Application of BCLAF1 in preparing drugs for treating and reversing resistance of liver cancer cells to durvalumab

Abstract

The present invention discloses the application of a BCLAF1 inhibitor in the preparation of a drug for treating and reversing the resistance of liver cancer cells to durvalumab, belonging to the field of biomedicine. In the present invention, bioinformatics analysis and immunohistochemistry are used to analyze the expression of BCLAF1 in liver cancer specimens and adjacent tissues, and the relationship between BCLAF1 and prognosis is analyzed. Then, the plasmid sgBCLAF1 with stable knockout of BCLAF1 is introduced into liver cancer cells by CRISPR-Cas9 and plasmid transfection techniques to achieve knockdown of BCLAF1 in liver cancer cells. Finally, after activating Jurkat T cells with a human CD3 / CD28 T cell activator and co-culturing them with liver cancer cells, the PD-L1 blocker durvalumab is added, and flow cytometry and ELISA are used to detect the apoptosis, cell cycle and cytokine levels of Jurkat T cells respectively, demonstrating that sgBCLAF1 can be used to improve the sensitivity of liver cancer cells to durvalumab and reverse the resistance of liver cancer cells to durvalumab.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine. Specifically, it relates to the application of BCLAF1 in the preparation of drugs for treating and reversing the resistance of liver cancer cells to durvalumab. More specifically, it relates to the application of a BCL-2 related transcription factor 1 in the preparation of drugs for treating and reversing the resistance of liver cancer cells to the PD-L1 blocker durvalumab. Background Art

[0002] In recent years, an immune checkpoint inhibitor, durvalumab, has achieved remarkable research results in clinical trials of solid malignant tumors. It is a selective and highly affinity human IgG1 monoclonal antibody that can block the binding of PD-L1 to PD-1 and CD80, enabling T cells to recognize and kill tumor cells, and has obvious curative effects on many tumors such as lung cancer, breast cancer, and cholangiocarcinoma. However, studies have found that liver cancer cells are resistant to durvalumab, greatly limiting the clinical application of durvalumab in liver cancer, yet its mechanism remains unclear.

[0003] In summary, the research and development of a drug for reversing the resistance of tumor cells to durvalumab will provide ideas and solutions for the research and development of tumor-targeted therapeutic drugs, and at the same time have great social and economic benefits. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for reversing the resistance of liver cancer cells to durvalumab in view of the problem that the liver cancer treatment drug durvalumab often produces resistance during treatment, which may lead to the failure of immunotherapy and even the recurrence of the disease.

[0005] To solve the above technical problem, the present invention provides the application of a BCLAF1 inhibitor in the preparation of drugs for treating and reversing the resistance of liver cancer cells to durvalumab. The BCLAF1 inhibitor is a molecule or preparation that reduces the expression level of BCLAF1 in liver cancer cells. BCLAF1, namely BCL-2 related transcription factor 1, is located on chromosome 6q23.3, includes 18 exons, and can encode 920 amino acids. The significant features of the BCLAF1 structure include an arginine-serine (RS)-rich domain, a basic leucine zipper (bZIP) domain located within the RS domain, and a MYB DNA-binding domain. Studies have shown that BCLAF1 mainly promotes the occurrence and development of liver cancer by regulating multiple signal pathways represented by HIF-1α.

[0006] Preferably, the molecule or preparation that reduces the expression level of BCLAF1 in liver cancer cells includes gene knockout RNA, and the gene knockout RNA is sgBCLAF1.

[0007] Preferably, the sg BCLAF1 has the following interfering sequences:

[0008] Sense strand: 5’-ACGAAGTGAACCGCTCGTTTGTTTTAGAGCTAGAAATAGCAAGTTAA-3’,

[0009] Antisense strand: 5’-ACAAACGAGCGGTTCACTTCGTCAAACAAGGCTTTTCTCCAAGG-3’.

[0010] Furthermore, the present invention also provides an sgRNA for targeted knockout of BCLAF1, the sequence of its sense strand is shown as SEQ ID No.1, and the sequence of its antisense strand is shown as SEQ ID No.2.

[0011] Furthermore, the present invention also provides a method for verifying that sg BCLAF1 reverses the resistance of liver cancer cells to durvalumab, which is characterized by including the following technical means:

[0012] (1): Use bioinformatics analysis and immunohistochemistry to analyze the expression of BCLAF1 in liver cancer specimens and adjacent tissues, and analyze the relationship between BCLAF1 and prognosis;

[0013] (2): Use CRISPR-Cas9 and plasmid transfection technologies to introduce plasmids with negative control, overexpression, and stable knockout of BCLAF1 into liver cancer cells to achieve overexpression and knockdown of BCLAF1 in liver cancer cells;

[0014] (3): Activate Jurkat T cells with a human CD3 / CD28 T cell activator and co-culture them with liver cancer cells, add the PD-L1 blocker durvalumab, and detect the apoptosis, cell cycle, and cytokine levels of Jurkat T cells by flow cytometry and ELISA respectively.

[0015] Preferably, the liver cancer cells are HepG2 and SK-Hep1 cells.

[0016] Preferably, the plasmid for overexpressing BCLAF1 is OEBCLAF1, and the sequence of BCLAF1 in the OEBCLAF1 plasmid is shown as SEQ ID No.3.

[0017] Preferably, the plasmid for stably knocking out BCLAF1 is cas9-BCLAF1, the sequence of the sense strand of cas9-BCLAF1 is shown as SEQ ID No.1, and the sequence of the antisense strand of cas9-BCLAF1 is shown as SEQ ID No.2

[0018] Advantages of the present invention: The method of the present invention provides a new basis for immunotherapy for malignancies in which durvalumab is used as an immunotherapeutic drug for tumors and is prone to drug resistance, and provides a new scientific basis for further improving tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a viewable diagram of the expression of BCLAF1 in liver cancer tissues and adjacent tissues from the TCGA and CPTAC databases in Example 2 of the present invention;

[0020] Figure 2 It is the immunohistochemical staining and immunohistochemical results for 105 pairs of fresh tissues of fresh primary liver cancer patients in Example 2 of the present invention;

[0021] Figure 3 It is a survival curve diagram of the expression of BCLAF1 and the overall survival of liver cancer patients based on the TCGA database and the CPTAC database in Example 2 of the present invention;

[0022] Figure 4 In Example 4 of the present invention, after interfering with and overexpressing BCLAF1 in liver cancer cells, the protein expression level indicates the success of interference and overexpression;

[0023] Figure 5 It is an experimental result diagram of the changes in flow cytometry and apoptosis levels after co-culturing liver cancer cells HepG2 after interfering with and overexpressing BCLAF1 with Jurkat T cells in Example 4 of the present invention;

[0024] Figure 6 It is an experimental result diagram of the changes in flow cytometry and apoptosis levels after co-culturing liver cancer cells SK-Hep1 after interfering with and overexpressing BCLAF1 with Jurkat T cells in Example 4 of the present invention;

[0025] Figure 7 It is an experimental result diagram of the cell cycle changes of Jurkat T cells detected after co-culturing liver cancer cells HepG2 after interfering with and overexpressing BCLAF1 with Jurkat T cells and adding or not adding durvalumab in Example 4 of the present invention.

[0026] Figure 8 It is an experimental result diagram of the cell cycle changes of Jurkat T cells detected after co-culturing liver cancer cells SK-Hep1 after interfering with and overexpressing BCLAF1 with Jurkat T cells and adding or not adding durvalumab in Example 4 of the present invention.

[0027] Figure 9This is the experimental result graph of detecting the changes in the levels of cytokines secreted by Jurkat T cells after co-culturing with HepG2 and SK-Hep1 hepatoma cells with interference and overexpression of BCLAF1 and adding or not adding durvalumab in Example 4 of the present invention. Detailed implementation manners

[0028] To make the above objects, features, and advantages of the present invention more obvious and understandable, the following detailed description of the specific embodiments of the present invention is provided. It should be noted that the following embodiments are only used to illustrate the implementation methods and typical parameters of the present invention, and are not used to limit the parameter range described in the present invention. Reasonable changes derived therefrom are still within the protection scope of the claims of the present invention.

[0029] It should be noted that the endpoints and any values within the ranges disclosed in this article are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this article.

[0030] As described in the background art, although durvalumab has significant efficacy against many tumors such as liver cancer, breast cancer, and intestinal cancer, these cancer cells may all develop drug resistance to durvalumab, greatly limiting the clinical use of durvalumab. Therefore, it is necessary to develop a new drug to reverse the durvalumab resistance of liver cancer cells to improve the therapeutic effect of durvalumab.

[0031] To make the present invention easy to understand, in the specific embodiments of the present invention, BCLAF1 knockout is represented by BCLAF1-KO, and BCLAF1 overexpression is represented by BCLAF1-OE.

[0032] Example 1

[0033] Collection of liver cancer tissue specimens and immunohistochemical staining of liver cancer

[0034] Collection of liver cancer tissue specimens

[0035] The sources of liver cancer tissue specimens collected in this example: 105 pairs of human liver cancer tissue samples included in the study were diagnosed as liver cancer by the Ningbo Clinical Pathology Diagnosis Center from January 1, 2021 to August 31, 2022. Among them, 50 pairs of formalin-fixed human liver cancer tissue specimens were from the Ningbo Clinical Pathology Diagnosis Center, and 55 pairs of fresh human liver cancer tissue specimens were from the Li Huili Hospital of Ningbo Medical Center.

[0036] a. Collection, processing and preservation of fresh human liver cancer tissue specimens: On the premise of meeting the inclusion criteria and not affecting the pathological diagnosis of the specimens, a dedicated person took liver cancer tissue and adjacent cancer tissue (within 1 - 3 cm beside the cancer tissue) within 30 minutes after the specimens were removed from the body, cut them into small pieces (0.5×0.5×0.4 cm), rinsed the residual blood on the specimens with physiological saline, put the specimens into specimen bottles, tightened the bottle caps, noted information such as patient information and collection date, and immediately put the specimen bottles into liquid nitrogen for preservation.

[0037] b. Collection, processing and preservation of formalin-fixed liver cancer tissue specimens: The formalin specimens were the remaining liver tissues after pathological diagnosis by hepatectomy, and were fixed in a sufficient amount of 10% neutral formalin solution at room temperature. Before collecting the specimens, an appropriate amount of 4% paraformaldehyde fixative was added to 1.8 mL specimen tubes. A professional pathologist took one piece of liver cancer and adjacent cancer tissue (0.5×0.5×1 cm) from each sample, respectively put them into 1.8 mL specimen tubes, submerged the specimens with the liquid, tightened the lids, stored them in a 4°C refrigerator for standby, and regularly changed the 4% paraformaldehyde fixative.

[0038] Inclusion criteria for liver cancer tissue specimens in this example: 1. The pathological diagnosis of the samples was clear, and it was diagnosed as primary hepatocellular carcinoma; 2. The patients did not receive preoperative radiotherapy or chemotherapy; 3. The patients had no other systemic malignancies in the past. Exclusion criteria were: 1. Complicated with other systemic malignancies; 2. Patients who had received radiofrequency ablation and destruction treatment before surgery; 3. Patients with infectious diseases such as syphilis and AIDS. All patients were treated by hepatectomy, and the specimens were obtained after the patients signed the informed consent. All human tissue specimens involved in this example were applied by the research group and were only used for laboratory research under the consent of the Human Ethics Committee of Ningbo University School of Medicine.

[0039] Immunohistochemical staining of liver cancer

[0040] The liver cancer tissue specimens used in this example were all samples collected in the aforementioned experiment, and the specific steps were as follows:

[0041] a. Fixation of liver cancer tissue: Use forceps to pick up the formalin-fixed liver cancer tissue samples onto the unfolded plastic wrap, cut them into tissue blocks of 0.5×0.5×0.2 cm size with a sterile scalpel, and soak them in 4% paraformaldehyde solution for more than 24 hours.

[0042] b. Rinsing: The tissue embedding cassettes were strung together with white cotton threads of the same length. Use sterile forceps to place the fixed tissues into the tissue embedding cassettes, tighten the embedding cassettes, mark the relevant sample information on the embedding cassettes with a pencil, put the embedding cassettes in a foam box, rinse them under running tap water for 5 minutes, and drain the water.

[0043] c. Dehydration: Gradually dehydrate the tissue embedding cassettes by sequentially placing them into glass cylinders containing ethanol solutions of different concentrations prepared in advance. Immediately cover the glass cylinder lids after the cassettes are placed in the glass cylinders, and leave the cotton threads outside the glass cylinders for easy cylinder replacement. Immerse in 75% ethanol solution for 1 h × 2 times; 80% ethanol solution for 1 h × 2 times; 95% ethanol solution for 1 h × 2 times; 100% ethanol solution for 1 h × 2 times. Finally, drain the ethanol solution on the embedding cassettes.

[0044] d. Clearing: Immerse the tissue embedding cassettes in xylene solution for 40 min × 2 times.

[0045] e. Wax infiltration: Preheat the wax cylinders containing paraffin for sectioning with a melting point of 58 - 60 °C in a 60 °C oven to melt. Sequentially place the tissue embedding cassettes into three wax cylinders in the oven, and soak in each wax cylinder for at least 1 h. The embedding cassettes need to be fully immersed in the liquid paraffin.

[0046] f. Embedding: Preheat the embedding machine in advance to melt the paraffin in the machine. After all the paraffin has melted, place the iron box for embedding into the left wax cylinder of the embedding machine, and take out the embedding cassettes from the wax cylinders in the oven and place them into the right wax cylinder of the embedding machine. Remove the embedding cassette, place the tissue in the center of the embedding iron box, fix the tissue with forceps, pour liquid paraffin into the embedding iron box to submerge the tissue block, remove the lid of the plastic embedding cassette, gently cover it on the embedding iron box, place it on a 4 °C freezing table to cool into a solid wax block, discard the embedding iron box, place the wax block in a sealed bag, and store it in a 4 °C refrigerator for later use.

[0047] g. Sectioning: Use a paraffin microtome to roughly section the tissue at 10 μm to remove the paraffin without tissue, and polish the surface of the wax block smoothly. Cut the tissue wax block into 4 μm thin sections, place them in a 40 °C water bath for flattening, select the thin sections with complete tissue and regular shape, pick up the tissue sections with a positively charged glass slide to make them adhere to the glass slide, and mark the relevant information of the specimen with a pencil at the end of the glass slide.

[0048] h. Baking the sections: Place the glass slides with attached tissue on a preheated 65 °C slide dryer for baking for 2 h.

[0049] i. Dewaxing and rehydration: Place the slide rack full of sections into xylene for soaking for 20 min; into absolute ethanol solution for soaking for 5 min × 2 times; into 95% ethanol solution for soaking for 5 min × 1 time; into 75% ethanol solution for soaking for 5 min × 1 time; finally, place the slide rack in deionized water for washing for 5 min.

[0050] j. Antigen retrieval: Dilute the 50× sodium citrate antigen retrieval solution with deionized water to 1× and shake well. Pour the 1× sodium citrate antigen retrieval solution into the pressure cooker, close the lid. Wait until the liquid in the pot boils, then place the slide rack with the sections into the pressure cooker, ensuring that the sodium citrate antigen retrieval solution completely submerges the sections. Tighten the lid, start timing after the pressure cooker steam valve begins to emit steam evenly. After 8 minutes, turn off the power of the induction cooker to end the heating. Place the pressure cooker in running tap water to cool down, open the lid, and let the paraffin sections cool to room temperature.

[0051] k. Washing: First, wash the sections thoroughly with deionized water for 5 minutes, then wash the sections with PBS buffer for 3 minutes, and repeat the washing 3 times.

[0052] l. Blocking endogenous peroxidase: Dilute the 10% hydrogen peroxide aqueous solution with deionized water to a concentration of 3%. Prepare it freshly each time. Place the paraffin sections in a wet box, add an appropriate amount of 3% hydrogen peroxide aqueous solution to the position of the tissue on the glass slide, close the lid of the wet box, and block at 37°C for 10 minutes.

[0053] g. Washing: Wash the sections with PBS phosphate buffer for 3 minutes × 3 times, and shake off the liquid on the glass slide.

[0054] m. Serum blocking: Place the washed sections in a wet box, add an appropriate amount of 10% donkey serum blocking solution to each section. After blocking at room temperature for 15 minutes, shake off the blocking solution on the sections.

[0055] n. Primary antibody incubation: Wipe off the blocking solution around the tissue, use a hydrophobic pen for immunohistochemical staining to draw a circle around the tissue, place the sections in a wet box, add an appropriate concentration of INF2 antibody dilution solution, make the antibody dilution solution submerge the tissue, close the lid of the wet box, and place it in the 4°C refrigerator overnight.

[0056] o. Washing: Wash the sections with PBS phosphate buffer for 3 minutes × 3 times.

[0057] p. Secondary antibody incubation: Dry the liquid around the tissue, add an appropriate concentration of HRP-labeled donkey secondary antibody to the tissue, make the antibody completely cover the tissue, close the lid of the wet box, and incubate at 37°C for 1 hour.

[0058] q. Washing: Wash the sections with PBS phosphate buffer for 3 minutes × 3 times.

[0059] m. DAB chromogenic reaction: Drop the DAB chromogenic solution onto the position of the tissue on the glass slide, place the glass slide on an inverted microscope to observe the tissue staining situation. When the staining intensity reaches the best, shake off the chromogenic solution, and wash with deionized water for 3 minutes × 3 times.

[0060] r. Hematoxylin counterstaining: Drop an appropriate amount of modified Lillie-Mayer hematoxylin staining solution on the tissue location on the glass slide, stain for 2 min, discard the staining solution, and place the glass slide rack with the glass slide under running tap water for 10 min to blue back.

[0061] s. Dehydration: Immerse the glass slide rack with the sections in 75% ethanol solution for 5 min × 1 time; immerse in 95% ethanol solution for 5 min × 1 time; immerse in absolute ethanol solution for 5 min × 3 times.

[0062] t. Clearing: Immerse the sections in xylene solution for 5 min × 2 times.

[0063] u. Sealing: Drop an appropriate amount of neutral balsam on the tissue location on the glass slide, pick up a cover glass with forceps and gently cover it on the glass slide, and let it dry in a fume hood to form a film.

[0064] v. Scoring: Place the sections on an inverted microscope and observe the tissue morphology, nuclear staining, and staining of the target protein under a low-power microscope and a high-power microscope in turn.

[0065] Example 2

[0066] Detect the expression of BCLAF1 in hepatocellular carcinoma cells and its prognostic relationship

[0067] Download and organize the RNAseq data of the STAR process of the TCGA-LIHC (hepatocellular carcinoma) project from the TCGA database (https: / / portal.gdc.cancer.gov) and extract the data in TPM format and clinical data. Download and organize the hepatocellular liver protein expression data and clinical data from the CPTAC database (https: / / cptac-data-portal.georgetown.edu / ). Use the survival package to perform a proportional hazards hypothesis test and perform a fitted survival regression, and the results are visualized using the survminer package and the ggplot2 package, and the results are as Figure 1 、 Figure 2 、 Figure 3 shown.

[0068] According to the immunohistochemical score of BCLAF1 in hepatocellular carcinoma tissues, it is divided into 3 groups: Negative (0 points), Low-positive, and High-positive. Use SPSS 26.0 to statistically analyze the clinicopathological characteristics of each patient in the 3 groups respectively. P < 0.05 is statistically significant, and the specific results are shown in Table 1.

[0069] Table 1 Immunohistochemical correlation analysis

[0070]

[0071]

[0072] Based on the immunohistochemical results of 105 pairs of liver cancer tissues in Table 1, the correlation analysis between BCLAF1 expression and the clinicopathological characteristics of liver cancer patients shows that liver cancer patients with high BCLAF1 expression have a higher level of microvascular invasion.

[0073] Example 3

[0074] BCLAF1 interference and overexpression

[0075] Cell culture

[0076] Human liver cancer cells HepG2 and SK-Hep1 were cultured in high-glucose DMEM medium containing 10% vol fetal bovine serum in a 5% CO2, 37 °C cell culture incubator.

[0077] Overexpression and interference

[0078] Using CRISPR-Cas9 and plasmid transfection techniques, plasmids of negative control, overexpression, and stable knockout of BCLAF1 were respectively introduced into hepatocellular carcinoma cells HepG2 and SK-Hep1. After 48 hours, the cells were lysed and proteins were extracted. The negative control plasmid was the CD513B-cas9 empty vector, the overexpression plasmid was a recombinant plasmid prepared by ligating the sequence in SEQ ID No. 3 to the pEnter vector, and the plasmid for stable knockout of BCLAF1 was a recombinant plasmid prepared by ligating the sense strand in SEQ ID No. 1 and the antisense strand in SEQ ID No. 2 to the CD513B-cas9 vector. The protein level of BCLAF1 in HepG2 and SK-Hep1 cells was detected by Western blot. The specific steps are as follows:

[0079] a. Total protein extraction: When the cell confluence reached 80% after plating, the cells were harvested, washed twice with PBS, digested with trypsin, and then washed again with PBS. Cell counting was performed, and 1*10 6 total proteins in cells were extracted with 30 μl lysis buffer (1 M Tris (PH7.4), 10% Triton-100, 1 M NaCl, 0.5 M EDTA and dd H2O), lysed at 4 °C on a rotary shaker for 15 min, centrifuged at 12,000 rpm / min for 10 min, and the supernatant was taken.

[0080] b. Electrophoresis: After preparing 10% separating gel and sealing it with water, prepare the separating gel. After it solidifies for 30 min, pull out the comb in the electrophoresis buffer. Take 5 μl of the sample for loading. First, perform electrophoresis at a constant current of 20 mA until the marker starts to separate, and then perform electrophoresis at a constant current of 40 mA until the target bands are separated.

[0081] c. Blotting: Place the negative electrode plate, sponge pad, filter paper, gel, PVDF membrane, filter paper, sponge pad, and positive electrode plate in sequence, connect the power supply, and perform blotting at a constant current of 220 mA.

[0082] d. Blocking and incubating with primary antibody: Block with 5% skim milk on a shaker at room temperature for 1 h, and incubate with the primary antibody against BCLAF1 overnight at 4 °C.

[0083] e. Washing the membrane and incubating with secondary antibody: Wash the membrane 4 times with TBST at room temperature for 5 min each time. Incubate with the horseradish peroxidase-labeled secondary antibody on a shaker at room temperature for 2 h. Wash the membrane 4 times again with TBST for 5 min each time, add the ECL luminescent solution, and perform film exposure in the darkroom.

[0084] Example 4

[0085] Cell co-culture

[0086] a. Seed HepG2 and SK-Hep1 cells into 6-well plates. After 1 day, when the cell density reaches about 70%-80% confluence, transfect the OEBCLAF1 or cas9-BCLAF1 plasmid into the liver cancer cells for 48 h.

[0087] b. Next, activate Jurkat T cells with a human CD3 / CD28 T cell activator (Proteintech, #KMS310), and add Jurkat T cells to the liver cancer cells at a ratio of Jurkat T: liver cancer cells = 5:1 for co-culture.

[0088] c. After co-culturing for 48 h, collect Jurkat T cells and the supernatant. Detect the apoptosis and cell cycle of Jurkat T cells in different groups using a flow cytometer, and measure the cytokine concentration secreted by Jurkat T cells in the supernatant using an ELISA kit.

[0089] d. For cell apoptosis, stain phosphatidylserine (PS) and DNA of the cells using an apoptosis kit containing Annexin V labeled with the fluorescent dye Phycoerythrin (PE) + nucleic acid dye Aminoactinomycin D (7-AAD) and an apoptosis kit containing Annexin V labeled with the fluorescent dye 5-isothiocyanate fluorescein (FITC) + nucleic acid dye propidium iodide (PI). Perform further data analysis and plotting using FlowJo software (v10.8.1).

[0090] e. For the cell cycle, PI staining of DNA was performed using a cell cycle kit. The stained cells were analyzed using a Beckman Cytoflex flow cytometer, and further data analysis and graphing were performed using FlowJo software (v10.8.1). The experimental results are as Figures 4 to 9 shown.

[0091] The technical results corresponding to the attached drawings of the present invention are as follows:

[0092] Figure 1 In A, B, C, and D, they are visual diagrams of the expression of BCLAF1 in liver cancer tissues and adjacent tissues from the TCGA and CPTAC databases, indicating that the expression of BCLAF1 mRNA and protein in liver cancer is significantly higher than that in adjacent tissues;

[0093] Figure 2 In A, it is a representative immunohistochemical staining diagram of 105 pairs of fresh tissues from patients with fresh primary liver cancer, Figure 2 In B, it is the immunohistochemical result of the expression of BCLAF1 in 105 pairs of fresh tissues from patients with fresh primary liver cancer, indicating that the expression of BCLAF1 protein in hepatocellular carcinoma is significantly higher than that in adjacent tissues;

[0094] Figure 3 In A, it is a survival curve diagram of the expression of BCLAF1 and the overall survival of liver cancer patients based on the TCGA database. In B of 3, it is a survival curve diagram of the expression of BCLAF1 and the overall survival of liver cancer patients based on the CPTAC database, indicating that liver cancer patients with high expression of BCLAF1 have a lower overall survival;

[0095] Figure 4 In A, in the liver cancer cell line HepG2, after interfering with and overexpressing BCLAF1, the protein expression level indicates the success of interference and overexpression, Figure 4 In B, in the liver cancer cell line SK-Hep1, after interfering with and overexpressing BCLAF1, the protein expression level indicates the success of interference and overexpression;

[0096] Figure 5 In A, it is the experimental result diagram of flow cytometry after co-culturing with Jurkat T cells after interfering with and overexpressing BCLAF1 in the liver cancer cell line HepG2, Figure 5 In B, in the liver cancer cell line HepG2, after interfering with and overexpressing BCLAF1 and co-culturing with Jurkat T cells, with or without durvalumab, the change in the apoptosis level of Jurkat T cells was detected;

[0097] Figure 6Figure A in [reference] shows the results of flow cytometry experiments after co-culturing Jurkat T cells with SK-Hep1 hepatoma cells in which BCLAF1 was interfered with and overexpressed. Figure 6 In Figure B in [reference], after co-culturing Jurkat T cells with SK-Hep1 hepatoma cells in which BCLAF1 was interfered with and overexpressed, the changes in the apoptotic level of Jurkat T cells were detected with or without durvalumab.

[0098] Figure 7 Figure A in [reference] and Figure 7 Figure B in [reference] show the results of experiments on the cell cycle changes of Jurkat T cells after co-culturing with HepG2 hepatoma cells in which BCLAF1 was interfered with and overexpressed, with or without durvalumab.

[0099] Figure 8 Figure A in [reference] and Figure 8 Figure B in [reference] show the results of experiments on the cell cycle changes of Jurkat T cells after co-culturing with SK-Hep1 hepatoma cells in which BCLAF1 was interfered with and overexpressed, with or without durvalumab.

[0100] Figure 9 In HepG2 and SK-Hep1 hepatoma cells, after co-culturing Jurkat T cells in which BCLAF1 was interfered with and overexpressed, with or without durvalumab, the changes in the levels of cytokines secreted by Jurkat T cells were detected. Among them, Figure 9 Figure A shows the level of interleukin-2 (IL-2), Figure 9 Figure B shows the level of interleukin-4 (IL-4), Figure 9 Figure C shows the level of interleukin-10 (IL-10), Figure 9 Figure D shows the level of interferon-γ (IFN-γ).

[0101] In the specific embodiments of the present invention, it is found that the highly expressed BCLAF1 gene in hepatocellular carcinoma is closely related to durvalumab resistance; the overexpression of BCLAF1 inhibits the activity of T cells co-cultured with hepatoma cells. Compared with the BCLAF1 overexpression group, the BCLAF1 overexpression + drug addition group reversed the inhibitory effect of BCLAF1 overexpression on T cell activity. This indicates that BCLAF1 inhibits the activity of T cells by antagonizing durvalumab, and it can be considered that cells with BCLAF1 overexpression are resistant to durvalumab. Using the BCLAF1 inhibitor sgBCLAF1 to specifically inhibit the expression of the BCLAF1 gene can greatly inhibit the activity and function of Jurkat T cells co-cultured with hepatoma cells; it can effectively enhance the sensitivity of hepatoma cells to durvalumab, thereby significantly improving the efficacy of the durvalumab drug and reducing drug resistance. On the contrary, opposite results were observed after specifically overexpressing the BCLAF1 gene.

[0102] Therefore, on the basis of the above research, the present invention proposes the BCLAF1 inhibitor sgBCLAF1. This inhibitor can significantly enhance the sensitivity of hepatoma cells to durvalumab after reducing the expression level of BCLAF1.

[0103] Although the present disclosure is disclosed as above, the protection scope of the present disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present disclosure, and these changes and modifications will all fall within the protection scope of the present invention.

Claims

1. A sgRNA for targeted knockout of BCLAF1, characterized in that, The sequence of its sense strand is shown in SEQ ID No.1, and the sequence of its antisense strand is shown in SEQ ID No.

2.

2. Use of the sgRNA targeting and knocking out BCLAF1 according to claim 1, characterized in that, The applications include: using the sgRNA to prepare a drug for reversing the resistance of liver cancer cells to durvalumab; using the sgRNA to prepare a drug for enhancing the sensitivity of liver cancer cells to durvalumab.

3. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the sgRNA as claimed in claim 1.

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