Application of FcγRIIA as a therapeutic target for glioblastoma

Abstract

The present invention relates to the application of FcγRIIA as a biomarker and therapeutic target for glioma, and belongs to the field of biomedical technology. The present invention finds that the immunoglobulin G receptor FcγRIIA is significantly highly expressed in glioma cells by analyzing the TCGA database. The use of shRNA to knock down the expression of FcγRIIA in glioma stem cells MES28, MGG8 and 3028 can inhibit the proliferation and activation of glioma stem cells, and affect the activity of proteins related to the PI3K / AKT signaling pathway. In mice transplanted with glioma stem cells, the expression of FcγRIIA was knocked down, and the tumors of the mice were significantly reduced and the survival period was significantly prolonged, indicating that the growth of glioma cells can be inhibited as the expression of FcγRIIA is reduced. FcγRIIA can be used as a biomarker and therapeutic target for glioma, providing a new development direction for the detection and treatment of glioma.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the application of FcγRIIA as a target for treating glioblastoma. Background Art

[0002] Cancer is one of the major causes of death in humans and a global problem faced by the whole world. Statistics from 2020 show that approximately 9.6 million people worldwide died of cancer, affecting people of all ages. The overall incidence and mortality of cancer in my country have continued to rise year by year in recent years, with an average of more than 10,000 people diagnosed with cancer every day. Cancer has become a major public health issue that seriously threatens the health of Chinese residents.

[0003] Glioma is an intracranial tumor originating from glial cells. It ranks first in incidence among primary brain tumors and is the most common malignant tumor in the brain, accounting for 40-50% of intracranial tumors. Malignant glioma has the highest mortality rate among refractory malignant tumors except pancreatic cancer. More than 95% of patients cannot survive for more than 5 years. It has the characteristics of high incidence, high recurrence rate, easy metastasis, high mortality rate and low cure rate. Glioma grows in an infiltrative manner, with unclear boundaries with normal brain tissue, difficult to resect, and prone to recurrence. The cause of glioma is still unclear, and may be related to factors such as tumor origin, genetic factors, biochemical environment, ionizing radiation, nitroso compounds, polluted air, bad living habits, and infection.

[0004] WHO divides gliomas into four types according to their increasing malignancy. The most malignant type is called glioblastoma (GBM), which is also the most malignant primary brain tumor in the central nervous system. The Cancer Genome Atlas (TCGA) divides glioblastoma into three subtypes based on transcriptional clustering analysis: proneural, classical, and mesenchymal. Glioblastoma has extremely strong radiotherapy resistance, which is closely related to the heterogeneity of tumor stem cells in GBM. Glioblastoma Stem Cells (GSCs) are a group of stem cells with self-renewal and multidirectional differentiation capabilities. The presence of GSCs leads to high heterogeneity, invasiveness, and malignant evolution in GBM tumors. During the unlimited proliferation of tumor stem cells, angiogenesis and central necrosis occur successively, which is likely to be an important cause of the suppression of anti-tumor immune response and resistance to cytotoxic therapy.

[0005] Although some progress has been made in the treatment of gliomas in recent years, surgical resection, chemoradiotherapy, and immune cell therapy are currently used in clinical practice. However, due to the invasive growth of gliomas and the lack of obvious boundaries with brain tissue, it is difficult to completely remove them except for those with small early tumors located in appropriate locations. The median survival of patients can only be increased to 15 months. In the past, people have conducted a series of exploratory studies on gliomas and obtained a large number of genome sequencing results. However, because the treatment mechanism of gliomas has not been clearly studied, the glioma markers that have been discovered have disadvantages such as low sensitivity and low specificity. Therefore, it is urgent to find new targets for glioma detection and treatment. Summary of the invention

[0006] To this end, the technical problem to be solved by the present invention is to overcome the lack of high-specificity detection and treatment targets for gliomas in the prior art. The present invention analyzes the TCGA database and finds that the immunoglobulin G receptor FcγRIIA is significantly highly expressed in glioma cells. Using shRNA to knock down the expression of FcγRIIA in glioma stem cells MES28, MGG8 and 3028 can inhibit the proliferation and activation of glioma stem cells, and affect the activity of proteins related to the PI3K / AKT signaling pathway.

[0007] The first object of the present invention is to provide the use of immunoglobulin G receptor FcγRIIA as a glioma biomarker.

[0008] Furthermore, the nucleotide sequence of FcγRIIA is shown in NCBI Gene ID: 2212.

[0009] The present invention uses the TCGA database and collected clinical tissue samples to analyze the expression difference of the FcγRIIA gene in glioma cells. The expression difference of the FcγRIIA gene has a significant correlation with glioma.

[0010] Furthermore, immunoblotting was used to verify that there was a significant difference in the expression level of FcγRIIA in normal brain tissue and glioma stem cells.

[0011] Furthermore, the glioma stem cells are clinically derived primary glioma stem cells MES28, MGG8 and 3028.

[0012] The second object of the present invention is to provide a glioma detection or prognosis evaluation kit, which comprises a reagent for detecting the expression level of FcγRIIA.

[0013] Furthermore, the glioma detection kit includes primers for amplifying FcγRIIA.

[0014] Furthermore, the primers include a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown as SEQ ID NO.1, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO.2.

[0015] Furthermore, the nucleotide sequence of the forward primer is 5'-ATCATTGTGGCTGTGGTCATTGC-3'.

[0016] Furthermore, the nucleotide sequence of the reverse primer is 5'-TCAGGTAGATGTTTTTATCATCG-3'.

[0017] Furthermore, the glioma detection kit also includes a component for PCR amplification.

[0018] The third object of the present invention is to provide a glioma therapeutic drug, which is designed with FcγRIIA as a target to reduce the expression of FcγRIIA.

[0019] The fourth object of the present invention is to provide a use of a preparation for knocking down FcγRIIA expression in the preparation of a drug for treating glioma.

[0020] Further, the formulation includes shRNA, which knocks down the mRNA target sequence of FcγRIIA.

[0021] Furthermore, the nucleotide sequence of the mRNA target sequence is shown in SEQ ID NO.3-4.

[0022] Furthermore, the nucleotide sequence of the mRNA target sequence is 5'-CATGAATTGCGCCTCAGATTT-3' (shown in SEQ ID NO. 3) or 5'-CCATGTCAACAGTAATAACTA-3' (shown in SEQ ID NO. 4).

[0023] Preferably, the nucleotide sequence of the mRNA target sequence is shown as SEQ ID NO.3.

[0024] Furthermore, the nucleotide sequence of the shRNA is shown in SEQ ID NO.5-6 or SEQ ID NO.7-8.

[0025] Furthermore, the nucleotide sequence of shFCGR2A-1 includes:

[0026] Forward sequence 5'-CCGGCATGAATTGCGCCTCAGATTTCTCGAGAAATCTGAGGCGCAATTCATGTTTTTTG-3' (shown in SEQ ID NO. 5);

[0027] Reverse sequence: 5′-GTACTTAACGCGGAGTCTAAAGAGCTCTTTAGACTCCGCGTTAAGTACAAAAAACTTAA-3′ (shown in SEQ ID NO. 6).

[0028] Furthermore, the nucleotide sequence of shFCGR2A-2 includes:

[0029] Forward sequence 5'-CCGGCCATGTCAACAGTAATAACTACTCGAGTAGTTATTACTGTTGACATGGTTTTT-3' (shown in SEQ ID NO. 7);

[0030] Reverse sequence 5'-GGTACAGTTGTCATTATTGATGAGCTCATCAATAATGACAACTGTACCAAAAATTAA-3' (shown in SEQ ID NO.8). Preferably, the nucleotide sequence of shRNA is shown in SEQ ID NO.5-6.

[0031] Furthermore, knocking down FcγRIIA expression in glioma cells significantly inhibited the expression of proteins related to the PI3K / AKT signaling pathway.

[0032] Furthermore, the above-mentioned shRNA was inserted using the pLKO.1 plasmid as a backbone.

[0033] Further, using Age I and EcoR I was used to digest the pLKO.1 plasmid.

[0034] Furthermore, a recombinant lentiviral vector pLKO.1-shFcγRIIA was obtained.

[0035] Furthermore, the recombinant lentiviral vector pLKO.1-shFcγRIIA and packaging plasmids pSPAX2 and pMD2.G were co-transfected into 293T cell lines to prepare lentivirus.

[0036] Beneficial effects of the present invention:

[0037] The present invention provides the application of FcγRIIA as a biomarker and therapeutic target for glioma. The present invention finds that the immunoglobulin G receptor FcγRIIA can be used as a detection and therapeutic target for glioma. The use of shRNA to knock down the expression of FcγRIIA in glioma stem cells MES28, MGG8 and 3028 can inhibit the proliferation and activation of glioma stem cells, and affect the activity of proteins related to the PI3K / AKT signaling pathway. The present invention also provides an application for amplifying FcγRIIA and a kit including the primer. The primer can be amplified by PCR technology to detect the expression amount of FcγRIIA in the sample, thereby achieving the purpose of detecting glioma. In addition, shRNA for knocking down the expression of FcγRIIA is provided, which can be used to prepare a glioma therapeutic drug. The present invention provides a basis and development direction for finding new and effective targeted treatment strategies for glioma, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0039] Figure 1 The TCGA database in Example 1 shows that the mRNA of FcγRIIA is highly expressed in glioma;

[0040] Figure 2 is the mRNA expression diagram of the FcγRIIA gene in glioma stem cells MES28, MGG8 and 3028 in Example 1;

[0041] Figure 3 is the protein electrophoresis diagram of FcγRIIA in MES28, MGG8 and 3028 in Example 1;

[0042] Figure 4 is the gene expression diagram of the knockdown of FcγRIIA gene in MES28, MGG8 and 3028 in Example 2;

[0043] Figure 5 This is a protein detection diagram of the knockdown of the FcγRIIA gene in MES28, MGG8 and 3028 in Example 2;

[0044] Figure 6 This is a diagram showing the proliferation of glioma stem cells MES28, MGG8 and 3028 detected for five consecutive days after knocking down the FcγRIIA gene in Example 3;

[0045] Figure 7 This is an experimental diagram of detecting the sphering of glioma stem cells MES28, MGG8 and 3028 for five consecutive days after knocking down the FcγRIIA gene in Example 3;

[0046] Figure 8 This is the limiting dilution graph of glioma stem cells MES28, MGG8 and 3028 detected for five consecutive days after knocking down the FcγRIIA gene in Example 3;

[0047] Fig. 9 is a signaling pathway diagram related to FcγRIIA in GSEA enrichment analysis in Example 4;

[0048] Fig.10 is the protein expression diagram of the PI3K / AKT signaling pathway in shFcγRIIA in Example 5;

[0049] Fig.11 This is the tumor formation in mice after the animals were intracranially transplanted with shFcγRIIA tumor cells in Example 6;

[0050] Fig.12 This is the survival curve of mice after intracranial transplantation of shFcγRIIA tumor cells in Example 6;

[0051] Fig.13 After intracranial inhibition of glioma stem cells and administration of FcγRIIA inhibitor in Example 6, the intracranial tumors of mice were reduced and the number of survival days was increased. DETAILED DESCRIPTION

[0052] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0053] Glioma stem cells MES28, MGG8 and 3028 are primary glioma stem cells derived from patients. They were obtained by Jeremy N. Rich's laboratory abroad through a material transfer agreement with Duke University in the United States and have been approved for publication in high-level international journals.

[0054] Example 1: FcγRIIA gene is highly expressed in glioblastoma

[0055] (1) Analysis of the Cancer Genome Atlas (TCGA) dataset revealed that Figure 1 As shown, the expression of FcγRIIA gene was significantly upregulated in glioblastoma compared with normal brain tissue controls.

[0056] (2) Extraction of mRNA: Glioma stem cells were collected, washed once with PBS after centrifugation, centrifuged at 1200 rpm for 5 min, supernatant discarded, Trizol (TakaRa) precooled at 4°C was added, and the tube was allowed to stand in an ice box for 5 min; chloroform was added at a ratio of 1:5, mixed by inversion, allowed to stand for 3 min, and centrifuged at 12000g for 15 min at 4°C; the supernatant was transferred to another RNase-free EP tube, an equal volume of isopropanol was added to precipitate RNA, allowed to stand at room temperature for 10 min, and centrifuged at 12000g for 10 min at 4°C; the isopropanol was carefully discarded, 600 μL of 75% ethanol was added to wash RNA, and centrifuged at 10000g for 10 min at 4°C; the ethanol was aspirated, the tube was dried at room temperature, diethyl pyrocarbonate (DEPC) solution was added to mix, and the RNA concentration was detected.

[0057] According to the FcγRIIA gene sequence of NCBI Gene ID: 2212, the primer sequence of FcγRIIA was designed, the forward sequence was 5'-ATCATTGTGGCTGTGGTCATTGC-3', and the reverse sequence was 5'-TCAGGTAGATGTTTTTATCATCG-3'.

[0058] The qRT-PCR reaction system (20 μL) included 2×ChamQ SYBR qPCR Master Mix 10 μL, Primer1 0.4 μL, Primer 2 0.4 μL, cDNA 2 μL, ddHO 2 O 7.2 μL.

[0059] The reaction procedure included pre-denaturation at 95°C for 30 s, cycle reaction at 95°C for 10 s, 60°C for 30 s, and 95°C for 15 s, for a total of 40 cycles, and melting curve at 60°C for 60 s and 95°C for 15 s.

[0060] like Figure 2 As shown, the FcγRIIA gene is highly expressed in the glioma stem cell lines MES28, MGG8, and 3028.

[0061] (3) Proteins were extracted from glioma stem cells MES28, MGG8 and 3028 cells, subjected to 10% SDS-PAGE vertical electrophoresis and electrotransferred to nitrocellulose membranes. The membranes were incubated with FcγRIIA antibody (Proteintech) at room temperature for 1 h, and then exposed to a gel imaging system (Bio-Rad) using a 1:5000 diluted HRP-goat anti-human IgG (Beijing Zhongshan Company) and an ECL luminescence kit (Pierce Company, USA).

[0062] like Figure 3As shown, FcγRIIA protein is highly expressed in different subtypes of glioma stem cells including MES28, MGG8 and 3028.

[0063] Example 2: FcγRIIA gene knockdown

[0064] The shFcγRIIA sequences were designed using mRNA 5'-CATGAATTGCGCCTCAGATTT-3' (shown in SEQ ID NO.3) and 5'-CCATGTCAACAGTAATAACTA-3' (shown in SEQ ID NO.4) as target sequences, namely shFCGR2A-1 and shFCGR2A-2, wherein:

[0065] The forward sequence of shFCGR2A-1 is 5′-CCGGCATGAATTGCGCCTCAGATTTCTCGAGAAATCTGAGGCGCAATTCATGTTTTTTG-3′ (shown in SEQ ID NO. 5), and the reverse sequence is 5′-GTACTTAACGCGGAGTCTAAAGAGCTCTTTAGACTCCGCGTTAAGTACAAAAAACTTAA-3′ (shown in SEQ ID NO. 6).

[0066] The forward sequence of shFCGR2A-2 is 5′-CCGGCCATGTCAACAGTAATAACTACTCGAGTAGTTATTACTGTTGACATGGTTTTT-3′ (shown in SEQ ID NO. 7), and the reverse sequence is 5′-GGTACAGTTGTCATTATTGATGAGCTCATCAATAATGACAACTGTACCAAAAATTAA-3′ (shown in SEQ ID NO. 8).

[0067] 1. Construction of shFcγRIIA gene

[0068] (1) shRNA plasmid ligation:

[0069] The shRNA primer annealing reaction system (50 μL) includes 5 μL of F primer, 5 μL of R primer, 5 μL of 10×NEB buffer, 5 μL of ddHO 2 O 35 μL.

[0070] The shRNA primer annealing reaction conditions were 95°C for 4 min and 70°C for 10 min.

[0071] pLKO.1 plasmid restriction enzyme digestion reaction system (50 μL) includes 6 μg of pLKO.1-TRC plasmid, 5 μL of 10×Cut SmartBuffer, Age I 1 μL, EcoR I 1 μL, ddH 2 O is calculated based on the volume of the plasmid.

[0072] Perform nucleic acid gel electrophoresis at 37°C for 2 h, 1% agarose gel, rated voltage 180V, electrophoresis for 15-20 min, take photos and observe under 300 nm ultraviolet light, cut out the bright band at the 7 kb position and recover it from the agarose gel, and measure the DNA concentration according to the GeneJET gel recovery kit protocol.

[0073] The primers and pLKO.1-TRC digested plasmid were connected. The shRNA plasmid connection reaction system (20 μL) included 2 μL of annealed primers, 20 ng of pLKO.1-TRC digested plasmid, and T 4 DNA ligase 1 μL, 10×T4 Ligation Buffer 2 μL, ddH 2 O is calculated based on the volume of the plasmid.

[0074] (2) Plasmid transformation

[0075] Take 30 μL of competent cells melted on ice, add 1 μL of target DNA / plasmid, use a pipette to repeatedly blow and mix, and place on ice for 30 minutes; heat shock in a 42°C water bath for 45 seconds, quickly transfer to an EP tube, and place on ice for 2 minutes; add 500 μL of antibiotic-free sterile LB medium to each tube, mix well, and place at 37°C, 200rpm shaker for 1 hour; centrifuge at 3000 rpm for 3 minutes, discard the supernatant, add 200 μL LB to resuspend the cells (if the transformed plasmid does not need to be concentrated), aspirate the transformed cells, drop them onto the LB plate containing AMP antibiotics, and spread them evenly with a coating rod; place upright in a 37°C incubator, invert the plate after 10 minutes, and culture overnight for 12-16 hours.

[0076] (3) Plasmid extraction

[0077] PCR amplification: According to the 20 μL PCR reaction system, a single colony was picked up with a sterile pipette tip as a template for PCR amplification. The reaction conditions were: 94°C for 5 min; 94°C for 30 s, 58°C for 40 s, 72°C for 45 s, 30 cycles; 72°C for 5 min. After the PCR was completed, 5 μL of the product was taken and the bands were detected by 1% agarose gel electrophoresis. Subsequently, the sequencing results of the positive clones were analyzed.

[0078] Plasmid extraction: Take the selected monoclonal colony after transformation and culture it in a shaking incubator at 37°C and 200 rpm; inoculate the glycerol bacteria into sterile LB medium containing AMP at a ratio of 1:200; directly pick the monoclonal colony into 5 mL of culture medium for culture; take the bacterial suspension and inoculate it into 200 mL sterile LB medium containing AMP at a ratio of 1:200 for expansion culture; collect the amplified bacterial precipitate by centrifugation at 4000 rpm for 10 min, and extract the plasmid according to the OMEGA plasmid extraction kit Protocol.

[0079] (4) Lentiviral packaging

[0080] 24 h before transfection, 2×10 6 Lenti-XTM293T cells were inoculated in a 10 cm cell culture dish and cultured with DMEM medium (containing 10% FBS) without antibiotics. Transfection was performed when the cell density reached about 80%.

[0081] The pLKO.1-shFcγRIIA and pLKO.1-shCONT plasmids and packaging plasmids pSPAX2 and pMD2.G were co-transfected into Lenti-XTM293T cells to prepare lentivirus. Preheat the DMEM / NBM complete medium and PBS buffer in a 37°C water bath in advance; the plasmid was melted at room temperature, oscillated and mixed, and then centrifuged for a few seconds using a handheld centrifuge; the mass ratio of pMD2.G to psPAX2 and the target plasmid was 1:3:4. Replace the 293T cells with new DMEM complete medium according to the steps for replacing the adherent cells to ensure that the 293FT cells are fully nourished; take the static mixture and slowly add it to the culture dish, 37°C, 5% CO 2 The cells were cultured for 12 h. After 48 h of medium replacement, the lentivirus was collected and the attached cells were blown off and collected in a centrifuge tube to obtain the pLKO.1-shFcγRIIA and pLKO.1-shCONT virus stock solutions.

[0082] II. FcγRIIA gene and protein expression in glioma stem cells in vitro

[0083] The pLKO.1-shFcγRIIA and pLKO.1-shCONT viruses were used to infect the glioma stem cells MES28, MGG8 and 3028 that needed to be knocked down. GSCs were collected, digested and counted according to the cell passage steps; the frozen virus was thawed at room temperature and centrifuged at 3000rpm for 5 minutes, and 4.5mL of viral supernatant was added to the culture dish. 1×106 cells were inoculated in a 10cm culture dish, and NBM culture medium was added to 10mL; after 12h, new NBM complete culture medium was replaced according to the cell replacement medium, and the cells were continued to be cultured for subsequent experiments.

[0084] The stably expressed cells were cultured and the total RNA and protein were extracted from the cell lysate. qPCR and Western blot were used to evaluate the FcγRIIA gene expression at the mRNA and protein levels. Figure 4 and 5 As shown, the expression levels of FcγRIIA gene and protein were detected in cells infected with FcγRIIA-shRNA and found to be significantly reduced, which indicates that lentiviral shRNA effectively inhibits the expression of FcγRIIA gene at the protein and mRNA levels in tumor cells.

[0085] Example 3: FcγRIIA gene knockdown affects the proliferation of glioma stem cells in vitro

[0086] Cell viability was measured in glioma stem cells infected with shFcγRIIA and shCONT. Cells were counted according to the cell counting procedure and 1 × 10 cells were seeded per well of a 96-well plate. 4 The cells were plated in 6 replicate wells per group in 96-well plates and supplemented with culture medium to 200 μL at 37°C with 5% CO. 2 Cultured in a constant temperature incubator; cell viability was detected on days 1, 3, 5, and 7 after plating; viability assay was performed according to the kit protocol, and luminescence values ​​were analyzed using Prism GraphPad software.

[0087] The number of cells forming spheres was analyzed in glioma stem cells infected with shFcγRIIA and shCONT. After measuring cell viability on the 7th day, the remaining 96-well plates were observed under a microscope, and the cells in each group of spheres were photographed and counted. Each group of cells was diluted in a gradient manner according to the number of cells inoculated per well of the 96-well plate, 1, 5, 10, 20, and 50 cells per well, 30 replicates per concentration, and the culture medium was added to 200 μL at 37°C. 5% CO 2 Culture in a constant temperature incubator. Continue to observe the sphering of cells in each well plate, and count them after the maximum concentration gradient of the control group forms spheroids; observe the 96-well plate under a microscope and record the number of spheroid wells in each group; the experimental results were analyzed using the website https: / / bioinf.wehi.edu.au / software / elda / .

[0088] like Figure 6 As shown in Figure 2, compared with the control group, knocking down the FcγRIIA gene in glioma stem cells MES28, MGG8, and 3028 significantly decreased cell viability. Figure 7 , Figure 8 As shown, knockdown of FcγRIIA gene expression affects glioma stem cell sphere formation.

[0089] Example 4: GSEA analysis of FcγRIIA is associated with the PI3K / AKT signaling pathway

[0090] like Fig. 9 As shown, KEGG enrichment and GSEA analysis of signaling pathways associated with FCGR2A expression in the TCGA GBM dataset showed that FcγRIIA was highly correlated with the PI3K / AKT signaling pathway.

[0091] Example 5: Knockdown of FcγRIIA affects proteins related to the PI3K / AKT signaling pathway

[0092] In glioma stem cells MES28, MGG8 and 3028 infected with shFcγRIIA and shCONT, exogenous IgG immunoglobulin was added to detect the activation of PI3K / AKT signaling pathway, and Western blot was used to detect the levels of activated AKT (p-AKT) and activated mTOR (p-mTOR) proteins. Fig.10 As shown, after knocking down the expression of the FcγRIIA gene, the expression levels of activated AKT (p-AKT) and activated mTOR (p-mTOR) proteins in glioma stem cells MES28, MGG8 and 3028 decreased accordingly, indicating that the FcγRIIA gene affects proteins related to the PI3K / AKT signaling pathway.

[0093] Example 6: Targeting FcγRIIA significantly inhibits intracranial tumor formation in mice

[0094] Glioma stem cells MES28, MGG8 and 3028 infected with shFcγRIIA and shCONT labeled with fluorescent dye were transplanted intracranially into immunodeficient nude mice to observe the growth of tumors in the brain of mice. Fig.11 and 12 As shown, the tumor size of mice transplanted with shFcγRIIA GSCs was significantly reduced ( Fig.11 ), the survival period was significantly prolonged ( Fig.12 ). GSCs and plasma cells PC were transplanted into the brain of mice to create a microenvironment in which GSCs and plasma cells promoted GBM progression; FcγRIIA inhibitor IV.3 was used to administer intracranial in situ twice a week on the third day after GSCs and microenvironment cells formed tumors. The tumor size of the mice was significantly reduced and the survival period was significantly prolonged ( Fig.13 ).

[0095] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. Use of a preparation for knocking down FcγRIIA expression in the preparation of a drug for treating glioblastoma, the preparation comprising shRNA, the shRNA knocking down the mRNA target sequence of FcγRIIA, the nucleotide sequence of the mRNA target sequence is shown in SEQ ID NO.3-4, and the nucleotide sequence of the shRNA is shown in SEQ ID NO.5-6 or SEQ ID NO.7-8.

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