Application of transcription factor CEBPZ in preparation of anti-tumor immune drugs

Abstract

The invention provides application of a transcription factor CEBPZ in preparation of an anti-tumor immune drug, relates to the field of molecular biology and tumor immunotherapy, and provides a novel PD-L1 regulation target (CEBPZ), and the transcription factor CEBPZ in breast cancer can positively regulate PD-L1 and can regulate immune escape of tumors. The invention shows that the transcription factor CEBPZ is a key transcription factor for regulating PD-L1. Firstly, after a transcription factor CEBPZ is knocked out in a breast cancer cell line SUM159 cell by using a CRISPR-Cas9 technology, the expression of PD-L1 is remarkably reduced, and meanwhile, immune escape of tumors can also be reduced. Experiment results of specific verification experiments show that the transcription factor CEBPZ is a key transcription factor for regulating PD-L1 and tumor immune escape, and has great potential in preparation of antitumor drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology and tumor immunotherapy, and in particular to the application of transcription factor CEBPZ in the preparation of anti-tumor immune drugs. Background Art

[0002] PD-L1 is a key molecule for tumor escape. It is usually highly expressed in a variety of cancer cells and cancer tissues. After PD-L1 binds to the immunosuppressive transmembrane protein PD-1 expressed on the surface of T cells, T cells cannot recognize tumor cells and kill them. The body's immune function is suppressed, causing the immune escape of tumor cells. At present, many antibodies targeting PD-L1 / PD-1 have been approved for the treatment of various tumors. However, the response rate of some patients is low, such as triple-negative breast cancer, with a treatment response rate of only about 20%. Pancreatic cancer has almost no response to immune checkpoint blockade therapy, and new regulatory targets are urgently needed to be developed.

[0003] A deep understanding of the immunological basis of treatment and the regulatory process of immune checkpoint molecules is the theoretical basis for further improving immune checkpoint blockade therapy. It is known that the expression of PD-L1 is precisely regulated at multiple levels, such as inflammatory signals (such as IFN-γ) and some transcription factors (such as STAT3, NF-κB), but the regulatory network has not yet been fully elucidated.

[0004] PD-L1 plays a key role in maintaining immune homeostasis. The transcription of PD-L1 is the basis for subsequent regulation at other levels, but the understanding of the transcriptional regulation of PD-L1 is still seriously insufficient. At present, the transcriptional regulation of PD-L1 is mainly concentrated in the promoter region of PD-L1, and many studies have studied the DNA sequence and related transcription factors of the promoter region of PD-L1. Summary of the invention

[0005] The purpose of the present invention is to solve the technical problem of insufficient response rate of PD-1 / PD-L1 inhibitors in the prior art.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] Application of transcription factor CEBPZ in the preparation of drugs for treating tumors.

[0008] Preferably, the transcription factor CEBPZ is a regulatory target of PD-L1.

[0009] Preferably, the drug is used to knock out the transcription factor CEBPZ or reduce the expression of the transcription factor CEBPZ.

[0010] Preferably, the drug also reduces the immune escape of the tumor.

[0011] The present application also provides a tumor treatment drug, which is used to knock out the transcription factor CEBPZ or reduce the expression of the transcription factor CEBPZ.

[0012] Preferably, the drug downregulates PD-L1 expression by knocking out the transcription factor CEBPZ or reducing the expression of the transcription factor CEBPZ, while reducing the immune escape of the tumor.

[0013] This application uses specific validation experiments to prove that the transcription factor CEBPZ plays a key role in the regulation of PD-L1. Therefore, knocking out the transcription factor CEBPZ will significantly reduce the expression of PD-L1 and reduce the immune escape of tumors. Based on this, we can further study new cancer diagnosis, treatment and drug development methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is an expression graph analyzing the expression level and survival curve of CEBPZ in breast cancer in a database in one embodiment of the present invention;

[0015] Figure 2 This is a comparative diagram of the expression of CEBPZ and PD-L1 in three wild-type breast cancer cell lines detected by WB in one embodiment of the present invention;

[0016] Figure 3 This is a comparison diagram of the knockout effect of CEBPZ in SUM159 verified by WB in one embodiment of the present invention;

[0017] Figure 4 This is a comparison chart of the protein expression changes of PD-L1 in sgCEBPZ detected by WB in one embodiment of the present invention;

[0018] Figure 5 This is a comparison diagram of changes in mRNA expression of PD-L1 in sgCEBPZ detected by WB in one embodiment of the present invention;

[0019] Figure 6 This is a comparison diagram of the expression changes of PD-L1 in sgCEBPZ detected by flow cytometry in one embodiment of the present invention;

[0020] Figure 7 This is a comparison diagram of the expression changes of PD-L1 in sgCEBPZ detected by immunofluorescence in one embodiment of the present invention;

[0021] Figure 8 This is a comparative diagram of the expression of PD-1 and IL-2 in unactivated and activated Jurkat detected by RT-QPCR in one embodiment of the present invention;

[0022] Fig. 9This is a comparison diagram of crystal violet-stained tumor cells after co-culture of sgCEBPZ and activated Jurkat cells in one embodiment of the present invention;

[0023] Fig.10 This is a comparison chart of IFN-γ and GZMB expression in T cells detected by RT-QPCR after co-culture in one embodiment of the present invention. DETAILED DESCRIPTION

[0024] The present invention is further described in detail below in conjunction with specific embodiments.

[0025] The use of transcription factor CEBPZ in the preparation of drugs for treating tumors. In one embodiment, the transcription factor CEBPZ serves as a regulatory target of PD-L1. Knocking out the transcription factor CEBPZ can significantly downregulate the expression of PD-L1 and also reduce the immune escape of the tumor.

[0026] Based on the above description, the present application also proposes a drug for treating tumors, and the drug is used to knock out the transcription factor CEBPZ or reduce the expression of the transcription factor CEBPZ.

[0027] The above contents are explained below in combination with specific verification experiments.

[0028] Example 1: Analysis of the expression level and survival curve of CEBPZ in breast cancer through database

[0029] In this example, the differential expression of the transcription factor CEBPZ in normal breast cells and breast cancer cells was first analyzed in the GEPIA database, and it was found that the expression of CEBPZ in breast cancer cells was slightly higher than that in normal breast cells ( Figure 1 -A).

[0030] In addition, in this example, a Kaplan-Meier survival curve was drawn to analyze the effect of CEBPZ expression on the survival rate of breast cancer patients. The results showed that the survival rate of patients with high CEBPZ expression was lower than that of patients with low CEBPZ expression ( Figure 1 -B).

[0031] These results indicate that the expression of transcription factor CEBPZ is crucial in breast cancer.

[0032] Example 2: Detection of the expression of CEBPZ and PD-L1 in three wild-type breast cancer cell lines

[0033] In this example, WB was used to detect the expression of CEBPZ and PD-L1 in three wild-type breast cancer cell lines, MDA-MB-231, SUM159 and MCF7, and it was found that the expression of CEBPZ and PD-L1 was positively correlated. In MDA-MB-231 and SUM159 cells with higher PD-L1 expression, CEBPZ expression was also higher, and in MCF7 cells with lower PD-L1 expression, CEBPZ expression was also lower ( Figure 2 ).

[0034] Example 3: Construction of sgCEBPZ knockout cell line and detection of PD-L1 expression changes in sgCEBPZ

[0035] In this example, sgCEBPZ knockout cell lines were first constructed in SUM159 cells using sgRNA designed using CRISPR-Cas9 technology ( Figure 3 ), and then use WB( Figure 4 )、RT-QPCR( Figure 5 ), flow cytometry ( Figure 6 )、Immunofluorescence( Figure 7 ) were used to detect the expression changes of PD-L1 in sgCEBPZ.

[0036] The results showed that the expression of PD-L1 decreased after knocking out CEBPZ, indicating that the transcription factor CEBPZ positively regulates PD-L1.

[0037] Example 4: Verification of the effect of transcription factor CEBPZ on tumor immune escape

[0038] In this example, a co-culture experiment was first performed to activate Jurkat T cells ( Figure 8 ) were co-cultured with sgCEBPZ. The results showed that in the co-culture experiment, the growth of sgCEBPZ was slower than that of the control ( Fig. 9 ), while the expression of IFN-γ and granzyme B in T cells increased ( Fig.10 ).

[0039] These results prove that after the knockout of transcription factor CEBPZ, tumor cells are more sensitive to T cells and the secretion capacity of T cells is enhanced, indicating that the transcription factor CEBPZ affects the immune escape of tumors.

[0040] The following are the experimental materials and related experimental steps used in the above examples:

[0041] 1. Experimental materials and sources

[0042]

[0043]

[0044] (II) Cell culture

[0045] The SUM159 cells used in the experiment were cultured in DMEM medium containing 10% fetal bovine serum and 1% double-antibody (penicillin and streptomycin mixture) and given 5% CO 2 The Jurkat cells used in the experiment were cultured in RPMI1640 medium containing 10% fetal bovine serum and 1% double antibody (penicillin and streptomycin mixture) and given 5% CO 2 , the temperature is 37℃.

[0046] (III) Cell transfection

[0047] ① The gene editing vector constructed using sgCEBPZ plasmid has puromycin resistance and can be used for positive clone cell screening.

[0048] ② Determine the minimum lethal dose of puromycin for normal cells, inoculate SUM159 cells into 6-well plates, with a cell density of about 70%, and culture them in serum-containing DMEM medium for 24 hours. Transfect the cells with the constructed gene editing vector at a transfection dose of 1ug / mL, change the medium after 6 hours, and continue culturing. After 48 hours, replace the medium containing puromycin for culturing. Change the medium every 2-3 days, and gradually increase the concentration of puromycin contained in the medium until the minimum lethal dose for normal cells is reached. After about 2-3 weeks, the screening cell line is obtained.

[0049] (IV) Protein immunoblotting

[0050] ① Total cell protein extraction

[0051] Aspirate the culture medium and wash the cells with PBS; add an appropriate amount of cell lysis buffer to the cells and add PMSF proteasome inhibitor before use; scrape the cells with a cell scraper and collect them into a clean EP tube; shake at 200 rpm on ice for 45 min; centrifuge at 12000 rpm for 15 min at 4°C, take the supernatant and add 5× protein loading buffer, boil in a metal bath at 105°C for 5 min, and store in a -80°C refrigerator.

[0052] ② Gel electrophoresis and protein immunoassay

[0053] 1) Prepare glue: clean the glass plates, add ultrapure water between the glass plates, and observe whether the liquid level drops. If the liquid level does not drop, pour out the water and absorb the remaining water with filter paper; prepare separation glue first, add TEMED at the end and mix quickly, slowly add separation glue with a 1000μL pipette, be careful not to have bubbles, and add isopropanol to seal the glue; wait for about 30 minutes for gelation, pour out the isopropanol, rinse with ultrapure water until it is odorless, and absorb the remaining water with filter paper; prepare concentrated glue, fill the remaining space, and insert a comb; wait for gelation, and remove the glue and the plate together.

[0054] 2) Electrophoresis: Clamp the gel plate and add electrophoresis solution to check for leaks; place it in the electrophoresis tank, unplug the comb, add marker and sample, pour electrophoresis solution into the outer box, covering the bottom to form a current path; select a suitable cover, connect the power supply, and keep the voltage at 80V; observe every 10-15 minutes, and when the marker is found to run away, change the voltage to 120V and continue.

[0055] 3) Transfer: The glue, PVDF membrane and filter paper should be kept moist, and there should be no bubbles on the glue. The PVDF membrane should be placed in methanol first, then in ultrapure water, and finally in the transfer solution in the order of cotton pad, filter paper, glue strip, PVDF membrane, filter paper and cotton pad; place the transfer clip in the transfer tank, black against black, and place the entire transfer box in ice water. The transfer conditions are set to a constant current of 300mA and 70min.

[0056] 4) Blocking: Prepare 5% skim milk powder with TBST. After the transfer, place the PVDF membrane in 5% skim milk powder for blocking. Shake at 50-60 rpm for 2 hours.

[0057] 5) Antibody incubation: discard the blocking solution, wash the membrane with TBST 90 for 5 min × 3 times; incubate with the corresponding primary antibody, shake at 50-60 rpm at 4°C overnight; recover the primary antibody, wash the membrane with TBST 90 for 5 min × 3 times; incubate with the corresponding secondary antibody, shake at 50-60 rpm for 2 h; wash with TBST 20 min × 3 times.

[0058] 6) Development: Prepare ECL developer and use it immediately. Develop the membrane on a chemiluminescence imager.

[0059] (V) Quantitative RT-PCR

[0060] ① Total RNA extraction from cells

[0061] 1) After trypsin digestion of cells, transfer to RNase-free EP tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, collect the cell pellet, add 1 mL Trizol and mix by pipetting repeatedly.

[0062] 2) The homogenized sample was placed at room temperature for 5 minutes to completely separate the nucleic acid-protein complex; 0.2 mL of chloroform was added to every 1 mL of Trizol lysis buffer, and the mixture was shaken vigorously for 15 seconds and placed at room temperature for 3 minutes.

[0063] 3) Centrifuge at 12,000 rpm for 15 min at 4°C and layer the sample; transfer the supernatant to a new EP tube, add an equal amount of isopropanol to precipitate RNA, and leave at room temperature for 10 min.

[0064] 4) Centrifuge at 12,000 rpm for 10 min at 4°C. After centrifugation, a gelatinous precipitate will appear on the sides and bottom of the tube. Remove the supernatant.

[0065] 5) Wash the RNA precipitate with 1 mL of 75% ethanol, centrifuge at 7500 rpm for 5 min at 4°C, remove the supernatant, and repeat this step twice.

[0066] 6) Leave the dried RNA precipitate at room temperature for about 5-10 minutes, add 20-100 mL of RNase-free ultrapure water, pipette several times, place at 55-60℃ for 10 minutes to dissolve the RNA, and store at -80℃.

[0067] ②Reverse transcription of RNA into cDNA

[0068] in accordance with II Reverse Transcriptase Instructions Reverse transcription of RNA to synthesize cDNA. The reaction system is as follows:

[0069]

[0070] ③RT-PCR

[0071] qRT-PCR amplification was performed according to the instructions of the AceQ qRT-PCR SYBR Green Master Mix kit:

[0072] 1) Place the eight-tube strip on ice, then add 5 μL of SYBR Green reagent, 0.2 μL of each upstream and downstream primers, and RNase-free ddH 2 O 2.6 μL and template cDNA 2 μL, and then mix thoroughly.

[0073] 2) Place the mixed reaction system into the qRT-PCR instrument and input the corresponding reaction parameters: 95℃15min, 95℃15sec, 60℃60sec, 40 cycles. Record and analyze the data. Calculate the gene expression using the 2-△△Ct method.

[0074] (VI) Flow cytometry

[0075] ① Digest with 200 μL of trypsin (observe under a microscope, the cells are completely rounded), terminate the digestion with 500 μL of DMEM containing 10% FBS, collect the cells by centrifugation, and ensure that the amount of cells in each tube is similar, about the amount of cells in a six-well plate.

[0076] ② Resuspend and wash once with 500μL PBS (to prevent cells from being sucked away, some liquid can be retained).

[0077] ③ Prepare the staining MIX in advance: (100μL PBS+0.25μL Ab)X(n+2) system and wrap it with tin foil.

[0078] ④Add mix to resuspend cells, wrap in tin foil and shake on ice for 30 minutes to avoid light.

[0079] ⑤ Resuspend and wash the cells once with 500 μL PBS, centrifuge, and keep the cell pellet (to prevent the cells from being sucked away, some liquid can be retained).

[0080] ⑥Add 300μL PBS, resuspend the cells, and test on the machine.

[0081] 7. Immunofluorescence

[0082] Prepare in advance: round coverslip, small tweezers, 24-well plate

[0083] 4% PFD (paraformaldehyde): Na 2 HPO 4 12H 2 O 73g, NaH 2 PO 4 ·2H 2 O 3.85 g, add 1000 mL ddH 2 O, heat to dissolve.

[0084] 0.2% TritonX-100: 10mL PBS+20μL TritonX-100

[0085] 1% BSA: 10 mL PBS + 0.1 g BSA

[0086] ① Use tweezers to place the round glass slide into a 24-well plate (the round cover glass has been disinfected with 75% alcohol), wash the round glass slide with PBS, and then inoculate the cells into the 24-well plate to a seeding density of 60%, and treat the cells with drugs for 24 hours.

[0087] ② Wash the cells three times with 500 μL of PBS in each well, each time for 5 min.

[0088] ③ Remove PBS, add 200 μL 4% paraformaldehyde to each well, and fix at room temperature for 15 minutes.

[0089] ④ Remove paraformaldehyde and wash the cells three times with 500 μL PBS per well, each time for 5 min; add 500 μL 1% BSA to each well and block at room temperature for 2 h.

[0090] ⑤ Remove the blocking solution, add 200 μL primary antibody (BSA dilution ratio is 1:200) to each well, and store in a 4°C refrigerator overnight.

[0091] ⑥ Recover antibodies, wash each well with 500 μL PBS for 5 min × 3 times, add 200 μL fluorescent secondary antibody, incubate at room temperature for 2 h, and protect from light.

[0092] ⑦ Remove the secondary antibody, wash each well with 500 μL PBS for 5 min × 3 times, add Hochest (1:200 PBS dilution), incubate at room temperature for 10 min, and avoid light.

[0093] ⑧ Remove Hochest and wash the cells 3 times with 500μL PBS, 5 min each time; drop a small drop of anti-fading agent on the slide, take the cover slip out of the well, and cover it face down on the anti-fading agent, making it lightly touch the slide (be careful of bubbles), and protect it from light.

[0094] ⑨ Place the slide in a dark place for 15 minutes, let it dry, and observe the fluorescence intensity and distribution under a fluorescence microscope.

[0095] 8. T cell co-culture

[0096] ① Jurkat T cells are suspension cells and cultured in 1640 medium. Jurkat T cells are expanded in advance for future use.

[0097] ② One day in advance, digest the control cells and established cells, count 3,000 cells, and plate them into six-well plates.

[0098] ③ Jurkat T cells were first activated with PHA at a concentration of 1.5 ng / mL for 4 h. After activation, the suspended Jurkat T cells aggregated into clumps. After being blown and mixed, a portion of the activated Jurkat T cells and the unactivated Jurkat T cells were aspirated, and total RNA was extracted. The expression of IL-2 and PD-1 was detected by PCR to determine whether the Jurkat T cells were successfully activated.

[0099] ④ Add activated Jurkat T cells to the tumor cells in the six-well plate and co-culture for 48 hours at a ratio of 1:10.

[0100] ⑤After 48 hours, the supernatant was aspirated, centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded to obtain the co-cultured Jurkat T cells. Total RNA was extracted for PCR detection of the expression changes of interferon-γ and granzyme B.

[0101] ⑥ After 48 h, aspirate the supernatant from the tumor cells in the six-well plate, wash the cells twice with PBS, fix them with 4% paraformaldehyde at room temperature for 20 min, discard the paraformaldehyde, add PBS and place them on a shaker to wash three times at 50 r for 5 min each time. Then add 1 mL of crystal violet and stain for 20 min, aspirate the crystal violet, add PBS and place them on a shaker to wash three times at 50 r for 5 min each time. Observe the proliferation of the tumor cells after co-culture.

[0102] (IX) Statistical analysis

[0103] All experiments were repeated at least three times. All data were expressed as mean ± standard deviation, and the significant differences between the experimental group and the control group were reflected by the method of analysis of variance. The analysis was performed using SPSS 21.0 software, and P < 0.05 was used to indicate that the difference was statistically significant.

[0104] Combined with the above specific verification experiments, it is proved in this application that the knockout of the transcription factor CEBPZ will reduce the expression of PD-L1 and also reduce the tumor immune escape. Furthermore, it can be proved that the transcription factor CEBPZ is a key transcription factor affecting the regulation of PD-L1 and tumor immune escape. A new regulatory target (CEBPZ) of PD-L1 is provided in this application, and a cancer immunotherapy strategy is developed based on this. Through the above technical solutions, this application solves the problem of insufficient response rate of existing PD-1 / PD-L1 inhibitors and has great potential in the preparation of anti-tumor drugs.

Claims

1. Application of transcription factor CEBPZ in the preparation of drugs for treating tumors.

2. The use of the transcription factor CEBPZ according to claim 1 in the preparation of a drug for treating tumors, characterized in that: The transcription factor CEBPZ is a regulatory target of PD-L1.

3. The use of the transcription factor CEBPZ according to claim 1 in the preparation of a drug for treating tumors, characterized in that: The drug is used for knocking out the transcription factor CEBPZ or reducing the expression of the transcription factor CEBPZ.

4. The use of the transcription factor CEBPZ according to claim 3 in the preparation of a drug for treating tumors, characterized in that: The drug also reduces the tumor's immune escape.

5. A tumor treatment drug, characterized in that: The drug is used for knocking out the transcription factor CEBPZ or reducing the expression of the transcription factor CEBPZ.

6. The tumor treatment drug according to claim 5, characterized in that: The drug downregulates PD-L1 expression by knocking out the transcription factor CEBPZ or reducing the expression of the transcription factor CEBPZ, while reducing the immune escape of the tumor.

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