CN116549643A - Application of over-expression PLUNC reagent and/or NLRP3 inhibitor in preparation of lung metastasis preparation for treating nasopharyngeal carcinoma - Google Patents
Application of over-expression PLUNC reagent and/or NLRP3 inhibitor in preparation of lung metastasis preparation for treating nasopharyngeal carcinoma Download PDFInfo
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Abstract
The invention discloses an application of an over-expressed PLUNC reagent and/or an NLRP3 inhibitor in preparing a lung metastasis preparation for treating nasopharyngeal carcinoma. The first finding is that NLRP3 inflammatory body activation promotes nasopharyngeal carcinoma lung metastasis, over-expression PLUNC inhibits NLRP3 inflammatory body activation induced nasopharyngeal carcinoma lung metastasis, and at the same time, over-expression PLUNC and NLRP3 inflammatory body activation inhibitor MCC950 combined can inhibit nasopharyngeal carcinoma lung metastasis to the greatest extent and delay tumor progression. The PLUNC combined with NLRP3 inflammatory corpuscle inhibitor treatment brings good news to nasopharyngeal carcinoma patients.
Description
Technical Field
The invention belongs to the technical field of nasopharyngeal carcinoma lung metastasis treatment, and particularly relates to application of an over-expressed PLUNC reagent and/or an NLRP3 inhibitor in preparation of a lung metastasis preparation for treating nasopharyngeal carcinoma.
Background
Nasopharyngeal carcinoma originates from the nasopharyngeal epithelium, a common malignancy of the head and neck. Nasopharyngeal carcinoma has a very unique geographical distribution, and it is generally believed that differences in eating habits, lifestyle and exposure to deleterious environmental factors may be the root cause of geographic differences in nasopharyngeal carcinoma incidence. Nasopharyngeal carcinoma is thought to be caused by interactions between EBV infection, which is considered the most common causative agent of nasopharyngeal carcinoma, genetic and environmental factors.
About 70% of the initial nasopharyngeal carcinoma is provided with headache symptoms, and most of the headache symptoms are migraine, cervical pain or craniofacial occipital pain, and early headache symptoms are intermittent attacks, and the headache symptoms may develop into continuous headache attacks along with the development of the illness state. Nasal discharge blood, tinnitus, hearing loss, lymphadenectasis and the like occur in the early stages of nasopharyngeal carcinoma. However, early nasopharyngeal carcinoma is hidden from the disease and is found to be in the middle and late stages of nasopharyngeal carcinoma. The clinical manifestation of advanced nasopharyngeal carcinoma is that tumor metastasis to cervical lymph nodes causes enlargement of cervical lymph nodes, tumor invasion to orbit or eyeball-related nerves causes vision disorder, visual field defect, double vision, eyeball protrusion and limited movement, and metastasis to lung causes respiratory symptoms such as cough, dyspnea and the like.
The World Health Organization (WHO) classifies nasopharyngeal carcinoma into three histological subtypes, keratinized squamous cell carcinoma, non-keratinized carcinoma and basal-like squamous cell carcinoma. Among them, non-keratinized cancer accounts for the vast majority of nasopharyngeal cancers in China, and can be further subdivided into differentiated and undifferentiated non-keratinized cancers. The clear pathological classification is crucial for accurately classifying and evaluating the optimal treatment scheme, and meanwhile, the stage of the disease is a key prognosis factor affecting the result and survival, so that a high-efficiency and accurate laboratory diagnosis method is needed for early screening of nasopharyngeal carcinoma patients, early detection of hidden patients and timely intervention are needed, and prognosis of the patients is improved. Magnetic resonance imaging is the most accurate method for determining local tumor stage, and is sensitive to describe small mucosa thickening, pharyngeal side and masticatory clearance involvement, skull base and cranial nerve infiltration, and the accuracy of detecting lymph node involvement is high. Positron Emission Tomography (PET) -CT has higher nasopharyngeal carcinoma recognition accuracy and sensitivity, and can provide important clues for diagnosis and treatment of cervical lymph node metastasis with unknown primary sites, in particular to biopsy of hidden nasopharyngeal carcinoma. And the PET-CT further improves the accuracy of lymph node stage, and is an optimal imaging method for detecting distant metastasis. Clinically, recurrence of patients with nasopharyngeal carcinoma after radiotherapy and chemotherapy is unavoidable, which is one of the main causes of poor prognosis of cancer treatment. Thus, a comprehensive understanding of the molecular mechanisms that promote nasopharyngeal carcinoma progression and metastasis may be helpful in developing more effective treatment strategies.
Nasopharyngeal carcinoma is a tumor highly sensitive to radiation and chemistry, and radiotherapy or radiotherapy-chemotherapy combination treatment is a main treatment method for patients with early and local advanced nasopharyngeal carcinoma. Although the combined radiotherapy and chemotherapy produces satisfactory survival rate, the clinical effect of single radiotherapy and chemotherapy is not satisfactory, and local recurrence and distant metastasis become a major obstacle for curing patients with nasopharyngeal carcinoma. Especially when the nasopharyngeal carcinoma patient develops lung metastasis progress, it is still a main cause of treatment failure of the nasopharyngeal carcinoma patient. Furthermore, the molecular mechanisms of nasopharyngeal carcinoma growth and metastasis remain largely unknown. Thus, there is a need for effective therapeutic strategies to treat patients with nasopharyngeal carcinoma.
Aiming at the treatment difficulty of nasopharyngeal carcinoma pulmonary metastasis, the invention discovers that PLUNC can inhibit the EMT, migration and invasion capacity of nasopharyngeal carcinoma cells, and further discovers that PLUNC can inhibit nasopharyngeal carcinoma pulmonary metastasis by inhibiting NLRP3 inflammatory body activation, and the combined action of over-expression PLUNC and NLRP3 inflammatory body activation inhibitor MCC950 can effectively inhibit nasopharyngeal carcinoma pulmonary metastasis, so that a potential treatment strategy is provided for nasopharyngeal carcinoma patients suffering from pulmonary metastasis.
In view of this, the present invention has been made.
Disclosure of Invention
The primary object of the invention is to provide the application of at least one of an over-expressed PLUNC reagent, a knocked-out NLRP3 reagent, an agent for inhibiting NLRP3 expression and an NLRP3 inflammatory corpuscle inhibitor in preparing a preparation for treating nasopharyngeal carcinoma and/or nasopharyngeal carcinoma lung metastasis. The application provides a new treatment method for nasopharyngeal carcinoma patients.
Further preferred, the over-expressed PLUNC agent is combined with an LRP3 inflammatory small body inhibitor to prepare a lung metastasis preparation for treating nasopharyngeal carcinoma and/or nasopharyngeal carcinoma.
In the experimental process, the over-expression PLUNC is found to inhibit lung metastasis of nasopharyngeal carcinoma, and the PLUNC is interfered to promote lung metastasis of nasopharyngeal carcinoma.
Meanwhile, the NLRP3 gene can be used as a potential target for treating nasopharyngeal carcinoma. The specific knockout NLRP3 gene can inhibit the progress of nasopharyngeal carcinoma, or the specific knockout NLRP3 can inhibit the EMT, the clonogenic capacity, the migration and the invasive capacity of nasopharyngeal carcinoma cells, thereby obviously inhibiting the lung metastasis of the nasopharyngeal carcinoma cells.
Inhibition of nasopharyngeal carcinoma progression by over-expression of PLUNC in combination with knockout of NLRP 3.
Experiments prove that the over-expression PLUNC is combined with NLRP3 inflammatory corpuscle inhibitor to take NLRP3 inflammatory corpuscle activation as a target point in the action process. The mechanism research shows that PLUNC accelerates the protein degradation of NLRP3 by promoting the ubiquitination of NLRP3, thereby inhibiting the activation of NLRP3 inflammatory corpuscles and further inhibiting the lung metastasis of nasopharyngeal carcinoma cells induced by the activation of NLRP3 inflammatory corpuscles.
Still further, the NLRP3 inflammasome inhibitor is MCC950.
In the practice of the present invention, the inhibitor of NLRP3 inflammatory activation of the small body is MCC950.
However, it should be noted that the NLRP3 inflammatory small body inhibitor of the present invention is not limited to MCC950, and other NLRP3 inflammatory small body inhibitors are used to target the treatment of nasopharyngeal carcinoma, which is also the protection scope of the present invention.
The invention discovers for the first time that NLRP3 inflammatory body activation promotes nasopharyngeal carcinoma lung metastasis, over-expression PLUNC inhibits NLRP3 inflammatory body activation induced nasopharyngeal carcinoma lung metastasis, and at the same time, over-expression PLUNC and NLRP3 inflammatory body activation inhibitor MCC950 combined can inhibit nasopharyngeal carcinoma lung metastasis to the greatest extent and delay tumor progression. The PLUNC combined with NLRP3 inflammatory corpuscle inhibitor treatment brings good news to nasopharyngeal carcinoma patients.
The agent for inhibiting NLRP3 expression comprises: any of the following interference sequences:
NLRP3-RNAi(62041-1)：ccGTAAGAAGTACAGAAAGTA
NLRP3-RNAi(62042-1)：gcCTTCTTGGTAGGAGTGGAA
NLRP3-RNAi(62043-1)：taCCAAGACAGGTTTGACTAT。
respectively shown in SEQ ID.NO. 1-3.
A second object of the present invention is to provide a medicament for treating nasopharyngeal carcinoma, comprising at least one of an over-expressing PLUNC reagent, a knockout NLRP3 reagent, a reagent for inhibiting expression of NLRP3, and an NLRP3 inflammatory small body inhibitor.
Further preferred are agents comprising overexpressing PLUNC agents and NLRP3 inflammatory corpuscle inhibitors.
Still further, the NLRP3 inflammasome inhibitor is MCC950.
The agent for inhibiting NLRP3 expression comprises: any of the following interference sequences:
NLRP3-RNAi(62041-1)：ccGTAAGAAGTACAGAAAGTA
NLRP3-RNAi(62042-1)：gcCTTCTTGGTAGGAGTGGAA
NLRP3-RNAi(62043-1)：taCCAAGACAGGTTTGACTAT。
a third object of the present invention is to provide the use of an agent for detecting the expression of PLUNC and/or NLRP3 in the preparation of a diagnostic formulation for nasopharyngeal carcinoma and/or lung metastasis of nasopharyngeal carcinoma.
The invention has the following beneficial effects:
the invention proves that PLUNC (NCBI Gene ID: 51297) and NLRP3 (NCBI Gene ID: 114548) are targets for treating nasopharyngeal carcinoma or lung metastasis of nasopharyngeal carcinoma, and the progress of nasopharyngeal carcinoma can be inhibited by over-expressing PLUNC and jointly knocking out NLRP 3. Over-expression of PLUNC in combination with NLRP3 inflammatory corpuscle inhibitor acts on NLRP3 inflammatory corpuscle activation as target. The therapeutic effect of the over-expressed PLUNC combined with the NLRP3 inflammatory corpuscle inhibitor in nasopharyngeal carcinoma is proved, namely the PLUNC can inhibit the lung metastasis of nasopharyngeal carcinoma cells, the NLRP3 inflammatory corpuscle activation promotes the lung metastasis of nasopharyngeal carcinoma cells, the NLRP3 inflammatory corpuscle activation inhibitor can inhibit the lung metastasis of nasopharyngeal carcinoma cells, and the over-expressed PLUNC combined with the NLRP3 inflammatory corpuscle inhibitor can inhibit the lung metastasis of nasopharyngeal carcinoma cells to the greatest extent. The PLUNC combined with the NLRP3 inflammatory corpuscle inhibitor for treating the lung metastasis of the nasopharyngeal carcinoma provides a new treatment idea for the nasopharyngeal carcinoma patients.
Drawings
In order to more clearly illustrate the technical solution of the implementation of the present invention, the following brief description of the drawings is provided.
Fig. 1: overexpression of PLUNC inhibits EMT, migration and invasion of nasopharyngeal carcinoma cells.
Fig. 2: interfering with PLUNC promotes EMT, migration and invasion of nasopharyngeal carcinoma cells.
Fig. 3: interfere with the ability of NLRP3 gene to inhibit the EMT and clonogenic ability of nasopharyngeal carcinoma cells.
Fig. 4: interference with NLRP3 gene inhibits nasopharyngeal carcinoma cell migration and invasion.
Fig. 5: treatment of nasopharyngeal carcinoma cells with LPS and ATP promotes nasopharyngeal carcinoma cell EMT, migration and invasion, and addition of NLRP3 inflammatory-body activation inhibitor MCC950 can inhibit nasopharyngeal carcinoma cell EMT, migration and invasion.
Fig. 6: overexpression of PLUNC inhibits the induction of EMT, migration and invasion of nasopharyngeal cancer cells by NLRP3 inflammatory platelet activation.
Fig. 7: overexpression of PLUNC inhibits EMT, migration and invasion of nasopharyngeal carcinoma cells, and overexpression of PLUNC in combination with NLRP3 inflammatory body activation inhibitor MCC950 can inhibit EMT, migration and invasion of nasopharyngeal carcinoma cells to the greatest extent.
Fig. 8: in vivo experiments prove that the over-expression of PLUNC can inhibit lung metastasis of nasopharyngeal carcinoma, and the over-expression of PLUNC combined with the NLRP3 inflammatory small body activation inhibitor MCC950 can inhibit the lung metastasis of nasopharyngeal carcinoma to the greatest extent.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
This example demonstrates that over-expression of PLUNC inhibits nasopharyngeal carcinoma cells (S18 and S26 cell lines, given away by the tumor hospital in the hunan province, from nasopharyngeal carcinoma patients, with significant nasopharyngeal carcinoma cell characteristics) EMT (EMT refers to the process of epithelial-mesenchymal transformation of cells, making cells more susceptible to lung metastasis), migration, and invasion in vivo.
1. Stably transfecting nasopharyngeal carcinoma cells over-expressing PLUNC.
And (3) paving: firstly preparing cell suspension with density of 3-5×10 4 Individual/ml, divided into 3 groups: the M group and the A group (adding A infection liquid, i.e. HiTransG A virus infection enhancing liquid, is a novel high molecular nonionic surfactant, and is also a cell protectant and a promoting absorbent)The infection efficiency of cells can be greatly improved, the infection condition of the cells is optimized by Ji Kai lentivirus), 100 μl of each suspension is added into a 96-well plate, a Control group (the specific grouping condition is shown in table 1, and 4 compound wells are arranged in each group), and the cells are cultured for 24 hours in a incubator for subsequent operation.
Lentiviruses (purchased from Ji Kai gene, gcGFP/over-expression/lentivirus/C-3 FLAG/Ubi Puromycin) were diluted to three concentration gradients in a volume of 100 μl: group I, group II, group III viruses at a concentration of 1X 10, respectively 6 TU/ml，1×10 7 TU/ml，1×10 8 TU/ml。
The supernatant after 24 hours of incubation in each well of step one was aspirated, washed 2 times with PBS, and the corresponding solutions were added according to Table 1, mixed well and incubated.
TABLE 1 Pre-infection experiment groups and infection conditions
The liquid in the 96-well plate is sucked and replaced by a complete culture medium after 12-16 hours of infection (the cell morphology is required to be observed continuously in the first experiment, and the liquid can be replaced 2-4 hours in advance if the cell is severely deformed).
Culturing was continued.
And (3) observing infection effect: after 72h of infection, morphology, fluorescence intensity and efficiency can be observed under a microscope. And selecting a group of corresponding infection conditions and MOI with good cell growth state and infection efficiency exceeding 80% as the basis of a formal infection experiment.
Preparation of the complete Medium to a Density of 3-5×10 4 2ml of each cell suspension was inoculated into a six-well plate and cultured at 37℃for 16-24 hours until the cell density was 20-30%. Cells are infected according to the infection MOI of lentivirus to cells and optimal infection conditions determined in advance. Pre-experiments determined that MOI of S18 and S26 cells were 50 and 100, respectively, with P-infection being the optimal infection condition. Discard six wellsThe original culture medium in the plate is added with 40 mu l P infection enhancing liquid to each hole, the corresponding volume of virus to be added is calculated according to the MOI of the cells and the purchased original virus titer and calculated according to a calculation formula, and finally, each Kong Peiji total volume is supplemented to 1ml by the complete culture medium. The calculation formula is as follows: MOI x cell mass = viral mass.
After 12-16 hours of cultivation, the medium is replaced with a new complete medium, followed by cultivation for 12-60 hours. The infection efficiency is observed about 72 hours after infection, and then cell screening is carried out by continuously feeding commercial puromycin (2 mug/ml) for about 2 weeks (the feeding concentration is continuously adjusted along with the cell morphology and screening time, and generally, the drug concentration can be reduced to less than half of the initial concentration when the subsequent fluorescence efficiency is slightly stable) until the fluorescence efficiency reaches more than 90%, and the construction of cell strains for stably transfecting PLUNC over-expression is completed.
The cell strain of the stably transfected PLUNC over-expression or control vector is collected into a centrifuge tube, lysate is added to extract total protein, and immunoblotting experiments are carried out to detect the expression level of the PLUNC in the cell strain, as shown in figure 1.
2. Immunoblotting experiments were used to confirm that overexpression of PLUNC inhibited nasopharyngeal carcinoma cell EMT.
The cell strain with the stable transfection PLUNC over-expression or the control vector is collected into a centrifuge tube, lysate is added to extract total protein, and immunoblotting experiments are carried out to detect the expression levels of EMT related molecules Zo-1, E-cadherin and Vimentin in the cell strain, and the expression levels are shown in figure 1.
Experimental results show that Zo-1 and E-cadherein are up-regulated and Vimentin is down-regulated after PLUNC is over-expressed, which indicates that over-expressed PLUNC can inhibit EMT of nasopharyngeal carcinoma cells.
3. Scratch experiments were used to confirm that over-expressed PLUNC inhibited nasopharyngeal carcinoma cell migration.
Cells over-expressing the PLUNC or containing the control vector are seeded in six well plates at a level of 100% cell density after 24 hours. The cells were cultured in DMEM medium containing 1% fbs by directly scratching with an autoclaved 200 μl pipette tip, washing 3 times with PBS, photographing under a microscope, recording the scratch width for 0 hours of scratching. After 24 hours, the scratch width of the scratch was recorded under a microscope for 24 hours. The width of the scratch at 0 and 24 was measured with Adobe illustrator and the difference in cell migration rate between the PLUNC over-expression group and the control group was quantitatively analyzed, see fig. 1.
The experimental result shows that after the PLUNC is over-expressed, the healing of scratches is inhibited, which indicates that the over-expressed PLUNC can inhibit the migration capacity of nasopharyngeal carcinoma cells.
4. The Transwell laboratory experiments were used to confirm that overexpression of PLUNC inhibited nasopharyngeal carcinoma cell invasion.
The matrigel is taken out from the minus 20 ℃ in advance and is placed in a refrigerator for dissolution at the temperature of 4 ℃. Pipette, centrifuge tube, transwell chamber 24-well plate, serum-free DMEM medium required for pre-chilling glue in advance. Matrigel was diluted with serum-free DMEM medium following medium: matrigel 1:4 ratio. 40 microliters of diluted matrigel was added to the upper chamber of the Transwell chamber 24-well plate. Placing the mixture in a 37 ℃ incubator for incubation for 4-5 hours. The remaining liquid in the plates was aspirated, 70 μl of serum-free medium light wash gel was added to each well, and the medium was aspirated. The nasopharyngeal carcinoma cells overexpressing PLUNC or containing the control vector were resuspended to a final concentration of 1X 10 with serum-free medium DMEM 5 Per ml, 100. Mu.l of cell suspension was added to the upper chamber, and 600. Mu.l of DMEM medium containing 20% FBS was added to the lower chamber. After culturing in an incubator for 24 to 48 hours, the upper chamber was taken out for crystal violet staining. After washing the cells 3 times with PBS, cells inside the cells were gently scraped off with a cotton swab, and the cells were placed on a microscope for photographing recording, see FIG. 1.
The experimental results show that after the PLUNC is over-expressed, the capacity of inhibiting the nasopharyngeal carcinoma cells from penetrating through the cell is achieved, which indicates that the over-expressed PLUNC can inhibit the invasion capacity of the nasopharyngeal carcinoma cells.
Example 2
This example demonstrates that interfering with PLUNC promotes lung metastasis in nasopharyngeal carcinoma cells.
1. Transient transfection was constructed to interfere with the nasopharyngeal carcinoma cells of the PLUNC.
Two nasopharyngeal carcinoma cells (S18 and S26) with good states are respectively digested, blown and uniformly mixed to prepare cell suspension, the cell suspension is paved into a six-hole plate, the plate is rocked in the cross direction to uniformly distribute the cells, the cells are placed into an incubator for culture overnight, and when the cells in each hole are fused to about 70%, the cells can be used for transfection.
Two sterile EP tubes were prepared per well of transfection, and the first tube was filled with: 250 μl DMEM,5 μl P2000; the second tube was charged with: 250ul of DMEM, 2. Mu.g of the corresponding plasmid DNA. Mixing, incubating for 5min at room temperature, mixing the solutions in the two tubes uniformly, incubating for 20min at room temperature, and adding the mixed solution into the corresponding holes. After all wells were transfected, they were gently swirled and mixed and placed in an incubator. After 6-8h the liquid in the wells was changed to a medium containing 10% serum.
After 48 hours, fluorescence intensity was observed under a microscope to determine transfection efficiency. The cell lines transiently transfected with the PLUNC interference or control vector are collected into a centrifuge tube, lysate is added to extract total protein, and immunoblotting experiments are carried out to detect the expression level of the PLUNC in the cell lines, as shown in FIG. 2.
2. Immunoblotting experiments were used to confirm that interfering PLUNC promoted nasopharyngeal carcinoma cell EMT.
The cell strain of the transient transfection PLUNC interference or the control vector is collected into a centrifuge tube, lysate is added to extract total protein, and immunoblotting experiments are carried out to detect the expression levels of EMT related molecules Zo-1, E-cadherein and Vimentin in the cell strain, and the expression levels are shown in figure 2.
After the experimental result shows that PLUNC is interfered, zo-1 and E-cadherin are down-regulated, and Vimentin is up-regulated, which shows that the interference of PLUNC can promote EMT of nasopharyngeal carcinoma cells.
3. Scratch experiments are used to confirm that interfering PLUNC promotes nasopharyngeal carcinoma cell migration.
Experimental results showed that the healing of scratches was promoted after the PLUNC interference, which indicates that the interference of the PLUNC can promote the migration ability of nasopharyngeal carcinoma cells, see fig. 2.
4. Transwell laboratory experiments were used to confirm that interfering PLUNC promotes nasopharyngeal carcinoma cell invasion.
Experimental results showed that the ability of the nasopharyngeal carcinoma cells to pass through the cells was promoted after the PLUNC interference, indicating that interfering with the PLUNC can promote the invasive ability of the nasopharyngeal carcinoma cells, see fig. 2.
Example 3
This example demonstrates the ability of interfering NLRP3 to inhibit EMT and clonogenic capacity of nasopharyngeal carcinoma cells.
1. Immunoblotting experiments prove that the interference NLRP3 inhibits the EMT of nasopharyngeal carcinoma cells.
Two nasopharyngeal carcinoma cells (S18 and S26) with good states are respectively digested, blown and uniformly mixed to prepare cell suspension, the cell suspension is paved into a six-hole plate, the plate is rocked in the cross direction to uniformly distribute the cells, the cells are placed into an incubator for culture overnight, and when the cells in each hole are fused to about 70%, the cells can be used for transfection.
Two sterile EP tubes were prepared per well of transfection, and the first tube was filled with: 250 μl DMEM,5 μl P2000; the second tube was charged with: 250ul of DMEM, 2. Mu.g of the corresponding plasmid DNA. Mixing, incubating for 5min at room temperature, mixing the solutions in the two tubes uniformly, incubating for 20min at room temperature, and adding the mixed solution into the corresponding holes. After all wells were transfected, they were gently swirled and mixed and placed in an incubator. After 6-8h the liquid in the wells was changed to a medium containing 10% serum.
After 48 hours, fluorescence intensity was observed under a microscope to determine transfection efficiency. The cell strain of the transient transfection NLRP3 interference or control vector is collected into a centrifuge tube, lysate is added to extract total protein, an immunoblotting experiment is carried out, and the expression levels of the EMT related molecules N-cadherin, E-cadherin and Vimentin in the cell strain are detected, as shown in figure 3.
After the experimental result shows that NLRP3 is interfered, N-cadherin and Vimentin are down-regulated, E-cadherin is up-regulated, which indicates that the interference NLRP3 can inhibit EMT of nasopharyngeal carcinoma cells.
The interference sequences are respectively:
NLRP3-RNAi(62041-1) ccGTAAGAAGTACAGAAAGTA
NLRP3-RNAi(62042-1) gcCTTCTTGGTAGGAGTGGAA
NLRP3-RNAi(62043-1) taCCAAGACAGGTTTGACTAT
2. the clonogenic assay was used to demonstrate that interfering NLRP3 inhibited the clonogenic capacity of nasopharyngeal carcinoma cells.
Two nasopharyngeal carcinoma cells (S18 and S26) with good states are respectively digested, blown and uniformly mixed to prepare cell suspension, the cell suspension is paved into a six-hole plate, the plate is rocked in the cross direction to uniformly distribute the cells, the cells are placed into an incubator for culture overnight, and when the cells in each hole are fused to about 70%, the cells can be used for transfection.
Two sterile EP tubes were prepared per well of transfection, and the first tube was filled with: 250 μl DMEM,5 μl P2000; the second tube was charged with: 250ul of DMEM, 2. Mu.g of the corresponding plasmid DNA. Mixing, incubating for 5min at room temperature, mixing the solutions in the two tubes uniformly, incubating for 20min at room temperature, and adding the mixed solution into the corresponding holes. After all wells were transfected, they were gently swirled and mixed and placed in an incubator. After 6-8h the liquid in the wells was changed to a medium containing 10% serum.
After 48 hours, fluorescence intensity was observed under a microscope to determine transfection efficiency. The transiently transfected NLRP3 interfering or interfering control cells were digested to make single cell suspension, and after calculation of cell density, 1000-2500 cells per well were plated into six well plates (total culture volume was 2.5 ml) to ensure complete cell dispersion distribution. The six-hole plate is placed in a cell incubator for continuous culture for 1-2 weeks, and liquid is changed every 3 days in the period, so that the force is required to be very gentle when the liquid is changed. After the cells formed macroscopic colonies, they were removed for crystal violet staining and photographed, see fig. 3.
Experimental results the ability of NLRP3 to inhibit the clonogenic potential of nasopharyngeal carcinoma cells was observed after interference.
Example 4
This example demonstrates that interfering NLRP3 inhibits nasopharyngeal carcinoma cell migration and invasion.
1. The Transwell chamber migration experiments were used to confirm that interfering NLRP3 inhibited nasopharyngeal carcinoma cell migration.
Transwell cell migration experiments differ from Transwell cell experiments in that no matrigel is required. The nasopharyngeal carcinoma cells overexpressing PLUNC or containing the control vector were resuspended to a final concentration of 1X 10 with serum-free medium DMEM 5 Per ml, 100. Mu.l of cell suspension was added to the upper chamber, and 600. Mu.l of DMEM medium containing 20% FBS was added to the lower chamber. After culturing in an incubator for 24 to 48 hours, the upper chamber was taken out for crystal violet staining. After washing the cells 3 times with PBS, the cells inside the cells were gently scraped off with a cotton swab, and the cells were placed on a microscope for recording by photographing, and the experimental results are shown in FIG. 4.
The experimental results show that after NLRP3 interference, the cells are inhibited from migrating to the lower chamber, and the interference on NLRP3 can inhibit the migration capacity of nasopharyngeal carcinoma cells.
2. The interference NLRP3 was confirmed to inhibit nasopharyngeal carcinoma cell invasion by using Transwell laboratory experiments.
The ability of the interfering NLRP3 to inhibit the penetration of nasopharyngeal carcinoma cells through the cell after the interference of NLRP3 was observed, which indicates that the interfering NLRP3 can inhibit the invasion ability of the nasopharyngeal carcinoma cells, as shown in the following figure 4.
Example 5
Lps+atp is a known activator useful in promoting activation of NLRP3 inflammatory bodies, this example demonstrates that lps+atp promotes activation of NLRP3 inflammatory bodies and promotes EMT, migration and invasion of nasopharyngeal carcinoma cells, and that the NLRP3 inflammatory body inhibitor MCC950 inhibits EMT, migration and invasion of nasopharyngeal carcinoma cells.
1. Immunoblotting experiments prove that the NLRP3 inflammatory corpuscle inhibitor can inhibit the EMT of nasopharyngeal carcinoma cells.
After the well-grown nasopharyngeal carcinoma cell lines S18 and S26 were digested, 30% of the cell suspension was spread in a small dish with a diameter of 60mm, and the dish was placed in an incubator for overnight culture. When the cell fusion degree reaches 65% -75%, preparing for adding medicine. The LPS, ATP, MCC950,950 stock was diluted with complete medium to the corresponding concentration (LPS concentration 1. Mu.g/ml, ATP concentration 5mM, MC 950 concentration 20. Mu.M) and used. The culture medium was discarded, washed 2 times with sterile PBS, and 3 ml of each diluted LPS, ATP, MCC and 950 solution was added after the residual PBS in the dish was blotted off, and the culture medium was further placed in a cell incubator to be incubated for 24 hours and taken out for subsequent experiments.
Collecting the cells after the drug addition treatment into a centrifuge tube, adding a lysate to extract total proteins, performing an immunoblotting experiment, and detecting the expression levels of EMT related molecules Zo-1, E-cadherein and Vimentin in cell strains, as shown in figure 5.
Experimental results showed that Zo-1 and E-cadherein were up-regulated and Vimentin was down-regulated after MCC950 was added, demonstrating that the NLRP3 inflammatory platelet inhibitor MCC950 was able to inhibit EMT of nasopharyngeal cancer cells.
2. Scratch experiments prove that NLRP3 inflammatory corpuscle inhibitor can inhibit nasopharyngeal carcinoma cell migration.
As shown in fig. 5, the results of the experiment show that MCC950 added inhibits healing of scratches, indicating that the NLRP3 inflammatory platelet inhibitor MCC950 can inhibit migration of nasopharyngeal carcinoma cells.
3. Transwell laboratory experiments prove that NLRP3 inflammatory corpuscle inhibitor can inhibit nasopharyngeal cancer cell invasion.
The ability of the NLRP3 inflammatory small body inhibitor MCC950 to inhibit the invasion of the nasopharyngeal carcinoma cells is shown in the experimental result of FIG. 5, wherein the MCC950 is added.
Example 6
This example demonstrates that NLRP3 inflammatory body activation promotes EMT, migration and invasion of nasopharyngeal carcinoma cells, and that overexpression of PLUNC inhibits EMT, migration and invasion of nasopharyngeal carcinoma cells induced by NLRP3 inflammatory body activation.
1. Immunoblotting experiments prove that the overexpression of PLUNC inhibits the nasopharyngeal carcinoma cell EMT induced by NLRP3 inflammatory corpuscle activation.
In the case of stable over-expressed PLUNC or nasopharyngeal carcinoma cells containing the control vector, the LPS+ATP treatment (LPS concentration 1. Mu.g/ml, ATP concentration 5 mM) was added or not, and the total protein was collected for immunoblotting analysis, see FIG. 6.
The experimental result shows that LPS+ATP promotes the EMT of the nasopharyngeal carcinoma cells, and that the overexpression PLUNC inhibits the EMT of the nasopharyngeal carcinoma cells promoted by LPS+ATP.
2. Scratch experiments are adopted to prove that the over-expression PLUNC inhibits the nasopharyngeal carcinoma cell migration induced by NLRP3 inflammatory corpuscle activation.
The experimental result in fig. 6 shows that the LPS+ATP promotes the migration ability of the nasopharyngeal carcinoma cells, and the over-expression PLUNC inhibits the migration of the nasopharyngeal carcinoma cells promoted by the LPS+ATP.
3. Transwell laboratory experiments were used to confirm that over-expression of PLUNC inhibited NLRP3 inflammatory body activation-induced nasopharyngeal cancer cell invasion.
The experimental result in fig. 6 shows that the capability of LPS+ATP to promote the invasion of nasopharyngeal carcinoma cells is observed, and the overexpression of PLUNC inhibits the LPS+ATP to promote the invasion of nasopharyngeal carcinoma cells.
Example 7
This example demonstrates that overexpression of PLUNC in combination with MCC950 inhibits EMT, migration and invasion of nasopharyngeal carcinoma in vitro.
1. Immunoblotting experiments were used to confirm that overexpression of PLUNC in combination with MCC950 inhibited nasopharyngeal carcinoma cell EMT in vitro.
In nasopharyngeal carcinoma cells stably overexpressing PLUNC or containing control vector, with or without MCC950 treatment (MCC 950 concentration of 20. Mu.M), total protein was collected for immunoblotting analysis, see FIG. 7.
Experimental results the MCC950 was observed to inhibit the nasopharyngeal carcinoma cell EMT, and the overexpression of PLUNC further inhibited the nasopharyngeal carcinoma cell EMT.
2. Scratch experiments were used to demonstrate that overexpression of PLUNC in combination with MCC950 inhibited nasopharyngeal carcinoma cell migration in vitro.
Fig. 7 shows that MCC950 has been observed to inhibit the ability of nasopharyngeal carcinoma cells to migrate, and that overexpression of PLUNC further inhibits nasopharyngeal carcinoma cell migration.
3. The use of Transwell laboratory experiments demonstrated that overexpression of PLUNC in combination with MCC950 inhibited nasopharyngeal carcinoma cell invasion in vitro.
Fig. 7 shows that MCC950 has been observed to inhibit the ability of nasopharyngeal carcinoma cells to invade, and that overexpression of PLUNC further inhibits nasopharyngeal carcinoma cell invasion.
Example 8
This example demonstrates that overexpression of PLUNC in combination with MCC950 inhibits lung metastasis of nasopharyngeal carcinoma cells in vivo.
The S18 cells over-expressing PLUNC or containing the control vector were injected into 4 week old female nude mice via the tail vein, two groups of which were intraperitoneally injected with MCC950 (8 mg/kg) and the control group given PBS, after 8 weeks, the mice were euthanized. The lung tissue of nude mice is taken out to observe lung metastasis focus, the lung metastasis of the single over-expressed PLUNC group is less than that of the blank vector group, and the over-expressed PLUNC combined MCC950 treatment has the best inhibition effect on the lung metastasis of nasopharyngeal carcinoma. Quantitative maps of lung metastasis nodules more intuitively demonstrate this result. Hematoxylin-eosin staining showed that over-expression of PLUNC in combination with MCC950 treatment effectively inhibited nasopharyngeal carcinoma lung metastasis, manifested as minimal lung metastasis area and minimal lung nodule number, with the results shown in figure 8.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. Use of at least one of an over-expressed PLUNC reagent, a knockout NLRP3 reagent, a reagent that inhibits expression of NLRP3, and an NLRP3 inflammatory small inhibitor in the preparation of a lung metastasis preparation for treating nasopharyngeal carcinoma and/or nasopharyngeal carcinoma.
2. The use according to claim 1, wherein the over-expressed PLUNC reagent is formulated in combination with an LRP3 inflammatory small body inhibitor for the treatment of nasopharyngeal carcinoma and/or nasopharyngeal carcinoma lung metastasis.
3. The use according to claim 1 or 2, wherein the NLRP3 inflammasome inhibitor is MCC950.
4. The use according to claim 1, wherein the agent that inhibits NLRP3 expression comprises: any of the following interference sequences:
NLRP3-RNAi(62041-1)：ccGTAAGAAGTACAGAAAGTA
NLRP3-RNAi(62042-1)：gcCTTCTTGGTAGGAGTGGAA
NLRP3-RNAi(62043-1)：taCCAAGACAGGTTTGACTAT。
5. a medicament for treating nasopharyngeal carcinoma, comprising at least one of an over-expressed PLUNC reagent, a knockout NLRP3 reagent, a reagent that inhibits expression of NLRP3, and an NLRP3 inflammatory body inhibitor.
6. The medicament of claim 5, comprising over-expressing a PLUNC agent and an NLRP3 inflammatory body inhibitor.
7. The medicament of claim 5 or 6, wherein the NLRP3 inflammasome inhibitor is MCC950.
8. The agent of claim 5, wherein the agent that inhibits NLRP3 expression comprises: any of the following interference sequences:
NLRP3-RNAi(62041-1)：ccGTAAGAAGTACAGAAAGTA
NLRP3-RNAi(62042-1)：gcCTTCTTGGTAGGAGTGGAA
NLRP3-RNAi(62043-1)：taCCAAGACAGGTTTGACTAT。
9. application of a reagent for detecting PLUNC and/or NLRP3 expression in preparing a diagnosis preparation for nasopharyngeal carcinoma and/or nasopharyngeal carcinoma lung metastasis.
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