Use of an inhibitor in the manufacture of a medicament for inhibiting migration and / or invasion of ovarian cancer cells

Abstract

The application discloses an application of an inhibitor in preparation of a reagent for inhibiting migration and / or invasion of ovarian cancer cells, and relates to the technical field of biological medicines.The application of the inhibitor in preparation of the reagent for inhibiting migration and / or invasion of ovarian cancer cells, wherein the inhibitor is an LAMA5 protein N-glycosylation modification inhibitor; the LAMA5 protein N-glycosylation modification inhibitor is selected from PNGase F enzyme, a calicheamicin or a mutant targeting an N-glycosylation site of the LAMA5 gene.A method for screening an anti-ovarian cancer candidate active substance by taking LAMA5 protein N-glycosylation modification as a target is provided.The application discloses a complete molecular regulation axis of LAMA5 protein N-glycosylation modification for promoting migration and invasion of ovarian cancer cells through a cytokine-cytokine receptor interaction channel, and provides a new target and intervention strategy for research and development of an anti-metastasis drug for ovarian cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of an inhibitor in the preparation of a reagent for inhibiting the migration and / or invasion of ovarian cancer cells. Background Technology

[0002] Ovarian cancer is the deadliest type of gynecological malignancy, primarily due to its high mortality rate caused by widespread intraperitoneal implantation and distant organ metastasis. Tumor metastasis is a complex, multi-step pathological process. Activation of epithelial-mesenchymal transition (EMT), cytoskeleton remodeling, and abnormal activation of inflammatory chemotactic signals in the tumor microenvironment are considered the three key drivers that enable tumor cells to acquire the ability to migrate and invade, detach from the primary tumor, and spread to distant sites. Laminin, as an important component of the extracellular matrix, plays a crucial role in mediating cell-matrix adhesion, regulating cell migration, and signal transduction. LAMA5, the α5 subunit of laminin, has been abnormally expressed in various malignant tumors, and its expression level is closely related to the prognosis of cancer patients. Therefore, providing an inhibitor for the preparation of reagents to inhibit the migration and / or invasion of ovarian cancer cells has significant scientific value and clinical application prospects for elucidating new mechanisms of ovarian cancer metastasis and developing novel anti-tumor strategies targeting glycosylation modifications. Summary of the Invention

[0003] In view of this, the present invention addresses the deficiencies of the existing technology, and its main objective is to provide an inhibitor for the application in the preparation of reagents for inhibiting the migration and / or invasion of ovarian cancer cells. It reveals the complete molecular regulatory axis of LAMA5 protein N-glycosylation modification promoting the migration and invasion of ovarian cancer cells through the cytokine-cytokine receptor interaction pathway, providing new targets and intervention strategies for the development of anti-metastatic drugs for ovarian cancer.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: The use of an inhibitor in the preparation of a reagent for inhibiting the migration and / or invasion of ovarian cancer cells, said inhibitor being an inhibitor of N-glycosylation modification of the LAMA5 protein.

[0005] As a preferred embodiment, the LAMA5 protein N-glycosylation modification inhibitor is selected from PNGase F enzyme, tunicamycin, or a mutant targeting the N-glycosylation site of the LAMA5 gene.

[0006] As a preferred embodiment, the LAMA5 protein N-glycosylation modification inhibitor is tunicamycin.

[0007] As a preferred embodiment, the mutant is a mutant with the N-glycosylation site of the Δ3-4 domain of the LAMA5 protein missing.

[0008] As a preferred embodiment, the reagent is either a reagent for reducing the N-glycosylation level of LAMA5 protein or a reagent for inhibiting the activity of the cytokine-cytokine receptor interaction pathway.

[0009] As a preferred embodiment, the reagent used to inhibit the activity of the cytokine-cytokine receptor interaction pathway inhibits the activity of the pathway by downregulating the expression of Csf3, CXCL2, and CXCL3 proteins in the pathway.

[0010] As a preferred embodiment, the reagent used to inhibit the activity of the cytokine-cytokine receptor interaction pathway inhibits the activity of the pathway by upregulating the expression of IL20Ra protein.

[0011] As a preferred embodiment, the ovarian cancer cells are the Ho-8910 cell line or the A2780 cell line.

[0012] A method for screening candidate active substances against ovarian cancer targeting N-glycosylation modification of LAMA5 protein includes the following steps: First, the active substance to be tested is brought into contact with ovarian cancer cells expressing LAMA5 protein; Second, the N-glycosylation modification level of LAMA5 protein, the activity of the cytokine-cytokine receptor interaction pathway, and the cell migration and invasion ability in the ovarian cancer cells were detected. Third, if the test compound reduces the N-glycosylation modification level of LAMA5 protein, inhibits the activity of the cytokine-cytokine receptor interaction pathway, and reduces cell migration and invasion ability, then it is judged to be a candidate drug.

[0013] As a preferred embodiment, the indicators for detecting the activity of the cytokine-cytokine receptor interaction pathway in step three include detecting the downregulation of the expression levels of Csf3, CXCL2, and CXCL3 proteins, and / or detecting the upregulation of the expression level of IL20Ra protein.

[0014] Compared with existing technologies, this invention has significant advantages and beneficial effects. Specifically, as can be seen from the above technical solution: This invention reveals for the first time that LAMA5 protein has N-glycosylation modification in ovarian cancer cells, confirming that N-glycosylation modification is a necessary condition for LAMA5 protein to exert its function of promoting the migration and invasion of ovarian cancer cells. Wild-type LAMA5 with complete glycosylation can significantly promote the migration and invasion of ovarian cancer cells, while its metastatic ability is completely lost after removing the N-glycosylation site mutation. This breaks through the limitation of existing technologies that only focus on the expression level of LAMA5, and elevates the research perspective from "expression level" to a new dimension of "modification state". It provides an application of an inhibitor in the preparation of reagents for inhibiting the migration and / or invasion of ovarian cancer cells, and provides a method for screening anti-ovarian cancer candidate drugs with LAMA5 protein N-glycosylation modification as a target. This provides a new intervention strategy and experimental basis for developing anti-metastatic drugs for ovarian cancer that target protein glycosylation modification, and has important clinical application value and drug development prospects.

[0015] To more clearly illustrate the structural features and effects of the present invention, a detailed description is provided below in conjunction with the accompanying drawings and specific embodiments. Attached Figure Description

[0016] Figure 1 The diagram shows the N-glycosylation modification of LAMA5 in this invention; (A) a diagram of LAMA5 mutation sites predicted by bioinformatics; (B) the expression and statistical graph of LAMA5 in different ovarian cancer cell lines; (C) the change in molecular weight of LAMA5 in Ho-8910 and A2780 cell lines after treatment with PNGase F; and (D) the change in molecular weight of LAMA5 in Ho-8910 and A2780 cell lines after treatment with tunicamycin.

[0017] Figure 2 The diagram shows the inhibition of ovarian cancer cell invasion and migration by tunicamycin according to the present invention; (A) Schematic diagram of scratch assay; (B) Schematic diagram of migration assay; (C) Schematic diagram of invasion assay; (D) Expression and statistical graph of EMT-related proteins after de-glycosylation.

[0018] Figure 3 The diagram shows the inhibition of ovarian cancer cell invasion and migration by de-glycosylated LAMA5 according to the present invention; (A) Schematic diagram of N-glycosylation mutation pattern; (B) Change in molecular weight of LAMA5 after lentiviral transfection; (C) Schematic diagram of scratch assay; (D) Schematic diagram of migration assay; (E) Schematic diagram of invasion assay; (F) Schematic diagram of expression of EMT-related proteins after de-glycosylation; (G) Statistical graph of EMT-related proteins after de-glycosylation. Figure 4This invention provides a schematic diagram illustrating how de-N-glycosylated LAMA5 inhibits ovarian cancer cell invasion and migration via the cytokine-cytokine receptor interaction pathway; (A) differentially expressed gene diagram; (B) KEGG pathway enrichment analysis diagram; (C) schematic diagram of pathway-related protein expression after de-N-glycosylation; (D) schematic diagram of pathway-related protein expression after the addition of tunicamycin. (E and F) are both confocal cytoskeleton. Detailed Implementation

[0019] The present invention is as follows Figure 1 As shown in Figure 4, an inhibitor is used in the preparation of a reagent to inhibit the migration and / or invasion of ovarian cancer cells. This inhibitor is an N-glycosylation modification inhibitor of the LAMA5 protein.

[0020] The LAMA5 protein N-glycosylation modification inhibitor is selected from PNGase F enzyme, tunicamycin, or mutants that target the N-glycosylation site of the LAMA5 gene.

[0021] The inhibitor of N-glycosylation modification of the LAMA5 protein is tunicamycin.

[0022] This mutant is a mutant with the N-glycosylation site of the Δ3-4 domain of the LAMA5 protein missing.

[0023] The reagent is used to reduce the N-glycosylation level of LAMA5 protein or to inhibit the activity of the cytokine-cytokine receptor interaction pathway.

[0024] This reagent, used to inhibit the activity of the cytokine-cytokine receptor interaction pathway, suppresses the pathway activity by downregulating the expression of Csf3, CXCL2, and CXCL3 proteins in the pathway.

[0025] This reagent, used to inhibit the activity of the cytokine-cytokine receptor interaction pathway, suppresses the activity of this pathway by upregulating the expression of IL20Ra protein.

[0026] The ovarian cancer cells are either the Ho-8910 cell line or the A2780 cell line.

[0027] A method for screening candidate active substances against ovarian cancer targeting N-glycosylation modification of LAMA5 protein includes the following steps: First, the active substance to be tested is brought into contact with ovarian cancer cells expressing LAMA5 protein; Second, the study examined the N-glycosylation level of LAMA5 protein, the activity of the cytokine-cytokine receptor interaction pathway, and the cell migration and invasion ability in these ovarian cancer cells. Third, if the test compound reduces the N-glycosylation modification level of LAMA5 protein, inhibits the activity of the cytokine-cytokine receptor interaction pathway, and reduces cell migration and invasion ability, then it is judged as a candidate drug.

[0028] In step three, the indicators for detecting the activity of the cytokine-cytokine receptor interaction pathway include detecting the downregulation of the expression levels of Csf3, CXCL2, and CXCL3 proteins, and / or detecting the upregulation of the expression level of IL20Ra protein.

[0029] Example 1: Application of an inhibitor in the preparation of a reagent for inhibiting the migration and / or invasion of ovarian cancer cells. Experimental methods: Cell culture: Cell resuscitation: (1) Take out the cryovial containing cells from the -80℃ freezer and put it into a clean plastic glove; (2) Melt in a 37°C water bath; (3) Add the frozen cell solution to a centrifuge tube containing 2 mL of DEME medium containing 10% FBS; (4) Centrifuge at 1000 r for 5 min; (5) Discard the supernatant, add 4 mL of complete culture medium to resuspend, transfer to a culture flask, mark the cell information and date with a marker pen, mix in an upside-down figure "8", observe under a microscope, and incubate in a 37℃ constant temperature incubator. Change the medium after 24 h.

[0030] Cell passage: (1) Observe the growth status of cells in the culture flask under a microscope; (2) Discard the supernatant, wash twice with PBS, add 1 mL of trypsin for digestion, and neutralize with complete culture medium; (3) Centrifuge at 1000 r for 5 min; (4) Discard the supernatant, resuspend the complete culture medium, transfer it to two culture flasks, mix by turning the mixture in a figure-eight shape, observe under a microscope, and place in an incubator.

[0031] Cell cryopreservation: (1) Cells with a confluence rate of 80%-90% observed under a microscope can be cryopreserved as needed; (2) Irradiate the ultra-clean workbench with ultraviolet light for 30 minutes and then disinfect with 75% alcohol; (3) Remove the cryovial and label it with cell information and date; (4) Remove the culture flask containing the cells, discard the old culture medium, wash twice with PBS, add 1 mL of trypsin to digest for 1.5-2 min, add complete culture medium to neutralize, gently blow to mix, and transfer to a centrifuge tube; (5) Centrifuge at 1000 rpm for 5 minutes; (6) Discard the supernatant, add 1 mL of serum-free cell cryopreservation solution, gently mix by blowing, transfer to a cryovial, and seal with sealing film. Place in a cryovial box and store at -80°C.

[0032] Lentiviral transfection: (1) Transfection can be performed when cells are in the logarithmic growth phase as observed under a microscope; (2) Remove the old culture medium, wash twice with PBS, digest with trypsin, neutralize with complete culture medium, and centrifuge at 1000 r for 5 min; (3) Discard the supernatant and resuspend in 1 mL of complete culture medium; (4) Take another 100 μl of cell suspension and add it to 900 μl of complete culture medium and mix well (if there are too few cells, you can count them directly without dilution). (5) Remove the counting plate, wipe it with an alcohol swab, and cover it with a coverslip; (6) Add 10 μl of cell suspension to the gap between the coverslip and the slide; (7) Observe under a microscope and count the total number of cells in the four quadrants using a cell counter (counting principle: count the top, not the bottom, count the left, not the right). Calculation formula: Cell concentration of the cell suspension to be tested (cells / mL) = (average number of cells in the four quadrants) × 10^4 × dilution factor; (8) Adjust the cell density by adding 2 mL of cell suspension to a 6-well plate, with 5 × 10^5 cells per well, and then incubate the 6-well plate in an incubator. (9) Cell transfection was performed on the afternoon of the second day. The old culture medium was discarded, and the cells were washed twice with PBS. Transfection buffer was prepared: 1 mL LDMMEM + 1 μL polybrene per well. Different amounts of GSTO1 shRNA lentivirus were added according to the multiplicity of infection (MOI) value of each cell line. Calculation formula: Lentiviral addition amount (μL) = (Number of cells exposed to lentivirus × MOI) / Lentiviral titer; (10) After 12-16 h of transfection, discard the transfection solution in the well and replace it with 2 mL of complete culture medium. Observe the transfection efficiency under a fluorescence microscope after 72 h. (11) After the culture flask is fully grown, it is transferred to 2 flasks. When the cells grow to about 70%-80% of the bottom of the flask, puromycin is added for screening.

[0033] (12) Continue until the number of cells with green fluorescence observed under the microscope is greater than 95% of the total number of cells under the microscope.

[0034] Western blot assay for proteins: Total protein extraction: (1) Under a microscope, the cell confluence was found to be above 90%; (2) Take the cells to be extracted, discard the old culture medium, wash 3 times with PBS, stand the culture flask upright, and thoroughly aspirate the remaining PBS; (3) Lysis mixture: 1 mL RIPA lysis buffer + 10 μl PMSF; (4) Add 800 μl of lysis mixture to each flask of cells, place the cell culture flask on ice and lyse for 30 min; scrape off the cells in one direction with a scraper; (5) First, turn on the low temperature high speed centrifuge, adjust the temperature to 4℃ for pre-cooling, transfer the scraped cells and lysis mixture to a 1.5mL centrifuge tube, set the centrifuge to 12000r for 30min; aspirate the protein supernatant into a new centrifuge tube; (6) Take 2 μl of protein solution, measure the protein concentration using NanoDrop, measure the volume of protein solution in the remaining centrifuge tube, add 1 / 4 volume of protein loading buffer, and mix thoroughly; (7) Set the temperature to 100℃ for 5 minutes using a metal thermostat.

[0035] Electrophoresis: (1) Fix the gel glass plate on the electrophoresis rack, add more electrophoresis solution than the thin glass plate to the two gels, and slowly pull out the lane comb; add 5 μl of protein marker to the first lane on the left, and add 10 μl of protein mixture to each of the remaining lanes; (2) Place the electrophoresis rack in the electrophoresis tank and add electrophoresis buffer according to the scale on the tank. Connect the electrophoresis apparatus and set the following settings: (1) 60V electrophoresis for 30 min, (2) 150V electrophoresis for 50 min. Observe the position of each molecular weight indicator band on the marker. When the position of bromophenol blue is close to the bottom of the gel, the electrophoresis can be terminated.

[0036] Transfer membrane: (1) Cut a PVDF membrane to a suitable size, immerse it in methanol for 5 min to activate it, and at the same time put two thick filter papers and a buffer pad into the transfer solution; take out the gel after electrophoresis, cut the gel according to the size of the target molecular weight, and include at least the part above and below the target molecular weight. (2) Place the black side of the transfer clamp at the bottom, and then place the buffer pad, filter paper, gel, PVDF membrane, filter paper, and buffer pad in sequence. Use a roller to remove air bubbles between the gel and the PVDF membrane. Place the transfer clamp in the transfer rack, then place it in the transfer tank. Place an ice pack in the transfer tank, and then place the transfer tank in ice water. (3) Connect to electrophoresis and set the transfer time according to the molecular weight of the target protein; constant current: 400mA.

[0037] Blocking and antibody incubation: (1) Blocking: Prepare TBST blocking liquid with 5% skim milk powder, and wash the PVDF membrane after transfer with TBST twice, 5 min / time; place the membrane in the blocking liquid and block it on a shaker at room temperature for 2 hours, and wash with TBST for 30 min / 6 times; (2) Primary antibody incubation: Prepare 5% BSA TBST antibody dilution solution, dilute the primary antibody according to the dilution concentration in the antibody instructions, place the PVDF membrane in the primary antibody dilution solution, incubate overnight at 4°C or at room temperature on a shaker for 2 hours, and wash with BST for 30 minutes / 6 times. (3) Secondary antibody incubation: Prepare 5% BSA TBST antibody dilution solution, dilute the secondary antibody at a ratio of 1:10000, incubate at room temperature on a shaker for 2 hours, and wash with TBST for 30 minutes / 6 times; (4) Development: Take the ECL ultraluminescence reagent kit, add solution A and solution B in a test tube at a 1:1 ratio and mix well. Place the PVDF membrane on filter paper to remove residual TBST, place it in a glass dish, add an appropriate amount of developing solution, and expose and develop it in a chemiluminescence analyzer. Select a band with a suitable exposure time for preservation. Analyze the grayscale value using ImageJ software.

[0038] Trasnwell migration experiments: (1) Remove the cell culture flask, observe the cell state, and digest the cells; centrifuge at 1000r for 5min; (2) Discard the supernatant and resuspend in 1 mL of culture medium; (3) Count the cells under a microscope to achieve a cell concentration of 5×10^4 / mL; (4) Add 600 μL of complete culture medium containing 20% ​​FBS to each well of a 24-well plate, seed 200 μL of cell suspension in the upper chamber of a Trasnwell chamber, and incubate for 24 h in an incubator; (5) Carefully wash out the liquid on the upper layer of the chamber, immerse the chamber in PBS for 5 minutes, and repeat 3 times; carefully remove the cells on the upper layer of the chamber that have not penetrated the pores using a cotton swab; (6) Fix; (7) Staining.

[0039] Matrigel invasion experiment: (1) Take out the Matrigel matrix and let it melt slowly at 4°C; (2) Dilute the melted matrix gel with the basal culture medium at a ratio of 1:5 to prepare the working concentration; (3) Take 50 μL of working concentration of matrix gel and drop it vertically into the middle of the upper chamber. Gently shake the culture plate to spread it evenly in the upper chamber. Then put the culture plate into a 37°C incubator to solidify for 30 min. (4) Remove the culture plate and add 600 μL of complete culture medium containing 20% ​​FBS to the wells of the culture plate; (5) Remove the cell culture flask, observe the cell state, and digest the cells; centrifuge at 1000r for 5min; (6) Discard the supernatant, add serum-free DMEM to resuspend the cells, count them using a cell counting chamber, and dilute to 5 × 10^4 cells per milliliter; (7) Take 200 μL of the diluted cell suspension and carefully add it vertically to the upper chamber, then incubate in an incubator for 24 hours; (8) Aspirate the liquid from the upper chamber, wash with PBS, and carefully remove the cells from the upper chamber with a cotton swab; (9) Fixed; (10) Staining.

[0040] Wound healing experiment: (1) Draw two horizontal lines evenly on the bottom surface of the 6-hole plate with a ruler; (2) Remove the cell culture flask, observe the cell state, and digest the cells; centrifuge at 1000r for 5min; (3) Discard the supernatant and resuspend the cells in basal culture medium; (4) According to the principle that the cells in each culture flask with 90% confluence can be seeded into one 6-well plate, seed the cell suspension into the 6-well plate, mix it in the figure-eight method, and put it into the incubator for culture; (5) Observe the growth of cells. When the cell confluence reaches 90%, use the thin end of a 200ul pipette tip to draw two vertical lines along the direction perpendicular to the horizontal line. (6) Wash with PBS, replace with fresh culture medium, and continue culturing; (7) The photo taking time was 0h and 24h respectively. Before taking the photo, the PBS was used to clean and replace the culture medium.

[0041] Transcriptome sequencing analysis: Cell samples were treated with TRIzol, and RNA from the total samples was isolated and purified according to the manufacturer's operating protocol. Sequencing was performed by Hangzhou Lianchuan Biotechnology Co., Ltd. using the Illumina Novaseq 6000 platform. After obtaining the sequencing data, the data underwent strict quality control, followed by transcriptome comparison, gene expression level studies, differential gene expression analysis, enrichment analysis, and other analyses.

[0042] Phalloid peptide staining: Storage liquid preparation: YF@ dye / Rhodamine-labeled phalloidin: Take an appropriate amount of methanol or sterile water to dissolve the lyophilized powder in the tube and prepare a 200T / mL stock solution (add 1.5 mL of liquid for 300T dye and 0.25 mL of liquid for 50T dye).

[0043] Fixed cell staining: (1) Wash the cells three times with PBS.

[0044] (2) Fix the cells with PBS solution containing 4% formaldehyde and fix at room temperature for 20 min.

[0045] (3) Wash the cells three times with PBS.

[0046] (4) Permeabilize the cells with PBS solution containing 0.4% Triton X-100 at room temperature for 10 min. (5) Wash the cells with PBS 3 times.

[0047] (5) Dilute 1-5 μL of fluorescently labeled phalloidin stock solution with 200 μL PBS, add it to a coverslip or well, incubate at room temperature for 20 min, and then stain.

[0048] (6) Wash the cells with PBS 2-3 times.

[0049] (7) Fluorescence microscopy observation. The labeled phalloidin has good photostability, and the sample can be imaged in PBS. However, for the best results, an anti-fluorescence quencher can also be used for observation.

[0050] Discussion of Results: LAMA5 exhibits N-glycosylation modification: To determine the presence of N-glycosylation modification in LAMA5, bioinformatics was first used to predict the N-glycosylation modification sites in LAMA5 (see...). Figure 1 (A). Subsequently, LAMA5 expression was detected in different ovarian cancer cell lines (see [reference needed]). Figure 1 (See Figure B). The results showed that LAMA5 expression was higher in the Ho-8910 and A2780 cell lines. To further validate the bioinformatics results, PNGase F (see Figure B) was used. Figure 1 (C) and tunicamycin (see Figure 1 Treatment of Ho-8910 and A2780 cell lines with D) resulted in a decrease in the molecular weight of LAMA5 from 400 kDa to 385 kDa. These results indicate that LAMA5 undergoes N-glycosylation modification.

[0051] Acetaminophen inhibits the invasion and migration of ovarian cancer cells Through scratch test (see) Figure 2 (A) showed that the scratch gaps in both the NC and DMSO control groups significantly decreased with increasing culture time; treatment with tunicamycin to inhibit N-glycosylation significantly slowed the scratch healing rate of both ovarian cancer cell types. Furthermore, Transwell migration and invasion assays (see...) Figure 2 B, Figure 2 Treatment with tunicamycin (C) significantly reduced the number of cells that passed through the chamber. Furthermore, the expression of EMT-related proteins was also detected (see [reference needed]). Figure 2 The results (D) showed that the expression of the epithelial marker E-Cadherin protein was upregulated, while the expression levels of the mesenchymal markers N-Cadherin, Snail, and Vimentin proteins were significantly downregulated. This suggests that inhibiting N-glycosylation modification can reverse the EMT process in ovarian cancer cells. In summary, inhibiting N-glycosylation modification weakens the migration and invasion abilities of ovarian cancer cells, and simultaneously inhibits the EMT epithelial-mesenchymal transition process.

[0052] De-glycated LAMA5 inhibits the invasion and migration of ovarian cancer cells. To verify these results, the N-glycosylation modification of different domains of LAMA5 was knocked down (see...). Figure 3 (A). Western blot was used to verify the change in LAMA5 protein molecular weight after lentiviral transfection (see [link]). Figure 3 In the B-cell mutant, the molecular weight and amount of LAMA5 protein at the Δ3-4 mutant site were significantly reduced, demonstrating that the site mutation successfully removed the N-glycosylation modification of LAMA5. Scratch assay (see...) Figure 3 The results (C) showed that, compared with the NC group and the WT wild-type group, the scratch healing rate of cells in the Δ3-4 mutant group was significantly slower. Transwell migration and invasion assays (see [link to assay]) Figure 3 (D, E) indicated that compared with the NC group and the WT wild-type group, the number of cells passing through the chamber was significantly reduced in the Δ3-4 mutant group. Furthermore, the expression of EMT-related proteins (see D, E) was also significantly reduced. Figure 3 (China F) also and Figure 2 The results were consistent with those in the study. N-glycosylation modification of LAMA5 is key to promoting ovarian cancer cell migration, invasion, and EMT; removal of N-glycosylated LAMA5 can inhibit ovarian cancer cell migration and invasion, and reverse the EMT process.

[0053] De-glycosylated LAMA5 inhibits the invasion and migration of ovarian cancer cells through the cytokine-cytokine receptor interaction pathway. Further investigation was conducted into the mechanism by which LAMA5 N-glycosylation affects the invasion and migration of ovarian cancer cells. Transcriptomic sequencing was used to identify differentially expressed genes (see...). Figure 4(A) and enrichment analysis via the KEGG pathway (see [link]). Figure 4 (B) indicates the most significant difference in the cytokine-cytokine receptor interaction pathway. Subsequently, mutants were examined (see...). Figure 4 (C) and after the addition of tunicamycin (see C) Figure 4 Protein expression in the Δ3-4 mutant and tunicamycin-added groups was analyzed, revealing significantly decreased Csf3, CXCL2, and CXCL3 protein expression levels, while significantly increased IL20Ra protein expression. This suggests that LAMA5-dependent N-glycosylation modification activates the cytokine-receptor interaction pathway. Subsequently, only phalloidin staining was used to observe the cytoskeleton (see...). Figure 4 Chinese E, Figure 4 In the NC and WT wild-type groups, the cytoskeleton fibers were abundant, and pseudopodia and filamentous processes were well-developed. However, in the Δ3-4 mutant deglycosylation mutant group, F-actin cytoskeleton rearrangement was inhibited, filamentous pseudopodia were significantly reduced, and cell invasion-related cytoskeleton structures were weakened. Similar changes were observed in the tunicamycin addition group. Both models demonstrate that N-glycosylation modification can regulate F-actin cytoskeleton remodeling in ovarian cancer cells.

[0054] Tumor invasion and metastasis are the core reasons for the high mortality rate of ovarian cancer. EMT activation, cytoskeleton remodeling, and inflammatory chemotactic signals in the tumor microenvironment are the three key drivers of tumor metastasis. This application elucidates a novel mechanism by which N-glycosylation modification regulates the malignant progression of ovarian cancer, focusing on post-translational modifications of the LAMA5 protein.

[0055] As a key laminin in the extracellular matrix, LAMA5 has been the subject of much previous research, with its overall expression level largely focused on its correlation with tumor prognosis. However, the molecular mechanisms by which its post-translational modifications regulate tumor function remain unclear. This application first demonstrates that LAMA5 exhibits typical N-glycosylation modifications in ovarian cancer cells, and that glycosylation directly maintains the normal molecular weight and stable expression of LAMA5 protein, suggesting that N-glycosylation is an important prerequisite for LAMA5 protein maturation, localization, and functional activation.

[0056] Functional phenotypic experiments revealed that the metastatic function of LAMA5 is strictly dependent on N-glycosylation modification: wild-type LAMA5 with intact glycosylation significantly promoted the migration and invasion of ovarian cancer cells, while the metastatic ability was completely lost after mutation of the glycosylation site. This indicates that simply high protein expression is not enough to exert oncogenic function, and glycosylation modification is the key switch for LAMA5 to exert its biological function.

[0057] At the mechanistic level, EMT is a core program for tumor cells to acquire the ability to migrate and invade. This application demonstrates that LAMA5 glycosylation can significantly activate Snail-mediated EMT, reduce cell adhesion, enhance intercellular matrix properties, and promote tumor cell detachment and invasion. Simultaneously, cytoskeleton staining results confirm that LAMA5 glycosylation can drive F-actin aggregation and rearrangement and promote invasive pseudopodia formation, explaining the morphological basis of glycosylation's enhanced tumor metastatic ability from the perspective of cell motility structure.

[0058] KEGG enrichment analysis indicated that the differentially expressed genes in this study were concentrated in the cytokine-cytokine receptor interaction pathway. This pathway is a classic pathway for tumor microenvironment inflammation activation, chemokine recruitment, and distant tumor metastasis. Our application validated that LAMA5 glycosylation upregulates inflammatory chemokines such as Csf3, CXCL2, CXCL3, and IL20Ra, suggesting that LAMA5 glycosylation can reshape the tumor microenvironment by activating the inflammatory chemokine signaling network, further amplifying tumor malignant progression signals. Conversely, glycosylation deletion can significantly silence this pathway, inhibit inflammation and chemokine signal transduction, and block the tumor metastasis cascade.

[0059] Based on the results of this application, a complete regulatory axis is proposed: LAMA5 N-glycosylation modification → activation of cytokine-receptor interaction pathways → promotion of F-actin scaffold remodeling → activation of EMT program → promotion of ovarian cancer cell migration and invasion. This application elucidates a novel mechanism by which LAMA5 regulates ovarian cancer progression from multiple dimensions, including protein modification, molecular pathways, cell structure, and cell function, providing new evidence for ovarian cancer metastasis mechanisms and intervention strategies targeting glycosylation modification.

[0060] The key design focus of this invention is that it reveals for the first time that LAMA5 protein undergoes N-glycosylation modification in ovarian cancer cells, confirming that N-glycosylation modification is a necessary condition for LAMA5 protein to exert its function of promoting ovarian cancer cell migration and invasion. Wild-type LAMA5 with intact glycosylation can significantly promote ovarian cancer cell migration and invasion, while its pro-metastatic ability is completely lost after removing the N-glycosylation site mutation. This breaks through the limitation of existing technologies that only focus on LAMA5 expression levels, elevating the research perspective from "expression level" to a new dimension of "modification state." The invention also provides the application of an inhibitor in the preparation of reagents for inhibiting ovarian cancer cell migration and / or invasion, and provides a method for screening anti-ovarian cancer drug candidates targeting LAMA5 protein N-glycosylation modification. This provides a new intervention strategy and experimental basis for developing anti-metastatic drugs for ovarian cancer that target protein glycosylation modification, and has significant clinical application value and drug development prospects.

[0061] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. The use of an inhibitor in the preparation of a reagent for inhibiting the migration and / or invasion of ovarian cancer cells, characterized in that: The inhibitor is an inhibitor of N-glycosylation modification of LAMA5 protein.

2. The application according to claim 1, characterized in that: The LAMA5 protein N-glycosylation modification inhibitor is selected from PNGase F enzyme, tunicamycin, or mutants targeting the N-glycosylation site of the LAMA5 gene.

3. The application according to claim 1, characterized in that: The inhibitor of N-glycosylation modification of the LAMA5 protein is tunicamycin.

4. The application according to claim 2, characterized in that: The mutant is a mutant with the N-glycosylation site of the Δ3-4 domain of the LAMA5 protein missing.

5. The application according to claim 2, characterized in that: The reagents are either reagents for reducing the N-glycosylation level of LAMA5 protein or reagents for inhibiting the activity of the cytokine-cytokine receptor interaction pathway.

6. The application according to claim 5, characterized in that: The reagents used to inhibit the activity of the cytokine-cytokine receptor interaction pathway suppress the activity of the pathway by downregulating the expression of Csf3, CXCL2, and CXCL3 proteins in the pathway.

7. The application according to claim 5, characterized in that: The reagent used to inhibit the activity of the cytokine-cytokine receptor interaction pathway inhibits the activity of the pathway by upregulating the expression of IL20Ra protein.

8. The application according to claim 5, characterized in that: The ovarian cancer cells are either the Ho-8910 cell line or the A2780 cell line.

9. A method for screening candidate active substances against ovarian cancer targeting N-glycosylation modification of LAMA5 protein, characterized in that: Includes the following steps: First, the active substance to be tested is brought into contact with ovarian cancer cells expressing LAMA5 protein; Second, the N-glycosylation modification level of LAMA5 protein, the activity of the cytokine-cytokine receptor interaction pathway, and the cell migration and invasion ability in the ovarian cancer cells were detected. Third, if the test compound reduces the N-glycosylation modification level of LAMA5 protein, inhibits the activity of the cytokine-cytokine receptor interaction pathway, and reduces cell migration and invasion ability, then it is judged to be a candidate drug.

10. The method according to claim 9, characterized in that: In step three, indicators for detecting the activity of the cytokine-cytokine receptor interaction pathway include detecting the downregulation of expression levels of Csf3, CXCL2, and CXCL3 proteins, and / or detecting the upregulation of expression levels of IL20Ra protein.

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