Application of matrix factor POCMM1 as ovarian cancer diagnosis marker

Abstract

The preparation method comprises the following steps: firstly, screening out No.3 polypeptide with the most obvious tumor cell migration promoting effect from supernatant proteins of cancer-related fibroblast (CAF) cells of primary tumor lesions and metastatic tumor lesions of an ovarian cancer patient, and naming the No.3 polypeptide as POCMM1; scratch experiments confirm that the POCMM1 promotes migration of ovarian cancer cells and has concentration dependence, and further prove that the matrix factor POCMM1 promotes development of tumors and has the potential of serving as a biomarker for ovarian cancer diagnosis. The expression level of the matrix factor POCMM1 in serum of an ovarian cyst patient, an ovarian cancer patient without metastasis and an ovarian cancer patient with metastasis is subsequently detected, and compared with the expression level of the matrix factor POCMM1 in the serum of the ovarian cyst patient, the expression level of the matrix factor POCMM1 in the serum of the ovarian cancer patient is increased; the expression in serum of a metastatic ovarian cancer patient is the highest, so that the matrix factor POCMM1 can be confirmed to be used as a biomarker for ovarian cancer diagnosis.

Description

Technical Field

[0001] This invention relates to the field of medical diagnostic technology, specifically to the application of stromal factor POCMM1 as a diagnostic biomarker for ovarian cancer. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Ovarian cancer (OC) is one of the most common malignant tumors in women and a leading cause of cancer death worldwide in recent years. Despite significant progress in medical technology and understanding of ovarian cancer, its incidence and mortality rates remain high; currently, ovarian cancer is the leading cause of death among gynecological malignancies. Ovarian cancer exhibits strong malignant biological behavior, is prone to peritoneal implantation metastasis, and 75% of patients even develop extraovarian disseminated lesions. Currently, the 5-year survival rate for early-stage ovarian cancer with conventional treatment is as high as 90%, however, the 5-year survival rate for advanced-stage ovarian cancer patients, even with multiple aggressive treatments, is less than 20%. Therefore, early diagnosis and treatment of ovarian cancer are particularly important to improve long-term survival and quality of life. However, early-stage ovarian cancer patients often lack specific symptoms and signs; current diagnosis relies heavily on physical examination, imaging examinations, laparoscopy, cytology, hematology, biomarker detection, and pathological biopsy. While each method has its advantages, they are either invasive or lack sufficient sensitivity or specificity to meet clinical needs. Therefore, finding biomarkers with high sensitivity and specificity for non-invasive early diagnosis of ovarian cancer is a pressing clinical challenge.

[0004] Matrix factors are biologically active components or derivatives of the extracellular matrix (ECM) that regulate cell behavior, tissue repair, and immune responses. Tumor formation and progression are highly dependent on matrix remodeling; therefore, changes in certain matrix factors may occur in the early stages of malignant transformation of cancer cells, even before traditional markers rise, offering potential for early diagnosis. Summary of the Invention

[0005] To overcome the above problems, this invention provides the application of stromal factor POCMM1 as a diagnostic biomarker for ovarian cancer.

[0006] To achieve the above technical objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a biomarker for the diagnosis of ovarian cancer, said biomarker being matrix factor POCMM1, the amino acid sequence of which is shown in SEQ ID NO. 1.

[0007] A second aspect of the invention provides the use of reagents for detecting the biomarkers described in the first aspect in the preparation of products for ovarian cancer diagnosis.

[0008] In one or more embodiments, the product includes a protein chip, a reagent kit, or a formulation.

[0009] In one or more embodiments, the reagent is a reagent for detecting the biomarker in a sample by enzyme-linked immunosorbent assay (ELISA), protein chip, immunoblotting, or microfluidic immunoassay.

[0010] Preferably, the sample is serum, plasma, interstitial fluid, or urine.

[0011] Preferably, the reagent is an antigen for detecting the biomarker; more preferably, the reagent is an antigen of matrix factor POCMM1.

[0012] A third aspect of the present invention provides a product for the diagnosis of ovarian cancer, the product comprising reagents for detecting the biomarkers described in the first aspect.

[0013] In one or more embodiments, the product includes a protein chip, a reagent kit, or a formulation.

[0014] In one or more embodiments, the reagent is a reagent for detecting the biomarker in a sample by enzyme-linked immunosorbent assay (ELISA), protein chip, immunoblotting, or microfluidic immunoassay.

[0015] Preferably, the sample is serum, plasma, interstitial fluid, or urine.

[0016] Preferably, the reagent is an antigen for detecting the biomarker; more preferably, the reagent is an antigen of matrix factor POCMM1.

[0017] The beneficial effects of this invention are as follows: (1) In this invention, peptides that are specifically highly expressed in CAF supernatant of metastatic tumor tissue were first screened from the supernatant of cancer-associated fibroblasts (CAF) cells of primary and metastatic tumor lesions of ovarian cancer patients. Ten peptides with the highest abundance and the most significant intergroup differences were selected, chemically synthesized, and their functions were preliminarily explored. The study found that peptide No. 3 had the most significant effect on promoting tumor cell migration and named it POCMM1. The scratch assay further confirmed that POCMM1 promotes the migration of ovarian cancer cells in a concentration-dependent manner, thus proving that matrix factor POCMM1 promotes tumor development. Matrix factor POCMM1 has the potential to be a biomarker for the diagnosis of ovarian cancer. Subsequently, the expression level of matrix factor POCMM1 in the serum of patients with ovarian cysts, patients with non-metastatic ovarian cancer, and patients with metastatic ovarian cancer was detected. Compared with the serum of patients with ovarian cysts, the expression of matrix factor POCMM1 in the serum of patients with ovarian cancer was increased. Its expression was the highest in the serum of patients with metastatic ovarian cancer. Therefore, it can be confirmed that matrix factor POCMM1 can be used as a biomarker for the diagnosis of ovarian cancer.

[0018] (2) This invention provides new serum biomarkers for the early diagnosis and metastasis of ovarian cancer, and provides theoretical basis and technical support for the future precision diagnosis and targeted intervention of ovarian cancer. It has broad prospects for clinical translation and industrial application potential.

[0019] (3) The test sample of the kit of the present invention is serum, which can avoid invasive diagnosis. The risk of ovarian cancer can be obtained by obtaining serum through minimally invasive means and testing. It requires less blood, causes less pain to the test subjects, and has high compliance. Moreover, it is simple to operate and the test results are obtained in a short time, which has broad market prospects and social benefits. Attached Figure Description

[0020] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0021] Figure 1 The study investigated the role of secreted proteins from primary cancer-associated fibroblasts (CAF) cells extracted from primary and metastatic ovarian cancer lesions in the migration of ovarian cancer cells. In the figure, A represents the collection process of secreted proteins from different CAF cells, B represents the results of the Transwell experiment, and C represents the quantification of B. Figure 2The basic characteristics of peptides in the less than 10 kDa range were analyzed. A shows the distribution of peptides with different molecular weights less than 10 kDa in CAF supernatant; B shows the GO analysis of 416 peptides specifically highly expressed in CAF supernatant of metastatic tumor tissue (P<0.05, Fold change>2); C shows the KEGG pathway analysis of 416 peptides specifically highly expressed in CAF supernatant of metastatic tumor tissue (P<0.05, Fold change>2); and D shows the predicted ranking of peptide bioactivity. Figure 3 For the preliminary functional screening of candidate peptides, A is the Transwell assay to detect the effect of candidate peptides on the migration ability of ovarian cancer cells; B is the homologous sequence alignment of peptide 3 among different species; C is the Gly-Ile cleavage site of peptide 3, which is a metalloproteinase cleavage site; D is the mass spectrometry identification of matrix factor POCMM1; E is the structure diagram of POCMM1 peptide. Figure 4 This study provides preliminary functional validation of the stromal factor POCMM1. A represents the results of the Transwell assay; B represents the quantification of A; C represents the effect of the scratch assay on the migration ability of POCMM1 in ovarian cancer cells; and D represents the quantification of C. Figure 5 This is a preliminary detection of POCMM1 in serum. In this diagram, A represents the successful establishment of the ELISA system; B represents the quantification of A; and C represents the difference in POCMM1 expression in serum from patients with ovarian cysts, non-metastatic ovarian cancer, and metastatic ovarian cancer detected by ELISA. OC-N: No Metastasis Ovarian Cancer; OC-M: Metastasis Ovarian Cancer. Detailed Implementation

[0022] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0023] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0024] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0025] Outgrowth method for isolating primary fibroblasts (CAF): Fresh surgical tissue is collected and rapidly placed in PBS containing 1% penicillin-streptomycin. The tissue is then transported to the laboratory on ice in sterile cell preservation tubes. Further processing is performed within 30 minutes of tissue removal. First, the fresh tissue specimen is thoroughly washed three times with PBS buffer containing 1% penicillin-streptomycin. Using pre-autoclaved ophthalmic scissors, the washed tissue is cut into 1 mm cubes. 3 Fresh tissue blocks were evenly placed into six-well plates using sterile forceps, spaced approximately 0.5 cm apart. 500 μL of DMEM / F-12 complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin was added to each well, and the plates were then incubated at 37 °C with 5% CO2. The next day, the plates were replaced, and the tissue blocks were placed in new six-well plates for continued culture (this step is optional). The DMEM / F-12 complete medium containing 10% FBS and 1% penicillin-streptomycin was then replaced every two days, 2 mL per well. When synaptic cells were observed to have grown out of the tissue blocks and reached 80%–90% binding, the cells were trypsinized and transferred to cell culture flasks / dishes. The resulting primary fibroblasts were considered generation 1 and passaged in a 37 °C, 5% CO2 cell culture incubator. After identification by α-SMA immunofluorescence staining, cells from generations 3 to 5 and 7 were used for subsequent studies.

[0026] Example 1 Primary cancer-associated fibroblasts (CAFs) were extracted from the primary and metastatic tumor lesions of 12 pairs of ovarian cancer patients collected in the previous period. Supernatants from different CAF cells were then cultured and collected. Figure 1 (A) Considering the rich variety and high abundance of proteins in CAF secretions, and the dynamic correlation between the CAF secreted proteome and changes in the tumor microenvironment, it is suggested that they may have important biological activities. Due to the large differences in molecular weight and abundance among secreted proteins, direct mass spectrometry analysis may lead to an overly strong background of high-abundance proteins that may mask some low-abundance functional proteins. Therefore, a filtration method was used to determine the molecular regions of functional proteins before mass spectrometry identification.

[0027] CAF culture supernatant was filtered using different molecular ultrafiltration membranes (with molecular weight cutoffs of <10 KDa, 10~30 KDa, 30~50 KDa, and >50 KDa, respectively). Figure 1 (B), extracting proteins from different molecular regions.

[0028] The results showed that proteins of different molecular weights secreted by CAFs all had varying degrees of cell migration-promoting effects. Among them, the components in the range of less than 10 kDa (approximately 100 amino acids) showed the strongest effect in promoting tumor migration, and the protein effect from the metastatic CAF group was significantly stronger than that from the primary lesion group. Figure 1 (C)

[0029] Peptidomics analysis was performed on components smaller than 10 kDa extracted from the CAF supernatant of primary and metastatic lesions. A total of 1432 peptides were identified, with molecular weights mainly concentrated in the range of 0–3 kDa. Figure 2 (A). 416 peptides specifically highly expressed in CAF supernatant of metastatic tumor tissues (P<0.05, Fold change>2) were analyzed using GO (Geneontology) and KEGG pathways. The results showed that these peptides are functionally involved in biological processes such as cell invasion, cell-cell adhesion, and ECM receptor interaction, as well as signaling pathways such as FAK-Src and PI3K-Akt. Figure 2 The results (B and C) suggest that these peptides are closely related to tumor metastasis. Further analysis of the peptides' bioactivity was conducted using a website (http: / / bioware.ucd.ie / ~compass / biowareweb / ). Figure 2 (D), and focus on the top 20% of peptides, from which 10 peptides with the highest abundance and the most significant inter-group differences were selected for chemical synthesis and preliminary functional exploration.

[0030] The effects of 10 selected peptides on tumor cell migration were examined using Transwell assays, and the results are as follows: Figure 3 As shown in Figure A, the results indicate that both polypeptide 3 (amino acid sequence as shown in SEQ ID NO. 1: IRGDKGEPGEKGPRGLPGLKGHNGLQGLPG) and polypeptide 5 (amino acid sequence: GVPKELLTHPSGLEGLPL) promote tumor migration. Among them, polypeptide 3 showed the most significant effect.

[0031] Bioinformatics analysis showed that the precursor protein of polypeptide 3 is Collagen I, an extracellular matrix protein, located at amino acids 1004-1033 of its C-terminus; its molecular weight is 2992.39 Da; ProtParam analysis showed an instability coefficient of 15.33 and a lipid index of 65, indicating that the polypeptide has good stability and lipophilicity; polypeptide 3 is highly conserved among human, mouse, and rat species. Figure 3(B) The above analysis shows that polypeptide 3 is a stable and evolutionarily conserved functional polypeptide. Further analysis of the polypeptide's production mechanism reveals that Collagen proteins can be cleaved by metalloproteinases to form active fragments called matrix factors, which participate in the regulation of tumor biological functions. The cleavage site of polypeptide 3 is located precisely at the Gly-Ile cleavage recognition site of metalloproteinases. Figure 3 (C). Therefore, polypeptide number 3 was named matrix factor POCMM1 (Pro-Ovarian Cancer Metastasis Matrikine 1).

[0032] Chemically synthesized peptide 3 standard, further analyzed by mass spectrometry in CAF supernatant, showed the presence of endogenous peptide 3. Figure 3 (D), structural analysis shows that it has 3 α-helices, possessing structural stability to support biological functions ( Figure 3 (E).

[0033] Further functional validation of the stromal factor POCMM1 revealed that its tumor migration-promoting effect was concentration-dependent. Figure 4 (A). To clarify the effect of POCMM1 on the migration of ovarian cancer cells, a scratch assay was used to find that POCMM1 promoted the migration distance of ovarian cancer cells ( ). Figure 4 (B)

[0034] Scratch assay: After trypsin digestion, the cell count of both the experimental and control groups was 2 × 10⁻⁶ cells. 5 Cells per well were placed in a six-well plate and incubated in a 37°C incubator containing 5% carbon dioxide. After 24 h, the bottom of the six-well plate was scratched with a 200 μL pipette tip, keeping the scratch width as consistent as possible. At the same time, different concentrations of POCMM1 peptide were added to the cell supernatant. The scratched areas were photographed under a microscope at 0 h, 24 h, and 48 h after scratching, and the changes in the scratches were analyzed.

[0035] In summary, the matrix factor POCMM1 was identified through peptidomics screening, and functional experiments confirmed that it can promote the migration of ovarian cancer cells.

[0036] Example 2 Serum samples were collected from 15 patients with ovarian cysts, 10 patients with ovarian cancer that had not yet metastasized, and 15 patients with ovarian cancer that had metastasized. The expression of matrix factor POCMM1 in the serum of different patients was detected.

[0037] The expression level of POCMM1 in serum was detected by enzyme-linked immunosorbent assay (ELISA). It was found that the expression of POCMM1 in the serum of ovarian cancer patients was increased compared with that of patients with ovarian cysts; and its expression was highest in the serum of patients with metastatic ovarian cancer. Figure 5 (B) The above results demonstrate at the clinical level the close correlation between POCMM1 and the occurrence and metastasis of ovarian cancer.

[0038] The sequence of the POCMM1 polypeptide antigen is IRGDKGEPGEKGPRGLPGLKGHNGLQGLPG.

[0039] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A biomarker for the diagnosis of ovarian cancer, characterized in that, The biomarker is matrix factor POCMM1, and the amino acid sequence of matrix factor POCMM1 is shown in SEQ ID NO.

1.

2. The use of the reagent for detecting the biomarker of claim 1 in the preparation of products for ovarian cancer diagnosis.

3. The application as described in claim 2, characterized in that, The products include protein chips, reagent kits, or formulations.

4. The application as described in claim 2, characterized in that, The reagent is used to detect the biomarkers in the sample by enzyme-linked immunosorbent assay (ELISA), protein chip, immunoblotting, or microfluidic immunoassay.

5. The application as described in claim 4, characterized in that, The sample may be serum, plasma, interstitial fluid, or urine.

6. The application as described in claim 4, characterized in that, The reagent is an antigen for detecting the biomarker; preferably, the reagent is an antigen of matrix factor POCMM1.

7. A product for the diagnosis of ovarian cancer, characterized in that, The product includes reagents for detecting the biomarkers of claim 1.

8. The product as described in claim 7, characterized in that, The products include protein chips, reagent kits, or formulations.

9. The product as described in claim 7, characterized in that, The reagent is used to detect the biomarkers in the sample by enzyme-linked immunosorbent assay (ELISA), protein chip, immunoblotting, or microfluidic immunoassay.

10. The product as described in claim 9, characterized in that, The sample may be serum, plasma, interstitial fluid, or urine; Alternatively, the reagent may be an antigen for detecting the biomarker; preferably, the reagent may be an antigen of matrix factor POCMM1.

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