Application of DNAJC19 and PD-L1 as markers in the preparation of drugs for preventing and / or treating tumors and drugs

Abstract

The present invention discloses the use of DNAJC19 and PD-L1 as markers in the preparation of drugs for preventing and / or treating tumors, and the drugs, belonging to the field of molecular biotechnology. The drugs are anti-resistance drugs for platinum-based chemotherapy agents for lung cancer. Preferably, the drugs include DNAJC19 inhibitors and PD-L1 inhibitors, and the DNAJC19 inhibitors and PD-L1 inhibitors are administered simultaneously or separately. The present invention further clarifies another potential new mechanism of cisplatin resistance in human advanced driver gene-negative non-small cell lung cancer, providing a theoretical basis for its combined chemotherapy and immunotherapy.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, and specifically to the use of DNAJC19 and PD-L1 as markers in the preparation of drugs for preventing and / or treating tumors, and drugs. Background Art

[0002] Cisplatin-based chemotherapy is currently an important treatment for advanced driver gene-negative non-small cell lung cancer. However, widespread cisplatin resistance is a major cause of treatment failure. Previously, it was generally believed that resistance was a mechanism that prevented cancer cells from being effectively eliminated by chemotherapy drugs. However, the underlying mechanism remains unclear.

[0003] In recent years, the changes and roles of the tumor immune landscape after the acquisition of drug resistance have received increasing attention. The tumor immune landscape refers to the overall description of the immune system's response and interactions with tumor cells during tumor development. It includes various immune cells such as macrophages (Mφ), lymphocytes, monocytes, and dendritic cells (DCs), as well as immune checkpoint molecules such as programmed cell death protein-1 (PD-1), programmed cell death ligand-1 (PD-L1), and cytotoxic T lymphocyte antigen 4 (CTLA-4). Many of these immune cells and immune checkpoint molecules are considered potential biomarkers. Research on the tumor immune landscape helps to understand tumor cell resistance from the perspective of tumor immunity and provide new ideas for the treatment of drug-resistant tumors.

[0004] However, the above situation still exists in the prevention and / or treatment of lung cancer. Therefore, it is hoped that through research related to tumor immune landscape, treatment options for drug-resistant tumors can be found from the perspective of tumor immunity. Summary of the Invention

[0005] The purpose of the present invention is to provide a use of DNAJC19 and PD-L1 as markers in the preparation of drugs for preventing and / or treating tumors, providing a theoretical basis for the combined treatment of lung cancer patients with chemotherapy and immunotherapy.

[0006] The present invention is achieved through the following technical solutions:

[0007] The use of DNAJC19 and PD-L1 as markers in the preparation of a drug for preventing and / or treating tumors, wherein the drug is a drug for combating drug resistance to platinum-based chemotherapy agents for lung cancer. Platinum-based chemotherapy agents include cisplatin, carboplatin, and other platinum-based chemotherapy agents.

[0008] Furthermore, the drugs include DNAJC19 inhibitors and PD-L1 inhibitors.

[0009] Furthermore, the DNAJC19 inhibitor and the PD-L1 inhibitor are administered simultaneously or separately.

[0010] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0011] Furthermore, the medicine also includes at least one pharmaceutically acceptable additive.

[0012] Furthermore, the DNAJC19 inhibitor and PD-L1 inhibitor are used to prepare drugs for inhibiting the proliferation, migration or chemotherapy resistance of cancer cells.

[0013] A platinum-based anti-resistance drug for lung cancer for preventing and / or treating tumors, comprising a DNAJC19 inhibitor and a PD-L1 inhibitor.

[0014] Furthermore, the PD-L1 inhibitor is used to inhibit the expression of PD-L1 in vivo in the prevention and / or treatment of platinum-resistant lung cancer; the DNAJC19 inhibitor is used to inhibit the expression of DNAJC19 in vivo in the prevention and / or treatment of platinum-resistant lung cancer.

[0015] The use of DNAJC19 and PD-L1 as markers in the preparation of drugs that inhibit the proliferation, migration or chemotherapy resistance of cancer cells.

[0016] Application of DNAJC19 and PD-L1 as markers in the preparation of drugs to enhance the sensitivity of anti-lung cancer drugs.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0018] 1. The present invention proposes that the DNAJC19 / PD-L1 signaling interaction inhibits apoptosis, which is another potential mechanism causing cisplatin resistance in NSCLC. This allows the use of DNAJC19 and PD-L1 as markers in the preparation of drugs for the prevention and / or treatment of tumors, wherein the drugs are platinum-based anti-resistance drugs for lung cancer, providing a theoretical basis for the combined immunotherapy and platinum-based treatment of advanced lung cancer, and even finding new indicators for predicting efficacy.

[0019] 2. In the present invention, another potential new mechanism of cisplatin resistance in human advanced driver gene-negative non-small cell lung cancer is further clarified, providing a theoretical basis for its combined chemotherapy and immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 These are morphological diagrams of the parental cell lines, colony-forming and radiation-resistant cell lines of A549 and NCI-H1299, respectively.

[0021] Figure 2 These are confocal laser scanning microscopy images of NSCLC cells in each group after single radiotherapy or during radiotherapy resistance.

[0022] Figure 3This is a graphic representation of the results of detecting cell viability of radioresistant cell lines using the CCK8 assay.

[0023] Figure 4 This is a graphic representation of the results of a cloning experiment to detect the clonal proliferation ability of radiation-resistant cells.

[0024] Figure 5 This is a graphic representation of the results of the Transwell assay to detect the cell migration ability of radiation-resistant cell lines.

[0025] Figure 6 The figure shows the results of flow cytometry detection of apoptosis in radiation-resistant cell lines.

[0026] Figure 7 This figure shows the expression of DNAJC19 and PD-L1 proteins in radioresistant cells detected by Western blotting.

[0027] Figure 8 This is a confocal laser scanning microscopy image of PD-L1 protein expression in NSCLC tumor cells analyzed by immunofluorescence. Green represents PD-L1 and blue represents DAPI.

[0028] Figure 9 This is a graphic representation of the results of analyzing the expression of DNAJC19 and PD-L1 proteins in NSCLC patients using the GEPIA database.

[0029] Figure 10 This is a survival analysis diagram of the relationship between DNAJC19 and PD-L1 and the survival prognosis of NSCLC patients.

[0030] Figure 11 This is a graphic representation of the DNAJC19 transient transfection test results.

[0031] Figure 12 This is a graphic representation of the results of CCK8 assay to detect the viability of A549 and H1299 cells in different groups.

[0032] Figure 13 This is a graphic representation of the results of a cloning experiment to detect the cell cloning and proliferation capabilities of different groups.

[0033] Figure 14 It is a graphic representation of the results of transwell assay for the migration ability of A549 and H1299 cells in different groups.

[0034] Figure 15 The figure shows the results of flow cytometry detection of the apoptosis ability of A549 and NCI-H1299 cells in different groups.

[0035] Figure 16This is a graphic representation of the results of Western blotting experiments to detect the expression of DNAJC19 and PD-L1 proteins in different groups. DETAILED DESCRIPTION

[0036] The present invention will be further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto.

[0037] Example 1

[0038] This example uses the current research on the application of DNAJC19 and PD-L1 as markers in the preparation of drugs for preventing and / or treating tumors as an example to further illustrate this solution.

[0039] The mainstay of treatment for locally advanced non-small cell lung cancer (NSCLC) is radiotherapy combined with platinum-based chemotherapy, supplemented with immunotherapy. Tyrosine kinase inhibitors (TKIs) and programmed death-1 / programmed death-ligand 1 (PD-1 / PD-L1) inhibitors, in particular, have improved overall survival in NSCLC patients. Radiotherapy can also induce PD-L1 expression in the tumor microenvironment, thereby reducing the sensitivity of anti-tumor immune responses. Combined with previous studies showing elevated DNAJC19 expression in NSCLC patients compared with normal tissue, this clinical data analysis also demonstrates that elevated PD-L1 or DNAJC19 expression is associated with poor prognosis in NSCLC patients, suggesting the possibility that overexpression or mutation of DNAJC19 and PD-L1 may influence tumor development and progression. DNAJC19 and PD-L1 may influence tumor cell radiosensitivity. Therefore, it is hypothesized that knocking down DNAJC19 combined with PD-L1 inhibition may enhance radiosensitivity in human NSCLC.

[0040] 1. Materials and sources involved in the experiment

[0041] 1. Experimental cells

[0042] Human alveolar adenocarcinoma basal epithelial cells (A549 cells) and human non-small cell lung cancer cell line (NCI-H1299) were used in this experiment and were purchased from the Cell Resource Bank of the Chinese Academy of Sciences (Shanghai, People's Republic of China).

[0043] A549 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBs), 50 mg / L streptomycin and 50 mg / L penicillin. NCI-H1299 cells were also cultured in RPMI1640 medium containing 10% fetal bovine serum (FBS), 50 mg / L streptomycin and 50 mg / L penicillin. Both A549 and NCI-H1299 are adherent cells. After adding culture medium, they were cultured in an incubator at 37°C, 5% CO2 and saturated humidity.

[0044] 2. Experimental Materials

[0045] The main reagents and antibodies and their sources are as follows:

[0046] DMEM high-glucose medium, from GIBCO; PMI-1640 medium, from GIBCO; fetal bovine serum (FBS), from GIBCO; DPBs, from Corning; 0.25% trypsin, from GIBCo; rapid cell freezing solution, from Solarbio; penicillin / streptomycin, from Solarbio; 4% paraformaldehyde, from Sangon Biotech (Shanghai) Co., Ltd.; 0.1% crystal violet stain, from Sinopharm Chemical Reagent Co., Ltd.; RIPA lysis buffer, from Beyotime; BCA protein concentration assay kit, from Beyotime; anti-DNA JC19, from Proteintech Group, China; anti-PD-L1, from Abcam, UK; HE staining kit, from Solarbio; Annexin V-FITC / PI apoptosis detection kit, from eBioscience; 5X protein loading buffer, from Solarbio; Cell Counting CCK-8 kit, Solarbio; skim milk powder (blocking), Coolaber; primary antibody diluent, Beyotime; fluorescent secondary antibody diluent, Beyotime; EDTA antigen retrieval solution (50X, pH 9.0), Solarbio; neutral gum mounting medium, Solarbio; DS-PAGE protein electrophoresis buffer (dry powder), Solarbio; 10X TBS buffer, Solarbio; secondary antibody diluent, Solarbio; 5% BSA, Solarbio; anhydrous ethanol, Solarbio; xylene, Solarbio.

[0047] The main experimental instruments and equipment and their sources are as follows:

[0048] -20°C, 4°C, and -80°C refrigerators, Haier Co., Ltd.; biological safety cabinets, Shanghai Zhenzi Chuang Electrical Purification Equipment Co., Ltd.; CO2 incubator, BIO-LAB; flow cytometer, BD; centrifuge, Thermo Fisher Scientific (China) Co., Ltd.; AIRTECH clean bench, Sujing Group Antai Company; low-temperature high-speed centrifuge, Changsha Xiangzhi Centrifuge Instrument Co., Ltd.; constant temperature water bath, Guohua Electric Co., Ltd.; culture shaker, Guohua Electric Co., Ltd.; electronic balance, FA2004, Shanghai Liangping Instrument Co., Ltd.; small animal irradiator, Precision X-ray Irradiation; cell freezing liquid nitrogen tank, YDS-65-216, Chart / Golden Phoenix; Western blot system (power supply, electrophoresis tank, transfer tank), Shanghai Tianneng; optical microscope, TS100, Nikon Eclipse; paraffin slicer ThermoFisher; pathology tissue bleaching and drying apparatus Changzhou Zhongwei Electronic Instrument Co., Ltd.; pathology tissue embedding and freezing table Changzhou Zhongwei Electronic Instrument Co., Ltd.; PCR instrument Bio-Rad, USA; sDs.PAGE protein electrophoresis instrument Shanghai Tianneng; full spectrum analysis flow cytometer Cytek Aurora; protein transfer instrument Shanghai Tianneng; PvDF membrane millipore; stabilized voltage electrophoresis instrument Shanghai Tianneng; microplate reader Tecan infinite.

[0049] 2. Experimental Methods

[0050] 2.1 Some basic cell processing methods involved in the experiment

[0051] 2.1.1 Cell culture

[0052] Both A549 and NCI-H1299 cell lines exhibit adherent growth. Under conventional culture conditions, A549 cells were cultured in high-glucose (4.5 g / L D-glucose) DMEM medium enriched with 10% fetal bovine serum (FBS), containing 100 U / ml penicillin and 100 U / ml streptomycin. NCI-H1299 cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 100 U / ml penicillin, and 100 U / ml streptomycin. Both cell lines were then maintained in a constant temperature environment of 37°C, 5% CO2, and 80% humidity. Tumor cell growth and morphology were observed daily, and any contamination was checked. Fresh culture medium was promptly replaced to ensure normal cell growth. When cells reached logarithmic phase and occupied 80%-90% of the flask density, they were washed, digested, and passaged using 0.25% trypsin solution. When the cell count and condition meet the desired requirements, digest and collect all the cells. After centrifugation, count the number of viable cells using trypan blue exclusion under a microscope. Prepare a cell suspension based on the desired cell number for the next step of the experiment.

[0053] 2.1.2 Cell line recovery

[0054] (1) Preparation of culture medium: Take out high-glucose DMEM, RPMI-1640 culture medium and fetal bovine serum from a 4°C refrigerator, place them in a clean bench, and after ultraviolet irradiation for 30 minutes, mix the culture medium and serum in a ratio of 9:1 to prepare a mixed culture medium containing 10% fetal bovine serum. Shake and mix thoroughly before use;

[0055] (2) Take out the A549 and NCI-H1299 cell lines frozen in the liquid nitrogen tank, quickly place the cryovials into a pre-prepared 37°C constant temperature water bath, and thaw them quickly in about 40 seconds. Then aspirate the cryovials into a 15ml centrifuge tube. Then place the centrifuge tube in a centrifuge and centrifuge at a speed of 1200 rpm, a temperature of 4°C, and a centrifugation time of 5 minutes.

[0056] (3) After centrifugation, pour out the supernatant and add 5 ml of fresh culture medium to the centrifuge tube.

[0057] After gently pipetting to mix, pipette the resuspended cell suspension into a T25 plastic culture flask. Use the figure-of-eight or figure-of-Piece (P-P) method to ensure even distribution of cells within the flask. Observe the cells under a microscope to ensure uniform distribution and assess cell number, density, and condition. Finally, place the flask containing the cell suspension in a CO2 incubator for cell culture.

[0058] 2.1.3 Cell passaging

[0059] (1) Remove the culture flask from the incubator and carefully observe the growth status, morphology, and density of A549 and NCI-H1299 cells. Start cell passaging when the cells cover 80%-90% of the flask wall.

[0060] (2) Place all necessary laboratory supplies under ultraviolet light in a clean bench for 20-30 minutes to ensure sterility throughout the entire process. Before subculturing, discard the old culture medium in the T25 flask and add 2-3 ml of sterile 1X PBS buffer to wash the cells 2-3 times.

[0061] (3) Using a pipette, gently draw up 500 μL of trypsin solution and place the culture flask horizontally, ensuring that the trypsin is evenly coated on the bottom of the culture flask to fully cover the tumor cells. Then, return the culture flask to the incubator to allow the digestion process to proceed.

[0062] (4) Observe the cell digestion process under a microscope. If the cells begin to float or become round, or if you gently shake the culture bottle from side to side or even tap the side of the culture bottle, you will see some adherent cells fall off the bottom of the bottle. If these phenomena are observed, stop the cell digestion process immediately.

[0063] (5) Use a pipette to repeatedly aspirate the digested suspension in the culture flask and gently blow the bottom of the culture flask to ensure that the A549 and NCI-H1299 cells are completely detached from the bottom of the culture flask.

[0064] (6) Next, transfer the cell suspension to a 15 ml centrifuge tube and place it in a low-temperature high-speed centrifuge. Set the centrifuge speed to 1000-1300 rpm and the temperature to 4°C.

[0065] (7) After centrifugation, discard the supernatant and inject 1 ml of culture medium into the centrifuge tube. Use a pipette to repeatedly extract and mix. Then, gently transfer the thoroughly mixed cell suspension to a T75 culture flask. Observe the cell status and density under a microscope. Finally, return the culture flask to an environment with 5% CO2, 80% humidity, and a constant temperature of 37°C to continue cell culture.

[0066] 2.1.4 Cell line cryopreservation

[0067] (1) Observe the status of the cells in the culture flask. When the cell coverage area reaches 80% to 90% of the bottom of the culture flask, prepare the cells for freezing. Replace the culture medium with fresh complete medium one day before freezing.

[0068] (2) Spray the culture flask to be frozen with 75% alcohol for disinfection and place it in a clean bench. Discard the waste liquid and add 1 ml of PBS buffer to the culture flask. Gently shake the culture flask to clean the adherent cells. Then add an appropriate amount of 0.25% trypsin to digest the cells. When the cells begin to float or become round and reduce in size, immediately add an appropriate amount of complete culture medium to terminate the cell digestion process.

[0069] (3) To ensure that the cells are completely separated from the bottom of the culture flask, use a pipette to repeatedly blow the cell suspension to mix it thoroughly. Then transfer the A549 or NCI-H1299 cell suspension to a low-temperature high-speed centrifuge and set the speed to 1000 rpm. Centrifuge for 3 minutes at room temperature.

[0070] (4) After centrifugation, discard the upper layer of culture medium again and add 1 ml of rapid cell freezing solution. Mix thoroughly by pipetting and transfer to a 1.5 ml cryovial. Seal the tube with sealing glue and label with cell-related information. First, freeze the cryovial in a -80°C freezer overnight. Then, by gradually lowering the temperature, transfer them to a container filled with liquid nitrogen for long-term storage.

[0071] 2.1.5 Cell Count

[0072] (1) Gently remove the A549 and NCI-H1299 culture bottles from the incubator and place them in a clean bench. Then, pour out the old culture medium and wash them 2 to 3 times with sterile PBS (phosphate buffered saline) to ensure the cleanliness of the cells. Then add an appropriate amount of trypsin to digest and separate the cells. During the digestion process, pay attention to the changes in cell morphology to avoid over-digestion. After the digestion is completed, add complete culture medium to terminate the digestion;

[0073] (2) Remove the cell counting glass and clean it with alcohol. Add the centrifuged cells to the culture medium to dilute into 5 ml of cell suspension. After mixing the cell suspension, use a pipette to transfer 20 μl to a 1.5 ml EP tube. Add 180 μl of trypan blue and mix well. Take 10 μl of the mixed stained cell suspension and drop it into the counting chamber covered with a coverslip, taking care to avoid bubbles.

[0074] (3) Under a microscope, the cell concentration can be calculated as ten times the total number of cells in the four corner squares of the counting slide divided by 4×10 4 Unit per milliliter;

[0075] (4) According to the requirements of subsequent experiments, cells are configured to the corresponding concentration.

[0076] 2.1.6 Construction of radioresistant tumor cell lines

[0077] (1) A549 and NCI-H1299 were observed under a microscope and grown to a cell confluence of 80%-90%. They were irradiated with a sublethal dose (6 Gy). Fresh culture medium was immediately replaced after irradiation. The cells were placed in a constant temperature incubator at 37°C, 5% CO2, and 80% humidity for further culture. When the surviving cells grew again to a cell confluence of 80%-90%, they were passaged to become first-generation subline cells. The cells were further cultured in sterile culture flasks until the cell confluence was 80%-90%. They were irradiated with a sublethal dose (6 Gy) again. Fresh culture medium was immediately replaced and cultured for further culture. The surviving cells were passaged again and irradiated. This process was repeated. After A549 and NCI-H1299 cell lines received 5 or more sublethal doses of irradiation, the cell growth rate slowed down. After the growth ability stabilized, irradiation was stopped, thereby obtaining their tumor radiation-resistant cell lines.

[0078] (2) irradiation of A549 and NCI-H1299 cell lines using 320 kV X-rays from a small animal irradiator;

[0079] (3) Grouping of radiotherapy resistance was carried out according to the requirements of subsequent experiments.

[0080] 2.2 Introduction to specific experimental methods

[0081] 2.2.1 Cell transfection experiment

[0082] (1) One day in advance, plate the cells in a 6-well plate at a density of 5×10 5 During the experiment, the cell morphology and cell density were observed under an inverted microscope. Cell transfection could be performed when the cell confluence reached 30%-50%.

[0083] (2) Following the transfection protocol manual, dilute the siRNA using DEPC water. In a 1.5 ml Eppendorf tube containing 250 μl of serum-free growth medium, mix the siRNA and Lipo3000X at a 1:1 ratio. Allow the mixture to stand at room temperature for 5 minutes. Add the freshly diluted siRNA, mix thoroughly, and allow the mixture to stand at room temperature for 20 minutes.

[0084] (3) Pour out the old culture medium in the 6-well plate, add PBS to wash, and then slowly drip the transfection reagent siRNA mixture into the 6-well plate along the well wall. After thorough shaking, place in a 37°C constant temperature incubator for continued cultivation. After 6 hours, replace the original culture medium with one containing 10% FBS serum and continue to culture. After 48 hours, digest again, centrifuge, collect cells and extract protein. After protein quantification, perform Western blotting to detect transfection efficiency.

[0085] (4) Based on the WB test results, select the reagent with the highest transfection efficiency and repeat the transient transfection for subsequent experiments;

[0086] The siRNA sequences used in this experiment to detect transient transfection efficiency are shown in Table 1.

[0087] Table 1: siRNA sequences

[0088]

[0089] 2.2.2 CCK8 cell viability assay

[0090] (1) First, remove the culture flask from the incubator and place it in a clean bench. Discard the original culture medium and wash the cells twice with sterile PBS buffer. Then, use an appropriate amount of trypsin containing 0.25% EDTA to digest the A549 or NCI-H1299 cells. When the cells are observed to increase in size and become round, add fresh culture medium to terminate the digestion process.

[0091] (2) Then, by counting the cells, a cell suspension of 2000 cells per well was prepared. Then, 100 μl of the cell suspension was evenly dropped into a 96-well plate. Three parallel wells were set up for each group of cells.

[0092] (3) The evenly seeded A549 and NC1-H1299 cells were placed in a constant temperature incubator and incubated for 24 hours. The cells were observed to be evenly attached to the wall. When the confluence reached 80%-90%, the culture was terminated and the prepared CCK8 solution was added. The solution was prepared into a mixed solution according to the concentration ratio of culture medium: CCK-8 at 9:1. During the addition process, bubbles were avoided. A control group was set up at the same time. Then the culture was continued in the incubator for 0.5h-2h. The culture was terminated when the color of the culture medium turned orange-yellow.

[0093] (4) Take out the 96-well plate, place it on a horizontal shaker and shake it at a constant speed for 10 seconds, then put it into a microplate reader to detect the OD value of the absorbance at 450 nm and record the data. At the same time, use Graph Prism 9.0 to analyze the cell proliferation of each group of A549 and NCI-H1299 and quantify the data.

[0094] (5) Cell viability formula: Cell viability (%) = (experimental group - blank control) / (negative control - blank control) × 100%.

[0095] 2.2.3 Cell cloning experiment

[0096] (1) When A549 and NCI-H1299 cells in the culture flask showed logarithmic growth under a microscope, the cells were collected by digestion and centrifugation;

[0097] (2) After adding fresh culture medium, take 10 μl of cell suspension for cell counting and prepare the cell suspension again at a concentration of 500 cells per well;

[0098] (3) In the cloning experiment, 2 ml of complete culture medium was first injected into a 6-well plate, and then the plate was seeded at a density of 500 cells per well. After ensuring that the cells were evenly dispersed, the 6-well plate was returned to the 37°C incubator and the cells were allowed to grow for two weeks. During this period, the state and morphology of A549 and NCI-H1299 cells were regularly observed by optical microscopy. The culture medium was replaced every three days. Cloning was terminated when cell colonies were formed and the number of clones in the cells exceeded 50.

[0099] (4) After the colony formation, the 6-well plate was washed twice with PBS, and 4% paraformaldehyde was slowly added along the well wall to fix the plate at room temperature for 20 min.

[0100] (5) After discarding the fixative, slowly drip 0.1% crystal violet stain along the well wall, allowing it to cover the cells, and stain at room temperature for 30 minutes. Then, gently rinse off the crystal violet stain with running water and allow the 6-well plate to air dry at room temperature.

[0101] (6) Observe and take photos under an inverted microscope to count the number of cloned cells;

[0102] (7) Repeat the above experiment 3 times.

[0103] 2.2.4 Transwell experiments

[0104] (1) The day before the experiment, the original culture medium was replaced with serum-free culture medium to allow A549 and H1299 cells to experience a starvation period of at least 12 hours;

[0105] (2) After A549 and NCI-H1299 cells were collected by digestion and centrifugation as described above, the cells were counted under a microscope and the cell density was adjusted to 1×10 per well. 5 Pieces / ml;

[0106] (3) Place the 24-well plate in a clean bench and irradiate with ultraviolet light for 30 minutes. Add 600 μl of complete culture medium containing 15% serum to the lower chamber of the 24-well plate. Then, slowly add 200 μl of A549 and NCI-H1299 cell suspensions adjusted to the cell density to the upper chamber. After fully flattening, place the plate in a 37°C cell incubator and continue culturing for 1 to 2 days.

[0107] (4) Closely observe the cell migration during the period. After the cell culture reaches the predetermined standard, remove the upper chamber and slowly rinse it with PBS solution twice. Then add 4% paraformaldehyde and let the cells fix for 20 minutes at room temperature;

[0108] (5) After fixation, the upper chamber was removed and immersed in PBS solution twice. The inner wall of the upper chamber was then gently wiped with a cotton swab. Under optical microscopy, the chamber after each experimental group was inverted and five fields of view were randomly selected for photographing and counting. Finally, statistical methods were used to quantify and compare the data.

[0109] (6) Each experiment was repeated three times.

[0110] 2.2.5 Cell apoptosis assay

[0111] (1) Remove the A549 and NCI-H1299 cell culture flasks from the incubator, wash with PBS buffer, add EDTA-free trypsin for digestion, and collect the cells into a 15 ml centrifuge tube;

[0112] (2) Place the centrifuge tube in a low-temperature high-speed centrifuge, set the centrifugal force to 500g, the temperature to 4°C, and centrifuge for 5 minutes;

[0113] (3) After centrifugation, discard the supernatant, add PBS buffer to wash the cells twice, and place the centrifuge tube in a low-temperature high-speed centrifuge again, set the centrifugal force to 400g, and centrifuge for 5 minutes;

[0114] (4) After centrifugation again, discard the upper buffer and add Binding Buffer to resuspend A549 or NCI-H1299 into cell suspension. Count the cells to make the cell suspension concentration reach 5×10 6 Pieces / ml;

[0115] (5) When preparing a single-positive tube, first take out 200 μl of the cell suspension and inject it into two different flow tubes, and mark the tube walls. In one single-positive tube, add an appropriate amount of fluorescently labeled Annexin V dye, and in the other single-positive tube, add an appropriate amount of PI dye. Then gently shake to evenly distribute the dye and incubate at room temperature in the dark for 20 minutes.

[0116] (6) After the cell suspension is sieved, it is detected using a flow cytometer;

[0117] (7) In this experiment, all apoptosis results are presented with Annexin V as the horizontal axis and PI as the vertical axis. The upper left quadrant represents cells that have been mechanically damaged, the lower left quadrant represents normal tumor cells, the upper right quadrant represents cells in late apoptosis or necrosis, and the lower right quadrant represents cells in the early stage of apoptosis;

[0118] 2.2.6 Western blotting (WB)

[0119] 2.2.6.1 Cell protein extraction

[0120] (1) Protein extraction: Discard the old cell culture medium and wash the culture flask three times with pre-chilled PBS. After aspirating the PBS, add sufficient pre-prepared protein lysis buffer (RIPA lysis buffer containing 1% PMSF) and mix thoroughly to ensure that the cells are in full contact with the lysis buffer.

[0121] (2) Place the culture flask on ice for 30 minutes to lyse the cells, ensuring that A549 and NCI-H1299 cells are fully exposed to the pre-prepared protein lysis buffer. Then, use a cell scraper to scrape the cells and collect the cell suspension into a 1.5 ml Eppendorf tube using a pipette.

[0122] (3) Place the Eppendorf tube in a pre-cooled high-speed centrifuge and centrifuge at 4°C and 12,000 rpm for 30 min.

[0123] (4) Store the supernatant in a new Eppendorf tube, label it, and keep 1-10 μl for protein concentration determination. The rest should be stored in a -80°C refrigerator.

[0124] 2.2.6.2 BCA protein quantification

[0125] (1) According to the requirements of the downstream experiment, prepare the working solution volume and mix reaction solution A and reaction solution B at a ratio of 50:1 (A solution (BC A Reagent) and B solution (Cu1 + Reagent)) to prepare a sufficient working solution.

[0126] (2) Dilute the standard protein sample according to the ratio specified in the instructions to obtain 10 groups of standard samples with different concentrations. Then, add 20 μl of the concentration standard sample to a 96-well plate and create a standard curve using the 10 different groups.

[0127] (3) Add 100 μl of pre-made working solution to the 96-well plate to be tested;

[0128] (4) Mix 2 μl of protein suspension and 18 μl of PBS and add to the working solution;

[0129] (5) Place the 96-well plate in a 37°C incubator and incubate for 30 min. Use a microplate reader to measure the absorbance at 562 nm for each well after shaking for 10 s. Calculate the protein concentration of each sample based on the previously prepared standard curve and balance the concentrations of each protein sample.

[0130] 2.2.6.3 Preparation of SDS-PACE (polyacrylamide) gel

[0131] (1) Take out 2 ml of lower gel solution and 2 ml of lower gel buffer, add them to a 15 ml centrifuge tube, and mix thoroughly;

[0132] (2) Add 40 μl of the modified coagulant to the mixed 15 ml centrifuge tube and mix thoroughly again;

[0133] (3) Immediately pipette the remixed suspension into the glue-making glass plate, and keep the distance between the liquid surface of the suspension and the upper edge of the short glass plate 0.5 to 1 cm longer than the tooth comb. Use a pipette to slowly inject distilled water into the remaining gap between the glass plates until the liquid surface reaches the upper edge of the glass plate, completely covering the lower layer of glue;

[0134] (4) When a broken line is observed between the lower layer of glue and the distilled water (about 15 minutes), the lower layer of glue has solidified and the distilled water in the upper layer is poured out;

[0135] (5) Take out 0.5 ml of the upper gel solution and 0.5 ml of the colored upper gel buffer, add them to a 15 ml centrifuge tube and mix thoroughly;

[0136] (6) Add 10 μl of the modified coagulant to the mixed 15 ml centrifuge tube and mix thoroughly again;

[0137] (7) Immediately pipette the remixed suspension into the glass plate until the liquid level reaches the top edge of the glass plate, and insert the tooth comb;

[0138] (8) When the remaining upper layer of gel in the centrifuge tube solidifies (about 15 minutes), the gel preparation is complete.

[0139] Table 2: Glue making configuration table

[0140]

[0141] 2.2.6.4 Protein electrophoresis

[0142] (1) Take out the previously quantified protein sample and add 5x loading buffer to it. After thorough mixing, denature at 100°C for 10 min.

[0143] (2) Prepare electrophoresis buffer: Add 14.4 g of glycine, 3 g of Tris-base, and 10 ml of 10% SDS to a 1 L graduated cylinder, and finally add ddH2O to make up to 1 L.

[0144] (3) Take two glass plates and clamp them on the electrophoresis stand to form an upper tank. Use the electrophoresis tank to fix the gel plate and add an appropriate amount of electrophoresis fluid to prevent the gel from drying out. After the upper sample cools down, pull the sample comb out of the gel plate.

[0145] (4) Add Iodizing Buff to the first and last wells, and add Protein Maker to the adjacent wells. Then add 20 μl of protein sample to the other loading wells.

[0146] (5) Electrophoresis: Connect the electrophoresis instrument to the electrophoresis tank using the red-to-red and black-to-black method. Set the constant voltage to 70 V and run the electrophoresis for about 25 minutes. Then adjust the voltage to 100 V and continue the electrophoresis. Stop the electrophoresis when the bromophenol blue reaches the bottom of the gelatin plate.

[0147] 2.2.6.5 Protein transfer

[0148] (1) Prepare transfer buffer: Prepare 1000 ml of transfer buffer by mixing 100 ml of 10× transfer buffer, 100 ml of anhydrous ethanol, and 700 ml of distilled water. Pre-cool the mixture in a 4°C refrigerator.

[0149] (2) Take the pre-cooled transfer solution out of the 4°C refrigerator and pour it into the prepared transfer tank. Then, cut the PVDF membrane with scissors according to the volume of the gel prepared previously. Soak the PVDF membrane in methanol in advance until the PVDF membrane becomes transparent, then place it in the transfer solution for use;

[0150] (3) Then we stacked the sandwich in the order of cathode-sponge pad-filter paper-gel-PVDF membrane-filter paper-sponge pad-anode, rolled out the bubbles with a roller, and then placed it in the transfer tank electrode;

[0151] (4) Place the transfer apparatus in a 4°C ice chamber and perform transfer at a constant current of 300 mA for 60 minutes.

[0152] 2.2.6.6 Closure

[0153] (1) Prepare TBST solution: Add 100 ml of 10× TBST solution and 900 ml of distilled water to a 1 L graduated cylinder and mix thoroughly.

[0154] (2) Prepare 5% skim milk (blocking solution): Use an electronic balance to measure 5 g of skim milk powder and add 5 g of skim milk + 100 ml of TBST solution to a 200 ml graduated cylinder. Vortex to mix thoroughly.

[0155] (3) After the transfer is completed, place the PVDF membrane in a blocking box filled with blocking solution to completely cover the PVDF membrane and block on a shaker at room temperature for 1 hour;

[0156] 2.2.6.7 Incubation with primary antibody

[0157] After blocking, wash three times with TBST on a shaker at room temperature for 5 minutes each time. Cut the PVDF membrane according to the molecular weight of the target protein, dilute the primary antibody according to the instructions, soak the PVDF membrane in the diluted primary antibody, and incubate at 4°C overnight.

[0158] 2.2.6.8 Incubation with secondary antibody

[0159] After the primary antibody incubation is complete, wash the PVDF membrane three times with TBST on a shaker at room temperature for 5 minutes each time. Select a secondary antibody of the appropriate species based on the source of the primary antibody. Dilute the secondary antibody against the target protein in TBST and transfer it to the antibody incubation box. Shake at room temperature for 1 hour.

[0160] 2.2.6.9 Exposure and Development

[0161] After the secondary antibody incubation is complete, discard the secondary antibody using a pipette and wash the PVDF membrane three times with TBST solution on a shaker at room temperature for 5 minutes each time. Prepare the ECL kit by mixing chemiluminescent solutions A and B (1:1), add an appropriate amount of luminescent solution to the PVDF membrane, and analyze using a chemiluminescence analyzer.

[0162] 2.2.7 Immunohistochemistry (IHC)

[0163] (1) Dewaxing: Place the paraffin sections in a 65°C slide dryer for 60 min. After drying, immediately immerse the sections in xylene in the following order: xylene I, xylene II, and xylene III for 15 min each.

[0164] (2) Hydration: The slides were then hydrated in a gradient of alcohols: 100% alcohol for 5 min, 90% alcohol for 5 min, 80% alcohol for 5 min, and 70% alcohol for 5 min, and finally rinsed gently with distilled water for 2 min.

[0165] (3) Antigen repair: Place the slides in an antigen repair box and completely immerse the slides in 1X pH 9.0 Tris-EDTA antigen repair solution, so that the antigen repair solution completely covers the slides. Then perform high-temperature repair in a microwave oven. Preheat at 100°C for 4 minutes, then high-temperature repair for 3 minutes, and cool for 5 minutes. Repeat this process 3 times. Cool naturally to room temperature and wash with PBS buffer 3 times, 5 minutes each time.

[0166] (4) Block endogenous peroxidase: Use an immunohistochemistry pen to draw a circle around the tissue to prevent liquid loss. Once the water blocking circle is drawn, 3% H2O2 can be dripped into the tissue. After incubation at room temperature for 10 minutes, rinse in PBS solution three times, each time for 5 minutes.

[0167] (5) Serum blocking: After washing, add goat serum blocking solution to the water spring and block at room temperature for 30 minutes;

[0168] (6) Primary antibody incubation: According to different antibody requirements, add the primary antibody of immunohistochemistry indicator at a dilution ratio of 1:200, incubate in a refrigerator at 4°C overnight, and rinse in PBS solution three times, each time for 5 minutes;

[0169] (7) Secondary antibody incubation: After the primary antibody incubation and washing is completed, dilute the secondary antibody according to the enzyme-labeled secondary antibody of the same species, add 50 μl to each slide and incubate at room temperature for 30 minutes. After the incubation is completed, wash with PBS three times, each time for 5 minutes;

[0170] (8) DAB color development: Prepare fresh DAB color development solution first, then use filter paper to absorb the secondary antibody on the glass slide, drop the DAB color development solution onto the glass slide, and control the reaction time under a microscope at room temperature to ensure that the color development time of the same group of specimens is consistent. Then rinse with tap water and PBS to stop the color development;

[0171] (9) Counterstaining: After counterstaining with hematoxylin for 5 minutes, rinse with tap water for 5 minutes;

[0172] (10) Dehydration and clearing: Place the stained sections in 70% alcohol, 80% alcohol, 90% alcohol for 2 minutes each, and 100% alcohol for 10 minutes. Use xylene I, xylene II, and xylene III for 5 minutes each to clear the sections.

[0173] (11) Sealing: After the slices are transparent, seal them with neutral resin. After the resin is dried, place them under a microscope for observation and photographic record.

[0174] 2.2.8 Immunofluorescence (IF)

[0175] (1) Cell preparation: Clean the slide with detergent, then rinse with tap water, then soak the slide in 75% alcohol for 10 minutes, take out the slide, pick it up with tweezers, place it on an alcohol burner to dry, and place it in a culture dish that has been irradiated with ultraviolet light in advance. 5 Cells) were evenly added onto the slide and placed in a 37°C, 5% CO2 constant temperature incubator overnight until the cell density reached 75%-85%.

[0176] (2) Wash the glass slide with cells three times with PBS, each time for 3-5 minutes.

[0177] (3) Fixation: Fix the slides with 4% paraformaldehyde at room temperature for 15-30 minutes, and then rinse the slides with PBS three times, each time for 3-5 minutes.

[0178] (4) Permeabilization: 0.5% Triton X-100 (PBS) was used for permeabilization at room temperature for 20 min.

[0179] (5) Blocking: Wash the slides with PBS three times, each time for 3-5 minutes. Drain the PBS with absorbent paper, add normal goat serum to the slides, and block at room temperature for 30 minutes.

[0180] (6) Incubation with primary antibody: Absorb the original blocking solution with absorbent paper, add a sufficient amount of diluted primary antibody to each slide and place it in a humidified box, incubate at 4°C overnight.

[0181] (7) Add fluorescent secondary antibody: Wash the slide again with PBS for 3 times, each time for 3-5 minutes. After absorbing the excess liquid on the slide with absorbent paper, add diluted fluorescent secondary antibody in the dark. Incubate at room temperature in a wet box for 1 hour. Then wash with PBS for 3 times, each time for 3-5 minutes.

[0182] (8) Counterstaining: Add DAPI-containing mounting medium to the slide and incubate in the dark for 5 minutes.

[0183] (9) Observe and collect images under a fluorescence microscope.

[0184] 2.2.9 Bioinformatics Analysis

[0185] This part of the experiment first compared DNAJC19 genes with significant differential expression in lung cancer tissue using the TCGA database. Gene set analysis (GESA) was performed using the MSigDB Collections database. GEPIA was then used to analyze NSCLC survival data under varying DNAJC19 expression.

[0186] 2.3 Statistical Methods

[0187] The experimental data in this invention were processed and analyzed using SPSS 11.0, GraphPad Prism 8, and the R language "ggplot package". Survival curves were drawn using the Kaplan-Meier method, and differences in patient survival time in different DNAJC19 and PD-L1 protein expression groups were analyzed using the Log-rank method. Experimental data are expressed as mean ± standard error (Mean ± SEM). The comparison between the two sample means was performed using the independent sample t-test. Fisher's exact test, spearman test, and chi-square test were used to compare variables between groups. P < 0.05 was considered statistically significant.

[0188] 3. Experimental Results and Analysis

[0189] 3.1 Radioresistance affects tumor cell characteristics and DNAJC19 and PD-L1 expression

[0190] 3.1.1 Cultivation of radiation-resistant strains

[0191] To investigate the radioresistance of tumor cells to ionizing radiation, two human non-small cell lung cancer cell lines, A549 and NCI-H1299, were revived and cultured in vitro from the cell cryopreservation bank of Sichuan Cancer Hospital. Both cell lines were seeded into T25 culture flasks and cultured in either Dulbecco's Modified Eagle's Medium (DMEM) or Medium 1640 (10% fetal bovine serum) supplemented with 10% fetal bovine serum (FBS) and 100 U / ml penicillin and streptomycin, respectively, in a 37°C, 5% CO2 incubator. Cells were passaged when they reached 80%-90% confluence. When the passaged cells reached 80%-90% confluence, they were irradiated with 6 Gy of X-rays at 320 kV using a small animal irradiator. After irradiation, the cells were returned to the cell culture room, disinfected with 75% alcohol, and immediately replaced with the original culture medium in a laminar flow hood. The cells were then incubated overnight in a constant temperature incubator before being passaged and cryopreserved. Continue to culture the tumor cells in this way until the degree of confluence is about 50% and then stop irradiation. During the irradiation process, the cells decrease logarithmically. When the total irradiation dose reaches 24Gy, the cells grow slowly. It takes about 1-2 months to grow again until the confluence is 80%-90% of the cell colonies. When the irradiation dose reaches 30Gy, observe the cell growth under an inverted microscope and the degree of confluence is 50%, then stop radiotherapy, and continue to change the fluid, passage, and cryopreservation. It was found that after 30Gy irradiation, the tumor cells increased logarithmically when they were passaged and cultured again. Compared with the parent cell line (WT), the radiation-resistant cell line (RT) grew faster. The morphology of the parent cell line, colony formation and radiation-resistant cell line of A549 and NCI-H1299 can be seen. Figure 1 , among which, Figure A is the morphology of A549 parental cells (A549WT); B is the colony formation of A549; C is the morphology of A549RT radiation-resistant cells (A549RT); Figure D is the morphology of NCI-H1299 parental cells (NCI-H1299WT); E is the colony formation of NCI-H1299; F is the morphology of NCI-H1299RT radiation-resistant cells (NCI-H1299RT).

[0192] 3.1.2 High Expression of γ-Phosphorylated Histone H2AX in Radiation-Resistant Cell Lines Verifies Radioresistance. Initially, we cultured A549 and NCI-H1299 parental and radiation-resistant cell lines. To verify the radiation resistance of these cultured radiation-resistant cell lines, we examined changes in γ-phosphorylated histone H2A.X foci. H2A.X, a member of the histone H2A family, is involved in maintaining genomic stability and repairing DNA damage. Radiation-induced double-strand breaks in tumor cells, upon which phosphorylated H2A.X helps cells identify and localize DNA damage and recruit other repair proteins to these sites. This study still used two cell lines, A549 and H1299. At the same time, the resistant strains and parental cell lines of these two cell lines were irradiated with 6 Gy respectively. After re-irradiation, they were divided into 8 groups: A549WT, A549WT+R, A549RT, A549RT+R, H1299WT, H1299WT+R, H1299RT, and H1299RT+R. Immunofluorescence was used to observe the expression of DNA damage marker γH2AX foci in the resistant cell lines.

[0193] After the resistant cell lines were irradiated again with the same dose, γ-H2AX still expressed highly, which verified the radiation resistance of the radiation-resistant cells. The formation of DNA damage marker γH2A.X foci in each group of NSCLC cells after single radiotherapy or during radiotherapy resistance was observed by immunofluorescence. Figure 2 . Figure 2 In the figure, red represents γH2AX and blue represents DIPI; A represents the formation of γH2AX foci in each A549 group; B represents the formation of γH2AX foci in each NCI-H1299 group.

[0194] The results showed that γ-H2AX was expressed in the nucleus and the number of γ-H2AX foci in the A549 / H1299 radiation-resistant cell line was more than that in the parental cell line.

[0195] 3.1.3 Radioresistance affects tumor cell proliferation

[0196] In order to study the proliferation characteristics of radioresistant cell lines, the cell viability of two radioresistant cell lines, A549 and NCI-H1299, was detected by CCK8 assay compared with the parental cell lines. The experiment was divided into four subgroups, namely A549WT, A549RT, H1299WT, and H1299RT. The results refer to Figure 3 . Figure 3Figure 1: A comparison of the proliferation capacity of the A549 parental strain (WT) and the resistance cell line (RT); Figure 2: B comparison of the proliferation capacity of the NCI-H1299 parental strain (WT) and the resistance cell line (RT). **** indicates P < 0.0001; *** indicates P < 0.001; ** indicates P < 0.01; * indicates P < 0.05; ns indicates P > 0.0.

[0197] The study confirmed that both A549 and NCI-H1299 cell lines had stronger proliferation abilities than the parental NSCLC cell line. Over time, both the parental and resistant cell lines showed increased proliferation, but the resistant cell lines showed greater proliferation compared to the parental cell lines. The enhanced viability of the radiation-resistant cell lines suggests that the proliferation ability of radiation-resistant cell lines is time-dependent.

[0198] In addition, this study used plate cloning assay to verify the above phenomenon. The radiation-resistant cell line and the parent cell line were inoculated in 6-well plates, with the inoculation number of 5×10 2 The number of clones of the two radiation-resistant cell lines A549 and NCI-H1299 was still higher than that of the parental cell line reference Figure 4 . Figure 4 Figure 1: A plots the cloning ability of the parental strains (WT) of A549 and NCI-H1299 and the resistant cell line (RT); B plots the cloning ability of the parental strain (WT) of A549 and the resistant cell line (RT); and C plots the cloning ability of the parental strain (WT) of NCI-H1299 and the resistant cell line (RT). **** indicates P < 0.0001; *** indicates P < 0.001; ** indicates P < 0.01; * indicates P < 0.05; ns indicates P > 0.05.

[0199] The results showed that compared with the parental cell line, the resistant strain had enhanced radiation resistance and clone formation ability, which increased the number of clone colonies formed by the tumor cell line.

[0200] 3.1.4 Radioresistance affects tumor cell migration ability

[0201] Migration ability is one of the main characteristics of NSCLC cells. To further compare the migration ability of radiation-resistant cell lines with that of parental cell lines, the Transwell assay was used to detect two cell lines, A549 and H1299. The number of tumor cells that migrated downward after 24 hours was compared with their parental cell lines, thereby exploring the migration ability of radiation-resistant cell lines. The results are referenced. Figure 5 . Figure 5Figure 1: A comparison of the migration abilities of the parental A549 and NCI-H1299 strains (WT) and the resistant strain (RT); Figure 2: B comparison of the migration abilities of the parental A549 strain (WT) and the resistant strain (RT); Figure 3: C comparison of the migration abilities of the parental NCI-H1299 strain (WT) and the resistant strain (RT). **** indicates P < 0.0001; *** indicates P < 0.001; ** indicates P < 0.01; * indicates P < 0.05; ns indicates P > 0.05.

[0202] The results showed that compared with the untreated parental cell line, both resistant strains A549 and H1299 showed stronger migration ability.

[0203] 3.1.5 Radioresistance affects tumor cell apoptosis

[0204] Apoptosis is a cell death process that is independently and orderly controlled by multiple genes. To further explore the apoptotic ability of radioresistant strains and parental cell lines, this study used A549 and H1299 cell lines and used flow cytometry to detect the effect of radioresistance on the apoptotic ability of different cell lines. Figure 6 , Figure 6 In Figure 1, A shows the apoptotic capacity of the parental strain (WT) of A549 and NCI-H1299 compared with the resistance cell line (RT); B shows the apoptotic capacity of the parental strain (WT) of A549 compared with the resistance cell line (RT); C shows the apoptotic capacity of the parental strain (WT) of NCI-H1299 compared with the resistance cell line (RT). **** indicates P < 0.0001; *** indicates P < 0.001; ** indicates P < 0.01; * indicates P < 0.05; ns indicates P > 0.05.

[0205] The results showed that the ability of radiation-resistant cells to resist apoptosis gradually induced by the cumulative radiation dose became weaker, while the parental lung cancer cell line showed a stronger apoptosis ability. This also verified that radiation-induced tumor cells escaped apoptosis. These cells failed to repair DNA damage after cumulative radiation exposure, and showed a stronger survival ability in order to protect themselves from further radiation damage.

[0206] 3.1.6 Radioresistance affects the expression of DNAJC19 and PD-L1 in tumor cells

[0207] In this study, 1×10 6 The cells were cultured in a flask containing 10% fetal bovine serum and cell proteins were collected after 24 hours. The expression of DNAJC19 and PD-L1 proteins in the collected tumor cells was analyzed by Western blotting. The results were referenced to Figure 7 . Figure 7 In Figure 1, A shows the expression of DNAJC19 and PD-L1 proteins in the parental (WT) and resistant (RT) A549 and NCI-H1299 lines; B shows the expression of DNAJC19 and PD-L1 proteins in the parental (WT) and resistant (RT) A549 lines; and C shows the expression of proteins in the parental (WT) and resistant (RT) NCI-H1299 lines. **** indicates P < 0.0001; *** indicates P < 0.001; ** indicates P < 0.01; * indicates P < 0.05; ns indicates P > 0.05.

[0208] The results showed that the expression of DNAJC19 and PD-L1 in radioresistant tumor cells in the A549 cell line was higher than that in the parental cell line. These results were also confirmed in NCI-H1299 cells.

[0209] 3.2PD-L1 is highly expressed in radioresistant cell lines

[0210] PD-L1, as a ligand protein for PD1, is widely present on the surface of immune cells and certain non-immune cells. PD-L1 expression increases on the surface of tumor cells, inflammatory cells, and certain immune cells. Radiotherapy can induce the expression of PD-L1 in the tumor microenvironment and bind to the PD1 protein ligand, prompting tumor cells to escape immune function, thereby reducing the sensitivity of anti-tumor immune response. In order to find the relationship between PD-L1 and radiotherapy resistance in NSCLC tumor cells, this study first selected the A549 cell line, and then irradiated the tumor-sensitive cell lines and resistant cell lines with the same dose and divided them into groups: A549WT, A549WT+R, A549RT, and A549RT+R. Immunofluorescence was used to observe the expression of PD-L1 in sensitive cells and resistant cell lines, and at the same time, the expression of PD-L1 in radiation-resistant cells after re-irradiation was observed. The experimental results are as follows Figure 8 shown. Figure 8 In the figure, the two cell lines involved are grouped as A549WT, A549RT, A549WT+R, A549RT+R; H1299WT, H1299RT, H1299WT+R, H1299RT+R. A is a graphic representation of the PD-L1 expression of the A549 parental cell line (WT) and the resistant strain (RT); B is a graphic representation of the PD-L1 expression of the NCI-H1299 parental cell line (WT) and the resistant strain (RT).

[0211] The results showed that PD-L1 expression was higher in radiation-resistant cells than in unirradiated tumor cells. After a single dose (6 Gy) of irradiation, PD-L1 expression was significantly reduced in sensitive cells, while no significant decrease was observed in resistant cells after a second dose of radiation. Similar results were observed using immunofluorescence in the H1299 cell line.

[0212] 3.3 Bioinformatics Analysis

[0213] 3.3.1 Bioinformatics analysis revealed that DNAJC19 and PD-L1 are highly expressed in NSCLC patients

[0214] This study used the GEO database (https: / / portal.gdc.cancer.gov) to use paired sample point-line plots to intuitively reflect the expression levels of DNAJC19 and PD-L1 proteins in cancer tissues and adjacent normal tissues in various lung cancer tissue types. The data came from the GEO-LUSC (lung squamous cell carcinoma) and GEO-LUAD (lung adenocarcinoma) projects. The results were as follows: Figure 9 . Figure 9 In the figure, A is the expression of DNAJC19 in tumor tissue (Tumor) and surrounding normal tissue (Normal) in lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD); B is the expression of PD-L1 in tumor tissue (Tumor) and surrounding normal tissue (Normal) in lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD), **** indicates: P < 0.0001; *** indicates: P < 0.001; ** indicates: P < 0.01; * indicates: P < 0.05; ns indicates: P > 0.05.

[0215] The results showed that in patients with lung squamous cell carcinoma (LUSC), DNAJC19 expression was higher in tumor tissue than in normal tissue, while in patients with lung adenocarcinoma (LUAD), no significant difference in DNAJC19 expression was detected between tumor tissue and surrounding normal tissue (Figure A). Statistical analysis of PD-L1 gene differences revealed that PD-L1 expression was higher in cancer tissue than in surrounding normal tissue in both LUAD and LUSC. Combined with the above analysis, it was generally suggested that DNAJC19 and PD-L1 are highly expressed in NSCLC tumor tissue.

[0216] 3.3.2 Bioinformatics analysis found that the expression of DNAJC19 and PD-L1 is associated with the prognosis of NSCLC patients. Based on the above bioinformatics analysis results, to further explore whether DNAJC19 and PD-L1 are associated with the prognosis of NSCLC patients, this plan first analyzed the NSCLC survival analysis data under high and low expression of DNAJC19 and PD-L1 through the GEO database (https: / / portal.gdc.cancer.gov), and then verified it through the TCGA database. The results are as follows Figure 10 .

[0217] Figure 10 In the figure, A is from the GEO database, the Kaplan-Meier survival analysis results showed that the median survival time of the DNAJC19 high expression group was significantly higher than that of the low expression group (p=0.0067); B is from the TCGA database: the Kaplan-Meier survival analysis results showed that the median survival time of the DNAJC19 high expression group was significantly higher than that of the low expression group (p=0.011); C is from the GEO database, the Kaplan-Meier survival analysis results showed that the median survival time of the PD-L1 high expression group was significantly higher than that of the low expression group (p=0.0066); D is from the TCGA database: the Kaplan-Meier survival analysis results showed that there was no significant difference in median survival time between the PD-L1 high and low expression groups (p=0.33). It can be seen that Figure 10 Analysis showed that DNAJC19 and PD-L1 were associated with the survival prognosis of NSCLC patients.

[0218] 3.4 siRNA-DNAJC19 combined with PD-L1 inhibitors regulates NSCLC tumor cell characteristics

[0219] 3.4.1 Detection of DNAJC19 transient transfection efficiency

[0220] Both clinical data and bioinformatics analysis show that DNAJC19 is highly expressed in NSCLC tumor tissues compared with normal tissues. Previous cell functional experiments have verified that DNAJC19 is expressed in both lung cancer resistant strains and parental cell lines, but DNAJC19 is highly expressed in resistant strains compared with parental cell lines. The above experiments were combined to silence DNAJC19-siRNA to construct a DNAJC19 downregulation model to further understand the biological function of DNAJC19 in NSCLC cells. In A549 cell lines and H1299 cell lines, we transfected three siRNA downregulation targets siDNAJC19a, siDNAJC19b and siDNAJC19c and their corresponding negative controls si-NC. After 48 hours, we used Western blotting to test their transfection efficacy, and the results showed that Figure 11: siDNAJC19a and siDNAJC19c targets interfere with the expression of DNAJC19 better than siDNAJC19b.

[0221] 3.4.2 Silencing DNAJC19 and adding PD-L1 inhibitors can significantly reduce the ability of tumor cells to clone and proliferate. This study has previously verified that radioresistance can enhance the cloning, proliferation, and migration of NSCLC cells. To further study the effect of DNAJC19 protein on the characteristics of radioresistant NSCLC cells, the present invention continued to select two cell lines, A549 and NCI-H1299, and divided the tumor cells into five groups: control group, radiotherapy group, radiotherapy combined with siRNA-DNAJC19, radiotherapy combined with anti-PDL1 (PD-L1 inhibitor), and radiotherapy and siRNA-DNAJC19 combined with anti-PDL1. The PD-L1 inhibitor was selected as atezolizumab injection for human use. Through CCK8 experiment, the results are as follows Figure 12 . Figure 12 In the figure, A is the cell viability between the A549 parental cell line (WT) and the radiation-resistant cell line (RT) under radiation; B is the cell viability between the NCI-H1299 parental cell line (WT) and the radiation-resistant cell line (RT) under radiation, **** indicates: P < 0.0001; *** indicates: P < 0.001; ** indicates: P < 0.01; * indicates: P < 0.05; ns indicates: P > 0.05.

[0222] Depend on Figure 12 It can be seen that both siRNA-DNAJC19 and the addition of PD-L1 inhibitors can inhibit tumor cell viability. When siRNA-DNAJC19 and anti-PDL1 are used in combination, the ability to inhibit tumor cell proliferation is more significant.

[0223] The inventors continued to obtain the same results through plate cloning experiments. Figure 13 . Figure 13In the figure, A is the results of A549 resistant and sensitive cell clones, which are divided into 10 groups: A549WT, A549WT+R, A549WT+R+siRNA-DNAJC19, A549WT+R+anti-PDL1, A549WT+R combined with siRNA-DNAJC19 and anti-PDL1; A549RT, A549RT+R, A549RT+R+siRNA-DNAJC19, A549RT+anti-PDL1, A549RT+R combined with siRNA-DNAJC19 and anti-PDL1; B is the results of NCI-H1299 resistant and sensitive cell clones, which are divided into 10 groups: H129 9WT, H1299WT+R, H1299WT+R+siRNA-DNAJC19, H1299WT+anti-PDL1, H1299WT+R combined with siRNA-DNAJC19 and anti-PDL1; H1299RT, H1299RT+R, H1299RT+R+siRNA-DNAJC19, H1299RT+anti-PDL1, H1299RT+R combined with siRNA-DNAJC19 and anti-PDL1; C is a quantitative comparison of A549 resistant and sensitive cell lines under radiation; D is a quantitative comparison of H1299 resistant and sensitive cell lines under radiation. **** indicates: P < 0.0001; *** indicates: P < 0.001; ** indicates: P < 0.01; * indicates: P < 0.05; ns indicates: P > 0.05.

[0224] Depend on Figure 13 It can be seen that after the A549 / H1299 parental cell line and the resistant cell line were irradiated with 6Gy, the proliferation ability of the A549 / H1299 sensitive cell line decreased, while the proliferation ability of the A549 / H1299 resistant cell line was almost unaffected and remained strong. Subsequently, on the basis of radiotherapy, silencing the gene DNAJC19 or adding PD-L1 inhibitors respectively showed inhibitory effects and weakened proliferation ability, but the difference between the two was not significant. However, when siRNA-DNAJC19 and anti-PD-L1 were combined to inhibit each resistant cell line, they all showed a strong inhibitory effect, resulting in a sharp decrease in proliferation ability and increased radiosensitivity.

[0225] 3.4.3 Silencing DNAJC19 and inhibiting PD-L1 can reduce the migration ability of tumor cells. Through preliminary experiments, it was found that radiotherapy can affect the migration ability of tumor cells, and migration ability is one of the hallmark characteristics of NSCLC cells. To further verify the regulation of transiently transfected siRNA-DNAJC19 and the addition of PD-L1 inhibitors on the ability of NSCLC to resist cell migration and invasion, the present invention first selected the A549 cell line and divided the tumor cells into ten groups: A549WT / RT, A549WT / RT+R, A549WT / RT+R+siRNA-DNAJC19, A549WT / RT+R+anti-PDL1, and A549WT / RT+R combined with siRNA-DNAJC19 and anti-PDL1. The migration experiment results are shown in the figure. Figure 14 .

[0226] Figure 14 In the figure, A is the migration results of A549 parental cell line and radiation-resistant cell line under radiation, which are divided into 10 groups: A549WT, A549WT+R, A549WT+R+siRNA-DNAJC19, A549WT+R+anti-PDL1, A549WT+R combined with siRNA-DNAJC19 and anti-PDL1; A549RT, A549RT+R, A549RT+R+siRNA-DNAJC19, A549RT+anti-PDL1, A549RT+R combined with siRNA-DNAJC19 and anti-PDL1; B is the result of NCI-H1299 resistant and sensitive cell clones under radiation, divided into 10 groups: H1299WT, H1299WT+R, H1299WT+R+siRNA-DNAJC19, H1299WT+anti-PDL1, H1299WT+R combined with siRNA-DNAJC19

[0227] and anti-PDL1; H1299RT, H1299RT+R, H1299RT+R+siRNA-DNAJC19, H1299RT+anti-PDL1, H1299RT+R combined with siRNA-DNAJC19 and anti-PDL1; C is a grouped quantitative graph comparing the number of A549 resistant and sensitive cells migrating under radiation; D is a grouped quantitative graph comparing the number of H1299 resistant and sensitive cells migrating under radiation.

[0228] Depend on Figure 14It was shown that both siRNA-DNAJC19 and PD-L1 inhibition reduced tumor cell migration, with the siRNA-DNAJC19 combined with anti-PDL1 showing the best inhibition. To validate these results, the above experiment was repeated using NCI-H1299 cells, and the results were consistent with those obtained with the A549 cell line.

[0229] 3.4.4 Silencing DNAJC19 and inhibiting PD-L1 can promote tumor cell apoptosis

[0230] The present invention explores the regulation of siRNA-DNAJC19 and PD-L1 inhibitors on the apoptosis ability of NSCLC cells. Flow cytometry was used to detect 10 groups of A549 and H1299, including the control group, 6G re-irradiation group, siRNA-DNAJC19, anti-PDL1, and siRNA-DNAJC19 combined with anti-PDL1. The test results are as follows: Figure 15 .

[0231] Figure 15 Middle: A shows the apoptosis results of A549 parental cell line and radiation-resistant strain under radiation, which are divided into 10 groups: A549WT, A549WT+R, A549WT+R+siRNA-DNAJC19, A549WT+R+anti-PDL1, A549WT+R+siRNA-DNAJC19+anti-PDL1; A549RT, A549RT+R, A549RT+R+siRNA-DNAJC19, A549RT+anti-PDL1, A549RT+R+siRNA-DNAJC19+anti-PDL1; B shows the apoptosis results of NCI-H1299 parental cell line and radiation-resistant strain under radiation, which are divided into 10 groups:

[0232] H1299WT, H1299WT+R, H1299WT+R+siRNA-DNAJC19, H1299WT+anti-PDL1, H1299WT+R+siRNA-DNAJC19+anti-PDL1; H1299RT, H1299RT+R, H1299RT+R+siRNA-DNAJC19, H1299RT+anti-PDL1, H1299RT+R+siRNA-DNAJC19+anti-PDL1; C is a quantitative comparison of the apoptosis ratio of resistant and sensitive A549 cells under radiation; D is a quantitative comparison of the apoptosis ratio of resistant and sensitive H1299 cells under radiation. **** indicates: P < 0.0001; *** indicates: P < 0.001; ** indicates: P < 0.01; * indicates: P < 0.05; ns indicates: P > 0.05.

[0233] The experimental results showed that in NSCLC tumor cells, whether untreated parental cell lines or resistant cell lines that have undergone multiple radiotherapy tolerance, when the gene DNAJC19 was silenced or PDL1 was inhibited, the apoptosis ability of both cell lines showed an upward trend, and the siRNA-DNAJC19 combined with anti-PDL1 group had the strongest ability to promote tumor cell apoptosis.

[0234] 3.4.5 Silencing DNAJC19 can reduce DNAJC19 expression, but PDL1 expression is not significant after adding PD-L1 inhibitors. Previous studies have shown through radiotherapy that radioresistance promotes the viability, proliferation, and migration of tumor cells in NSCLC patients, while also promoting tumor cell apoptosis. Western blot experiments have shown that DNAJC19 is highly expressed in NSCLC radioresistant cell lines compared to the parental cell lines, and PD-L1 also has the same promoting effect on NSCLC-resistant cells. Therefore, the inventors of this approach envisioned whether tumor cells could reverse radioresistance after dual inhibition by knocking down the target proteins DNAJC19 and PD-L1, thereby promoting radiosensitization.

[0235] Therefore, the inventors used Western blot experiments to analyze the expression of DNAJC19 protein in A549 and H1299 cells after gene silencing. At the same time, the same tumor cells after PD-L1 expression inhibition were also verified. The results are as follows Figure 16 .

[0236] The experimental results showed that the expression of DNAJC19 in NSCLC tumor cells was reduced after gene silencing treatment, and the expression of PD-L1 was also downregulated after the addition of immunosuppressive doses. After dual inhibition treatment, A549 and H1299 cells both expressed low levels of DNACJ19 and PD-L1.

[0237] The above experiments show that DNAJC19 and PD-L1 are key molecules that induce NSCLC metastasis. Dual inhibition of siRNA-DNAJC9 combined with anti-PD-L1 can significantly reduce the proliferation, migration and clone formation abilities of tumor cells, thereby promoting tumor cell apoptosis and providing an opportunity for reversal of radiosensitization.

[0238] Combined with the above experiments, it can be seen that DNAJC19 and PD-L1 can be used as markers in the preparation of drugs for the prevention and / or treatment of tumors, wherein the drugs are drugs for combating drug resistance to platinum-based chemotherapy agents for lung cancer. Platinum-based chemotherapy agents include cisplatin, carboplatin, and other platinum-based chemotherapy agents.

[0239] Preferably, the drug includes a DNAJC19 inhibitor and a PD-L1 inhibitor.

[0240] Preferably, the DNAJC19 inhibitor and the PD-L1 inhibitor are administered simultaneously or separately.

[0241] Preferably, the drug further comprises a pharmaceutically acceptable carrier.

[0242] Preferably, the drug further comprises at least one pharmaceutically acceptable additive or excipient.

[0243] Furthermore, the DNAJC19 inhibitor and PD-L1 inhibitor are used to prepare drugs for inhibiting the proliferation, migration or chemotherapy resistance of cancer cells.

[0244] According to the above content, a platinum-based anti-resistance drug for lung cancer for preventing and / or treating tumors can be prepared, comprising a DNAJC19 inhibitor and a PD-L1 inhibitor.

[0245] Preferably, the PD-L1 inhibitor is used to inhibit the expression of PD-L1 in vivo in the prevention and / or treatment of platinum-resistant lung cancer; the DNAJC19 inhibitor is used to inhibit the expression of DNAJC19 in vivo in the prevention and / or treatment of platinum-resistant lung cancer.

[0246] In addition, DNAJC19 and PD-L1 can be used as markers in the preparation of drugs that inhibit the proliferation, migration or chemotherapy resistance of cancer cells.

[0247] In addition, DNAJC19 and PD-L1 can also be used as markers in the preparation of drugs that enhance the sensitivity of anti-lung cancer drugs.

[0248] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention shall fall within the scope of protection of the present invention.

Claims

1. Use of a DNAJC19 inhibitor in combination with a PD-L1 inhibitor in the preparation of a drug for enhancing the sensitivity of anti-lung cancer drugs, characterized in that: The drug is an anti-drug resistance drug for lung cancer platinum-based chemotherapy agents.

2. The use according to claim 1, characterized in that: The DNAJC19 inhibitor and the PD-L1 inhibitor are administered simultaneously or separately.

3. The use according to claim 1, characterized in that: The drug further includes a pharmaceutically acceptable carrier.

4. The use according to claim 1, wherein: The medicament further comprises at least one pharmaceutically acceptable additive.

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