CDKN2A and ISG15 interaction key site THR95 and application thereof

Abstract

The invention discloses a CDKN2A and ISG15 interaction key site THR95 and an application of the CDKN2A and ISG15 interaction key site THR95. The invention particularly relates to application of the key site in preparation of a product for diagnosing or inhibiting tumor immune escape. The invention provides a new target spot and a new method for diagnosis and treatment of tumor immune escape by researching key sites of interaction of CDKN2A and ISG15, and has important clinical application value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of lung cancer cell molecular regulation, and particularly relates to a CDKN2A and ISG15 interaction key site THR95 and an application thereof. BACKGROUND

[0002] Lung cancer is one of the malignant tumors with extremely high morbidity and mortality worldwide. In recent years, although certain progress has been made in the diagnosis and treatment of lung cancer, the overall prognosis is still poor, and tumor immune escape of tumor cells is an important reason for treatment failure and death of lung cancer patients. Tumor immune escape is a phenomenon in which tumor cells escape recognition and attack by the body's immune system, and is one of the important mechanisms of tumor occurrence, development and metastasis, which seriously affects the treatment effect and prognosis of tumor patients. For many years, scientific researchers have been committed to revealing the molecular mechanism of tumor immune escape in order to find effective diagnostic and therapeutic targets.

[0003] The interaction between programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) is an important immune checkpoint that mediates tumor immune suppression, has become one of the main targets of tumor immunotherapy, and inhibits the activation and function of T cells, prevents cytotoxic T cells from killing tumor cells, and leads to tumor immune escape. The immune checkpoint formed by the combination of ligand PD-L1 and its receptor PD-1 makes tumor cells escape the monitoring of the body's immune system, and the therapy of immune checkpoint inhibitors (ICIs) targeting PD-1 / PD-L1 has been widely used in clinical tumor treatment, bringing hope to tumor patients and achieving encouraging results. However, the effective rate of PD-1 / PD-L1 antibody therapy for tumors is about 20-30%, that is, 70-80% of tumor patients develop primary or secondary resistance to PD-1 / PD-L1 antibody therapy, therefore, finding new strategies for targeting inhibition of the PD-1 / PD-L1 immune checkpoint provides new methods for tumor ICIs therapy, and plays a very important role in improving the effect of tumor immunotherapy.

[0004] With the deepening of the research on tumor microenvironment and molecular mechanisms of tumor cells and immune cells interaction, it is found that the interaction between intracellular proteins plays a key regulatory role in tumor immune escape. CDKN2A (cyclin-dependent kinase inhibitor 2A) and ISG15 (interferon-stimulated gene 15) are important intracellular proteins, and the interaction between them and the specific mechanism of this interaction in tumor immune escape, especially the role of key sites, have not been fully studied and understood. In-depth exploration of the key sites of CDKN2A and ISG15 interaction and the function of the key sites in tumor immune escape are of great significance for developing more precise and effective tumor immune escape diagnosis and treatment methods. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a CDKN2A and ISG15 interaction key site THR95 and its application.

[0006] The technical scheme of the present application mainly includes the following contents: One of the purposes of the present application is to provide the application of the THR95 site of the ISG15 protein in preparing products for diagnosing or inhibiting tumor immune escape, the THR95 site being the amino acid at position 95 of the sequence shown in SEQ ID NO. 1.

[0007] The second purpose of the present application is to provide the application of the THR95 site of the SG15 protein in preparing products for diagnosing or reducing the PD-L1 level of tumor cells.

[0008] The third purpose of the present application is to provide a method for reducing the PD-L1 level of tumor cells, which comprises: causing point mutation of the THR95 site of the ISG15 protein of the tumor cells.

[0009] Further, the tumor includes lung cancer.

[0010] Further, the lung cancer includes non-small cell carcinoma.

[0011] Further, the lung cancer includes lung adenocarcinoma.

[0012] The fourth purpose of the present application is to provide a method for reducing the expression amount of ISG15 protein, which comprises: causing point mutation of the THR95 site of the ISG15 protein, the THR95 site being the amino acid at position 95 of the sequence shown in SEQ ID NO. 1.

[0013] The fifth object of the present application is to provide a method for reducing the inhibitory effect of ISG15 protein on CDKN2A protein, which comprises: causing a point mutation at the THR95 site of the ISG15 protein. The point mutation refers to the replacement, insertion or deletion of bases in the gene sequence of the ISG15 protein, which causes the threonine at the THR95 site of the protein to be replaced by other amino acids.

[0014] In the following specific embodiments of the present application, the amino acid sequence of SG15 is shown in SEQ ID NO. 1, the nucleotide sequence is shown in SEQ ID NO. 2, the amino acid sequence after mutation of the THR95 site is shown in SEQ ID NO. 3, and the nucleotide sequence is shown in SEQ ID NO. 4; the amino acid sequence of CDKN2A is shown in SEQ ID NO. 5, and the nucleotide sequence is shown in SEQ ID NO. 6.

[0015] The beneficial effects of the present application are: The determined key interaction site THR95 of CDKN2A and ISG15 provides a new specific molecular marker for the diagnosis of tumor immune escape. Based on this site, the detection reagent and kit can more accurately reflect the state of tumor immune escape, which is helpful for early diagnosis and disease monitoring of tumors.

[0016] Developing drugs to inhibit tumor immune escape by targeting the THR95 site provides a new strategy for tumor treatment. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 : The expression of epithelial marker E-cadherin and mesenchymal marker N-cadherin in each group of immunofluorescence detection experiment.

[0018] Figure 2 : Statistical analysis results of the expression of epithelial marker E-cadherin and mesenchymal marker N-cadherin in each group of immunofluorescence detection experiment, * represents P<0.05, and ** represents P<0.01.

[0019] Figure 3 : The expression of MMP2 and MMP9 related to cell invasion and migration in the Western blot experiment.

[0020] Figure 4 : Detection of CDKN2A, ISG15, JAK-STAT1 signaling pathway and PD-L1 signaling pathway enriched by proteomics analysis in each group of cells in the Western blot experiment.

[0021] Figure 5 : The tumor colony formation experiment effect diagram of each group of experiments with or without activated T cells.

[0022] Figure 6 : Statistical bar chart of tumor clone formation in each experimental group with or without activated T cells.

[0023] Figure 7 Statistical analysis results of IFN-γ levels in each group after co-culturing with activated T cells using ELISA assay.

[0024] Figure 8 Bar chart showing the apoptosis rate of Jurkat T cells in each group.

[0025] Figure 9 Statistical analysis of IL-2 levels in each group after co-culturing with Jurkat T cells using ELISA assay. Detailed Implementation

[0026] To better understand the technical content of this invention, the invention will be further described below with reference to specific embodiments and accompanying drawings. Unless otherwise specified, any reagents or pharmaceuticals involved in the following embodiments are commercially available products, and the experimental methods described are all conventional methods.

[0027] 1. Experimental Methods 1.1 Experimental Procedures for Cloning Cell culture conditions and grouping: 1) Cell culture: PC-9 cells were cultured in 1640 medium containing 10% FBS + 1% antibiotics.

[0028] 2) Take logarithmically grown cells and seed them in 6-well plates. After the cells adhere, perform the following grouping treatments: PC9: NC+T cells: PC9 cells transfected with NC cells and co-cultured with T cells wt-ISG95+ T cells: PC9 cells transfected with wt-ISG95 and co-cultured with T cells mut-ISG95 (THR95) + T cells: PC9 cells transfected with mut-ISG95 (THR95) were co-cultured with T cells. NC: PC9 cells transfected with NC wt-ISG95: PC9 cells transfected with wt-ISG95 mut-ISG95 (THR95): PC9 cells transfected with mut-ISG95 (THR95) Cell colony formation assay: (1) Take cells from each group in the exponential growth phase, digest them with 0.25% trypsin and pipette them into single cells, and suspend the cells in a complete culture medium containing 10% fetal bovine serum for later use. (2) Seed 200 cells per well of the cell suspension into 6-well plates containing 1 ml of culture medium at room temperature, and gently rotate to disperse the cells evenly. Incubate at 37°C in a cell culture incubator with 5% CO2 and saturated humidity for 2-3 weeks, changing the medium as needed during this period; (3) Observe frequently, and stop culturing when visible clones appear in the culture dish; (4) Discard the culture medium, carefully wash twice with PBS, add 1 ml of 4% paraformaldehyde to each well to fix the cells, and fix for 15 min; (5) Remove the fixative, add 1 ml of staining working solution, and stain at room temperature for 30 min; (6) Wash away the staining solution slowly with running water and air dry. (7) Cell photography: Mobile phone photography.

[0029] (8) Decolorization and OD value measurement: Take out the 6-well plate, add 1 ml of 10% acetic acid to soak it to decolorize it, and measure the absorbance (OD) value at 550 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Repeat the results three times.

[0030] 1.2 Cellular Immunofluorescence Cells were seeded into six-well plates containing slides for the experiment. After the experiment, the slides containing cells were removed, and the cells were gently washed three times with PBS. After washing, the cells were fixed with 95% ethanol for 25 min, and the slides were gently washed three times with PBS. The primary antibody for the target marker was then diluted according to the manufacturer's instructions and added to the slides, incubating overnight at 4°C. The next day, the slides were removed, gently and thoroughly washed three times to remove any residual primary antibody. The corresponding secondary antibody was diluted 1:1000 and added to the slides incubated with the primary antibody. The slides were incubated in the dark for 1 h, and then gently washed three times with PBS buffer to remove any excess secondary antibody residue. Finally, DAPI was added and incubated at room temperature for 15 min, followed by washing three times with methanol for 5 min each time. Finally, 1 ml of anti-fluorescence quencher was added, and the slides were immediately mounted and placed under a fluorescence microscope for further observation and photography.

[0031] 1.3 Western Blot (1) Protein sample preparation: a. After digesting the cells, transfer them to 1.5 mL EP tubes, wash twice with pre-cooled 1×PBS, discard the supernatant, add an appropriate amount of RIPA lysis buffer to the cell pellet, and lyse on ice for 30 min, mixing the lysis buffer with a shaker every 5 min for 5 sec.

[0032] b. Place the EP tube at 4 ℃ and centrifuge at 12000 rpm for 20 min.

[0033] c. Take out the centrifuged lysate, and use the BCA protein detection kit to determine the protein concentration of the lysate.

[0034] d. Use 6x protein loading buffer to configure the protein lysate into a protein sample.

[0035] (2) Preparation of protein concentration gel and separation gel: configure the appropriate concentration of protein gel according to the formula mentioned in the material.

[0036] (3) Electrophoresis: add enough electrophoresis liquid, and add the electrophoresis liquid from the middle of the two glass plates, and the amount of the electrophoresis liquid should exceed the glass plate. Add 50 μg of the above-mentioned heated denatured protein sample to the lane, and add the protein Marker to the appropriate lane. After running at 80 V for 30 minutes, use 120 V for constant voltage electrophoresis until the protein Loading Buffer moves to the lower edge of the gel (about 90 minutes).

[0037] (4) Membrane transfer: place the membrane transfer buffer in the -20 °C refrigerator for pre-cooling for 1 hour, immerse the PVDF membrane in methanol for 30 sec, and then wash it with ultrapure water, and place it in the membrane transfer buffer for 5 min. Gently pry open the glass plate in the membrane transfer buffer, and then pry open the gel. After removing the small glass plate, gently scrape off the concentration gel without breaking the separation gel. Place the separation gel on the PVDF membrane, align it with the membrane, press the filter paper to remove bubbles. According to the following order, load the membrane transfer clamp: black panel (negative), sponge, filter paper, gel, PVDF, filter paper, sponge, white panel (positive). Place the entire membrane transfer system on ice, and run at 100 V for 90 min (note that the current should not exceed 400 mA to prevent excessive heating).

[0038] (5) Blocking: after the membrane transfer is completed, take out the PVDF membrane, rinse it once with 1x TBST to remove residual membrane transfer liquid, immerse it in 5% skimmed milk prepared with 1x TBST, and block it at room temperature for 1 h.

[0039] (6) Primary antibody incubation: according to the instructions, dilute the antibody to the desired concentration with 5% TBST prepared with skimmed milk. Take the membrane out of the blocking solution, absorb it with filter paper, and place it with the protein side down in the antibody solution, and pull the four corners of the membrane to remove bubbles. Incubate at 4 °C overnight. The next day, place the membrane in 1x TBST and shake it for 3 times, 15 min each time. The main purpose is to wash away the non-specific binding of the primary antibody and antigen, and the effect of washing will affect the depth of the background.

[0040] (7) Secondary antibody incubation: According to the species origin of the primary antibody, select the appropriate secondary antibody, and dilute it to the appropriate concentration according to the instructions of the secondary antibody. Cover the PVDF membrane on the surface of the secondary antibody dilution, and incubate in a biochemical incubator at 37 °C for 1 hour. Take out the PVDF membrane and put it in 1xTBST, shake and wash for 2 times, 15 minutes each time.

[0041] (8) Luminescence development: Add an appropriate amount of ECL luminescence solution to the membrane, develop by imaging system, and determine the target band according to the position of the protein marker.

[0042] Band analysis: Texture (longitudinal stripes): The sample contains insoluble particles.

[0043] The band is diffused on both sides: the amount of sample is too much.

[0044] The band is a smiley face, the reason: the gel is not evenly cooled, and the middle is not cooled well.

[0045] The band is a frown, the reason: there are air bubbles at the bottom, and the device is not suitable.

[0046] 1.4 Cell flow detection method First, prepare the cell suspension, wash it 3 times with sterile PBS buffer solution, and then add trypsin digestion solution for 3-5 min. Observe the cell digestion under a light microscope, then place it in a centrifuge with parameters 1000 rmp for 5 min. After centrifugation, remove the supernatant and resuspend the cells with buffer solution. Adjust the cell density to 3x10 6 . Prepare 6 EP tubes of sample, add 0.1 ml of cell suspension to each tube, and mark the antibodies used for each. The negative control adds antibody IgG-FITC, IgG-PE; other experimental groups add mouse anti-human antibodies CD3-PE, CD8-PE, GZMB-PE, and mix them gently with a gun. Wrap them with tin foil to avoid light. Incubate in a 4 °C refrigerator for 30 min, then start the machine detection. When detecting flow cytometry apoptosis, add 5 μL Annexin V-EGFP, mix, then add 5 μL Propidium Iodide, mix, and react for 5-15 min at room temperature in the dark before starting the machine detection.

[0047] 2 Experimental results 2.1 In order to obtain the binding conformation of CDKN2A and ISG15 and find out the key residues of their interaction, the binding model and binding free energy of CDKN2A and ISG15 were predicted and analyzed by HawkDock protein-protein docking and MM / GBSA module, and the binding conformation of the two and the key residues of the interaction were obtained.

[0048] To further verify the function of the key residues described above, we constructed ISG15 overexpression plasmids (Flag-WT) and corresponding point mutation overexpression plasmids (Flag-LEU114, Flag-LEU145, Flag-THR95); CDKN2A highly expressed lung adenocarcinoma cell line PC9 was transfected respectively, and the experimental groups were: Control, NC PC9, ISG15 PC9, LEU114 PC9, LEU145 PC9, and THR95 PC9.

[0049] The EMT (epithelial-mesenchymal transition), invasion, and migration of lung adenocarcinoma cells were detected by immunofluorescence and Western blot experiments. First, the EMT-related indicators (E-cadherin, N-cadherin) of each group of cells were detected by immunofluorescence, and the results showed that after overexpression of ISG15, E-cadherin decreased significantly, and N-cadherin expression increased significantly. Notably, it was found that the THR95 site mutation reversed the results significantly ( Figure 1 、 Figure 2 ); Western blotting technology also verified the above immunofluorescence results. Thus, the THR95 site residue is a key residue for ISG15 to play a key role.

[0050] Further, Western blot experiments were performed to detect the expression of MMP2 and MMP9, which are related to cell invasion and migration, in each group of cells. The results showed that after overexpression of ISG15, the expression of MMP2 and MMP9 increased significantly, and notably, the above results were significantly reversed after the THR95 site mutation ( Figure 3 ).

[0051] Further, CDKN2A, ISG15, and the JAK-STAT1 signaling pathway and PD-L1 signaling pathway enriched by proteomics analysis were detected in each group of cells. Western blotting was performed on the above indicators, and the results showed that after overexpression of ISG15, CDKN2A expression decreased, and after the THR95 site mutation, it was significantly reversed, while ISG15, p-JAK, p-STAT1, and PD-L1 expression increased significantly, and after the THR95 site mutation, the above indicators were significantly reversed ( Figure 4 ).

[0052] 2.2 PC9 cells with high CDKN2A expression were modified by THR95 point mutation and co-cultured with activated T cells and Jurkat T cells from healthy adults. The PC9 cells were co-cultured with T cells or not co-cultured, and the experimental groups were NC, ISG15, and THR95.

[0053] Firstly, tumor cell clone formation experiment was used to detect the difference of tumor cell clone formation in each group of the experiment, the results showed that the number of PC9 cell survival was significantly increased after overexpression of ISG15, while compared with ISG15 group, the number of survival was significantly reduced after THR95 point mutation, and the number of survival cells in each group was significantly reduced after co-culture with activated T cells Figure 5 、 Figure 6 IFN-γ is a cytokine produced by activated T cells that can promote the cytotoxicity of effector CD8 + T cells, and the level of IFN-γ in the supernatant of each group of cells was detected, the experimental results showed that the level of IFN-γ in the supernatant of cells was significantly reduced after overexpression of ISG15, while the reducing trend was significantly reversed after THR95 point mutation Figure 7}.

[0054] Further, flow cytometry was used to detect the apoptosis of T cells in each group after co-culture of tumor cells and Jurkat T cells, the experimental results showed that the apoptosis rate of T cells was significantly increased after overexpression of ISG15, while the apoptosis rate was significantly reversed after THR95 point mutation Figure 8 IL-2 is also a cytokine produced by activated T cells that can promote the cytotoxicity of effector CD8+ T cells, and the level of IL-2 in the supernatant of each group of cells was detected, the experimental results showed that the level of IL-2 in the supernatant of cells was significantly reduced after overexpression of ISG15, while the reducing trend was significantly reversed after THR95 point mutation Figure 9}. Combined with the above results, it is shown that the residue at THR95 site is a key residue for ISG15 to regulate tumor immune escape.

[0055] The above only describes some embodiments of the present application, and does not limit the present application, any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application, all belong to the protection scope of the present application.

Claims

1. The use of the THR95 site of the ISG15 protein in the preparation of products for diagnosing or inhibiting tumor immune escape, wherein the THR95 site is the 95th amino acid located in the sequence shown in SEQ ID NO.

1.

2. Application of the THR95 site of the ISG15 protein in the preparation of products for diagnosing or reducing PD-L1 levels in tumor cells.

3. A method for reducing PD-L1 levels in tumor cells, characterized in that, A point mutation was induced in the THR95 site of the ISG15 protein in tumor cells. The THR95 site is the 95th amino acid in the sequence shown in SEQ ID NO.

1.

4. The application according to any one of claims 1 to 2 or the method according to claim 3, characterized in that, The tumors include lung cancer.

5. The application according to claim 4, characterized in that, The lung cancer mentioned includes non-small cell lung cancer.

6. The application according to claim 4, characterized in that, The lung cancer mentioned includes lung adenocarcinoma.

7. A method for reducing the expression level of ISG15 protein, characterized in that, A point mutation was made at the THR95 site of the ISG15 protein, where the THR95 site is the 95th amino acid in the sequence shown in SEQ ID NO.

1.

8. A method for reducing the inhibitory effect of ISG15 protein on CDKN2A protein, characterized in that, A point mutation was made at the THR95 site of the ISG15 protein, where the THR95 site is the 95th amino acid in the sequence shown in SEQ ID NO.

1.

9. The method according to claim 3, claim 7 or claim 8, characterized in that, The mutation is that the amino acid at position 95 of the sequence shown in SEQ ID NO.1 is mutated to alanine.

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