Application of NLRP2 as a target in the diagnosis and / or treatment of ovarian cancer

Abstract

The present invention provides the use of NLRP2 as a target in the diagnosis and / or treatment of ovarian cancer, relating to the field of biomedicine. Through big data analysis of ovarian cancer, the present invention discovered that NLRP2 is highly expressed in ovarian cancer. It was found that knocking down NLRP2 in ovarian cancer cell lines significantly inhibited ovarian cancer cell growth. Targeted inhibition of NLRP2 can also significantly inhibit the growth and proliferation of tumor cells. Using NLRP2 as a therapeutic target for ovarian cancer and developing and preparing ovarian cancer therapeutic drugs provides new insights into the mechanisms and treatment strategies involved in the development and progression of ovarian cancer, enriches ovarian cancer detection indicators, and improves the accuracy of ovarian cancer screening.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of NLRP2 as a target in the diagnosis and / or treatment of ovarian cancer. Background Art

[0002] Ovarian cancer (OC) is the third most common female malignant tumor and has the highest mortality rate among gynecological malignancies. It is insidious in onset, prone to infiltration and metastasis, and has an extremely high recurrence rate, seriously threatening women's lives and health. The 5-year survival rate of patients with stage I ovarian cancer can exceed 90%, while the 5-year survival rate of patients with advanced ovarian cancer is only 39%. However, the ovaries are deep in the pelvic cavity, and ovarian lesions in the early stages often have no specific clinical symptoms. When they seek medical treatment due to symptoms, 70% of patients are already in the advanced stage. Therefore, ovarian cancer is a major challenge in both diagnosis and treatment. Therefore, exploring the key molecules in the development and progression of ovarian cancer is crucial for the diagnosis and treatment of ovarian cancer.

[0003] Tumor development and progression are closely linked to inflammatory responses. Inflammatory cells can kill pathogens, promote tissue repair, and inhibit tumor growth, thereby exerting a tumor-suppressing effect. Different mammals possess distinct NLRP protein members, each composed of three conserved domains: PYD, NACHT, and LRRs. These members exhibit distinct expression profiles and are primarily categorized as immune-related and reproductive-related proteins. Immune-related proteins include NLRP1, NLRP3, NLRP6, NLRP10, and NLRP12, which primarily function in the innate immune system. Reproductive-related proteins include NLRP2, NLRP4, NLRP5, NLRP7, NLRP8, NLRP9, NLRP11, NLRP13, and NLRP14, which primarily function in the mammalian reproductive system. NLRP2 is abundantly expressed in mouse oocytes during the germinal vesicle stage and plays a crucial role in oocyte maturation and early embryonic development. NLRP2 deficiency significantly inhibits early embryonic development in mice. NLRP2 acts as an inflammasome sensor. After detecting danger signals, it recruits apoptosis-associated speck-like protein (ASC) and Pro-caspase-1 to form an inflammasome complex. The inflammasome activates by causing the self-cleavage of caspase-1, and after activation, it processes Pro-IL-1β and Pro-IL-18 into their biologically active forms to promote inflammatory responses.

[0004] Therefore, it is particularly important to develop and prepare ovarian cancer therapeutic drugs to provide new ideas for exploring the mechanism research and treatment strategies in the occurrence and development of ovarian cancer, enrich ovarian cancer detection indicators, and improve the accuracy of ovarian cancer screening. Summary of the Invention

[0005] The present invention aims to provide an application of NLRP2 as a target in the diagnosis and / or treatment of ovarian cancer.

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

[0007] The present invention provides an application of a reagent for detecting NLRP2 expression in the preparation of a product for diagnosing ovarian cancer.

[0008] Preferably, the detection reagent diagnoses the development of ovarian cancer by detecting the expression level of NLRP2 in ovarian cancer tissue cells of a subject and comparing it with normal ovarian tissue cells.

[0009] Preferably, the expression level of NLRP2 in different subtypes of ovarian cancer tissue cells is higher than that in normal ovarian epithelial cells.

[0010] The present invention also provides an application of an NLRP2 inhibitor in the preparation of a drug for treating ovarian cancer.

[0011] Preferably, the inhibitor is selected from an NLRP2 gene antagonist, a shRNA that inhibits NLRP2 gene expression, or a gene editing vector that inhibits NLRP2 gene expression.

[0012] Preferably, the shRNA comprises the sequences SEQ ID NO.1 and SEQ ID NO.2 during synthesis.

[0013] Preferably, the inhibitor can target and inhibit NLRP2 expression, significantly inhibiting the growth and proliferation of ovarian cancer cells.

[0014] The present invention also provides a use of an ovarian cancer molecular marker NLRP2 in the preparation of a diagnostic reagent for diagnosing the prognosis and progression of ovarian cancer.

[0015] The present invention also provides a use of a reagent for detecting NLRP2 expression in the preparation of a product for evaluating the survival time of ovarian cancer patients.

[0016] Preferably, the high expression of NLRP2 is significantly negatively correlated with the survival time of ovarian cancer patients.

[0017] Compared with the prior art, the present invention has the following technical effects:

[0018] NLRP2 is an inflammasome receptor and a factor associated with inflammation. Studies have shown that NLRP2 is expressed in oocytes and is associated with oocyte development and early embryonic development. The present invention found that NLRP2 expression is significantly increased in human ovarian cancer tissue cells compared to normal tissue cells, and its expression is significantly negatively correlated with the survival prognosis of ovarian cancer patients. Inhibiting NLRP2 expression will significantly inhibit the growth and proliferation of ovarian cancer cells, suggesting that NLRP2 is a potential diagnostic or therapeutic target for ovarian cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 To analyze NLRP2 expression profile using The Cancer Genome Atlas (TCGA) dataset;

[0020] Figure 2 This is a diagram showing the expression of NLRP2 in different human cancer types;

[0021] Figure 3 The figure shows the expression of NLRP2 in different subtypes of ovarian cancer tissue cells. In the right figure, the horizontal axis 1 represents High Grade Serous, 2 represents Serous, 3 represents Clear cell, 4 represents Endornetrioid, 5 represents Mucinous, and 6 represents Mixed Germ Cell.

[0022] Figure 4 This is a graph showing the correlation between NLRP2 expression levels and 5-year progression-free survival (FPS) of ovarian cancer patients using Kaplan-Meier analysis;

[0023] Figure 5 This is a diagram showing the expression of NLRP2 in ovarian cancer cell lines detected by Western blot;

[0024] Figure 6 The figure shows the mRNA level of NLRP2 detected by RT-PCR in ovarian cancer cell lines;

[0025] Figure 7 Figure 2 is the expression of NLRP2 in SKOV3 cells after NLRP2 knockdown and NLRP2-sh virus infection;

[0026] Figure 8 This figure shows the short-term and long-term growth effects of NLRP2 knockdown on SKOV3 cells;

[0027] Figure 9 Figure 2 is the expression of NLRP2 in A2780 cells after NLRP2 knockdown and NLRP2-sh virus infection;

[0028] Figure 10This figure shows the short-term and long-term growth effects of knocking down NLRP2 on A2780 cells. DETAILED DESCRIPTION

[0029] The present invention provides an application of a reagent for detecting NLRP2 expression in the preparation of a product for diagnosing ovarian cancer.

[0030] In the present invention, the detection reagent diagnoses the development of ovarian cancer by detecting the expression level of NLRP2 in ovarian cancer tissue cells of a subject and comparing it with normal ovarian tissue cells.

[0031] In the present invention, the expression level of NLRP2 in different subtypes of ovarian cancer tissue cells is higher than that in normal ovarian epithelial cells. In the present invention, the expression of NLRP2 was analyzed using the Cancer Genome Atlas (TCGA) data set. Compared with normal ovarian tissue cells, NLRP2 was significantly more highly expressed in ovarian cancer tissue cells. Among different human cancer types, NLRP2 was significantly more highly expressed in ovarian cancer tissue cells. The expression level of NLRP2 in high-grade serous ovarian cancer cells was 2.2 times that of normal human ovarian epithelial cells, serous ovarian cancer was 3.2 times that of normal ovarian epithelial cells, human ovarian clear carcinoma was 4.5 times that of normal ovarian epithelial cells, endometrioid tumor was 3.3 times that of normal ovarian epithelial cells, and mucinous ovarian cancer was 3 times that of normal ovarian epithelial cells.

[0032] The present invention also provides an application of an NLRP2 inhibitor in the preparation of a drug for treating ovarian cancer.

[0033] In the present invention, the inhibitor is selected from NLRP2 gene antagonists, shRNA that inhibits NLRP2 gene expression, or gene editing vectors that inhibit NLRP2 gene expression.

[0034] In the present invention, the shRNA comprises the sequences SEQ ID NO.1 and SEQ ID NO.2 during synthesis.

[0035] In the present invention, the inhibitor can target and inhibit NLRP2 expression, significantly inhibiting the growth and proliferation of ovarian cancer cells.

[0036] The present invention also provides a use of an ovarian cancer molecular marker NLRP2 in the preparation of a diagnostic reagent for diagnosing the prognosis and progression of ovarian cancer.

[0037] The present invention also provides a use of a reagent for detecting NLRP2 expression in the preparation of a product for evaluating the survival time of ovarian cancer patients.

[0038] In the present invention, high NLRP2 expression is significantly negatively correlated with the survival time of ovarian cancer patients. Using Kaplan-Meier analysis, the present invention showed a significant correlation between NLRP2 expression levels and the 5-year progression-free survival (FPS) of ovarian cancer patients. High NLRP2 expression is associated with significantly reduced patient survival, suggesting that NLRP2 is an oncogene associated with the development and progression of ovarian cancer.

[0039] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0040] Example 1

[0041] Construction of plasmid vector pGreen-NLRP2-sh1

[0042] Construct the pGreen-NLRP2-sh1 vector and clone the shRNA sequence corresponding to the NLRP2 gene into the pGreenPuro vector. Design the pGreen-NLRP2-sh1 primers according to the primer design principles and the instructions for use provided by the pGreenPuro vector company SBI:

[0043] Table 1 pGreen-NLRP2-sh1 primer sequence information

[0044]

[0045] Example 2

[0046] Lentiviral packaging

[0047] The HIV lentiviral expression vector used in this experiment is pGreenPuro. The protein shell and components of the packaged genome are composed of pMD2.G and psPAX2 plasmids. The packaging steps are as follows:

[0048] (1) Plating 293TN cells: One day before transfection, digest 293TN cells to form a single cell suspension, count them using trypan blue, and plate 293TN cells at 2×10 6 The cells were inoculated into 6 cm cell culture dishes to make the cell density reach 50%. The cell density reached 70% to 80% during transfection the next day.

[0049] (2) Transfection: Prepare transfection reagent according to the following system, with a mass ratio of PEI: all plasmids = 3:1.

[0050] Table 2 Plasmid reagent information and dosage

[0051]

[0052] (3) In a biosafety cabinet, prepare four 1.5 mL centrifuge tubes and label them. Add 250 μL of Opti-MEM medium (without serum and double antibody) to each tube.

[0053] (4) The PEI concentration is 1 μg / μL. Add 24 μL of PEI reagent to each of the two tubes. Calculate the required volume of plasmid based on the actual concentrations of the packaging plasmid and the target plasmid. Add equal amounts of pMD2.G and psPAX2 to the other two tubes, and then add the target plasmid pGreenPuro or pGreen-NLRP2-sh1, respectively. Vortex for 15 seconds, centrifuge briefly for 15 seconds, and place in a biosafety cabinet at room temperature for 5 minutes.

[0054] (5) Slowly add the PEI mixture to the DNA mixture, pipette to mix, vortex for 15 seconds, briefly centrifuge for 15 seconds, and let stand at room temperature for 20 minutes.

[0055] (6) Remove the 293TN cells that were plated the day before from the CO2 incubator, mark the lid of the culture dish, carefully aspirate the cell culture medium in the 6 cm culture dish, and carefully and slowly add the above mixed reagent dropwise to the culture dish at a distance of less than 0.5 cm. Slowly tilt the culture dish up and down and left and right to allow the transfection reagent to completely cover the 293TN cells. Let it stand in the biosafety cabinet for 5 minutes.

[0056] (7) Add 2.5 mL of DMEM high-glucose medium containing serum and double antibodies to each culture dish.

[0057] (8) Medium exchange: After 24 hours, carefully remove the culture medium and replace it with 3 mL of fresh DMEM high-glucose medium containing serum and double-antibody. Observe under a fluorescence microscope that 293TN cells transfected with the pGreenPuro vector (containing GFP green fluorescent protein) exhibit green fluorescence.

[0058] (9) Virus collection: After 48 hours, collect the cell supernatant into a 15 mL centrifuge tube and temporarily store in a 4°C refrigerator. Add 3 mL of fresh DMEM high-glucose medium containing serum and double-antibody. After 72 hours, collect the cell supernatant into the same 15 mL centrifuge tube. If the virus is present in the supernatant, centrifuge the collected supernatant at 1000 rpm for 5 minutes, aliquot the virus, label it, and store it in a -80°C refrigerator.

[0059] Example 3

[0060] Construction of Ctrl-sh, NLRP2-sh1 cell lines

[0061] (1) SKOV3 and A2780 ovarian cancer cell lines in good condition were digested and seeded into 6 cm culture dishes at a density of 35%-50%. The density of cell attachment was about 55%-65% the next day.

[0062] (2) The next day, take out the pre-packaged pGreenPuro (Ctrl-sh) and pGreen-NLRP2-sh1 (NLRP2-sh1) viruses from the -80℃ freezer. After thawing, remove the culture medium from the culture dish and carefully add 1 mL of Ctrl-sh and NLRP2-sh1 lentivirus to SKOV3 and A2780 cells, respectively. Place the cells in a CO2 incubator for culture and add 2.5 mL of new culture medium after 6 h.

[0063] (3) When SKOV3 and A2780 cells are fully grown, they are digested and expanded. The normal RPMI-1640 medium is replaced with RPMI-1640 medium containing 2 μg / mL puromycin. The cells are screened and cultured for 2-3 days until there is no more cell death and almost all cells in the cell line infected with the knockdown virus express green fluorescence. The cells are collected and total protein is extracted. Using cells infected with Ctrl-sh as a control, Western blot analysis is performed to confirm that the target gene NLRP2 is knocked down. Subsequent experiments are then conducted to explore the effect of NLRP2 on the growth and proliferation of ovarian cancer cells.

[0064] Example 4

[0065] Trypan blue staining growth counting assay

[0066] (1) Cell plating. The control group (Ctrl-sh) and experimental group (NLRP2-sh1) cells in the logarithmic growth phase were digested and cell suspensions were prepared. After counting using a hemocytometer, the control group cells and experimental group cells were plated at 1×10 4 Each well of a 24-well plate was inoculated with 100 cells / well.

[0067] (2) Cell counting. The first day of cell plating was defined as the day after plating. Cells were then digested and collected on the 3rd, 5th, and 7th days for cell counting. Three wells of each group of cells were counted each time, using the same method of trypan blue staining combined with hemocytometer counting.

[0068] (3) Data processing: The data obtained by counting were used to create a growth curve using Excel and perform statistical analysis.

[0069] Example 5

[0070] Clone formation assay

[0071] (1) The cells in the control group (Ctrl-sh) and the experimental group (NLRP2-sh1) in the logarithmic growth phase were digested and counted after trypan blue staining. The cells of each group were seeded into 12-well plates at a density of 500 or 800 cells per well and cultured in a 37°C incubator. Fresh culture medium was replaced every 3-5 days.

[0072] (2) After 14 days of culture, differences in growth trends were observed between the cell groups treated with different methods. The culture medium was aspirated and the cells were gently rinsed 2-3 times with PBS.

[0073] (3) Gently add 1 mL of formaldehyde fixative along the wall of the well, being careful not to blow up the cells, and fix at room temperature for 20 minutes.

[0074] (4) Aspirate the fixative and add 500 μL of 0.1% crystal violet staining solution to each well. Stain at room temperature for 20 min.

[0075] (5) Aspirate the staining solution and gently rinse with deionized water 3-4 times until the deionized water becomes transparent and colorless.

[0076] (6) Absorb the deionized water and wait for the hole to dry naturally before taking photos and recording.

[0077] (7) Data processing: ImageJ software was used to analyze the cell clone formation ability and perform statistical analysis on the differences.

[0078] The results are analyzed as follows:

[0079] according to Figure 1 It can be seen that the expression of NLRP2 was analyzed using the Cancer Genome Atlas (TCGA) dataset, which showed that NLRP2 was significantly overexpressed in ovarian cancer tissue cells compared with normal ovarian tissue cells;

[0080] according to Figure 2 It can be seen that NLRP2 is significantly overexpressed in ovarian cancer tissues among different human cancer types;

[0081] according to Figure 3 It can be seen that NLRP2 is generally highly expressed in different subtypes of ovarian cancer tissue cells: the expression level of NLRP2 in high-grade serous ovarian cancer is 2.2 times that of normal human ovarian epithelial cells, serous ovarian cancer is 3.2 times that of normal human ovarian epithelial cells, human ovarian clear cell carcinoma is 4.5 times that of normal ovarian epithelial cells, endometrioid tumor is 3.3 times that of normal ovarian epithelial cells, and mucinous ovarian cancer is 3 times that of normal ovarian epithelial cells.

[0082] according to Figure 4High NLRP2 expression is significantly negatively correlated with survival in ovarian cancer patients, suggesting that NLRP2 is an oncogene involved in the development and progression of ovarian cancer. Kaplan-Meier analysis showed a significant correlation between NLRP2 expression and the five-year progression-free survival (FPS) of ovarian cancer patients. High NLRP2 expression was associated with significantly decreased survival.

[0083] according to Figures 5-6 It can be seen that the expression of NLRP2 in ovarian cancer cell lines was detected by Western blot, and NLRP2 was highly expressed in A2780 and SKOV3 cells; the mRNA level of NLRP2 in ovarian cancer cell lines was detected by RT-PCR, and NLRP2 was highly expressed in A2780 and SKOV3 cells.

[0084] according to Figures 7-10 It can be seen that knocking down NLRP2 in the ovarian cancer cell lines SKOV3 and A2780 with high NLRP2 expression, and using cell growth counting and clone formation experiments, it was found that NLRP2 knockdown significantly inhibited the growth of ovarian cancer cells, suggesting that NLRP2 is a potential therapeutic target for ovarian cancer.

[0085] Western blot was used to detect the efficiency of NLRP2 knockdown in SKOV3 cells. After infection with the NLRP2-sh1 virus, NLRP2 expression was significantly reduced. Growth count and colony formation experiments were performed to examine the effects of NLRP2 knockdown on the short-term and long-term growth of SKOV3 cells. Knockdown of NLRP2 inhibited the short-term growth of SKOV3 cells by 95% and colony formation by 74%.

[0086] Western blot was used to detect the efficiency of NLRP2 knockdown in A2780 cells. After NLRP2-sh1 virus infection, the expression of NLRP2 was significantly reduced. Growth counting and clone formation experiments were used to detect the effects of NLRP2 knockdown on the short-term and long-term growth of A2780 cells. After NLRP2 knockdown, the short-term growth of A2780 was inhibited by 80% and the clone formation was inhibited by 74%.

[0087] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Application of reagents for detecting NLRP2 expression in the preparation of ovarian cancer disease diagnostic products.

2. The use according to claim 1, characterized in that The detection reagent diagnoses the development of ovarian cancer by detecting the expression level of NLRP2 in ovarian cancer tissue cells of a subject and comparing it with normal ovarian tissue cells.

3. The use according to claim 2, characterized in that The expression level of NLRP2 in different subtypes of ovarian cancer tissue cells is higher than that in normal ovarian epithelial cells.

4. Application of reagents for detecting the expression of ovarian cancer molecular marker NLRP2 in the preparation of diagnostic reagents for ovarian cancer disease prognosis and progression.

5. Application of reagents for detecting NLRP2 expression in the preparation of products for assessing the survival time of ovarian cancer patients.

6. The use according to claim 5, characterized in that The high expression of NLRP2 is significantly negatively correlated with the survival time of ovarian cancer patients.

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