Application of PTX3 protein in auxiliary diagnosis and treatment of ovarian cancer peritoneal metastasis

Abstract

The invention relates to the technical field of medicines, in particular to application of PTX3 protein in auxiliary diagnosis and treatment of ovarian cancer peritoneal metastasis. Immunohistochemical experiments show that the expression of PTX3 in peritoneal metastasis tissue is obviously higher than that of primary tumor tissue, and the high expression of PTX3 is in positive correlation with clinical staging and metastasis conditions of patients. Through overexpression and knock-down experiments of PTX3, the overexpression of PTX3 significantly promotes the proliferation, migration and invasion ability of ovarian cancer cells, and the knock-down of PTX3 significantly inhibits the proliferation, migration and invasion ability of ovarian cancer cells. In-vitro experiments comprehensively verify the key effect of PTX3 in ovarian cancer peritoneal metastasis, the importance of PTX3 as a diagnosis and treatment target is defined, and a scientific basis is provided for formulation of clinical diagnosis and treatment schemes.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and specifically to the application of PTX3 protein in the auxiliary diagnosis and treatment of peritoneal metastasis of ovarian cancer. More specifically, it relates to an ovarian cancer diagnosis and treatment scheme using PTX3 (Pentraxin 3) as a therapeutic target. Background Art

[0002] Ovarian cancer is one of the most lethal gynecological malignancies, characterized by no obvious early symptoms. Most patients are already in the advanced stage when diagnosed, and the tumor has spread widely in the abdominal cavity. Among them, high-grade serous ovarian cancer is the most important and most aggressive histological subtype of ovarian cancer, accounting for about 70-80% of ovarian cancer-related deaths. Peritoneal metastasis is the main way ovarian cancer spreads and is also a key factor leading to patient death.

[0003] PTX3 is an important member of the long-chain pentose protein family and was originally considered to be a pattern recognition receptor. PTX3 exists in human body fluids and is an important component of the innate immune response. It has the functions of identifying pathogens, regulating inflammatory responses, and promoting tissue structure remodeling. Studies have found that PTX3 plays an important regulatory role in a variety of malignant biological behaviors of peritoneal metastasis of ovarian cancer, including promoting tumor cell migration, invasion, angiogenesis, and remodeling of the immune microenvironment. These processes are closely related to the occurrence of peritoneal metastasis and the prognosis of patients. Preliminary studies have shown that PTX3 mainly mediates signal transduction through TLR4 or CD44 receptors, further promoting the progression of peritoneal metastasis. Clinical studies have shown that ovarian cancer patients with high expression of PTX3 are more likely to have peritoneal metastasis and have a poor prognosis. However, the specific role of PTX3 in peritoneal metastasis of ovarian cancer and its deep molecular mechanism have not been fully elucidated.

[0004] Since the early symptoms of ovarian cancer are not obvious, most patients are already in the advanced stage when diagnosed, which makes treatment more difficult. The extensiveness of peritoneal metastasis and the chemotherapy resistance of cancer cells in metastatic lesions limit the efficacy of surgical resection and traditional chemotherapy, resulting in high recurrence rates and poor prognosis. Currently, targeted drugs for the treatment of ovarian cancer are relatively limited. Although PARP inhibitors can be used for maintenance treatment of patients with BRCA gene mutations and HRD-positive patients, they can only prolong the progression-free survival of platinum-sensitive patients and have poor efficacy in patients with platinum-resistant ovarian cancer. Therefore, there is an urgent need to develop more targeted therapeutic drugs for the combination treatment of patients with advanced ovarian cancer who are platinum-resistant or have peritoneal metastasis. Summary of the invention

[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to provide an application of PTX3 protein in the auxiliary diagnosis and treatment of peritoneal metastasis of ovarian cancer. The present invention aims to reduce the migration and invasion ability of cancer cells by inhibiting the function of PTX3, thereby effectively reducing the incidence of peritoneal metastasis. This method not only provides a new target for the treatment of ovarian cancer, but also provides new possibilities for improving the prognosis of patients.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0007] The invention discloses the application of PTX3 gene or its encoded protein in preparing a kit for diagnosing and screening peritoneal metastasis of ovarian cancer.

[0008] Furthermore, the kit for diagnosing and screening peritoneal metastasis of ovarian cancer uses PTX3 as a diagnostic marker.

[0009] Furthermore, the kit for diagnosing and screening peritoneal metastasis of ovarian cancer detects the expression level of PTX3 in surgically removed tissues of patients with peritoneal metastasis of ovarian cancer by western or quantitative PCR technology, and the degree of PTX3 expression is positively correlated with the incidence of peritoneal metastasis of ovarian cancer.

[0010] The present invention also discloses the use of a detection reagent for the expression level of the PTX3 gene or its encoded protein in the preparation of an auxiliary diagnosis product for peritoneal metastasis of ovarian cancer.

[0011] Furthermore, the product detects the expression level of PTX3 in a sample by reverse transcription PCR, real-time quantitative PCR, in situ hybridization, Northern blotting, chip, high-throughput sequencing platform, immunohistochemical staining or enzyme-linked immunosorbent assay.

[0012] Furthermore, the product is a chip, a preparation or a kit; and the sample is a tissue, serum or a cell.

[0013] The invention also discloses the use of an inhibitor of PTX3 protein expression level in preparing a medicine for treating peritoneal metastasis of ovarian cancer.

[0014] The invention also discloses the use of an inhibitor of PTX3 protein expression level in the preparation of a drug for inhibiting the proliferation, migration and invasion of cells in ovarian cancer.

[0015] Furthermore, the above-mentioned application is characterized in that the inhibitor of PTX3 protein expression level is a plasmid containing a nucleic acid molecule.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0017] 1. Revealing the key role of PTX3 in peritoneal metastasis of ovarian cancer for the first time: This invention first clarifies the key role of PTX3 in the peritoneal metastasis of ovarian cancer and its molecular mechanism, clarifies the core regulatory position of PTX3 in the occurrence and progression of peritoneal metastasis, and provides a new perspective for further research on the role of PTX3 in peritoneal metastasis of ovarian cancer. This discovery fills the gap in the existing technology on the mechanism of peritoneal metastasis of ovarian cancer, and is not limited to the study of ovarian cancer itself.

[0018] 2. Innovative peritoneal metastasis prediction method: This invention has established a method for predicting peritoneal metastasis of ovarian cancer based on PTX3 expression level, which can effectively predict the risk of peritoneal metastasis in patients and provide an important basis for clinical diagnosis and the formulation of individualized treatment plans. This method focuses on the early prediction of peritoneal metastasis, overcomes the shortcomings of the existing technology in the risk assessment of peritoneal metastasis of ovarian cancer, and provides new possibilities for improving the prognosis of patients with peritoneal metastasis.

[0019] 3. New molecular targets for early intervention of peritoneal metastasis: This invention identifies PTX3 as a key molecular target for peritoneal metastasis of ovarian cancer, providing a new direction for early warning and clinical intervention of peritoneal metastasis. Through treatment strategies targeting PTX3, it is expected to significantly inhibit the occurrence and progression of peritoneal metastasis, thereby improving the treatment effect and prognosis of patients. The proposal of this target focuses on the treatment needs of peritoneal metastasis, which is superior to the existing surgical and chemotherapy methods that only target the overall treatment of ovarian cancer, and makes up for the shortcomings of existing treatment methods in the intervention of peritoneal metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the expression level of PTX3 in primary ovarian cancer lesions and peritoneal metastases.

[0021] Figure 2 is the transfection efficiency after knockdown and overexpression of PTX3 in ovarian cancer cell lines. Among them, A is the expression of PTX3 in OVCAR3 after knockdown of PTX3 in ovarian cancer cell line OVCAR3; B is the expression of PTX3 in CAOV3 after knockdown of PTX3 in ovarian cancer cell line CAOV3; C is the expression of PTX3 in SKOV3 after overexpression of PTX3 in SKOV3; D is the expression of PTX3 in CAOV3 after overexpression of PTX3 in CAOV3.

[0022] Figure 3The effects of knockdown and overexpression of PTX3 on the migration and invasion of ovarian cancer cells. A shows the changes in the migration and invasion of OVCAR3 cells after knockdown of PTX3 in the ovarian cancer cell line OVCAR3; B shows the changes in the migration and invasion of CAOV3 cells after knockdown of PTX3 in the ovarian cancer cell line CAOV3; C shows the changes in the migration and invasion of SKOV3 cells after overexpression of PTX3 in the ovarian cancer cell line SKOV3; D shows the changes in the migration and invasion of CAOV3 cells after overexpression of PTX3 in the ovarian cancer cell line CAOV3.

[0023] Figure 4 The results of EdU cell proliferation experiment. A shows the change of proliferation ability of OVCAR3 cells after knocking down PTX3 in ovarian cancer cell line OVCAR3; B shows the change of proliferation ability of CAOV3 cells after knocking down PTX3 in ovarian cancer cell line CAOV3; C shows the change of proliferation ability of SKOV3 cells after overexpressing PTX3 in ovarian cancer cell line SKOV3; D shows the change of proliferation ability of CAOV3 cells after overexpressing PTX3 in ovarian cancer cell line CAOV3.

[0024] Figure 5 The results of CCK8 experiments. A shows the changes in OVCAR3 proliferation after knocking down PTX3 in the ovarian cancer cell line OVCAR3; B shows the changes in CAOV3 cell proliferation after knocking down PTX3 in the ovarian cancer cell line CAOV3; C shows the changes in SKOV3 cell proliferation after overexpressing PTX3 in the ovarian cancer cell line SKOV3; D shows the changes in CAOV3 cell proliferation after overexpressing PTX3 in the ovarian cancer cell line CAOV3.

[0025] Figure 6 The co-localization of PTX3, TLR4 and CD44 in ovarian cancer cell OVCAR3.

[0026] Figure 7Figure 2 shows the activation of key proteins of EMT and NF-κB pathway in ovarian cancer cells after knockdown and overexpression of PTX3. A shows the changes of E-cadherin, N-cadherin and vimentin proteins in OVCAR3 cells after knockdown of PTX3 in ovarian cancer cell line OVCAR3; B shows the changes of E-cadherin, N-cadherin and vimentin proteins in CAOV3 cells after knockdown of PTX3 in ovarian cancer cell line CAOV3; C shows the changes of E-cadherin, N-cadherin and vimentin proteins in SKOV3 cells after overexpression of PTX3 in SKOV3 cells; D shows the changes of E-cadherin, N-cadherin and vimentin proteins in CAOV3 cells after overexpression of PTX3 in ovarian cancer cell line CAOV3. E shows the changes in NF-κB and p-NF-κB proteins in OVCAR3 cells after knocking down PTX3 in the ovarian cancer cell line OVCAR3; F shows the changes in NF-κB and p-NF-κB proteins in CAOV3 cells after knocking down PTX3 in the ovarian cancer cell line CAOV3; G shows the changes in NF-κB and p-NF-κB proteins in SKOV3 cells after overexpressing PTX3 in the ovarian cancer cell line SKOV3; H shows the changes in NF-κB and p-NF-κB proteins in CAOV3 cells after overexpressing PTX3 in the ovarian cancer cell line CAOV3. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below with specific embodiments, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention.

[0028] Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0029] Example 1: Detection of PTX3 expression in ovarian cancer tissues.

[0030] The immunohistochemical staining of human ovarian cancer tissue sections specifically includes the following steps.

[0031] Step 1. Sample processing: The surgically removed ovarian cancer tissue specimens were fixed in 4% paraformaldehyde and 4 μm thick serial sections were prepared.

[0032] Step 2, immunohistochemical staining: dewaxing, rehydration and antigen repair were performed in sequence. After blocking with a blocking solution prepared with 5% fetal bovine serum, the primary antibody was incubated at 4°C overnight. After rewarming the next day, the secondary antibody was incubated at room temperature for 15 minutes, and horseradish peroxidase was incubated for 15 minutes. The Zhongshan Jinqiao kit was used for color development, hematoxylin counterstaining, and lithium carbonate neutralization was performed.

[0033] Step 3: Observation of results: Use a Leica DM3000 microscope to observe the sections and collect images.

[0034] Step 4. Interpretation of results: A semi-quantitative scoring method was used to multiply the staining intensity (0-3 points, corresponding to no staining to strong positive) and the proportion of positive cells (1-4 points, corresponding to ≤25% to >75%) to obtain the final expression score (0-12 points).

[0035] 1. Experimental materials.

[0036] 1.1 Instrument.

[0037] Electric constant temperature drying oven (HWL-70) -- (Tianjin Leboter Instrument Equipment Co., Ltd. - China); microscope (LEICA DM4000) -- (LEICA, Germany).

[0038] 1.2 Reagents.

[0039] 4% paraformaldehyde (Beijing Solebold Technology Co., Ltd., China); PBS buffer (BioInd, Israel); hematoxylin solution (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., China); immunohistochemistry DAB staining kit (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., China); PTX3 antibody (Abcam, USA); water was ultrapure water.

[0040] 2. Experimental method.

[0041] 2.1 Pre-processing of tissue sections.

[0042] The processed tissue sections were placed in a 42.5℃ constant temperature oven overnight, and then cooled at room temperature for 30 minutes. Then, dewaxing and hydration treatment was performed: the sections were placed in three xylene solutions (xylene I, II, and III) in sequence, and each immersion was for 15 minutes for dewaxing; then, they were hydrated in gradient ethanol in sequence: immersion in anhydrous ethanol I and II for 10 minutes, 90%, 80%, and 70% ethanol for 5 minutes each, and finally rinsed with distilled water 3 times, 3 minutes each time.

[0043] 2.2 Antigen processing and blocking.

[0044] The pre-prepared 3% hydrogen peroxide solution was added to the surface of the tissue section to inactivate endogenous peroxidase, incubated at room temperature for 20 minutes, and then rinsed with distilled water three times, 3 minutes each time. The sections were placed in sodium citrate repair solution and repaired in a pressure cooker for 1 hour. After natural cooling, the repair solution was discarded and rinsed with PBS buffer three times, 3 minutes each time. The sections were then placed in a wet box and blocked with blocking solution for 1 hour.

[0045] 2.3 Immunohistochemical staining.

[0046] First, add PTX3 antibody working solution to the slices and incubate overnight at 4°C. Take out the slices the next day, rewarm at room temperature for 30 minutes, and rinse with PBS buffer three times, 3 minutes each time. Incubate with secondary antibody and horseradish peroxidase in sequence: add the corresponding secondary antibody and horseradish peroxidase working solution respectively, incubate at room temperature for 15 minutes each time, and rinse with PBS buffer three times after each step, 3 minutes each time.

[0047] 2.4 Color development and counterstaining.

[0048] The substrate solution and DAB stock solution were mixed at a ratio of 1:20 as recommended in the instructions to prepare the color developing solution. The staining was observed under a microscope. The best staining time was when the slice first turned yellow. After the color development was completed, the slice was placed in the hematoxylin solution for 2 minutes, rinsed with water for 10 minutes, and the nuclear staining effect was observed under a microscope, followed by lithium carbonate blueing for 5 minutes.

[0049] 2.5 Dehydrate and seal the slides.

[0050] The sections were dehydrated in 70%, 80%, 90%, 100% I and 100% II gradient ethanol for 3 min, 3 min, 3 min, 5 min and 5 min respectively. After dehydration, they were dried and about 100 μL of neutral gum was added to each section, covered with a cover glass, and placed in a fume hood to dry naturally to obtain the desired sample.

[0051] 2.6 Interpretation of results

[0052] Interpretation of immunohistochemical staining results: 5 non-repetitive fields of view were randomly selected under a microscope and scored according to the staining results: no staining - 0 point, light yellow - 1 point, yellow or brown - 2 points, yellowish brown or dark brown - 3 points; staining area <5% - 0 point, 5%~25% - 1 point, 25%~50% - 2 points, 50%~75% - 3 points, >75% - 4 points. The two were multiplied to obtain the immunohistochemical score, and the average was calculated as the final score for each film.

[0053] 3. Experimental results.

[0054] 3.1 Immunohistochemistry results.

[0055] like Figure 1 As shown, the expression level of PTX3 was significantly upregulated in paired peritoneal metastatic lesions compared with primary ovarian cancer lesions.

[0056] Example 2: Functional experiment on the effect of PTX3 on ovarian cancer cells.

[0057] 1. Methods and materials

[0058] 1.1 Transfection.

[0059] 1.1.1 Experimental reagents.

[0060] Lipofectamine 3000 transfection reagent (Invitrogen, USA); PTX3 knockdown plasmid (Shanghai Genetech Co., Ltd., China); PTX3 overexpression plasmid (Hanheng Biotech (Shanghai) Co., Ltd., China).

[0061] 1.1.2 Experimental equipment.

[0062] 4℃ chromatography cabinet (Haier Group Corporation, China); biological safety cabinet (Haier Group Corporation, China); negative pressure pipette (Dalong Xingchuang Laboratory Instrument Co., Ltd., China).

[0063] 1.1.3 Experimental methods.

[0064] Select ovarian cancer cell lines grown to 50% density; Prepare the transfection reagent, specifically: slowly add 3.75 μL lipofectamine 3000 into 125 μL serum-free medium and let stand at room temperature for 5 min; Transfection was prepared as follows: 2.5 μg of plasmids of the experimental group and the control group were added to 125 μL of serum-free medium, mixed at a ratio of 1:1, and allowed to stand at room temperature for 20 minutes; The cells to be transfected were pretreated as follows: the culture medium was removed and washed twice with PBS; Add the mixture prepared in the previous step to the 6-well plate and add 1750 μL of complete medium; The transfected cells were cultured in a 37°C, 5% CO2 incubator for 24 hours.

[0065] 1.2 Transwell migration and invasion assay.

[0066] After 24 hours of transfection, the cells were washed with PBS and digested with trypsin. 5 The cells were inoculated at a density of 1:15 / 200 μL in the Transwell chamber (the invasion experiment required laying and solidifying 1:15 diluted matrix gel in the chamber pre-cooled at -20°C in advance), and 600 μL of complete culture medium was added to the lower chamber. After culturing for 24 hours, the cells were fixed with anhydrous methanol and stained with hematoxylin-eosin. After removing the cells that did not penetrate the membrane, the cells were observed and counted under a microscope.

[0067] 1.3 EdU and CCK8 proliferation assay.

[0068] 1.3.1 EdU experiment

[0069] Place the cells into a 6-well plate and inoculate 6×10 5 The cells were added with EdU working solution diluted 1:500 in complete medium and incubated for 2 hours. After being fixed with 4% paraformaldehyde for 15 minutes, they were washed with PBS containing 3% BSA, permeabilized with 0.3% TritonX-100, and incubated with Click reaction solution (including Click Reaction Buffer, CuSO4, Azide 594 and ClickAdditive Solution) at room temperature in the dark for 30 minutes. Finally, they were stained with DAPI and stored in the dark at 4°C and the images were taken.

[0070] 1.3.2 CCK8 experiment

[0071] The cells were digested with trypsin and counted 24 hours after transfection, and seeded in a 96-well plate (5 replicate wells in each group) at a density of 3000 cells / well. 10 μL of CCK-8 reagent was added at 4 hours (0h) and 24h, 48h, 72h, and 96h after seeding and incubated for 2 hours. The absorbance value was detected at a wavelength of 450nm using an enzyme reader, and the cell growth curve was drawn accordingly.

[0072] 2. Experimental results.

[0073] 2.1 Experimental results of transfection efficiency detection.

[0074] Depend on Figure 2 It can be seen that compared with the control group without knockdown, the expression of PTX3 was significantly downregulated after PTX3 knockdown (A, B); and the expression of PTX3 was significantly upregulated after PTX3 overexpression (C, D). * P <0.05,** P <0.01, **** P <0.0001.

[0075] 2.2 Transwell migration and invasion assay results.

[0076] Depend on Figure 3 It can be seen that compared with the control group, the migration and invasion abilities of ovarian cancer cells were significantly reduced after PTX3 knockdown (A, B); and the migration and invasion abilities of ovarian cancer cells were significantly enhanced after PTX3 overexpression (C, D). * P <0.05,** P <0.01,*** P <0.001.

[0077] 2.3EdU cell proliferation experiment results.

[0078] Depend on Figure 4 It can be seen that changes in the expression level of PTX3 significantly affect the proliferation ability of ovarian cancer cells.

[0079] 2.4 Results of CCK8 cell proliferation experiment.

[0080] Depend on Figure 5 It can be seen that compared with the control group, the proliferation ability of ovarian cancer cells was significantly reduced after knocking down PTX3 (A, B); after overexpressing PTX3, the proliferation ability of ovarian cancer cells was significantly enhanced (C, D). P >0.05,* P <0.05,** P <0.01,*** P <0.001.

[0081] Example 3: Study on the mechanism of PTX3 regulating the function of ovarian cancer cells.

[0082] 1. Methods and materials

[0083] 1.1 Immunofluorescence

[0084] 1.1.1 Experimental reagents.

[0085] Anti-fluorescence quenching mounting medium (Beijing Solebow Technology Co., Ltd., China); DAPI staining solution (ThermoFisher, USA); AlexaFluor fluorescent secondary antibody (ThermoFisher, USA).

[0086] 1.1.2 Experimental equipment: DM3000 microscope (Leica, Germany).

[0087] 1.1.3 Experimental methods.

[0088] The wax blocks of human ovarian cancer primary lesion tissue and paired peritoneal metastasis lesion tissue were first sliced ​​and baked, then dewaxed using xylene and a series of ethanol with decreasing concentrations. After rinsing with distilled water, the sections were placed in citrate buffer for high temperature and high pressure antigen retrieval.

[0089] Then, after washing with PBS, the key steps of tissue autofluorescence quenching, blocking solution incubation, primary antibody incubation, fluorescent secondary antibody incubation and DAPI staining were carried out in sequence. The primary antibody was incubated overnight at 4°C, and the fluorescent secondary antibody was incubated for 1 hour at room temperature in the dark. Finally, after rinsing with PBS, the slides were sealed with anti-fluorescence quencher containing DAPI, observed under a fluorescence microscope, and images were collected for analysis.

[0090] 1.2 Immunoblotting.

[0091] 1.2.1 Experimental reagents.

[0092] SDS-polyacrylamide gel kit (Nanjing Keygen Biotechnology, China); glycine solution (Beijing Solebio Technology Co., Ltd., China); SDS (BBI, Canada); total protein extraction kit (Nanjing Keygen Biotechnology, China); TBST buffer (Beijing Biomed, China); primary antibodies: PTX3 (Abcam, USA), E-cadherin (Signalway Antibody, USA), N-cadherin (Signalway Antibody, USA), vimentin (Wuhan Tri-Eagle Biotechnology Co., Ltd., China), NF-κB (Cell Signaling Technology, USA), phosphorylated NF-κB p65 (Cell Signaling Technology, USA), β-tubulin (Cell Signaling Technology, USA), CD44 (Cell Signaling Technology, USA), and TLR4 (Wuhan Tri-Eagle Biotechnology Co., Ltd., China); secondary antibodies (Zhongshan Jinqiao, China); luminescent solution (Nanjing Keygen Biotechnology, China).

[0093] 1.2.2 Experimental equipment.

[0094] Digitally controlled rocker shaker (SK-R330-Pro LCD)-(Beijing Dalong Xingchuang Experimental Instrument Co., Ltd., China); electrophoresis transfer system (Bio-RAD, USA); chemiluminescence analyzer (MicroChemi 4.2) DNR, Israel.

[0095] 1.2.3 Experimental methods.

[0096] Prepare the lysis buffer reaction system, including 0.4μL phosphatase inhibitor, 0.2μL PMSF, 0.04μL protease inhibitor and 40μL Lysis Buffer. After washing the cultured cells twice with pre-cooled PBS, add 45μL of the prepared lysis buffer to each well and place it in an ice box and shake it on a shaker for 20 minutes. Then collect the cells with a cell scraper, transfer them to a centrifuge tube and let them stand on ice for 10 minutes, centrifuge them at 4°C and 12000g for 20 minutes, and collect the supernatant for storage.

[0097] When performing protein quantification, first prepare a standard curve in a 96-well plate, set a 0-20μL protein standard solution gradient (corresponding to 0-10μg protein), and use ddH2O to make up the volume to 20μL. Add 18μL ddH2O and 2μL of the protein sample to be tested to the sample well, and then add 200μL BCA working solution to each well (the ratio of reagent A solution to B solution is 50:1). After incubating the 96-well plate at 37℃ for 30 minutes, use an ELISA reader to measure the absorbance at a wavelength of 562nm, and calculate the sample protein concentration using the standard curve.

[0098] Western Blot analysis uses conventional SDS-PAGE electrophoresis and membrane transfer techniques. According to the molecular weight of the target protein, a suitable concentration of separation gel (usually 10%) is selected, and the protein sample (15μL / well) treated at 100℃ for 5 minutes is subjected to electrophoresis separation (100V for 30 minutes and then adjusted to 120V for 50 minutes). The protein after electrophoresis is transferred to a methanol-activated PVDF membrane (100V for 60 minutes, 4℃).

[0099] The cells were then blocked with 5% skimmed milk powder at room temperature for 2 hours, and incubated with primary antibodies (4°C overnight) and secondary antibodies (room temperature for 1 hour) in sequence. The cells were washed thoroughly with TBST during and after the incubation, and finally developed using a chemiluminescent substrate for imaging.

[0100] 2. Results.

[0101] 2.1 Immunofluorescence experiment results.

[0102] Depend on Figure 6 It can be seen that PTX3 co-localizes with TLR4 and CD44 in ovarian cancer cells OVCAR3.

[0103] 2.2 Western Blot experimental results.

[0104] Depend on Figure 7 It can be seen that knocking down PTX3 in ovarian cancer cells inhibits the activation of EMT and key proteins in the NF-κB pathway; overexpression of PTX3 can promote the activation of EMT and key proteins in the NF-κB pathway. P >0.05,* P <0.05,** P <0.01,*** P <0.001, **** P <0.0001.

[0105] 3. Discussion.

[0106] Studies have shown that the role of PTX3 in cancer is affected by multiple factors, including cell origin, cancer type, and tumor microenvironment characteristics. There is evidence that PTX3 is upregulated in ovarian cancer tissues, suggesting that it may be used as a biomarker for the diagnosis and prognosis of ovarian cancer. However, the mechanism of action of PTX3 in peritoneal metastasis of ovarian cancer has not yet been fully elucidated.

[0107] Through in-depth research, the present invention found that overexpression of PTX3 can significantly promote the proliferation, migration and invasion of ovarian cancer cells, and its expression level is closely related to the progression of peritoneal metastasis of ovarian cancer. Further studies have shown that PTX3 can not only directly promote malignant biological behavior, but also co-localize with the TLR4 receptor on the surface of ovarian cancer cells, activate epithelial-mesenchymal transition and NF-κB signaling pathway, thereby further promoting peritoneal metastasis of ovarian cancer. This reflects the dual cancer-promoting effect of PTX3 in the process of peritoneal metastasis of ovarian cancer.

[0108] More importantly, the present study confirmed that inhibiting the expression of PTX3 in ovarian cancer cells can effectively inhibit their various malignant biological behaviors during peritoneal metastasis, including cell proliferation, migration and invasion. This not only provides an important theoretical basis and experimental basis for PTX3 as a therapeutic intervention target for ovarian cancer peritoneal metastasis, but also provides a new idea for the development of a therapeutic strategy for ovarian cancer peritoneal metastasis targeting PTX3 and its receptor. Through targeted intervention of PTX3, it is expected to achieve effective inhibition of ovarian cancer peritoneal metastasis, which has significant clinical application prospects.

[0109] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of PTX3 gene or its encoded protein in the preparation of a kit for the diagnosis and screening of peritoneal metastasis of ovarian cancer.

2. The use according to claim 1, characterized in that: The kit for diagnosing and screening peritoneal metastasis of ovarian cancer uses PTX3 as a diagnostic marker.

3. The use according to claim 2, characterized in that: The kit for diagnosing and screening peritoneal metastasis of ovarian cancer detects the expression level of PTX3 in surgically removed tissues of patients with peritoneal metastasis of ovarian cancer by western or quantitative PCR technology. The degree of PTX3 expression is positively correlated with the incidence of peritoneal metastasis of ovarian cancer.

4. Use of a detection reagent for the expression level of the PTX3 gene or its encoded protein in the preparation of an auxiliary diagnosis product for peritoneal metastasis of ovarian cancer.

5. The use according to claim 4, characterized in that: The product detects the expression level of PTX3 in a sample by reverse transcription PCR, real-time quantitative PCR, in situ hybridization, Northern blotting, chip, high-throughput sequencing platform, immunohistochemical staining or enzyme-linked immunosorbent assay.

6. The use according to claim 5, characterized in that: The product is a chip, a preparation or a kit; the sample is a tissue, serum or a cell.

7. Application of inhibitors of PTX3 protein expression levels in the preparation of drugs for the treatment of peritoneal metastasis of ovarian cancer.

8. Use of inhibitors of PTX3 protein expression levels in the preparation of drugs for inhibiting the proliferation, migration and invasion of ovarian cancer cells.

9. The use according to claim 7 or 8, characterized in that: The inhibitor of PTX3 protein expression level is a plasmid containing a nucleic acid molecule.

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