate tissues is lower than that of a cancer group, it is indicated that the LARS1 protein has a certain relationship with occurrence and development of prostate cancer, and through research on the relationship between the LARS1 protein and prostate cancer, the diagnosis rate of patients is increased, an operation is early found, and the prostate cancer is developed. And non-invasive reexamination and medication guidance of urine after treatment are also facilitated, urine self-test of a patient is facilitated, and the survival rate of the patient is increased.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of a reagent for detecting LARS1 protein in the preparation of a product for diagnosing prostate cancer. Background Art

[0002] Prostate cancer (PCa) is a common malignant tumor in men, with its incidence increasing in recent years. Patients with advanced prostate cancer and those with metastases have a high mortality rate. When serum prostate-specific antigen (PSA) levels are elevated, definitive diagnosis of prostate cancer currently relies on a needle biopsy, which is difficult to diagnose in some patients and lacks effective biomarkers. The complexity of clinical testing and the hidden nature of clinical manifestations of prostate cancer result in a low rate of early diagnosis, posing a greater challenge to its treatment. Therefore, improving the accuracy and sensitivity of early prostate cancer diagnosis is crucial.

[0003] LARS1 is a member of the aminoacyl-tRNA synthetase I family and is a protein-coding gene. LARS1The LARS1 protein encoded by the gene is widely expressed throughout the body and is an important enzyme in protein synthesis. The enzyme it encodes can catalyze the connection between L-leucine and tRNA, and activate the rapamycin target complex (mTORC1) to control protein synthesis, metabolism, autophagy and cell growth.In addition, LARS1 has been shown to have an important impact on the occurrence and development of various tumors and may become a potential therapeutic target, including colon cancer (Cho JG, Park SJ, Han SH, Park JI. PGC-1αRegulates Cell Proliferation, Migration, and Invasion by Modulating Leucyl-tRNA Synthetase 1 Expression in Human Colorectal Cancer Cells. Cancers(Basel). 2022;15(1):159), lung cancer (Lee SH, Kim EY, Han JM, Han G, Chang YS.Combination of the LARS1 Inhibitor, BC-LI-0186 with a MEK1 / 2 InhibitorEnhances the Anti-Tumor Effect in Non-Small Cell Lung Cancer. Cancer ResTreat. 2023;55(3):851-864), osteosarcoma (Chen W, Lin Y, Jiang M, Wang Q, Shu Q.Identification of LARS as an essential gene for osteosarcoma proliferation through large-Scale CRISPR-Cas9 screening database and experimental verification. J Transl Med. 2022;20(1):355. Published 2022 Aug 12), breast cancer (Passarelli MC, Pinzaru AM, Asgharian H, et al. Leucyl-tRNA synthetase is atumour suppressor in breast cancer and regulates codon-dependent translationdynamics. Nat Cell Biol. 2022;24(3):307-315) and many other diseases.At present, there are no reports on the connection between LARS1 protein and the occurrence and development of prostate cancer. Through the study of the relationship between LARS1 protein and prostate cancer, a theoretical basis can be provided for prostate cancer screening and prognosis prediction, and treatment can be guided. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide the use of a reagent for detecting LARS1 protein in the preparation of a product for diagnosing prostate cancer.

[0005] The present invention achieves the above-mentioned purpose through the following technical solutions: As a first aspect of the present invention, a reagent for detecting LARS1 protein is used in the preparation of a product for diagnosing prostate cancer.

[0006] A further improvement is that the amino acid sequence of the LARS1 protein is shown in SEQ ID No. 1, and its GeneBank accession number is Q9P2J5.

[0007] A further improvement is that the reagent is a binding agent that specifically binds to the LARS1 protein, an oligonucleotide probe that specifically recognizes the gene encoding the LARS1 protein, or a primer pair that specifically amplifies the gene encoding the LARS1 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 LARS1 protein, and the protein detection kit includes reagents, chips, probes, colloidal gold or test paper for detecting the expression level of LARS1 protein in a sample to be tested.

[0010] A further improvement is that the sample to be tested is any one of urine, blood, tissue or cells.

[0011] The present invention has the following beneficial effects: Urine proteome analysis revealed that LARS1 protein was highly expressed in the urine of prostate cancer patients, with a higher content than that in the normal male group. Further immunohistochemical analysis of archival wax blocks of prostate cancer patients and adjacent normal prostate tissues showed that LARS1 protein was strongly positively expressed in prostate cancer tissue, while the staining intensity in normal prostate tissue was weaker than that in the prostate cancer group. LARS1 protein has a certain relationship with the occurrence and development of prostate cancer, can be used to assist in the diagnosis of prostate cancer, and can provide guidance for the treatment of prostate cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1The expression of the LARS1 protein provided by the present invention in prostate cancer tissue and normal prostate tissue. In the figure, Figures AB are from the same prostate cancer patient: Figure A shows positive expression of LARS1 protein in prostate cancer tissue (black arrow), and the normal prostate tissue adjacent to the cancer has a lower degree of staining than the prostate cancer (red arrow); Figure B shows strong positive expression of prostate cancer under a high-power microscope (400x), with predominantly cytoplasmic staining, partially accompanied by nuclear staining. DETAILED DESCRIPTION

[0013] 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.

[0014] 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.

[0015] 1. Urine proteome analysis In October 2024, 20 ml of morning urine was collected from 10 patients clinically diagnosed with prostate cancer before surgery. 5 normal men were also collected as a control group for analysis. T-tests were used to analyze differences in the levels of related proteins in urine. Differences were considered statistically significant when P < 0.05. The analysis process was as follows: (1) Protein extraction 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 minutes, and the supernatant was collected. The samples were centrifuged at 17000g for 10 minutes, and an equal volume of methanol and 1 / 4 volume of chloroform were added to the supernatant. The samples were shaken for 15 seconds and then placed at room temperature for 5 minutes. The samples were centrifuged at 12000g for 15 minutes at room temperature, and the supernatant was discarded. An equal volume of methanol was added, shaken for 15 seconds, and the samples were centrifuged at 12000g for 15 minutes 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.

[0016] (2) Enzymatic hydrolysis with pancreatic enzymes Equal amounts of protein from each sample were enzymatically digested. The volume was adjusted to the same value with lysis buffer, followed by the addition of dithiothreitol (DTT) to a final concentration of 5 mM and reduction at 56°C for 30 min. 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 min. Urea was diluted with TEAB to ensure a concentration below 2 M. Trypsin was added at a 1:50 ratio (protease: protein, m / m) and digestion was allowed to proceed overnight. Trypsin was then added at a 1:100 ratio (protease: protein, m / m) and digestion continued for 4 h.

[0017] (3) Liquid chromatography-mass spectrometry analysis 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 and 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. After separation by the ultra-high performance liquid chromatography system, 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.

[0018] Using urine proteome analysis, a series of studies including protein extraction, trypsin hydrolysis, and liquid chromatography-mass spectrometry analysis found that there was a difference in the expression of LARS1 protein in the urine of the prostate cancer group and the normal male group (P<0.001). The content of LARS1 protein was high in the prostate cancer group. The results are shown in Table 1.

[0019] Table 1. Comparison of LARS1 protein levels in urine between prostate cancer and normal male groups ; Note: C1 to C5 are normal male groups; P1 to P10 are prostate cancer groups.

[0020] 2. Immunohistochemical Analysis Archival paraffin blocks from 30 prostate cancer patients and 10 adjacent normal prostate tissues were collected between May 2017 and November 2024. LARS1 polyclonal antibody (lot number TA387844) purchased from Origene was diluted to a titer of 1:300, and immunohistochemistry was performed using the EnVision one-step method. 4-μm thick sections were dewaxed and hydrated, followed by heat antigen retrieval with EDTA and development with DAB. Statistical analysis was performed using SPSS 25.0 software, and the Fisher Scientific data were analyzed. ， s exact probability test method was used for analysis.

[0021] Immunohistochemistry revealed that LARS1 was positive in all 30 prostate cancer cases, with stained cells ranging from 70.0% to 100.0%, with strong positivity being the predominant finding. Low expression was also observed in a small number of translucent cells within four tumors. Normal prostate tissue also expressed LARS1, but to a lesser degree than in prostate cancer (P<0.05). This suggests that LARS1 is associated with the growth and development of normal prostate tissue and the development and progression of prostate cancer. Furthermore, LARS1 was also observed to be expressed in surrounding nerve fibers, striated muscle, and urothelium.

[0022] like Figure 1 Figures AB show the same patient with prostate cancer. Figure A shows strong expression of LARS1 protein in prostate cancer tissue (black arrow). Expression is also observed in adjacent normal prostate tissue, but the staining intensity is weaker than in the prostate cancer tissue (red arrow). Figure B shows a high-magnification image (400x) of a patient with strong positive expression of LARS1. The staining is primarily cytoplasmic, with some nuclear staining.

[0023] 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. Use of a reagent for detecting LARS1 protein in the preparation of a product for diagnosing prostate cancer.

2. The use according to claim 1, characterized in that The amino acid sequence of the LARS1 protein is shown in SEQ ID No.

1.

3. The use according to claim 1, characterized in that The reagent is a binding agent that specifically binds to the LARS1 protein, an oligonucleotide probe that specifically recognizes the gene encoding the LARS1 protein, or a primer pair that specifically amplifies the gene encoding the LARS1 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 includes a specific binding agent for LARS1 protein, and the protein detection kit includes reagents, chips, probes, colloidal gold or test paper for detecting the expression level of LARS1 protein in a sample to be tested.

6. The use according to claim 5, characterized in that The sample to be tested is any one of urine, blood, tissue or cells.

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