Application of PRMT5 inhibited NK-92 cell in preparation of medicine for treating colorectal cancer

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses application of PRMT5 inhibited NK-92 cells in preparation of medicines for treating colorectal cancer, and the preparation process of the PRMT5 inhibited NK-92 cells comprises the following steps: inoculating the NK-92 cells to a culture medium containing 1-3 [mu] m < 1 > / L of EPZ015666, and culturing to obtain the PRMT5 inhibited NK-92 cells. Compared with NK-92 cells, the PRMT5 inhibited NK-92 cells provided by the invention have the advantages that the tumor cell killing capability is remarkably improved, and the tumor volume and weight can be remarkably inhibited.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of PRMT5-inhibited NK-92 cells in the preparation of drugs for treating colorectal cancer. Background Art

[0002] Cancer is a major public health problem worldwide and the second leading cause of death. Among them, colorectal cancer (CRC) is the digestive tract malignant tumor with the highest incidence rate. According to research reports, colorectal cancer is one of the three cancers with the most global incidence cases, second only to breast cancer and lung cancer, and the mortality rate ranks second, second only to lung cancer.

[0003] Colorectal cancer has a hidden onset. In the early stage, only fecal occult blood positivity is often seen, and then the following clinical manifestations may occur: changes in defecation habits and fecal shape, abdominal pain, rectal and abdominal masses, and a series of systemic conditions, etc. Complications are seen in the advanced stage, mainly including intestinal obstruction, intestinal bleeding, and related complications caused by peritoneal metastasis of the cancer.

[0004] The development of modern biomedicine has brought more options for the treatment of colorectal cancer, including surgical resection, radiotherapy, chemotherapy, immunotherapy, targeted therapy, and extensive surgery and local ablation therapy for metastatic lesions, etc. These treatment methods effectively inhibit the development of CRC and prolong the survival period of patients. However, due to the hidden onset, the vast majority of colorectal cancer patients are in the middle and advanced stages at the time of initial diagnosis and lose the valuable opportunity for radical treatment such as surgical resection and local ablation.

[0005] With the rapid progress of tumor immunotherapy in recent years, studies have shown that immune checkpoint sites exist not only in T lymphocytes but also in natural killer (NK) cells. NK cells are an important part of the body's innate immune system, with dual functions of cytotoxicity and immune regulation, and play an important role in the body's anti-tumor immune response. A large number of studies have shown that compared with the normal colonic epithelial microenvironment, the infiltration number and killing function of NK cells are significantly down-regulated in CRC, and CRC patients with a relatively large number of tumor-infiltrating NK cells have a good prognosis, suggesting that NK cells play an important anti-tumor immune role in CRC, but most are in a state of exhaustion and dysfunction, resulting in poor anti-tumor effects. Therefore, it is necessary to explore a way to improve the curative effect of NK-92 cells in the treatment of colorectal cancer. Summary of the Invention

[0006] In order to explore and develop a way to improve the curative effect of NK-92 cells in the treatment of colorectal cancer, the present invention provides the application of PRMT5-inhibited NK-92 cells in the preparation of drugs for treating colorectal cancer. The PRMT5-inhibited NK-92 cells provided by the present invention have significantly improved killing ability against tumor cells compared with NK-92 cells, and can significantly inhibit tumor volume and weight.

[0007] The present invention provides the use of PRMT5-inhibited NK-92 cells in the preparation of drugs for treating colorectal cancer.

[0008] In the present invention, NK-92 cells were successfully transformed by inhibiting the expression of the PRMT5 gene in NK-92 cells to obtain PRMT5-inhibited NK-92 cells (E-NK-92 cells). Compared with NK-92 cells, E-NK-92 cells change the functional exhaustion state of NK cells, the phenotype changes, the expression of inhibitory receptors NKG2A and IL-1R8 decreases, which can prevent NK cell exhaustion, maintain NK cell activity, and significantly improve the killing ability of tumor cells, and can significantly inhibit tumor volume and weight.

[0009] Furthermore, the PRMT5-inhibited NK-92 cells are obtained by targeting and inhibiting the expression of the PRMT5 gene in NK-92 cells.

[0010] Furthermore, the method for targeting and inhibiting the expression of the PRMT5 gene in NK-92 cells is as follows: by adding a PRMT5 inhibitor during the culture of NK-92 cells to target and inhibit the PRMT5 gene in NK-92 cells, the transformed PRMT5-inhibited NK-92 cells are obtained.

[0011] Furthermore, the PRMT5 inhibitor is EPZ015666.

[0012] Furthermore, the preparation process of PRMT5-inhibited NK-92 cells is as follows: inoculating NK-92 cells into a medium containing 1 μmol / L to 3 μmol / L EPZ015666 for culture to obtain PRMT5-inhibited NK-92 cells, namely E-NK-92 cells.

[0013] Furthermore, the concentration of EPZ015666 in the medium is 1 μmol / L.

[0014] Furthermore, the said E-NK-92 cells are used for the preparation of drugs for inhibiting tumor volume and weight.

[0015] Furthermore, the targeting and inhibition of the expression of the PRMT5 gene in NK-92 cells is a gene editing method: designing interfering RNAs targeting and inhibiting or knocking out the PRMT5 gene, and using the interfering RNAs to transform NK-92 cells to obtain PRMT5-inhibited NK-92 cells.

[0016] The present invention also provides the use of a PRMT5 inhibitor in the preparation of a synergist for drugs for treating colorectal cancer with NK-92 cells, and the PRMT5 inhibitor is EPZ015666.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] The present invention successfully transforms NK-92 cells by inhibiting the expression of the PRMT5 gene in NK-92 cells to obtain PRMT5-inhibited NK-92 cells (E-NK-92 cells). Compared with NK-92 cells, E-NK-92 cells change the functional exhaustion state of NK cells, the phenotype changes, the expression of inhibitory receptors NKG2A and IL-1R8 decreases, effectively preventing NK cell exhaustion, maintaining NK cell activity, and significantly improving the killing ability of tumor cells, and can significantly inhibit tumor volume and weight.

[0019] The anti-tumor effect of PRMT5-inhibited NK cells on colorectal cancer is enhanced. The cytotoxic molecules granzyme B or IFN-γ released by PRMT5-inhibited NK cells increase, directly killing colorectal cancer cells. In addition, in the colorectal cancer microenvironment, the expression of activating receptors on the surface of PRMT5-inhibited NK cells increases, and the expression of inhibitory receptors decreases, which enhances the activity of NK cells.

[0020] The research and development of PRMT5 inhibitors may bring great market prospects, especially in the field of tumor immunotherapy, improving the treatment effect and reducing the treatment cost. At the same time, by improving the effect of existing immunotherapy, the overall cost and medical burden of cancer treatment may be reduced. If the PRMT5-related NK cell treatment strategy is successfully translated into clinical application, it will bring considerable economic benefits and business opportunities to the biomedical industry. Description of the Drawings

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0022] Figure 1 Effect of PRMT5 inhibitor on the proliferation of NK-92 cells;

[0023] In the figure, A is the detection of the PRMT5 inhibition level in NK-92 cells by Western Blot after treatment with different concentrations of PRMT5 inhibitor (EPZ015666) for two days;

[0024] Figure B is a statistical chart of the effect of PRMT5 inhibitor on the proliferation of NK-92 cells, ****P<0.0001.

[0025] Figure 2Effect of PRMT5 inhibitor on the phenotype of NK-92 cells; Isotype isotype control, NK-92 cell is the control group, E-NK-92 is the NK-92 cell inhibited by PRMT5, which is the experimental group;

[0026] In the figure, A is the flow cytometry diagram of the expression of CD56 in NK-92 cells inhibited by PRMT5;

[0027] B is the flow cytometry diagram of the expression of CD16 in NK-92 cells inhibited by PRMT5;

[0028] C is the flow cytometry diagram of the expression of NKG2D in NK-92 cells inhibited by PRMT5;

[0029] D is the flow cytometry diagram of the expression of NKG2A in NK-92 cells inhibited by PRMT5;

[0030] E is the flow cytometry diagram of the expression of IL-1R8 in NK-92 cells inhibited by PRMT5;

[0031] F is the statistical chart of the expression of CD16, CD56, NKG2A, NKG2D, and IL-1R8 in NK-92 cells inhibited by PRMT5.

[0032] Figure 3 Effect of NK-92 cells inhibited by PRMT5 on the killing effect on colorectal cancer, *P<0.05, **P<0.01, ****P<0.0001;

[0033] In the figure, A is the killing effect of NK-92 cells and NK-92 cells inhibited by PRMT5 on SW620 under the condition of co-culturing different numbers of NK-92 cells with SW620 detected by CCK-8; SW620 with NK-92 indicates co-culture of NK-92 cells with SW620, and sw620 with E-NK-92 indicates co-culture of NK-92 cells inhibited by PRMT5 with SW620;

[0034] B is the killing effect of NK-92 cells and NK-92 cells inhibited by PRMT5 on HCT116 under the condition of co-culturing different numbers of NK-92 cells with HCT116 detected by CCK-8; HCT116 with NK-92 indicates co-culture of NK-92 cells with HCT116, and HCT116 with E-NK-92 indicates co-culture of NK-92 cells inhibited by PRMT5 with HCT116.

[0035] Figure 4Effect of PRMT5 inhibition on the phenotypic transformation of NK-92 cells after co-culture with colorectal cancer cell line SW620; Isotype isotype control, NK-92 cell is the control group, E-NK-92 is the NK-92 cell with PRMT5 inhibition, which is the experimental group;

[0036] In the figure, A is the flow cytometry plot of CD56 expression in NK-92 cells after PRMT5 inhibition after co-culture with colorectal cancer cell line SW620;

[0037] B is the flow cytometry plot of CD16 expression in NK-92 cells after PRMT5 inhibition after co-culture with colorectal cancer cell line SW620;

[0038] C is the flow cytometry plot of NKG2D expression in NK-92 cells after PRMT5 inhibition after co-culture with colorectal cancer cell line SW620;

[0039] D is the flow cytometry plot of NKG2A expression in NK-92 cells after PRMT5 inhibition after co-culture with colorectal cancer cell line SW620;

[0040] E is the flow cytometry plot of IL-1R8 expression in NK-92 cells after PRMT5 inhibition after co-culture with colorectal cancer cell line SW620;

[0041] F is the flow cytometry statistical chart of the expression of each marker (CD16, CD56, NKG2A, NKG2D and IL-1R8) in NK-92 cells after PRMT5 inhibition after co-culture with colorectal cancer cell line SW620.

[0042] Figure 5 Effect of PRMT5 inhibition on the phenotypic transformation of NK-92 cells after co-culture with colorectal cancer cell line HCT116; Isotype isotype control, NK-92 cell is the control group, E-NK-92 is the NK-92 cell with PRMT5 inhibition, which is the experimental group;

[0043] In the figure, A is the flow cytometry plot of CD56 expression in NK-92 cells after PRMT5 inhibition after co-culture with colorectal cancer cell line HCT116;

[0044] B is the flow cytometry plot of CD16 expression in NK-92 cells after PRMT5 inhibition after co-culture with colorectal cancer cell line HCT116;

[0045] C is the flow cytometry plot of NKG2D expression in NK-92 cells after PRMT5 inhibition after co-culture with colorectal cancer cell line HCT116;

[0046] D is a flow cytometry plot of the effect of PRMT5 inhibition on NKG2A expression in NK-92 cells after co-culture with colorectal cancer cell line HCT116;

[0047] E is a flow cytometry plot of the effect of PRMT5 inhibition on IL-1R8 expression in NK-92 cells after co-culture with colorectal cancer cell line HCT116;

[0048] F is a flow cytometry statistical plot of the effect of PRMT5 inhibition on the expression of various markers (CD16, CD56, NKG2A, NKG2D, and IL-1R8) in NK-92 cells after co-culture with colorectal cancer cell line HCT116.

[0049] Figure 6 shows the killing effect of PRMT5-inhibited NK-92 cells (E-NK cells) on colorectal cancer in vivo;

[0050] In the figure, A shows the effect of PRMT5-inhibited NK-92 cells (E-NK cells) on the size of colorectal cancer tumors in vivo;

[0051] B shows the effect of PRMT5-inhibited NK-92 cells (E-NK cells) on the weight of colorectal cancer tumors in vivo, *P<0.05, indicating a significant difference;

[0052] C shows the effect of PRMT5-inhibited NK-92 cells (E-NK cells) on the volume of colorectal cancer tumors in vivo, **P<0.01, indicating a highly significant difference.

[0053] Figure 7 shows the plasmid maps of the control plasmid and the plasmid overexpressing PRMT5;

[0054] In the figure, A shows the map of the control plasmid (pCDH-ef1 a-COGFP-PURO-SFFV-MCS-WPRE);

[0055] B shows the map of the PCDH-EF1a-CoGFP-P2A-Puro-SFFV-PRMT5-3MYC-WPRE vector (the plasmid overexpressing PRMT5).

[0056] Figure 8 shows the establishment of the NK92 cell line overexpressing PRMT5 and its effect on the proliferation of NK cells;

[0057] A shows the cell status of NK92 cells transfected with lentiviruses containing the control plasmid and the plasmid overexpressing PRMT5 under bright and dark fields;

[0058] B shows the protein content of PRMT5 detected by Western blotting in NK92-Vector and NK92-PRMT5 cells;

[0059] C shows the proliferation of NK-92 cells at different times of PRMT5 overexpression; ***P<0.001.

[0060] Figure 9 This is the effect of PRMT5 overexpression on the phenotype of NK92 cells;

[0061] In the figure, A is a histogram of the expression of CD16, CD56, SIGIRR, NKG2A, NKG2D, and TIGIT in NK92 cells detected by flow cytometry; the green line represents "IsoType", the blue line represents "NK92-Vector", and the purple line represents "NK92-PRMT5";

[0062] B is a statistical chart of the expression of CD16, CD56, SIGIRR, NKG2A, NKG2D, and TIGIT in NK92 cells detected by flow cytometry; *P<0.05, **P<0.01.

[0063] Figure 10 Overexpression of PRMT5 in vitro inhibits the killing effect of NK92 cells on CRC cells;

[0064] In the figure, A is the killing activity of NK92-Vector or NK92-PRMT5 cells against HCT116 cells measured by CCK8 at a specified NK:HCT116 (1:1) ratio at 6h, 12h, and 24h;

[0065] B is the killing activity of NK92-Vector or NK92-PRMT5 cells against SW620 cells measured by CCK8 at a specified NK:SW620 (1:1) ratio at 6h, 12h, and 24h, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0066] Figure 11 This is the phenotypic change of NK92 cells after co-culture with cancer cells detected by flow cytometry;

[0067] In the figure, A is a flow cytometry plot of CD56, CD16, NKG2D, NKG2A, IL-1R8, and TIGIT in NK-92 cells after co-culture with colorectal cancer HCT116; Isotype is the isotype control, NK92-vector is the control group, and NK92-PRMT5 is the NK-92 cells with PRMT5 overexpression, which is the experimental group;

[0068] Figure B shows the flow cytometry graphs of CD56, CD16, NKG2D, NKG2A, IL-1R8, and TIGIT in NK-92 cells after co-culture with colorectal cancer HCT116, *P<0.05, **P<0.001;

[0069] Figure C shows the flow cytometry graphs of CD56, CD16, NKG2D, NKG2A, IL-1R8, and TIGIT in NK-92 cells after co-culture with colorectal cancer SW620; Isotype is the isotype control, NK92-vector is the control group, and NK92-PRMT5 is the NK-92 cells with overexpressed PRMT5, which is the experimental group;

[0070] Figure D shows the flow cytometry statistical graphs of CD56, CD16, NKG2D, NKG2A, IL-1R8, and TIGIT in NK-92 cells after co-culture with colorectal cancer SW620, *P<0.05, **P<0.001.

[0071] Figure 12 Shows the effect of NK92 cells with overexpressed PRMT5 on anti-tumor;

[0072] In the figure, Figure A shows the apparent graphs of the dissected tumors of nude mice injected with PBS, nude mice injected with NK-92 cells, and nude mice injected with NK-92 cells with overexpressed PRMT5;

[0073] Figure B shows the statistical graphs of the weights of the dissected tumors of nude mice injected with PBS, nude mice injected with NK-92 cells, and nude mice injected with NK-92 cells with overexpressed PRMT5;

[0074] Figure C shows the changes in the volumes of the tumors of nude mice injected with PBS, nude mice injected with NK-92 cells, and nude mice injected with NK-92 cells with overexpressed PRMT5 over days, *P<0.05. Specific implementation manners

[0075] The following describes the specific implementation manners of the present invention in detail, but it should be understood that the protection scope of the present invention is not limited by the specific implementation manners. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0076] Example 1: A kind of NK cell and its preparation method and application.

[0077] I. Effects of PRMT5 inhibitor on phenotypic transformation of NK cells and enhancement of the anti-tumor effect of NK cells in the colorectal cancer microenvironment

[0078] 1. Effects of PRMT5 inhibitor on the proliferation of NK-92 cells.

[0079] NK cell culture: Purchase the human NK cell line NK-92 cells (product number: CL-0530) from Procell. After the cells arrive, first loosen the bottle cap, place the bottle upright and let it stand still for 5 hours until the cells sink to the bottom. Carefully aspirate the upper culture medium into a 15 mL centrifuge tube, leaving 8 mL of culture medium in the bottle. Then place it in an incubator at 37 °C with 5% CO2 for continued culture. During the culture process, fresh NK-92 medium (special medium for NK-92 cells purchased from Procell, product number: CM-0530) with a volume of 40% needs to be supplemented every 60 hours. If the turbidity of the culture medium increases significantly and the cell density reaches 85%, the centrifugation optimization passage method is immediately used for passage. First, collect the cells and the culture medium into a 15 mL centrifuge tube, then centrifuge at 800 rpm / min for 3 minutes at room temperature. After removing the supernatant, resuspend the cells with complete medium, adjust the density as needed, and then aliquot the cells into new culture containers in an appropriate ratio and continue to culture after adding fresh medium.

[0080] Take NK-92 cells in the logarithmic growth phase, set a control group and experimental groups. The control group is NK-92 cells cultured normally, and the experimental groups are NK-92 cells cultured with NK-92 cell special medium (special medium for NK-92 cells purchased from Procell, product number: CM-0530) containing 1 μmol / L, 3 μmol / L, and 5 μmol / L of PRMT5 enzyme activity inhibitor (EPZ015666) respectively. After culturing for two days, collect the cells, lyse them, extract proteins, and use Western blot to detect the inhibitory effect of different concentrations of EPZ015666 on the PRMT5 enzyme activity in NK-92 cells.

[0081] Protein Extraction: After the PRMT5 enzyme activity inhibitor had acted for two days, the cells were collected into 15 mL centrifuge tubes respectively, centrifuged at 800 rpm / min for 3 minutes, the supernatant was discarded, resuspended with PBS, the cells were pipetted after resuspension, transferred to EP tubes, centrifuged at 2500 rmp / min for 5 min, the supernatant was removed, and the excess PBS was aspirated away (the cells could not be aspirated). 60 mL of lysis buffer was added to each EP tube, and lysed for 50 min, pipetting and mixing every 10 minutes. A pre-cooled centrifuge at 4°C was used to centrifuge at 13000 r for 5 minutes, and the supernatant was aspirated into a new 1.5 mL EP tube, and the sample information and date were labeled. The protein concentration was measured using the BCA kit method. According to the number of standard products and samples, the BCA reagent and Cu reagent were mixed at a ratio of 50:1 to prepare the BCA working solution (prepared and used immediately). In a 96-well plate, 200 μL of BCA working solution, 18 μL of buffer and 2 μL of sample were added to each well as the experimental group, and 200 μL of BCA working solution and 18 μL of buffer were added at the same time as the control group. The plate was incubated at 37°C for 25 minutes, and the absorbance was measured at a wavelength of 562 nm using a microplate reader. The protein concentration was calculated according to the standard curve and marked on the EP tube. After the protein concentration measurement was completed, the protein was boiled. It was mixed according to the ratio of 6×loading buffer to protein of 1:5, mixed well and boiled at 100°C for 10 minutes, then cooled to room temperature and stored at -20°C for a long time.

[0082] Western Blot: The prepared samples were taken for SDS-PAGE electrophoresis, and the electrophoresis was terminated when the phenol blue ran out of the gel at 100 V. The proteins separated after electrophoresis were electrotransferred to the PVDF membrane at a constant current of 220 mA for 2 hours, with ice bath throughout the process. Then the PVDF membrane was immersed in 5% non-fat milk powder and blocked at room temperature for 2 h. Incubated with the primary antibody and the blocked PVDF membrane overnight at 4°C; washed the membrane with TBST for 5 min×5 times, then added the HRP-labeled secondary antibody and incubated at room temperature for 1 h; washed the membrane with TBST for 5 min×5 times, and then detected by ECL luminescence in a UVP chemiluminescence imager.

[0083] The results are as Figure 1 shown. 1 μmol / L, 3 μmol / L, and 5 μmol / L EZP015666 had obvious inhibitory effects on the PRMT5 activity of NK-92 cells ( Figure 1 Figure A), and with the continuous action of 1 μmol / L EZP015666 on NK-92, the proliferation of NK-92 cells slowed down ( Figure 1 Figure B).

[0084] 2. Effects of PRMT5 inhibitors on the phenotype of NK-92 cells.

[0085] The CD56 and CD16 of effector molecules of NK-92 cells, the inhibitory receptors SIGIRR (IL-1R8) and NKG2A, and the activating receptor NKG2D were detected by flow cytometry respectively. NK-92 cells in the logarithmic growth phase were taken and seeded in a 6-well culture plate at a density of 5×10 5 cells per well. Subsequently, the experiment was divided into three groups: the experimental group (added with the special medium for NK-92 cells containing 1 μmol / L EPZ015666), the blank control group (added with the same volume of the special medium for NK-92 cells without inhibitor), and the isotype control group (added with the same volume of the special medium for NK-92 cells without inhibitor). After culturing for 2 days, the cells of each group were collected respectively, the supernatant was discarded by centrifugation, the cells were washed with PBS, and the cells were resuspended after centrifugation again. Subsequently, CD16 (1:600), CD56 (1:500), SIGIRR (1:600), NKG2A (1:600), and NKG2D (1:500) were diluted according to the instructions. The corresponding specific primary antibodies were added to the experimental group and the control group, and incubated in the dark at 4 °C for 35 minutes. After incubation, the supernatant was discarded by centrifugation, the cells were washed with PBS again, and collected by centrifugation. The required secondary antibody dilution solution was prepared according to the instructions (prepared freshly and stored in the dark), and 500 μL of the secondary antibody dilution solution was added to the experimental group, the blank control group, and the isotype control group respectively, and incubated for 30 minutes in the dark. After incubation, the supernatant was discarded by centrifugation again, the cells were washed with PBS, the supernatant was discarded, and the cells were resuspended with PBS. The cells were filtered through a 70 μm cell strainer. After treatment, each 1.5 ml centrifuge tube should contain about 150 μL of cell suspension, and the samples were labeled and then injected into the flow cytometer for detection. The data were collected and statistically analyzed and processed using professional analysis software.

[0086] The results were as Figure 2 shown. The results showed that the expressions of the effector molecule CD16, the inhibitory receptors NKG2A and IL-1R8, and the activating receptor NKG2D of NK-92 cells after PRMT5 inhibition were higher than those of wild-type NK-92 cells, but the expression of the effector molecule CD56 of NK-92 cells after PRMT5 inhibition was lower than that of wild-type NK-92 cells.

[0087] 3. Effect of PRMT5-inhibited NK-92 cells on the killing effect on colorectal cancer.

[0088] Treatment of NK-92 cells: NK-92 cells in the logarithmic growth phase were taken and set into two groups: the first group was NK-92 cells cultured normally, and the second group was NK-92 cells cultured with the special medium for NK-92 cells containing 1 μmol / L EPZ015666 for two days.

[0089] Culture of human colorectal cancer cell lines SW620 and HCT116: The colorectal cancer cell lines in this invention are SW620 (product number: CL-0225) and HCT116 (product number: CL-0096) purchased from Procell. After receiving the cells at room temperature, take photos in time to record whether there is liquid leakage or bottle body damage. Do not open the culture bottle cap first. Place the cells in the cell culture incubator and let them stand for 3 hours to stabilize the cell state. If the growth density of the cells is below 60% under the microscope, aspirate the perfusion medium in the culture bottle until there is only 7 mL of complete medium left in the culture bottle, and then put it back into the cell culture incubator for further culture. If the cell growth density reaches over 70%, the cells can be passaged. Aspirate the original culture medium, add about 2 mL of PBS, gently shake the culture bottle to wash the cells, aspirate the PBS and discard it, add 7 mL of complete medium (for SW620 cells, use the special medium for SW620 cells purchased from Procell, product number: CM-0225A; for HCT116 cells, use the special medium for HCT116 cells, product number: CM-0096), shake well, and place it in a 37°C constant temperature and 5% CO2 incubator for culture.

[0090] Select two colorectal cancer cells, HCT116 and SW620, as target cells respectively. Seed HCT116 and SW620 cells into 96-well plates. When they grow to 55%, co-culture the colorectal cancer cell lines with NK-92 cells or NK92 cells after PRMT5 inhibition. First, co-culture 1×10 3 、3×10 3 、5×10 3 、7×10 3 、9×10 3 、11×10 3 NK-92 cells and NK92 cells after PRMT5 inhibition with HCT116 and SW620 cells respectively for 6 hours, and then use CCK8 reagent to detect the activity of tumor cells. Aspirate the NK-92 cells, wash them with PBS, and replace them with complete medium containing 10% CCK8. Place the 96-well plate in a 37°C and 5% CO2 constant temperature incubator for dark incubation for 2.5 hours. After incubation, use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance value (OD value) of each well at a wavelength of 450 nm.

[0091] The results are as Figure 3 shown. For SW620 cells, the killing effect of NK-92 cells after PRMT5 inhibition is stronger than that of NK-92 cells ( Figure 3 A); the same result appears in HCT116 cells ( Figure 3 B).

[0092] 4. Effect of PRMT5 inhibition on the phenotype of NK-92 cells after co-culture with colorectal cancer cells

[0093] After co - culturing with colorectal cancer cells, the expressions of CD56, CD16, SIGIRR (IL - 1R8), NKG2A, and NKG2D in NK - 92 cells and cells after PRMT5 inhibition were detected by flow cytometry respectively.

[0094] The results are as Figure 4 shown. After co - culturing with SW620 cells: The results showed that there were no significant differences in the expressions of CD56 and CD16 in NK - 92 cells and NK - 92 cells after PRMT5 inhibition. The expression of the NK cell activating receptor NKG2D increased in NK - 92 cells after PRMT5 inhibition, while the expressions of the inhibitory receptors NKG2A and IL - 1R8 decreased in NK - 92 cells after PRMT5 inhibition.

[0095] The results are as Figure 5 shown. After co - culturing with HCT116 cells: The results showed that there were no significant differences in the expressions of CD56, CD16, and IL - 1R8 in NK - 92 cells and NK - 92 cells after PRMT5 inhibition. The expression of the NK cell activating receptor NKG2D increased in NK - 92 cells after PRMT5 inhibition, while the expression of the inhibitory receptor NKG2A decreased in NK - 92 cells after PRMT5 inhibition. It can be seen that after co - culturing with colorectal cancer cells, PRMT5 inhibition can cause phenotypic transformation of NK - 92 cells.

[0096] 5. PRMT5 - inhibited NK - 92 cells (E - NK - 92 cells) can enhance the killing effect on colorectal cancer in vivo.

[0097] Mouse xenograft tumor model: BALB / c - nu male nude mice were selected as experimental subjects. Tumor cells of each group were harvested under sterile conditions, resuspended with PBS, and 5×10 6 human colorectal cancer HCT116 cells (total volume of 0.2 mL) were injected subcutaneously into the inguinal side of mice. After the formation of cell solid tumors, the short diameter and long diameter of the tumors were measured daily, and the tumor volume was calculated using the following formula: V = d 2 ×D / 2 (d: short diameter of the tumor, D: long diameter of the tumor).

[0098] Immunotherapy of nude mice: When the volume of the largest tumor in nude mice reached about 25 mm 3 , the nude mice were randomly divided into two groups (n = 3), and cell immunotherapy was started. NK - 92 cells were injected via the tail vein. The control group was injected with wild - type NK - 92 cells, and the experimental group was injected with NK - 92 cells treated with EPZ015666 for two days. First, the cells were collected under sterile conditions and resuspended with PBS, at a concentration of 5×10 6Cells per injection, with a total volume of 200 μL, were injected once every three days for a total of 4 times. At the same time, the status of the nude mice was observed, the short and long diameters of the tumors were measured, and the tumor volume was calculated using a formula. When the tumor volume of the nude mice reached 400 mm 3 , the nude mice were anesthetized, sacrificed by cervical dislocation, and the tumors were dissected and weighed. The growth curve of the tumors was plotted with time as the horizontal axis and tumor volume as the vertical axis.

[0099] The results were as Figure 6 shown. The tumors of the nude mice injected with PRMT5-inhibited NK-92 grew more slowly than those of the nude mice injected with NK-92 (as Figure 6 shown in C). After dissection, the tumors were removed. The tumors of the nude mice injected with PRMT5-inhibited NK-92 were significantly smaller than those of the nude mice injected with NK-92 cells (as Figure 6 shown in A, Figure 6 shown in B). This also demonstrated that inhibiting PRMT5 enhanced the antitumor effect of NK-92 cells in vivo.

[0100] II. Effects of PRMT5 overexpression on the phenotypic transformation of NK cells and the attenuation of the antitumor effect of NK cells in the colorectal cancer microenvironment.

[0101] 1. Establishment of an NK92 cell line with PRMT5 overexpression and its effect on the proliferation of NK cells.

[0102] An empty plasmid Lenti-SFFV-CoGFP-Puro ( Figure 7 shown in A) and a PRMT5 overexpression plasmid PCDH-EF1 a-CoGFP-P2A-Puro-SFFV-PRMT5-3MYC-WPRE ( Figure 7 shown in B) were purchased from QinKe Biotechnology Co., Ltd.

[0103] The packaging plasmids pMD2.G, psPAX2 and the target plasmids (empty plasmid or PRMT5 overexpression plasmid) were transfected into 293T cells with the assistance of Lipofectamine 3000 reagent to prepare lentiviruses carrying the empty plasmid and the PRMT5 overexpression plasmid respectively. The prepared lentivirus solutions carrying the empty plasmid and the PRMT5 overexpression plasmid were transfected into NK92 cells respectively to obtain an NK92 cell line with PRMT5 overexpression with GFP fluorescence (abbreviated as NK92-PRMT5) and an NK92 cell line transfected with the virus carrying the empty plasmid (abbreviated as NK92-Vector).

[0104] The results were as Figure 8As shown in Figure D, the protein expression levels of PRMT5 in NK92-PRMT5 cells and NK92-Vector cells were detected by Western blot. It can be seen that the protein expression level of PRMT5 in NK92-PRMT5 cells was significantly higher than that in NK92-Vector cells.

[0105] The results were as Figure 8 shown in Figure E. By detecting the effect of PRMT5 overexpression on the long-term proliferation of NK92-Vector cells and NK92-PRMT5 cells using a CCK8 kit, it was found that: on the 1st, 2nd, and 3rd days, the cell proliferation rate of NK92-PRMT5 was significantly higher than that of the NK92-Vector group.

[0106] 2. Effects of PRMT5 overexpression on the phenotype of NK92 cells.

[0107] To study whether overexpression of PRMT5 in NK92 cells has an impact on the phenotype of NK92 cells, in the present invention, flow cytometry was used to detect the expression of CD56, CD16, CD56, SIGIRR (IL-1R8), NKG2A, NKG2D, and TIGIT in NK92-Vector cells and NK92-PRMT5 cells respectively.

[0108] The results were as Figure 9 shown. There was no significant difference in the expression of CD56, CD16, and NKG2A between NK92-PRMT5 and NK92-Vector cells. However, the expression of these molecules such as TIGIT, NKG2D, and SIGIRR was significantly higher in NK92-PRMT5 cells with PRMT5 overexpression than in the control group NK92-Vector. It can be seen that overexpression of PRMT5 will change the phenotype of NK92 cells.

[0109] 3. Overexpression of PRMT5 inhibits the killing effect of NK92 cells on CRC cells in vitro.

[0110] To study whether overexpression of PRMT5 has an impact on the toxicity of NK92 cells, in the present invention, two cell lines (HCT116 and SW620) were selected as target cells and co-cultured with NK92 cells respectively. First, NK92-Vector cells and NK92-PRMT5 cells were co-cultured with HCT116 and SW620 cells for 6 hours respectively, and then the CCK8 reagent was used to detect the activity of tumor cells.

[0111] The results were as Figure 10As shown, for HCT116, the cytotoxicity of NK92-PRMT5 cells against it was significantly lower than that of NK92-Vector; the same result was obtained in SW620 cells.

[0112] 4. Flow cytometry was used to detect the phenotypic changes of NK92 cells after co-culture.

[0113] To study whether overexpression of PRMT5 in NK92 cells affects the phenotype of NK92 cells after co-culture with tumor cells, in this invention, flow cytometry was used to detect the expression of CD56, CD16, CD56, SIGIRR (IL-1R8), NKG2A, NKG2D, and TIGIT in NK92-Vector cells and NK92-PRMT5 cells respectively.

[0114] The results are as Figure 11 shown. After co-culture with HCT116 cells: The results showed that there were no significant differences in the expression of CD56, CD16, and NKG2A between NK92-PRMT5 and NK92-Vector cells. However, the expression of the two molecules TIGIT and SIGIRR was significantly higher in NK92-PRMT5 cells with overexpressed PRMT5 than in the control group NK92-Vector. And the expression of NKG2D was significantly lower in NK92-PRMT5 cells with overexpressed PRMT5 than in the control group NK92-Vector.

[0115] After co-culture with SW620 cells: The results showed that there were no significant differences in the expression of CD56, CD16, NKG2A, TIGIT, and SIGIRR between NK92-PRMT5 and NK92-Vector cells. And the expression of NKG2D was significantly higher in NK92-PRMT5 cells with overexpressed PRMT5 than in the control group NK92-Vector.

[0116] 5. Overexpression of PRMT5 in vivo inhibits the antitumor function of NK92 cells.

[0117] To detect whether overexpression of PRMT5 affects the antitumor function of NK92 in vivo, BALB / C nude mice were selected and HCT116 tumor cells were subcutaneously injected on day 0. When the average size of the tumor reached 50 mm 3 , the treatment group started intravenous injection of NK92 cells via the tail vein.

[0118] The results are as Figure 12As shown, the tumor volume of the mice inoculated with NK92-Vector cells was significantly reduced compared with that of the mice in the PBS group (P<0.01); while there was no difference in the tumor volume and weight between the mice inoculated with NK92-PRMT5 cells and those in the PBS group, and the tumor volume and weight of the mice inoculated with NK92-Vector cells were significantly reduced compared with those of the mice inoculated with NK92-PRMT5 cells (P<0.5). The same result was obtained for the tumor size. It can be seen that the overexpression of PRMT5 in NK92 cells (NK92-PRMT5 cells) by tail vein injection significantly inhibited the original anti-tumor function of NK92 cells.

[0119] Although the preferred embodiments of the present invention have been described, additional changes and modifications can be made to these embodiments by those skilled in the art once they learn the basic creative concept.

[0120] Obviously, those skilled in the art can make various changes and deformations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and deformations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and deformations.

Claims

1. Use of PRMT5-inhibited NK-92 cells in the preparation of drugs for treating colorectal cancer.

2. Use of the PRMT5-inhibited NK-92 cells according to claim 1 in the preparation of a medicament for treating colorectal cancer, characterized in that, PRMT5-inhibited NK-92 cells are obtained by targeting and inhibiting the expression of the PRMT5 gene in NK-92 cells.

3. Use of the PRMT5-inhibited NK-92 cells according to claim 2 in the preparation of a medicament for treating colorectal cancer, characterized in that, The method for targeting and inhibiting the expression of the PRMT5 gene in NK-92 cells is as follows: By adding a PRMT5 inhibitor during the culture of NK-92 cells to target and inhibit the PRMT5 gene in NK-92 cells, the modified PRMT5-inhibited NK-92 cells are obtained.

4. Use of the PRMT5-inhibited NK-92 cells according to claim 3 in the preparation of a medicament for treating colorectal cancer, characterized in that, The PRMT5 inhibitor is EPZ015666.

5. Use of the PRMT5-inhibited NK-92 cells according to claim 4 in the preparation of a medicament for treating colorectal cancer, characterized in that, The preparation process of PRMT5-inhibited NK-92 cells is: Inoculate NK-92 cells into a medium containing 1 μmol / L to 3 μmol / L EPZ015666 for culture to obtain PRMT5-inhibited NK-92 cells, that is, E-NK-92 cells.

6. Use of the PRMT5-inhibited NK-92 cells according to claim 5 in the preparation of a medicament for treating colorectal cancer, characterized in that, The concentration of EPZ015666 in the medium is 1 μmol / L.

7. Use of the E-NK-92 cell according to any one of claims 1 to 6 in the preparation of a medicament for treating colorectal cancer, characterized in that, The described E-NK-92 cells are used in the preparation of drugs for inhibiting tumor volume and weight.

8. Use of the PRMT5-inhibited NK-92 cells according to claim 2 in the preparation of a medicament for treating colorectal cancer, characterized in that, Targeting and inhibiting the expression of the PRMT5 gene in NK-92 cells is a gene editing method: Design interfering RNAs that target and inhibit or knockout the PRMT5 gene, and use the interfering RNAs to modify NK-92 cells to obtain PRMT5-inhibited NK-92 cells.

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