Application of BNLF2b gene in gastric cancer

Abstract

The present invention belongs to the field of medicine, and specifically relates to the application of the BNLF2b gene in gastric cancer. Experiments of the present invention show that overexpression of BNLF2b can significantly inhibit the growth of EBV-positive gastric cancer cells, and the tumor volume and weight of the AGS / EBV cell group with overexpressed BNLF2b are significantly smaller than those of the control group. At the same time, BNLF2b enhances the antigen presentation ability by upregulating HLA-I expression, activates the anti-tumor immune response of cytotoxic T cells, and blocks the immune escape mechanism of tumor cells by inhibiting the expression of JAK2 and PD-L1. Western blot analysis confirmed that the protein expression levels of JAK2 and PD-L1 in the BNLF2b overexpression group were significantly reduced, while the HLA-I expression level was significantly increased. These findings provide an important basis for the development of novel treatment strategies based on the BNLF2b gene and have broad clinical application prospects.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to the application of BNLF2b gene in gastric cancer. Background Art

[0002] Gastric cancer is one of the malignant tumors with high morbidity and mortality rates worldwide. It is the fifth most common cancer and the third leading cause of cancer death in the world. Although some progress has been made in the diagnosis and treatment of gastric cancer in recent years, its prognosis is still poor, especially for patients with advanced gastric cancer, whose 5-year survival rate is less than 30%. Currently, the main treatments for gastric cancer include surgical resection, chemotherapy, and radiotherapy, but these methods have limited therapeutic effects on advanced patients and are often accompanied by serious side effects. In addition, the high heterogeneity and complex tumor microenvironment of gastric cancer make it difficult for traditional treatments to meet the needs of personalized treatment, and new treatment strategies are urgently needed.

[0003] In recent years, immunotherapy, as an emerging anti-tumor method, has shown significant therapeutic effects in many cancers. However, its application in gastric cancer still faces many challenges. First, gastric cancer cells often evade immune surveillance by downregulating the expression of major histocompatibility complex (MHC) molecules (such as HLA-I), resulting in decreased antigen presentation ability, thereby weakening the anti-tumor immune response of T cells. Secondly, the overexpression of immunosuppressive molecules (such as PD-L1) in the tumor microenvironment further inhibits the activity of T cells and promotes tumor immune escape. Therefore, how to effectively enhance antigen presentation ability and block immune escape mechanisms has become a key issue that needs to be urgently addressed in the field of gastric cancer immunotherapy.

[0004] Epstein-Barr virus (EBV) infection is closely related to the occurrence and development of some gastric cancers, and about 10% of gastric cancer cases are EBV positive. EBV-positive gastric cancer has unique molecular characteristics and clinical pathological manifestations, which provides a potential target for the development of specific immunotherapy for EBV-related antigens. However, the current research on immunotherapy for EBV-positive gastric cancer is still in its early stages, and an effective strategy that can simultaneously enhance antigen presentation ability and block immune escape mechanisms has not yet been found. Therefore, it is of great clinical significance to explore the mechanism of action of EBV-related proteins in gastric cancer and develop new therapeutic methods based on their functions. Summary of the invention

[0005] In view of the shortcomings of the existing problems, the object of the present invention is to provide an application of the BNLF2b gene in gastric cancer.

[0006] The technical solution adopted by the present invention to solve the technical problem is:

[0007] In a first aspect, the present invention protects the use of the BNLF2b gene or the protein encoded by the BNLF2b gene in treating gastric cancer.

[0008] In a second aspect, the present invention protects the use of a substance overexpressing the BNLF2b gene in the following (A1) or (A2):

[0009] Use in the preparation of a product for treating gastric cancer;

[0010] Use in the preparation of a product for inhibiting the proliferation and / or growth of gastric cancer cells.

[0011] In a specific embodiment, the gastric cancer is EBV-positive gastric cancer.

[0012] In a specific embodiment, the gastric cancer cells are EBV-positive gastric cancer cells.

[0013] In a specific embodiment, the substance overexpressing BNLF2b is a recombinant vector containing the BNLF2b gene.

[0014] In a more specific embodiment, the backbone vector of the recombinant vector is GV341.

[0015] In a specific embodiment, the product is a drug.

[0016] In a more specific embodiment, the drug further contains other pharmaceutically acceptable carriers.

[0017] In a third aspect, the present invention also provides a method for treating gastric cancer, which is achieved by overexpressing BNLF2b.

[0018] Advantageous Effects

[0019] The application of the BNLF2b gene provided by the present invention in gastric cancer has the following advantageous effects compared with the prior art:

[0020] By constructing an EBV-positive gastric cancer research model based on BNLF2b overexpression, the present invention reveals the dual functions of BNLF2b in inhibiting tumor progression and activating anti-tumor immunity, achieving remarkable effects. Experimental data show that overexpression of BNLF2b can significantly inhibit the growth of EBV-positive gastric cancer cells. In in vivo experiments, the tumor volume and weight of the AGS / EBV cell group overexpressing BNLF2b are significantly smaller than those of the control group.

[0021] Meanwhile, BNLF2b enhances antigen presentation ability by upregulating HLA-I expression, activates the anti-tumor immune response of cytotoxic T cells, and blocks the immune escape mechanism of tumor cells by inhibiting the expression of JAK2 and PD-L1. Western blot analysis confirms that the protein expression levels of JAK2 and PD-L1 in the BNLF2b overexpression group are significantly reduced, while the HLA-I expression level is significantly increased.

[0022] Compared with the prior art, the present invention has significant advantages in simulating the human immune system, revealing the mechanism of BNLF2b, shortening the experimental cycle, and supporting the development of treatment strategies. By optimizing the experimental procedures and shortening the experimental cycle, the present invention reduces the usage of experimental animals and experimental costs, and is more environmentally friendly and economical. In summary, the present invention provides an efficient and accurate tool and method for the research and treatment of EBV-related gastric cancer, and has important scientific value and clinical application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Flow chart of siRNA knockdown.

[0024] Figure 2 Gene expression levels after BNLF2b knockdown, where Figure 2 Panel A in shows the gene expression levels 24 h after BNLF2b knockdown, Figure 2 Panel B in shows the gene expression levels 48 h after BNLF2b knockdown.

[0025] Figure 3 Relationship diagram between cell survival rate and concentration logarithm at different puromycin concentrations.

[0026] Figure 4 BNLF2b regulates the expression of JAK2, PD-L1 and HLA-I in AGS / EBV cells; among them, Figure 4 The RT-qPCR results in Panel A show that the present invention successfully established a cell line with stable overexpression of BNLF2b; Figure 4 Panel B in shows the effect of overexpression of BNLF2b on the gene expression of JAK2; Figure 4 Panel C in shows the effect of overexpression of BNLF2b on the gene expression of CD274; Figure 4 Panel D in shows the representative flow cytometry histogram of HLA-1 expression after overexpression of BNLF2b; Figure 4 Panel E in shows the statistical analysis of HLA-1 expression level after overexpression of BNLF2b; Figure 4 Panel F in shows the representative flow cytometry histogram of HLA-1 expression after knockdown of BNLF2b; Figure 4 Panel G in shows the statistical analysis of HLA-1 expression level after knockdown of BNLF2b.

[0027] Figure 5 Results of mouse experiments on BNLF2b, where Figure 5 Panel A in shows a schematic diagram of the experimental process for evaluating the effect of BNLF2b on tumors in animal models; Figure 5 Panel B in shows the line graph of mouse body weight changes; Figure 5 Panel C in shows the line graph of tumor volume changes;Figure 5 Figure D in [it] is a direct shot of a tumor camera; Figure 5 Figure E in [it] is a Western Blot of the protein expressions of JAK2 and PD-L1 in a tumor tissue sample of a mouse overexpressing BNLF2b; Figure 5 Figure F in [it] is a statistical chart of the Western Blot of the protein expression of PD-L1 in a tumor tissue sample of a mouse overexpressing BNLF2b; Figure 5 Figure G in [it] is a statistical chart of the Western Blot of the protein expression of JAK2 in a tumor tissue sample of a mouse overexpressing BNLF2b. Specific implementation manners

[0028] The present invention will be further described in detail below in conjunction with embodiments. Reagents or instrument and equipment not indicating the manufacturer are regarded as conventional products that can be purchased on the market.

[0029] The human EBV-positive gastric cancer cell line AGS / EBV was purchased from the Biological Cell Laboratory of Central South University (China, Hunan). After the cells arrived, they were amplified and immediately aliquoted and cryopreserved. All cell lines were identified by STR and used within 6 months, and the passage times were controlled between 15 and 20 generations. Mycoplasma contamination was excluded using the MycAway™ Plus-Color one-step mycoplasma detection kit (40612ES, Yeasen Biotech). The cell lines were cultured in RPMI-1640 medium (KGL1501-500, KeyGen Biotech) containing 10% fetal bovine serum (C2910-0500, Viva Cell) and 1% penicillin-streptomycin. All cells were maintained in an incubator at 37 °C and 5% CO 2 2.

[0030] Human peripheral blood mononuclear cells (PBMC), product number: FPB004F-C, were purchased from Shanghai Aoneng Biotechnology Co., Ltd.

[0031] 6-8-week-old SPF-grade female severe combined immunodeficiency mice C-NKG, weighing 22±2 g, were purchased from Cyagen Biosciences (Suzhou) Co., Ltd. and raised in the SPF-grade barrier facility of the Experimental Animal Center of China Pharmaceutical University. In a 12-hour light / dark cycle (07:30-19:30 light, 5 mice per group, and the mice were raised in each individually ventilated cage; 19:30-7:30 dark), the room temperature (23±2 °C) and relative humidity (40-50%) were controllable. The mice were marked before grouping and then randomly divided into different groups by an independent person. We used 6 mice per group. We ensured that the experimental groups were balanced in terms of animal age and weight. All experimental animal operations complied with the standards of the Experimental Animal Welfare and Ethics Review Committee of China Pharmaceutical University.

[0032] Example 1 Determination of BNLF2b knockdown conditions

[0033] Suzhou GenePharma Co., Ltd. purchased the BNLF2b triple guarantee one siRNA package, which contains the negative control siNC and the positive control siPC (siGAPDH). The specific sequences are shown in Table 1. The siRNA powder was centrifuged at 4000 r / min, and after adding an appropriate amount of DEPC water, the concentration was adjusted to 40 μM. It was vortexed and mixed well to dissolve the powder in the DEPC water.

[0034] The knockdown process is as Figure 1 shown. Seed a 6-well plate with 15×10^4 cells / well and culture for 18 h. Transfection was carried out according to the GP-transfecr-mate instruction manual (product number: G04008, Suzhou GenePharma Co., Ltd.). First, prepare the diluted solutions of siRNA with different fragments and the corresponding diluted transfection reagent. Mix gently with a pipette gun, let it stand at room temperature for 5 min, then slowly add the diluted transfection reagent to the siRNA diluted solution. After gently mixing with a pipette gun and standing at room temperature for 15 min (during the standing period, change the medium in the 6-well plate to 1.6 mL of preheated fresh medium / well), the transfection complex was obtained. The transfection complex was added dropwise to the 6-well plate. After 6 h, change the medium to fresh complete medium. At 24 h and 48 h after transfection, the gene expression level was detected by RT-qPCR method (the primers are shown in Table 2). The knockdown efficiency is as Figure 2 shown. Select the siBNLF2b-3 fragment transfected for 48 h for subsequent experimental research. Note: The optimal diluted transfection reagent volume and siRNA dosage obtained from the previous experimental optimization for AGS / EBV cells are 7.5 μL and 300 pmol, that is, 7.5 μL of siRNA solution with a concentration of 40 μM.

[0035]

[0036]

[0037] Example 2 Construction of BNLF2b overexpression stable lentivirus

[0038] The BNLF2b overexpression sequence was optimized by Shanghai Sangon Biotech Co., Ltd. mainly based on the human BNLF2b sequence and supplemented by the mouse sequence. The optimized gene sequence is:

[0039] gctagcGccaccATGCGGCCCGGCCGGCCCCTGGCCGGCTTCTACGCCACCCTGCGGAGGAGCTTCCGGAGGATGTCCAAGCGGAGCAAAAACAAGGCCAAGAAGGAAAGGGTGCCTGTGGAGGATAGGCCCCCCACCCCCATGCCCACAAGCCAGCGGCTGATCAGGCGGAACGCCCTGGGGGGCGGGGTGAGGCCCGACGCCGAGGACTGCATCCAGAGGTTCCACCCCCTGGAGCCCGCCCTGGGGGTGAGCACAAAGAACTTCGATCTGCTGAGCCTGAGGTGCGAGCTGGGGTGGTGCGGCTAAaagctt, as shown in SEQ ID NO:1;

[0040] The AA sequence is:

[0041] ASATMRPGRPLAGFYATLRRSFRRMSKRSKNKAKKERVPVEDRPPTPMPTSQRLIRRNALGGGVRPDAEDCIQRFHPLEPALGVSTKNFDLLSLRCELGWCG*, as shown in SEQ ID NO:2.

[0042] The lentivirus with stable overexpression of BNLF2b was constructed by Shanghai GeneChem Co., Ltd. Specific information of the vector: The cloning vector is GV341, and the vector element order is Ubi-MCS-3FLAG-SV40-puromycin; Specific information of the lentivirus: 50 µL, titer / concentration is 3.5×10^8 TU / mL, 7 tubes, the actual total amount is 1.225×10^8 TU, stored in a -80 °C refrigerator.

[0043] Example 3 Construction of a stable cell line with overexpression of BNLF2b (AGS / EBV-BNLF2b-OE)

[0044] A 96-well plate, with a cell density of 5×10^4 cells / mL and 5000 cells per well. After the cells adhered, puromycin was added. 1 µL of 5 mg / mL puromycin was diluted to 0.5 mL to obtain 10 µg / mL puromycin; 10 µg / mL puromycin was diluted to different concentrations: 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 µg / mL, and the medium was changed. Fresh puromycin-containing medium was changed every day for the next 4 days. On the 5th day, the minimum puromycin concentration that could remove 80 - 100% of the cells was obtained using the MTT method. This concentration was used for the subsequent stable cell line screening steps. The puromycin IC50 of human EBV-positive gastric cancer cell line AGS / EBV cells was calculated to be 0.2065 µg / mL by the MTT method, and R 2 = 0.9720 (as Figure 3 shown), the minimum puromycin concentration of 0.4 µg / mL that could remove 80 - 100% of the cells was selected as the screening concentration for the stable cell line, and 1 / 10 of the concentration, i.e., 0.04 µg / mL, was used as the maintenance concentration for the stable cell line.

[0045] For a 48-well plate, 1 / 10 of the cells in a large dish that were confluent were diluted to 10 mL, with 0.2 mL per well. The next day, after the cells adhered, 200 µL of fresh medium was changed, and 8 µL of 25× infection reagent A (product number: REVG003, Haijikai Gene Medicine Technology Co., Ltd.) was added to each well. Then, the corresponding volume of virus was added to each well (virus volume = (MOI × number of cells) / virus titer, MOI was calculated as 10, the number of cells per well in the 48-well plate was calculated as 10000, and the virus titer was 3.5×10^8 TU / mL); the cells were cultured at 37°C for 12 - 16 h, then changed to complete medium and continued to be cultured; gene expression detection could be performed by qPCR ( Figure 4 Figure A in

[0046] Example 4 In vitro study on the function of BNLF2b gene

[0047] To deeply explore whether BNLF2b exerts anti-tumor immune effects by inhibiting JAK2 and PD-L1, overexpression and knockdown experiments of BNLF2b were respectively carried out in human EBV-positive gastric cancer cell line AGS / EBV cells. The qPCR results showed that overexpression of BNLF2b significantly inhibited the expression of JAK2 and PD-L1 ( Figure 4Figures B and C in). Additionally, given that HLA-I plays a crucial role in the antigen presentation process and its upregulation can activate the anti-tumor immune response and inhibit tumor progression, we further investigated the possible effect of BNLF2b on HLA-I expression. The results of flow cytometry showed that overexpression of BNLF2b could upregulate the expression of HLA-I ( Figure 4 Figures D and E in), while knockdown of BNLF2b showed a phenomenon of downregulating HLA-I expression ( Figure 4 Figures F and G in). These findings together suggest that overexpression of EBV-encoded BNLF2b may synergistically promote the anti-tumor immune response by simultaneously inhibiting JAK2, reducing the expression level of PD-L1, and increasing HLA-I expression. This series of findings provides a theoretical basis and experimental support for further research on BNLF2b as a viral gene with potential important value in cancer treatment, and also reveals a new perspective on its mechanism of action and therapeutic potential.

[0048] Example 5 Inhibitory effect of BNLF2b overexpression in the PBMC-CDX model in vivo

[0049] To further investigate whether overexpression of BNLF2b can inhibit the progression of EBV-related gastric cancer in vivo, we established a stable cell line with overexpression of BNLF2b (AGS / EBV-BNLF2b-OE) using the AGS / EBV cell line, and at the same time established a control group (AGS / EBV-Vector-OE). C-NKG mice were randomly divided into two groups: the AGS / EBV-Vector-OE group and the AGS / EBV-BNLF2b-OE group. In the experiment, first, human peripheral blood mononuclear cells (hPBMCs) were injected intravenously, and then the corresponding tumor cell line was injected subcutaneously the next day. The mice were continuously observed, and their physiological status and tumor progression were recorded. After the mice reached the end-point criteria, they were euthanized, and various tissue samples were collected to further evaluate the anti-tumor immune effect of BNLF2b in vivo (the experimental flow chart is as shown in Figure 5 Figure A in).

[0050] By observing and recording the tumor growth curve ( Figure 5 Figure C in) and taking pictures of the tumors of tumor-bearing mice and the pictures after tumor dissection ( Figure 5 Figure D in), we found that the cell line with overexpression of BNLF2b significantly inhibited tumor growth in the humanized EBV-positive xenograft model. Figure 5 The growth curve of the body weight of the mice in Figure B shows that there is no significant change in the body weight of the mice, indicating that overexpression of BNLF2b does not have a toxic effect on the mice.

[0051] Furthermore, we performed Western blot analysis on tumor tissues, and the results showed that BNLF2b could effectively inhibit the protein expression levels of JAK2 and PD-L1 in vivo ( Figure 5 Figures E-G in

[0052] ). This finding strongly suggests that BNLF2b may exert a potential anti-gastric cancer immune effect in vivo through its functions of inhibiting JAK2 and PD-L1. The protection scope of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be conceived by those skilled in the art are included in the present invention, and the appended claims are taken as the protection scope.

Claims

1. Use of a substance that overexpresses the BNLF2b gene in the following (A1) or (A2): (A1) Application in the preparation of products for treating gastric cancer; (A2) Use in the preparation of products for inhibiting the proliferation and / or growth of gastric cancer cells; in, The amino acid sequence of the protein encoded by the BNLF2b gene is shown in SEQ ID NO: 2; The gastric cancer is EBV-positive gastric cancer, and the gastric cancer cells are EBV-positive gastric cancer cells; The substance for overexpressing BNLF2b is a recombinant vector containing the BNLF2b gene; The product described is a drug.

2. The use according to claim 1, characterized in that: The backbone vector of the recombinant vector is GV341.

3. The use according to claim 1, characterized in that: The medicine also contains other pharmaceutically acceptable carriers.

Images