Application of BNLF2b gene in gastric cancer

Abstract

The invention belongs to the field of medicines, and particularly relates to application of a BNLF2b gene in gastric cancer. Experiments show that BNLF2b overexpression can significantly inhibit the growth of EBV positive gastric cancer cells, and the tumor volume and weight of an AGS / EBV cell group of the overexpression BNLF2b are significantly smaller than those of a control group. Meanwhile, the BNLF2b enhances the antigen presentation capability by up-regulating the expression of HLA-I, 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 proves that the expression level of JAK2 and PD-L1 proteins of a BNLF2b overexpression group is remarkably reduced, and the expression level of HLA-I is remarkably improved. The discovery provides an important basis for developing a novel treatment strategy based on the BNLF2b gene, and has a wide clinical application prospect.

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 provides a substance for protecting overexpression of the BNLF2b gene for use in the following (A1) or (A2):

[0009] (A1) Application in the preparation of products for treating gastric cancer;

[0010] (A2) 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 that overexpresses 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 specific embodiments, the product is a pharmaceutical.

[0016] In a more specific embodiment, the medicament 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] Beneficial Effects

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

[0020] The present invention has achieved remarkable results by constructing an EBV-positive gastric cancer research model based on overexpression of BNLF2b, revealing the dual functions of BNLF2b in inhibiting tumor progression and activating anti-tumor immunity. Experimental data showed 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 were significantly smaller than those of the control group.

[0021] At the same time, BNLF2b enhanced antigen presentation ability by upregulating HLA-I expression, activated the anti-tumor immune response of cytotoxic T cells, and blocked the immune escape mechanism of tumor cells by inhibiting the expression of JAK2 and PD-L1. Western blot analysis confirmed that the expression levels of JAK2 and PD-L1 proteins in the BNLF2b overexpression group were significantly reduced, while the expression level of HLA-I was 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 steps and shortening the experimental cycle, the present invention reduces the use of experimental animals and experimental costs, and is more environmentally friendly and economical. In summary, the present invention provides efficient and accurate tools and methods for the research and treatment of EBV-related gastric cancer, which has important scientific value and clinical application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 siRNA knockdown flow chart.

[0024] Figure 2 The gene expression levels after BNLF2b knockdown, including Figure 2 Figure A shows the gene expression level after BNLF2b knockdown for 24 h. Figure 2 Figure B shows the gene expression level after BNLF2b knockdown for 48 h.

[0025] Figure 3 The relationship between cell survival rate and the logarithm of concentration under different concentrations of puromycin.

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

[0027] Figure 5 The results of the mouse experiments on BNLF2b, among which, Figure 5 Figure A shows a schematic diagram of the experimental process for evaluating the effect of BNLF2b on tumors in animal models; Figure 5 Figure B is a line graph of the weight changes of mice; Figure 5 Figure C in the figure is a line graph of tumor volume changes; Figure 5 Figure D in the figure is a direct-view image of the tumor by camera; Figure 5 Figure E is a Western Blot diagram of JAK2 and PD-L1 protein expression in tumor tissue samples of BNLF2b-overexpressing mice; Figure 5 Figure F is a statistical graph of the Western Blot graph of PD-L1 protein expression in tumor tissue samples of BNLF2b-overexpressing mice; Figure 5 Figure G is a statistical graph of the Western Blot graph of JAK2 protein expression in tumor tissue samples of BNLF2b-overexpressing mice. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below with reference to the examples. The reagents or instruments used without indicating the manufacturer are all 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 (Hunan, China). Upon arrival, the cells were amplified and immediately aliquoted for cryopreservation. All cell lines were authenticated by STR and used within 6 months, with the number of passages controlled at 15 to 20. Mycoplasma contamination was excluded using the MycAway™ Plus-Color One-Step Mycoplasma Detection Kit (40612ES, Yisheng Bio). The cell lines were cultured in RPMI-1640 medium (KGL1501-500, Keygen Biotechnology) 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% CO2.

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

[0031] SPF female severe combined immunodeficient mice C-NKG aged 6 to 8 weeks, weighing 22 ± 2 g, were purchased from Saiye (Suzhou) Biotechnology Co., Ltd. and raised in the SPF barrier facility of the Experimental Animal Center of China Pharmaceutical University. Mice were housed in individually ventilated cages in a 12-h light / dark cycle (07:30-19:30 light, 5 mice per group; 19:30-7:30 dark), room temperature (23 ± 2 °C) and relative humidity (40-50%) were controlled. 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 were in accordance with the standards of the Experimental Animal Welfare Ethics Review Committee of China Pharmaceutical University.

[0032] Example 1 Determination of BNLF2b knockdown conditions

[0033] Suzhou Genema Gene Co., Ltd. purchased the BNLF2b three-in-one siRNA package, which contained negative control siNC and positive control siPC (siGAPDH), and the specific sequences are shown in Table 1. The siRNA powder was centrifuged at 4000 r / min, and an appropriate amount of DEPC water was added to make the concentration 40 μM, and the powder was vortexed to dissolve in the DEPC water.

[0034] The knock-down process is as follows Figure 1 As shown, 6-well plates were plated, 15×10^4 cells / well were cultured for 18 h, and transfection was performed according to the GP-transfecr-mate instruction manual (Cat. No.: G04008, Suzhou Jima Gene Co., Ltd.). First, siRNA dilutions of different fragments and corresponding transfection reagent dilutions were prepared, and the dilutions were gently mixed with a pipette. After standing at room temperature for 5 min, the transfection reagent dilution was slowly added to the siRNA dilution. After gently mixing with a pipette, the cells were allowed to stand at room temperature for 15 min (during the standing period, 1.6 mL of preheated fresh culture medium / well was replaced for the 6-well plate cells) to obtain the transfection complex, which was added dropwise to the 6-well plate. After 6 h, the medium was replaced with fresh complete culture medium. The gene expression level was detected by RT-qPCR method (primers are shown in Table 2) at 24 h and 48 h after transfection. The knockdown efficiency is shown in Table 2. Figure 2 As shown, the siBNLF2b-3 fragment was selected for transfection for 48 h for subsequent experimental studies. Note: The optimal dilution volume of transfection reagent for AGS / EBV cells obtained by previous experiments was 7.5 μL and the amount of siRNA was 300 pmol, that is, 7.5 μL of 40 μM siRNA solution.

[0035]

[0036]

[0037] Example 2 Construction of BNLF2b overexpression stable lentivirus

[0038] The BNLF2b overexpression sequence was obtained by Shanghai Sangon Biotechnology Co., Ltd. based on the BNLF2b sequence optimized with human as the main component and mouse as the auxiliary component. The optimized gene sequence is:

[0039] gctagcGccaccATGCGGCCCGGCCGGCCCCTGGCCGGCTTCTACGCCACCCTGCGGAGGAGCTTCCGGAGGATGTCCAAGCGGAGCAAAAACAAGGCCAAGAAGGAAAGGGTGCCTGTGGAGGATAGGCCCCCCACCCCATGCCCACAAGCCAGCGGC TGATCAGGCGGAACGCCCTGGGGGGCGGGGTGAGGCCCGACGCCGAGGACTGCATCCAGAGGTTCCACCCCCTGGAGCCCGCCCTGGGGGTGAGCACAAAGAACTTCGATCTGCTGAGCCTGAGGTGCGAGCTGGGGTGGTGCGGCTAAaagctt, such as SEQ Shown as ID NO:1;

[0040] The AA sequence is:

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

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

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

[0044] 96-well plates, 5×10^4 cells / mL, 5000 cells / well, puromycin was added after the cells adhered, 1 µL 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 of puromycin: 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 culture medium was replaced every day for the next 4 days. On the 5th day, the MTT method was used to obtain the minimum puromycin concentration that could remove 80-100% of the cells. This concentration was used for the subsequent stable cell screening step. The IC50 of puromycin for human EBV-positive gastric cancer cell line AGS / EBV cells was calculated by the MTT method to be 0.2065 µg / mL, R 2 =0.9720 (e.g. Figure 3 As shown in the figure, the minimum puromycin concentration of 0.4 µg / mL that can eliminate 80-100% of the cells was selected as the stable cell line screening concentration, and 1 / 10 concentration, i.e. 0.04 µg / mL, was selected as the stable cell line maintenance concentration.

[0045] In a 48-well plate, dilute 1 / 10 of the cells in a large dish full of cells to 10 mL, 0.2 mL / well. After the cells adhered to the wall the next day, replace with 200 µL of fresh culture medium, add 8 µL of 25× infection reagent A (Cat. No.: REVG003, Haijikai Gene Medicine Technology Co., Ltd.) to each well, and then add the corresponding volume of virus to each well (virus volume = (MOI×cell number) / virus titer, MOI is calculated as 10, the number of cells per well of the 48-well plate is calculated as 10, and the virus titer is 3.5×10^8 TU / mL); culture at 37℃ for 12-16 h, replace with complete culture medium, and continue to culture; qPCR can be used to detect gene expression ( Figure 4 Figure A shows that the overexpression was successful). Alternatively, the cell line can be expanded by continuing to culture and adding puromycin for selection for one week, and then maintaining the concentration of puromycin to obtain an overexpressing stable cell line.

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

[0047] In order to further explore whether BNLF2b exerts anti-tumor immune effects by inhibiting JAK2 and PD-L1, we performed overexpression and knockdown experiments of BNLF2b in human EBV-positive gastric cancer cell line AGS / EBV cells. qPCR results showed that overexpression of BNLF2b significantly inhibited the expression of JAK2 and PD-L1 ( Figure 4In addition, given that HLA-I plays a key role in antigen presentation and its upregulation can activate anti-tumor immune responses and inhibit tumor progression, we further investigated the possible effects of BNLF2b on HLA-I expression. Flow cytometry results showed that overexpression of BNLF2b could upregulate HLA-I expression ( Figure 4 Figure D and Figure E in the figure), knockdown of BNLF2b showed a phenomenon of down-regulating HLA-I expression ( Figure 4 F and G in Figure 1). Together, these findings suggest that overexpression of EBV-encoded BNLF2b may synergistically promote anti-tumor immune responses by simultaneously inhibiting JAK2, reducing PD-L1 expression levels, 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 value in cancer treatment, and also reveals new perspectives on its mechanistic role and therapeutic potential.

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

[0049] To further study whether BNLF2b overexpression can inhibit the progression of EBV-related gastric cancer in vivo, we used the AGS / EBV cell line to establish a stable cell line (AGS / EBV-BNLF2b-OE) that stably overexpresses BNLF2b, and at the same time established a control group (AGS / EBV-Vector-OE). C-NKG mice were randomly divided into two groups: AGS / EBV-Vector-OE group and AGS / EBV-BNLF2b-OE group. In the experiment, human peripheral blood mononuclear cells (hPBMCs) were first injected intravenously, and then the corresponding tumor cell lines were injected subcutaneously the next day. The mice were continuously observed, and their physiological state and tumor progression were recorded. After the mice reached the endpoint 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 shown in the figure). Figure 5 (as shown in Figure A).

[0050] By observing and recording the tumor growth curve ( Figure 5 C in the figure), taking pictures of tumors in tumor-bearing mice and pictures after the tumors were removed ( Figure 5 (D) We found that BNLF2b-overexpressing cell lines significantly inhibited tumor growth in a humanized EBV-positive xenograft model. Figure 5 The mouse weight growth curve in Figure B shows that there is no significant change in the mouse weight, indicating that overexpression of BNLF2b does not have a toxic effect on 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 This finding strongly suggests that BNLF2b may exert a potential anti-gastric cancer immune effect in vivo by inhibiting the functions of JAK2 and PD-L1.

[0052] The protection content 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 thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

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 a product for inhibiting the proliferation and / or growth of gastric cancer cells.

2. The use according to claim 1, characterized in that: The gastric cancer is EBV-positive gastric cancer.

3. The use according to claim 1, characterized in that: The gastric cancer cells are EBV-positive gastric cancer cells.

4. The use according to claim 1, characterized in that: The substance for overexpressing BNLF2b is a recombinant vector containing the BNLF2b gene.

5. The use according to claim 4, characterized in that: The backbone vector of the recombinant vector is GV341.

6. The use according to claim 1, characterized in that: The product described is a drug.

7. The use according to claim 6, characterized in that: The medicine also contains other pharmaceutically acceptable carriers.

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