阅读说明：本技术 Tak1抑制剂抑制pdl1的检测方法及其在制备抗pdl1药物中的应用 (Detection method for PDL1 inhibited by TAK1 inhibitor and application of detection method in preparation of anti-PDL 1 drugs ) 是由 范义辉 毛仁芳 马盼盼 金鑫鑫 于 2020-12-14 设计创作，主要内容包括：本发明提供一种TAK1抑制剂抑制PDL1的检测方法,包括如下步骤：(1)探究肿瘤细胞中PDL1分子的表达调控机制：利用小分子抑制剂处理肿瘤细胞,分别通过基因水平和蛋白水平来表征PDL1的表达；(2)检测肿瘤细胞中ERK的磷酸化水平；(3)验证TAK1抑制剂对肿瘤细胞PDL1的抑制作用：用TAK1抑制剂处理肿瘤细胞后,用免疫荧光技术检测细胞中PDL1的蛋白表达水平。本发明采用的TAK1抑制剂是一种具有良好开发前景的潜在的抗肿瘤药物,在基因水平明显下调PDL1的表达,有望为临床上治疗乳腺癌提供可行性和理论依据。(The invention provides a detection method for inhibiting PDL1 by a TAK1 inhibitor, which comprises the following steps: (1) the expression regulation mechanism of PDL1 molecule in tumor cells is explored: treating tumor cells with a small molecule inhibitor, characterizing the expression of PDL1 by gene level and protein level, respectively; (2) detecting the level of phosphorylation of ERK in the tumor cell; (3) the inhibition effect of the TAK1 inhibitor on tumor cell PDL1 is verified: after treating tumor cells with the TAK1 inhibitor, the protein expression level of PDL1 in the cells was detected by immunofluorescence technique. The TAK1 inhibitor adopted by the invention is a potential anti-tumor drug with good development prospect, the expression of PDL1 is obviously reduced at the gene level, and the feasibility and theoretical basis are hopefully provided for clinically treating breast cancer.)

1. A detection method for detecting PDL1 inhibited by TAK1 inhibitor, which is characterized by comprising the following steps:

(1) the expression regulation mechanism of PDL1 molecule in tumor cells is explored: treating tumor cells with a small molecule inhibitor, characterizing the expression of PDL1 by gene level and protein level, respectively;

(2) detecting the level of phosphorylation of ERK in the tumor cell;

(3) the inhibition effect of the TAK1 inhibitor on tumor cell PDL1 is verified: after treating tumor cells with the TAK1 inhibitor, the protein expression level of PDL1 in the cells was detected by immunofluorescence technique.

2. The method for detecting PDL1 inhibition by TAK1 inhibitor according to claim 1, wherein in step (1), the small molecule inhibitor is TAK1 inhibitor 5Z 7.

3. The detection method for detecting PDL1 inhibition by TAK1 inhibitor according to claim 1, wherein the tumor cells are breast cancer cell lines SUM159 and MDA-MB-231.

4. The method for detecting PDL1 inhibited by TAK1 inhibitor according to claim 1, wherein the small molecule inhibitor in step (1) is TAK1 inhibitor 5Z7, and the treatment concentration is 0, 1 and 3 μ M.

5. The method for detecting PDL1 inhibition by TAK1 inhibitor according to claim 1, wherein the expression of PDL1 protein level and gene level is detected by Western Blot method and real-time quantitative PCR method in step (1).

6. The method for detecting PDL1 inhibition by TAK1 inhibitor according to claim 1 or 5, wherein the step (1) comprises the following steps:

(1-1) resuscitating the cells: taking out the freezing tube from-80 deg.C, directly soaking in warm water of 37 deg.C, and shaking to melt it as soon as possible; taking out the frozen tube from the water bath at 37 ℃, adding 1mL of complete culture medium, and uniformly mixing; centrifuging at 1000rpm for 3min, and discarding supernatant; adding 10% FBS-containing DMEM medium to resuspend the cells, uniformly inoculating in a 60mm culture dish, and 5% CO₂Standing and culturing in an incubator at 37 ℃; replacing the culture solution once the next day, and continuing culturing; when the cell density reaches 90%, the cells are digested by pancreatin, the number of the paved holes is determined according to subsequent experiments, and the cells are continuously cultured;

(1-2) treatment of TAK1 inhibitor 5Z 7: the concentration of the selected treatment is 0, 1 and 3 mu M in sequence, and the treatment time is 24 h;

(1-3) Western blotting

(1-3-1) preparation of protein sample: preparing a lysis buffer solution and placing the lysis buffer solution in an ice bath for later use; washing cells by precooling PBS, transferring the cells to a sterilized centrifuge tube, centrifuging and removing a supernatant; resuspending the cells with a lysate containing PMSF, and lysing for 30min on ice bath; centrifuging at 4 deg.C for 15min, and completely sucking supernatant; after mixing the protein with 5 × Loading buffer, the ratio of 4: 1, boiling in 95 deg.C metal bath for 5min, and storing at-20 deg.C;

(1-3-2) electrophoresis glue running and film transferring color development: boiling the protein sample again for 5min at 105 ℃; preparing 10% separation gel according to the formula of the protein gel, preparing 5% concentrated gel on the upper layer, and adding 10 μ L protein sample into each hole; adjusting the voltage of the electrophoresis apparatus to 80V, and adjusting the voltage of the electrophoresis apparatus to 120V again when the bromophenol blue indicator reaches the lower layer separation gel; according to a standard protein marker, a sample area to be inspected is cut off; cutting PVDF membrane into size same as cut protein gel, and balancing in membrane buffer for 3 min;

the stacking sequence is as follows: performing wet rotation for 1h at 300mA for a power supply anode, filter paper, a PVDF (polyvinylidene fluoride) membrane, gel, filter paper and a power supply cathode; putting the PVDF membrane in 5% skimmed milk powder, and sealing for 2 h; rinsing the PVDF membrane for 3min by TBST, adding milk or primary antibody diluted by primary antibody diluent, and incubating overnight at 4 ℃; rinsing with TBST for 5min x 3 the next day, adding appropriate amount of secondary antibody, and incubating at room temperature for 2 h; finally, TBST rinsing is carried out for 10min x 3, and ECL reagent is used for developing color;

(1-4) real-time quantitative PCR

(1-4-1) extraction of total RNA of cells: total RNA was extracted from the above treated cells by Trizol method as follows: discarding culture solution of cells, washing once by using PBS, adding pancreatin to digest cells and collecting in an EP tube without RNA enzyme; adding 1mL of RNA isolator, and standing at room temperature for 5 min; adding 200 μ L chloroform, shaking vigorously for 15s, and standing at room temperature for 3 min; centrifuging at 12000r at 4 deg.C for 15min, separating into three layers, carefully sucking the upper layer into a new EP tube without RNase, adding 0.5ml isopropanol, turning upside down, mixing, and standing at-20 deg.C for 2 hr; centrifuging at 12000r at 4 deg.C for 10min to obtain white precipitate at the bottom of the tube; discarding the supernatant, adding 1mL of precooled 75% ethanol, 7500r, centrifuging at 4 ℃ for 5min, and discarding the supernatant as much as possible; drying and precipitating in a fume hood for 10-30min, adding 30 μ L DEPC water to dissolve RNA, heating in 65 deg.C water bath for 5min, measuring concentration, and storing at-80 deg.C;

(1-4-2) cDNA Synthesis and qPCR detection: the RNA was inverted after the above measurement to synthesize cDNA as follows:

removing genome: preparing the following mixed solution in a centrifugal tube of RNase-free, wherein the template RNA is 1 mug; 4 XgDNA wiper Mix, 4 μ L; RNase-free ddH₂Supplementing O to 16 μ L, gently blowing and beating with a pipette, and mixing uniformly at 42 deg.C for 2 min;

preparing a reverse transcription reaction system: directly adding 4 mu L of 5 XHiScript II qRT SuperMix II into the reaction tube in the first step;

carrying out reverse transcription reaction: the product was diluted 8-fold with water at 50 ℃ for 15min and 85 ℃ for 5s, stored at-20 ℃ and then tested for gene expression levels of PDL1 and PDL 2.

7. The method for detecting PDL1 inhibition by TAK1 inhibitor according to claim 6, wherein the step (1-4) is performed by gene transcription level detection, RT-PCR primers PDL1 and PDL2 are designed and verified in SUM159 and MDA-MB-231 cells, respectively; the primers are respectively as follows:

PDL1-RT-F：GTAGCACTGACATTCATCTTC；

PDL1-RT-R：TTCCTTCCTCTTGTCACGCTC；

PDL2-RT-F：CATAGCCACAGTGATAGCCCT；

PDL2-RT-R： GGCTCCCAAGACCACAGGTTC。

8. the method for detecting PDL1 inhibition by TAK1 inhibitor according to claim 1 or 5, wherein the immunofluorescence assay of step (3) is as follows:

growing tumor cells on a circular cover glass placed in a 24-hole cell culture plate, wherein the inoculation density reaches 60%, and the cells are treated by adding medicine for 24 hours; cells were washed 3 times with 500 μ L PBS per well for 5min each time; then adding 200ul of 4% paraformaldehyde into each hole, and fixing for 15min at room temperature; removing paraformaldehyde, washing cells with 500 μ L PBS for 5min each time for 3 times; adding 500 mu L of 1% BSA into each hole, and blocking for 2h at room temperature; removing the blocking solution, adding 200 μ L primary antibody per well, and standing overnight at 4 deg.C; remove the antibody, wash the cells 3 times with 500 μ Ι _ of PBS per well, 5min each time; adding 200 mu L of fluorescent secondary antibody into each hole, and incubating for 2h at room temperature; remove the secondary antibody, wash the cells 3 times with 500 PBS per well for 5min each time; add Hochest into each hole, room temperature 10min, 500 u L PBS wash cells 3 times, each time 5 min; dropping a small drop of anti-fading agent on the glass slide, taking the glass cover out of the hole, covering the glass cover on the anti-fading agent in a downward mode, and enabling the glass cover to lightly contact the glass slide; the slide was left to air dry in the dark for 5min, at which time it was observed under a fluorescent microscope.

9. Application of a TAK1 inhibitor in preparing anti-PDL 1 medicines is provided.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a detection method for inhibiting PDL1 by a TAK1 inhibitor and application of the detection method in preparation of a PDL 1-resistant medicine.

Background

Programmed cell death receptor 1 (PD 1) is an important immunosuppressive molecule widely expressed on the surface of T cells, is an immunoglobulin superfamily, and is a transmembrane protein containing 268 amino acid residues. PD-1 primarily inhibits activated T cell activity in chronic inflammation, infection or cancer, and has two related ligands, Programmed cell death-ligand 1 (PD-L1) and Programmed cell death-ligand 2 (Programmed cell death 1 ligand 2, PD-L2). Generally, in a tumor microenvironment, tumor cells can highly express PD-L1 or PD-L2, and the anti-tumor activity of T cells is remarkably inhibited, so that the aim of immune escape is fulfilled. Among them, PD-1, also called as surface antigen differentiation 274 (CD 274) or B7 homolog (B7 homolog 1, B7-H1), is encoded by the CD274 gene on chromosome 9 under the control of interferon regulatory factor 1 (IRF 1) and signal transducer activated transcription 1 (STAT 1) response elements. It was found that PD-L1 is a 40kDa transmembrane protein, upregulated in a variety of malignancies, and is commonly expressed on the surface of tumor cells. For example, PD-L1 protein is highly expressed in tissues such as melanoma, renal cell carcinoma, non-small cell lung cancer, breast cancer, and prostate cancer. Breast Cancer (BC) has been one of the hot spots in the field of tumor research, with mortality rates leading to the median malignancy in women. Despite the ever-increasing incidence of breast cancer over the last several decades, its mortality rate has continued to decline due to the rapid development of diagnostic and therapeutic techniques. The clinical treatment is generally carried out by adopting methods such as operation, chemotherapy, targeted therapy and the like, but the clinical treatment effect is not ideal all the time due to the wide existence of the drug resistance problem of the tumor. For example, in triple negative breast cancer with higher malignancy, the treatment means is relatively deficient, the PD1 monoclonal antibody Pembrolizumab (MK-3475) is a humanized IgG4/k type anti-PD 1 monoclonal antibody with high selectivity, and in a KEYNOTE-12Ib phase clinical test aiming at recurrent/metastatic triple negative breast cancer, the Objective Remission Rate (ORR) is 18.5%, but adverse reactions including arthralgia, hypodynamia, nausea, myalgia and the like still occur.

Transforming growth factor Kinase 1 (TGF beta-Activated Kinase 1, TAK 1) is one of the mitogen-Activated protein Kinase kinase Kinase kinase (MAP 3K) family members. In 1995, Yamaguchi et al were first confirmed in yeast in vivo genetic selection experiments for MAPK signaling pathway and are important intracellular signaling molecules. The TAK1 inhibitor 5Z-7-oxozeaenol (5Z 7), an irreversible ATP competitive inhibitor, is mainly purified from fungal secretion. The research shows that the small molecule 5Z-7 can inhibit TAK1 or gene silencing to induce the cell death of Diffuse Large B Cell Lymphoma (DLBCL) in a large amount. Mechanistically, inhibition of TAK1 significantly reduced nuclear factor κ B (NF- κ B) activity, and inhibition of TAK1 was found to promote death of DLBCL cells by inhibiting chronic NF- κ B signaling.

Breast cancer is a heterogeneous disease, and the pathogenesis of breast cancer cannot be fully elucidated by the currently established risk factors. With the application of molecular biological methods such as signal pathway, apoptosis and the like in tumor research, breast cancer molecular targets and targeted therapy gradually become trends and hot spots for anti-breast cancer research. In view of the limitations of traditional therapeutic approaches, immunotherapy, with its high specificity and memory, has the potential to improve the survival and prognosis of tumor patients. Immune checkpoints are the main reason for hindering the body's immune cells from resisting tumors and are the chief culprit in the development of immune escape. At present, aiming at immune check points, a method of adopting a monoclonal antibody inhibitor is mainly adopted to improve the anti-tumor effect. PDL1 and PDL2 are core targets for current tumor immunotherapy, in particular immune checkpoint blockade therapy. Inhibiting PD-L1 and PD-L2 function, activating anti-tumor immunity of T cell, and killing tumor cell. The anti-tumor effect of the TAK1 inhibitor is widely developed in various tumor treatments, and the invention aims to research the application of the TAK1 inhibitor in the preparation of anti-PD-L1 drugs and provide more theoretical basis for the clinical practice of the TAK1 inhibitor.

Disclosure of Invention

The invention aims to provide a detection method for inhibiting PDL1 by a TAK1 inhibitor and application of the detection method in preparation of anti-PDL 1 drugs, and provide more theoretical bases for clinical practice of the TAK1 inhibitor.

In order to solve the above technical problem, an embodiment of the present invention provides a detection method for inhibiting PDL1 by a TAK1 inhibitor, including the following steps:

(1) the expression regulation mechanism of PDL1 molecule in tumor cells is explored: treating tumor cells with a small molecule inhibitor, characterizing the expression of PDL1 by gene level and protein level, respectively;

(2) detecting the level of phosphorylation of ERK in the tumor cell;

(3) the inhibition effect of the TAK1 inhibitor on tumor cell PDL1 is verified: after treating tumor cells with the TAK1 inhibitor, the protein expression level of PDL1 in the cells was detected by immunofluorescence technique.

Wherein in the step (1), the small molecule inhibitor is TAK1 inhibitor 5Z7, and the treatment concentration is 0, 1 and 3 mu M.

Wherein, the tumor cells are breast cancer cell lines SUM159 and MDA-MB-231.

Wherein, the Western Blot method and the real-time quantitative PCR method are adopted in the step (1) to detect the protein level and the gene level expression of PDL 1.

Wherein, the specific steps of the step (1) comprise:

(1-1) resuscitating the cells: taking out the freezing tube from-80 deg.C, directly soaking in warm water of 37 deg.C, shaking to melt as soon as possible (instant slow freezing); taking out the frozen tube from the water bath at 37 ℃, adding 1mL of complete culture medium, and uniformly mixing; centrifuging at 1000rpm for 3min, and discarding supernatant; adding 10% FBS-containing DMEM medium to resuspend the cells, uniformly inoculating in a 60mm culture dish, and 5% CO₂Standing and culturing in an incubator at 37 ℃; replacing the culture solution once the next day, and continuing culturing; when the cell density reaches 90%, the cells are digested by pancreatin, the number of the paved holes (6-hole plate) is determined according to the subsequent experiment, and the cells are continuously cultured;

(1-2) treatment of TAK1 inhibitor 5Z 7: the concentration of the selected treatment is 0, 1 and 3 mu M in sequence, and the treatment time is 24 h;

(1-3) Western blotting

(1-3-1) preparation of protein sample: preparing Lysis buffer (containing 10 μ L PMSF and 990 μ L lysine buffer) and placing in ice bath for later use; washing cells by precooling PBS, transferring the cells to a sterilized centrifuge tube, centrifuging and removing a supernatant; resuspending the cells in a lysate containing PMSF, and lysing for 30min on ice bath (shaker); centrifuging at 4 deg.C for 15min, and completely sucking supernatant (protein); after mixing the protein with 5 × Loading buffer, the ratio of 4: 1, boiling in 95 deg.C metal bath for 5min, and storing at-20 deg.C;

(1-3-2) electrophoresis glue running and film transferring color development: boiling the protein sample again for 5min at 105 ℃; preparing 10% separation gel according to the formula of the protein gel, preparing 5% concentrated gel on the upper layer, and adding 10 μ L protein sample into each hole; adjusting the voltage of the electrophoresis apparatus to 80V, and adjusting the voltage of the electrophoresis apparatus to 120V again when the bromophenol blue indicator reaches the lower layer separation gel; according to a standard protein marker, a sample area to be inspected is cut off; cutting PVDF membrane into size same as cut protein gel, and balancing in membrane buffer for 3 min;

the stacking sequence is as follows: performing wet rotation for 1h at 300mA for a power supply anode, filter paper, a PVDF (polyvinylidene fluoride) membrane, gel, filter paper and a power supply cathode; putting the PVDF membrane in 5% skimmed milk powder, and sealing for 2 h; rinsing PVDF membrane with TBST for 3min, adding milk or primary antibody (1: 1000) diluted by primary antibody diluent, and incubating overnight at 4 deg.C; rinsing with TBST for 5min x 3 the next day, adding appropriate amount of secondary antibody (1: 2000), and incubating at room temperature for 2 h; finally, TBST rinsing is carried out for 10min x 3, and ECL reagent is used for developing color;

(1-4) real-time quantitative PCR

(1-4-1) extraction of total RNA of cells: total RNA was extracted from the above treated cells by Trizol method as follows: discarding culture solution of cells, washing once by using PBS, adding pancreatin to digest cells and collecting in an EP tube without RNA enzyme; 1mL of RNA isolater (Novozam R401-01) was added and left at room temperature for 5 min; adding 200 μ L chloroform, shaking vigorously for 15s, and standing at room temperature for 3 min; centrifuging at 12000r at 4 deg.C for 15min, separating into three layers, carefully sucking the upper layer into a new EP tube without RNase, adding 0.5ml isopropanol, mixing by turning upside down, and standing at-20 deg.C for 2h (increasing precipitation); centrifuging at 12000r at 4 deg.C for 10min to obtain white precipitate at the bottom of the tube; discarding the supernatant, adding 1mL of precooled 75% ethanol (prepared by DEPC water), centrifuging at 7500r at 4 ℃ for 5min, and discarding the supernatant as much as possible; drying and precipitating in a fume hood for 10-30min, adding 30 μ L DEPC water to dissolve RNA, heating in 65 deg.C water bath for 5min, measuring concentration, and storing at-80 deg.C;

(1-4-2) cDNA Synthesis and qPCR detection: the RNA after the above measurements was inverted to synthesize cDNA according to the instructions of HiScript II Q RT SuperMix for qPCR (+ gDNA wrapper) (Norzan R223-01) as follows:

removing genome: preparing the following mixed solution in a centrifugal tube of RNase-free, wherein the template RNA is 1 mug; 4 XgDNA wiper Mix, 4 μ L; RNase-free ddH₂Supplementing O to 16 μ L, gently blowing and beating with a pipette, and mixing uniformly at 42 deg.C for 2 min;

preparing a reverse transcription reaction system: directly adding 4 mu L of 5 XHiScript II qRT SuperMix II into the reaction tube in the first step;

carrying out reverse transcription reaction: 15min at 50 ℃, 5s at 85 ℃, 8-fold dilution of the product in water, storage at-20 ℃ and subsequent detection of the gene expression levels of PDL1 and PDL2 by AceQ qPCR SYBR Green Master Mix (Novozak Q111-02) kit.

Further, the steps (1-4) are carried out by detecting the gene transcription level, RT-PCR primers of PDL1 and PDL2 are designed and verified in SUM159 cells and MDA-MB-231 cells respectively; the primers are respectively as follows:

PDL1-RT-F：GTAGCACTGACATTCATCTTC；

PDL1-RT-R：TTCCTTCCTCTTGTCACGCTC；

PDL2-RT-F：CATAGCCACAGTGATAGCCCT；

PDL 2-RT-R: GGCTCCCAAGACCACAGGTTC are provided. Wherein, the specific method of the immunofluorescence experiment in the step (3) is as follows:

growing tumor cells on a circular cover glass placed in a 24-hole cell culture plate, wherein the inoculation density reaches 60%, and the cells are treated by adding medicine for 24 hours; cells were washed 3 times with 500 μ L PBS per well for 5min each time; then adding 200ul of 4% paraformaldehyde into each hole, and fixing for 15min at room temperature; removing paraformaldehyde, washing cells with 500 μ L PBS for 5min each time for 3 times; adding 500 mu L of 1% BSA into each hole, and blocking for 2h at room temperature; removing blocking solution, adding 200. mu.L primary antibody (BSA dilution) to each well, and standing overnight at 4 ℃; removing antibody, washing cells with 500 μ LPBS for 5min 3 times per well; adding 200 mu L of fluorescent secondary antibody into each hole, and incubating for 2h at room temperature; remove the secondary antibody, wash the cells 3 times with 500 PBS per well for 5min each time; add Hochest (1: 200PBS dilution) to each well, wash cells 3 times for 5min at room temperature with 500. mu.L PBS; dropping a small drop of anti-fade agent on the slide, removing the cover slip from the well, covering it face down onto the anti-fade agent, and gently touching it (carefully bubbling) onto the slide; the slide was left to air dry in the dark for 5min, at which time it was observed under a fluorescent microscope.

The invention also provides application of the TAK1 inhibitor in preparation of anti-PDL 1 medicines.

The technical scheme of the invention has the following beneficial effects: the research of the invention finds that the TAK1 inhibitor 5Z7 can obviously reduce the protein level of PD-L1 in breast cancer cells SUM159 and MDA-MB-231, and the inhibition capability of the inhibitor is higher than that of SUM159 cells in triple negative breast cancer cell line MDA-MB-231 with stronger transfer capability. TAK1 also activates MAPKKs by phosphorylation, resulting in the activation of MAPKs (ERK). Phosphorylation modification is considered to be an important regulatory mechanism in TAK 1-dependent signal transduction, with 5Z7 reducing phosphorylated ERK at the protein level. The TAK1 inhibitor adopted by the invention is a potential anti-tumor drug with good development prospect, the expression of PDL1 is obviously reduced at the gene level, and the feasibility and theoretical basis are hopefully provided for clinically treating breast cancer.

Drawings

FIG. 1 is a graph showing the expression of WB-detected PDL1 in SUM159 and MDA-MB-231 cells treated with an inhibitor of TAK1 in accordance with the present invention;

FIG. 2 is a graph showing the expression of PDL1 detected by RT-PCR after SUM159 and MDA-MB-231 cells are treated with TAK1 inhibitor in the present invention;

FIG. 3 is a graph showing the expression of p-ERK detected by WB after treating SUM159 and MDA-MB-231 cells with TAK1 inhibitor in accordance with the present invention;

FIG. 4 is a graph showing the expression of PD-L1 in immunofluorescence assays after treating SUM159 cells with varying concentrations of TAK1 inhibitor in accordance with the present invention;

FIG. 5 is a graph showing the expression of PD-L1 in immunofluorescence assays after MDA-MB-231 cells were treated with varying concentrations of TAK1 inhibitor in accordance with the present invention.

Detailed Description

In order to make the technical problems, technical solutions and advantages of the present invention more apparent, the following detailed description is given with reference to the accompanying drawings and specific embodiments.

The invention provides a detection method for inhibiting PDL1 by a TAK1 inhibitor, which comprises the following steps:

(1) the expression regulation mechanism of PDL1 molecule in tumor cells is explored: treating tumor cells with a small molecule inhibitor, characterizing the expression of PDL1 by gene level and protein level, respectively;

(2) detecting the level of phosphorylation of ERK in the tumor cell;

(3) the inhibition effect of the TAK1 inhibitor on tumor cell PDL1 is verified: after treating tumor cells with the TAK1 inhibitor, the protein expression level of PDL1 in the cells was detected by immunofluorescence technique.

The invention also provides application of the TAK1 inhibitor in preparation of anti-PDL 1 medicines.

The technical solution of the present invention is described in detail below with reference to specific examples.

In order to obtain a new method for inhibiting PD-L1, the expression of PD-L1 in tumor cells is detected to be inhibited by different small molecule inhibitors, the TAK1 inhibitor 5Z7 treats the breast cancer cell lines SUM159 and MDA-MB-231, and the expression of PD-L1 and PD-L2 is obviously reduced. TAK1 inhibitor 5Z7 treated breast cancer cell lines SUM159 and MDA-MB-231. In the experiment, 5Z7 treatment concentrations were selected to be 0, 1 and 3. mu.M, and the expression of PDL1 protein level and gene level was detected by Western Blot method and real-time quantitative PCR (RTPCR) method. The specific experimental steps comprise:

(1-1) resuscitating the cells: taking out the freezing tube from-80 deg.C, directly soaking in warm water of 37 deg.C, shaking to melt as soon as possible (instant slow freezing); taking out the frozen tube from the water bath at 37 ℃, adding 1mL of complete culture medium, and uniformly mixing; centrifuging at 1000rpm for 3min, discardingClear liquid; adding 10% FBS-containing DMEM medium to resuspend the cells, uniformly inoculating in a 60mm culture dish, and 5% CO₂Standing and culturing in an incubator at 37 ℃; replacing the culture solution once the next day, and continuing culturing; when the cell density reached 90%, cells were digested with trypsin, the number of plated wells (6-well plate) was determined according to the subsequent experiment, and the culture was continued.

(1-2) treatment of TAK1 inhibitor 5Z 7: the concentration of the selected treatment is 0, 1 and 3 mu M in sequence, and the treatment time is 24 h.

(1-3) Western blotting

(1-3-1) preparation of protein sample: preparing Lysis buffer (containing 10 μ L PMSF and 990 μ L lysine buffer) and placing in ice bath for later use; washing cells by precooling PBS, transferring the cells to a sterilized centrifuge tube, centrifuging and removing a supernatant; resuspending the cells in a lysate containing PMSF, and lysing for 30min on ice bath (shaker); centrifuging at 4 deg.C for 15min, and completely sucking supernatant (protein); after mixing the protein with 5 × Loading buffer, the ratio of 4: sucking at a ratio of 1, boiling in metal bath at 95 deg.C for 5min, and storing at-20 deg.C.

(1-3-2) electrophoresis glue running and film transferring color development: boiling the protein sample again for 5min at 105 ℃; preparing 10% separation gel according to the formula of the protein gel, preparing 5% concentrated gel on the upper layer, and adding 10 μ L protein sample into each hole; adjusting the voltage of the electrophoresis apparatus to 80V, and adjusting the voltage of the electrophoresis apparatus to 120V again when the bromophenol blue indicator reaches the lower layer separation gel; according to a standard protein marker, a sample area to be inspected is cut off; the PVDF membrane is cut into the same size as the cut protein glue and is placed in a membrane buffer for balancing for 3 min.

The stacking sequence is as follows: performing wet rotation for 1h at 300mA for a power supply anode, filter paper, a PVDF (polyvinylidene fluoride) membrane, gel, filter paper and a power supply cathode; putting the PVDF membrane in 5% skimmed milk powder, and sealing for 2 h; rinsing PVDF membrane with TBST for 3min, adding milk or primary antibody (1: 1000) diluted by primary antibody diluent, and incubating overnight at 4 deg.C; rinsing with TBST for 5min x 3 the next day, adding appropriate amount of secondary antibody (1: 2000), and incubating at room temperature for 2 h; finally TBST rinse 10min x 3, developed using ECL reagent.

(1-4) real-time quantitative PCR: RT-PCR primers PDL1 and PDL2 are respectively designed by detecting gene transcription level and are respectively verified in SUM159 and MDA-MB-231 cells; the primers are respectively as follows:

PDL1-RT-F：GTAGCACTGACATTCATCTTC；

PDL1-RT-R：TTCCTTCCTCTTGTCACGCTC；

PDL2-RT-F：CATAGCCACAGTGATAGCCCT；

PDL 2-RT-R: GGCTCCCAAGACCACAGGTTC are provided. (1-4-1) extraction of total RNA of cells: total RNA was extracted from the above treated cells by Trizol method as follows: discarding culture solution of cells, washing once by using PBS, adding pancreatin to digest cells and collecting in an EP tube without RNA enzyme; 1mL of RNA isolater (Novozam R401-01) was added and left at room temperature for 5 min; adding 200 μ L chloroform, shaking vigorously for 15s, and standing at room temperature for 3 min; centrifuging at 12000r at 4 deg.C for 15min, separating into three layers, carefully sucking the upper layer into a new EP tube without RNase, adding 0.5ml isopropanol, mixing by turning upside down, and standing at-20 deg.C for 2h (increasing precipitation); centrifuging at 12000r at 4 deg.C for 10min to obtain white precipitate at the bottom of the tube; discarding the supernatant, adding 1mL of precooled 75% ethanol (prepared by DEPC water), centrifuging at 7500r at 4 ℃ for 5min, and discarding the supernatant as much as possible; drying the precipitate in a fume hood for 10-30min, adding 30 μ L DEPC water to dissolve RNA, heating in 65 deg.C water bath for 5min, measuring concentration, and storing at-80 deg.C.

(1-4-2) cDNA Synthesis and qPCR detection: the RNA after the above measurements was inverted to synthesize cDNA according to the instructions of HiScript II Q RT SuperMix for qPCR (+ gDNA wrapper) (Norzan R223-01) as follows:

removing genome: preparing the following mixed solution in a centrifugal tube of RNase-free, wherein the template RNA is 1 mug; 4 XgDNA wiper Mix, 4 μ L; RNase-free ddH₂Supplementing O to 16 μ L, gently blowing and beating with a pipette, and mixing uniformly at 42 deg.C for 2 min;

preparing a reverse transcription reaction system: directly adding 4 mu L of 5 XHiScript II qRT SuperMix II into the reaction tube in the first step;

carrying out reverse transcription reaction: 15min at 50 ℃, 5s at 85 ℃, 8-fold dilution of the product in water, storage at-20 ℃ and subsequent detection of the gene expression levels of PDL1 and PDL2 by AceQ qPCR SYBR Green Master Mix (Novozak Q111-02) kit.

The detection result of the protein level is shown in figure 1, and the result analysis shows that the TAK1 inhibitor 5Z7 can obviously inhibit the expression of PDL1 protein in SUM159 and MDA-MB-231 cells. Similarly, the results of the mRNA level detection are shown in FIG. 2, and the analysis of the results shows that the TAK1 inhibitor 5Z7 can significantly inhibit the transcription of pdl1 gene in SUM159 and MDA-MB-231 cells.

Previous results indicate that expression of PD-L1 and PD-L2 in SUM159 and MDA-MB-231 cells is dependent on activation of ERK in tumor cells. In addition, phosphorylation levels of ERK were measured before and after treatment with TAK1 inhibitor 5Z7 in both cells, and the results are shown in fig. 3, and the analysis of the results shows that TAK1 inhibitor 5Z7 was able to significantly inhibit phosphorylation levels of ERK in SUM159 and MDA-MB-231 cells.

In order to further verify the inhibition effect of the TAK2 inhibitor 5Z7 on tumor cells PD-L1, the protein expression level of PD-L1 in the cells is detected by an immunofluorescence technique. Next, the expression level of PDL1 was detected in SUM159 and MDA-MB-231 cells using immunofluorescence techniques, and 5Z7 treatment concentrations of 0, 3, and 5. mu.M were selected for the experiments. The detection result of SUM159 cells is shown in fig. 4, and the result analysis shows that after tam 1 inhibitor 5Z7 treats SUM159 cells, the immunofluorescence intensity is obviously weakened, which indicates that the protein level of PDL1 is obviously reduced. Similar experimental results were also confirmed in MDA-MB-231 cells, and the results are shown in FIG. 5.

The specific method of the immunofluorescence assay is as follows:

tumor cells were grown on round coverslips placed in 24-well cell culture plates, the seeding density reached 60%, and cells were treated with drug for 24 h. Cells were washed 3 times with 500 μ L PBS per well for 5min each time; then adding 200ul of 4% paraformaldehyde into each hole, and fixing for 15min at room temperature; removing paraformaldehyde, washing cells with 500 μ L PBS for 5min each time for 3 times; adding 500 mu L of 1% BSA into each hole, and blocking for 2h at room temperature; removing blocking solution, adding 200. mu.L primary antibody (BSA dilution) to each well, and standing overnight at 4 ℃; removing antibody, washing cells with 500 μ LPBS for 5min 3 times per well; adding 200 mu L of fluorescent secondary antibody into each hole, and incubating for 2h at room temperature; remove the secondary antibody, wash the cells 3 times with 500 PBS per well for 5min each time; add Hochest (1: 200PBS dilution) to each well, wash cells 3 times for 5min at room temperature with 500. mu.L PBS; dropping a small drop of anti-fade agent on the slide, removing the cover slip from the well, covering it face down onto the anti-fade agent, and gently touching it (carefully bubbling) onto the slide; the slide was left to air dry in the dark for 5min, at which time it was observed under a fluorescent microscope.

In conclusion, the TAK1 inhibitor 5Z7 can significantly inhibit the expression of both PDL1 gene and protein in breast cancer cells (mainly SUM159 and MDA-MB-231), and may reduce the transcription of PDL1 gene by reducing the phosphorylation level of ERK, thereby inhibiting the expression of PDL1 in tumor cells. Therefore, the TAK1 inhibitor 5Z7 can enhance the immunotherapy effect of T cells in breast cancer, can be used as a future therapy target, and the conclusion of the invention can provide necessary guiding significance for the treatment of breast cancer.

Inhibiting PD-L1 function in tumor cell, and activating anti-tumor immunity of T cell. Clinically, some patients responded better to anti-PD-L1 treatment. Currently anti-PD-L1 is mainly achieved by antibodies. The invention discovers that the small molecule inhibitor can inhibit the expression of PD-L1 in tumor cells, thereby obtaining the similar effect of anti-PD-L1. The TAK1 inhibitor 5Z-7-oxozeaenol is a potential anti-tumor drug with good development prospect, and is of great interest in various tumor treatments. According to the invention, in breast cancer cells SUM159 and MDA-MB-231, 5Z7 obviously inhibits the expression of PDL 1. Therefore, the TAK1 inhibitor can inhibit PD-L1 expression of tumor cells and activate anti-tumor immunity of T cells.

While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

<110> university of southeast Tong

<120> detection method for PDL1 inhibition by TAK1 inhibitor and application of detection method in preparation of anti-PDL 1 drugs

<141> 2020-12-14

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<213> Artificial Synthesis (Artificial Synthesis)

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