Application of targeting MTA1 highly expressed in glioma stem cells in treatment of glioblastoma

Abstract

The application provides application of MTA1 which is highly expressed in glioma stem cells (GSC) in treatment of glioblastoma (GBM). The application firstly proposes that GSC in GBM highly expresses MTA1 protein molecules, the highly expressed MTA1 is positively correlated with poor prognosis of GBM patients, and also promotes the stemness, cell proliferation and spheroid formation ability of GSC. Targeting the highly expressed MTA1 in GSC can inhibit the malignant progression of GBM. The application provides a new target and inhibitor for targeted treatment of GBM.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of targeting MTA1, which is highly expressed in glioma stem cells (GSCs), in the treatment of glioblastoma. More specifically, this invention relates to the application of targeting MTA1, which is highly expressed in glioma stem cells (GSCs), to inhibit the proliferation and spheroidization ability of glioma stem cells in the treatment of glioblastoma. Background Technology

[0002] Glioblastoma (GBM) is a primary malignant tumor, most commonly found in the central nervous system, accounting for approximately 16% of all primary brain tumors. Its distinctive growth location, highly invasive nature, and high rate of postoperative recurrence result in a median survival of only about 14 months and a five-year survival rate of less than 5% for GBM patients. The current standard treatment for GBM is surgical resection followed by surgical chemotherapy and radiotherapy. However, the overall prognosis remains poor, with a median survival of less than 2 years and a very low long-term survival rate. Therefore, there is an urgent need to develop new therapies to improve the prognosis of GBM patients.

[0003] GBM contains a group of GSCs with self-renewal capabilities. GSCs are a type of cell population in glioma with strong self-renewal capabilities, participating in many pathological processes such as tumor invasion, immune escape, and tumor angiogenesis, and are key targets for the treatment of GBM.

[0004] Previous studies have shown that MTA1 is upregulated in various malignant tumors, including breast cancer, prostate cancer, cervical cancer, liver cancer, and esophageal cancer. The expression level of MTA1 increases with the severity of the tumor. MTA1 promotes tumor cell proliferation by regulating the expression of cell cycle and proliferation-related genes.

[0005] However, the mechanism by which MTA1 mediates the malignant phenotype of glioblastoma is still unclear. Further in-depth research is needed to explore the role of MTA1 in the malignant phenotype of glioblastoma in order to develop drugs that target the key protein MTA1 in glioblastoma for clinical application. Summary of the Invention

[0006] This invention is the first to propose that glioma stem cells have a high level of MTA1 protein expression compared to non-stem glioma cells.

[0007] This invention also suggests that high expression of MTA1 is associated with poor prognosis in GBM patients. Therefore, MTA1 is a potential therapeutic target for glioblastoma.

[0008] This invention also first constructed a GSC cell line with MTA1 knockout, and first proposed that MTA1 plays a key role in maintaining the stemness and malignant phenotype of glioma stem cells. MTA1 can affect the stemness, proliferation and spheroidization ability of glioma stem cells.

[0009] Specifically, the present invention provides the following technical solutions:

[0010] On the one hand, the present invention provides the use of reagents for detecting MTA1 protein levels in the preparation of diagnostic and / or prognostic compositions or kits for glioblastoma, particularly glioma stem cells.

[0011] On the other hand, the present invention provides a diagnostic and / or prognostic composition or kit for glioblastoma, particularly glioma stem cells, characterized in that the composition or kit includes a reagent for detecting MTA1 protein levels.

[0012] In some implementations, antibodies are used as reagents to detect MTA1 protein levels.

[0013] On the other hand, the present invention provides the use of MTA1 inhibitors in the preparation of medicaments or pharmaceutical compositions for treating glioblastoma, particularly glioma stem cells.

[0014] On the other hand, the present invention provides the use of MTA1 inhibitors in the preparation of medicaments or pharmaceutical compositions that inhibit the proliferation and spheroidization ability of glioblastoma stem cells, particularly glioma stem cells.

[0015] On the other hand, the present invention provides a drug or pharmaceutical composition for inhibiting the proliferation and spheroidization ability of glioblastoma stem cells, particularly glioma stem cells, characterized in that the drug or pharmaceutical composition comprises an inhibitor of MTA1.

[0016] On the other hand, the present invention provides a medicament or pharmaceutical composition for treating glioblastoma, particularly glioma stem cells, characterized in that the medicament or pharmaceutical composition comprises an inhibitor of MTA1.

[0017] In some embodiments, the inhibitor of MTA1 is an sgRNA that reduces the level of MAT1 protein, preferably the sequence of which is shown in SEQ ID No. 1 or SEQ ID No. 2.

[0018] In some implementations, the sgRNA is introduced into a lentiviral vector.

[0019] Preferably, MTA1 inhibitors treat cancer by killing glioma stem cells by inhibiting their proliferation and spheroidization ability.

[0020] On the other hand, the present invention provides a method for distinguishing glioma stem cells from non-stem glioma cells, characterized in that the method includes detecting the MTA1 protein level of the cell to be tested and comparing it with the MTA1 protein level of a reference non-stem glioma cell; if the MTA1 protein level of the cell is higher than that of the reference non-stem glioma cell, the cell is identified as a glioma stem cell.

[0021] On the other hand, the present invention provides a method for treating glioblastoma, particularly glioma stem cells, the method comprising administering a therapeutically effective amount of an inhibitor of MTA1 to a subject.

[0022] definition

[0023] MTA1: Metastasis-associated protein 1, a subunit of the NuRD complex. A transcriptional co-regulator, it can act as both a transcriptional co-repressor and co-activator. As part of the histone deacetylase multiprotein complex (NuRD), it regulates the transcription of its target molecules by altering the acetylation state of target chromatin and the accessibility of cofactors to target DNA. It plays a crucial role in tumorigenesis, tumor invasion, and metastasis.

[0024] CCK-8: A reagent for easy and accurate measurement of the number of viable cells.

[0025] Spheroidization capacity: This includes the number and size of spheroids formed. The number of spheroids refers to the number of stem cell spheroids formed by tumor stem cells, and is one of the standards for measuring the stemness of tumor cells. The size of the spheroids refers to the size of the stem cell spheroids formed by tumor stem cells, and is also one of the standards for measuring the stemness of tumor cells.

[0026] Co-IP technology: Based on the specific interaction between antibodies and antigens, it uses target protein-specific antibodies to indirectly capture proteins that bind to specific target proteins, thereby identifying related protein-protein interactions in vivo.

[0027] Beneficial effects

[0028] Previous studies have shown that MTA1 is highly expressed in various human malignancies, including breast cancer, prostate cancer, cervical cancer, liver cancer, and esophageal cancer, and participates in tumorigenesis, development, and metastasis. Specifically, MTA1 may affect tumor invasiveness and metastasis by regulating cellular function and participating in signal transduction pathways. Furthermore, MTA1 is closely associated with high tumor recurrence rates and poor prognosis. MTA1 also promotes tumor cell proliferation by regulating the expression of cell cycle and proliferation-related genes.

[0029] This invention is the first to discover that MTA1 has a high protein expression level in glioma stem cells compared to non-stem glioma cells.

[0030] This invention is the first to discover that high expression of MTA1 in glioma stem cells is positively correlated with poor prognosis in GBM patients. Therefore, high expression of MTA1 is a very promising biomarker for assessing the prognosis of glioblastoma patients.

[0031] This invention is the first to discover that MTA1 plays a crucial role in the proliferation and spheroidization of glioma stem cells. By constructing a stable GSC cell line with MTA1 knockout, it was found that MTA1 knockout inhibited the proliferation and spheroidization of glioma stem cells. Therefore, this invention is the first to demonstrate that MTA1 plays a key role in promoting the proliferation and spheroidization of glioma stem cells. Thus, MTA1 is a very promising target for the treatment of glioblastoma. Attached Figure Description

[0032] Figure 1 The image shows the results of immunofluorescence assay to verify whether MTA1 is highly expressed in glioma stem cells at the same location as the stemness markers SOX2 and OLIG2, thus validating the correlation between MTA1 and the stemness of glioma stem cells.

[0033] Figure 2 The figure shows the results of Western blotting experiments to verify that MTA1 is highly expressed in glioma stem cells (GSCs) compared to non-stem glioma cells (NSTCs).

[0034] Figure 3 The results of the analysis of the CGGA and TCGA databases show that high expression of MTA1 is associated with poor prognosis of GBM.

[0035] Figure 4 The image shows the results of agarose gel electrophoresis to verify that the sgMTA1-1 and sgMTA1-2 plasmids are in the correct positions and that they are in a normal supercoiled structure.

[0036] Figure 5 The image shows the results of using Western blotting to verify the successful construction of GSC cell lines sgCTRL and sgMTA1.

[0037] Figure 6 This figure shows the results of using the CCK-8 reagent to investigate the effect of MTA1 knockout on glioma stem cell proliferation. Biological replicates of two cell lines were used in the experiment.

[0038] Figure 7A , Figure 7B and Figure 7C To investigate the effect of MTA1 on the spheroidization ability of glioma stem cells using sgMTA1 and sgCTRL cell lines, biological replicates of both cell lines were used in the experiment. Figure 7AThese are representative images selected; Figure 7B and 7C This is a statistical chart showing the results of counting the images. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0040] The main materials used in this invention are as follows:

[0041] Primary antibody Anti-MTA1 was purchased from Abcam; primary antibody Anti-SOX2 was purchased from Cell Signaling Technology and ProteinTech; primary antibody Anti-OLIG2 was purchased from Abcam; primary antibody Anti-GFAP was purchased from Biolegend; primary antibody Anti-α-tubulin was purchased from Cell Signaling Technology; secondary antibodies Goat Anti-Mouse IgG (H+L) HRP Conjugate and Goat Anti-Rabbit IgG (H+L) HRP Conjugate were purchased from TRANS; BCA protein quantification kit was purchased from Solario; and WB chemiluminescent substrates (Super Signal Westchemiluminescent substrates) were purchased from Biosharp.

[0042] The lysis buffer formulation is as follows:

[0043] 20mM beat-GPA (Texas Chemical (Shanghai) Chemical Industry Development Co., Ltd., G0097); 200mM PMSF (Sangon Biotech, A610425-0005); 50mM Tris (Sangon Biotech, A501492-0005); HCl (Sinopharm Chemical Reagent Co., Ltd., I04328); pH 7.4; 150mM NaCl (Biosharp, BS112-1kg); 2mM EDTA pH 8.0 (Biosharp, BL518A); 1% NP-40 (Sangon Biotech, A600385-0100); 0.1% SDS (BioFROXX, 3250GR500); 500mM Na3VO4 (Sangon Biotech, A600869-0100); 500mM NaF (Sangon Biotech, A600869-0100); Biotech, A500850); 1MDTT (BioFROXX, 111GR005); 1:50 Cocktails (RocheDiagnostics GmbH, 11873580001).

[0044] The statistical analysis involved in this invention was performed using GraphPad Prism statistical software. The t-test was used to compare the means of two groups, and the one-way ANOVA was used to compare the means of multiple groups. A p-value less than 0.05 was considered statistically significant.

[0045] Example 1: MTA1 is highly expressed at the same location as stem cell markers SOX2 and OLIG2 in glioma stem cells; glioma stem cell smear and immunofluorescence experiments were performed using two different glioma stem cell lines (GBM-1 GSC and GBM-2 GSC).

[0046] 1. The steps for the cell smear experiment are as follows:

[0047] (1) Prepare culture dishes and round glass slides: Add 1 coverslip to a 12-well plate (add the appropriate number of coverslips according to the staining requirements; the number of staining dishes can be changed, or a 6-well plate or a 6 cm culture dish can be used; NEST Corporation, catalog number 801011), and 3 replicates for each cell line.

[0048] (2) Diluting the matrix gel: Add 500 μL of matrix gel stock solution (thawed on ice; purchased from Corning Incorporated, catalog number: 354234) to 14.5 mL of ice-cold PBS (pre-cooled at 4°C) and mix well, i.e. dilute to 30 times (20, 30, or 40 times dilution is also acceptable).

[0049] (3) Solidification of matrix gel: Take 1 mL of the diluted matrix gel and add it to the 12-well plate prepared in step 1. Spread it evenly over the coverslip and the bottom of the culture dish, and place it in a 37℃ incubator to solidify for half an hour.

[0050] (4) Cell preparation: Digest cells and prepare 4.5 × 10⁴ cells. 5 Each 3 mL of NBM complete culture medium (containing 500 ml NBM and GlutaMAX) TM -1 (100×) 5 ml; NEAA (100×) 5 ml; Sodium pyruvate (100×) 5 ml; B27 (50×) 10 ml; EGF (1 mg / ml) 10 µl; FGF (1 mg / ml) 10 µl; Penicillin / streptomycin antibiotic solution (1000×) 50 µl) to suspend cells. Under a microscope, after the gel has solidified, the cell suspension is added along the side wall into the culture dish. After the cell suspension covers the bottom, it is placed in a 37℃ incubator for 24 h. The cells can be seen to sink to the bottom of the dish and spread out in a polar manner.

[0051] 2. The immunofluorescence assay procedure is as follows:

[0052] (1) Fixation: Discard the culture medium. Add 1 mL of paraformaldehyde and fix for 30 min. Wash 3 times with PBS for 5 min each time, shake at 60 rpm. Discard the PBS.

[0053] (2) Membrane disruption + blocking: 0.3% Triton X-100 (1% BSA, dissolved in PBS), add 500 μL of membrane disruption and blocking solution, incubate at room temperature for 1 h, and wash 3 times with PBS.

[0054] (3) Mix the antibodies of MTA1 and SOX2 separately with 1% BSA in PBS solution, incubate overnight at 4°C, and wash with PBS 3 times for 5 min each time after incubation.

[0055] (4) Incubate the fluorescent secondary antibody (the secondary antibody is an antibody that binds to the primary antibody and has a fluorescent group that can be excited by a laser) at room temperature for 2 hours. Hoechst and the secondary antibody can be incubated together (diluted to 0.01%). Wash three times with PBS. Keep out of light. After incubation, wash three times with PBS for 5 minutes each time.

[0056] (5) Use mounting medium (Sigma, F4680) to mount the slide to prevent fluorescence quenching.

[0057] (6) Use a fluorescence microscope for observation and imaging.

[0058] Figure 1The results showed that the protein level of MTA1 in glioma stem cell spheres was highly expressed at the same location as the stemness markers SOX2 and OLIG2, indicating that the expression of MTA1 is related to the stemness of glioma stem cells.

[0059] Example 2: Compared to non-stem glioma cells, MTA1 protein is specifically highly expressed in glioma stem cells.

[0060] To identify proteins specifically highly expressed in GSCs, three different glioma stem cell lines (GBM-1 GSC, GBM-2 GSC, and GBM-3 GSC) and their corresponding non-stem glioma cell lines (GBM-1 NSTC, GBM-2 NSTC, and GBM-3 NSTC) (see Zhou K, Yao YL, Ping YF, et al. VDAC2 interactions with PFKP to regulate glucose metabolism and phenotypicre programming of glioma stem cells. Cell Death Dis. 2018 Sep 24; 9(10):988) were cultured in NBM medium (Gibco, 12349-015) and 1640 medium (BI, C3010-0500), respectively. Several days later, glioma stem cells and non-stem glioma cells were collected. Western blotting was used to detect the stemness characteristics of glioma stem cells and the differentiation characteristics of non-stem glioma cells, as well as the expression of MTA1 in glioma stem cells and non-stem glioma cells. The Western blotting experimental procedure is as follows:

[0061] Preparation: Pre-cool the centrifuge at 4℃; remove the RIPA lysis buffer from the 20℃ freezer and thaw it on ice; prepare the spectrophotometer measuring cup: clean the inner wall once with alcohol and then once with ddH2O, and let it dry; dilute Coomassie Brilliant Blue at 5x concentration: 400μL per sample.

[0062] (1) Centrifuge the culture medium containing glioma stem cells and non-stem glioma cells at 1000 rpm for 3 min.

[0063] (2) Discard the supernatant and add an appropriate volume of protein lysis buffer RIPA according to the cell pellet (<1 million cells, 70 μL; 2 million to 3 million cells, 90 to 120 μL; 3 million cells, 120 μL). Lyse on ice for 15 min.

[0064] (3) Centrifuge at 4℃ and 12000rpm for 15 minutes. After completion, place the supernatant into a new EP tube.

[0065] (4) Quantify the protein concentration of the sample using the BCA method.

[0066] (5) Add 4 times the concentration of loading buffer (Tris-Cl 200mM (pH 6.8), SDS 8%, glycerol 40%, mercaptoethanol 4%, bromophenol blue 0.40%), in a 100℃ metal bath for 10 min, and store at 20℃.

[0067] Preparation: Electrophoresis buffer (Tris base 250mM, Glycine 1.92mM, SDS 35mM); Transfer buffer (Glycine 1.92M, Tris base 250mM, 20% volume methanol), adjust pH to 7.6 with HCl, reusable 3 to 4 times; TBST system (NaCl 2.74M, KCl 54mM, Tris base 380mM, 1 / 1000 volume Tween-20); 5% skim milk powder (40mL TBST, 2g milk powder); Antibody dilution (primary antibody 1:1000, secondary antibody 1:5000, using primary antibody dilution buffer or 1% BSA).

[0068] (1) Prepare 8% separating gel (prepare according to the instructions) and remove residual gel from the well before loading the sample.

[0069] (2) Add 4 μL of marker protein to the sample and 10 μg to the sample. Perform electrophoresis at the following voltages: 80 mV for 40 min; 110 mV for 80 min.

[0070] (3) Transfer membrane: Cut a 4cm×8cm PVDF membrane, pre-activate it in methanol, and install the electric transfer clamp in the order of black clamp-sponge-filter paper-adhesive-PVDF membrane-filter paper-sponge-white clamp. Perform constant current 300mA ice bath wet transfer for 120min; (Reference values: one adhesive: 1h; two adhesives: 1.5h; three adhesives: 2h; four adhesives: 2.5h).

[0071] (4) Blocking: Block with 5% skim milk for 1 hour, then wash three times with TBST (conventional washing solution) for 5 minutes each time.

[0072] (5) Membrane cutting: Cut the PVDF membrane according to the location of the target protein: α-tubulin 55kD, MTA1 80kD, SOX2 34kD, OLIG2 32kD, GFAP 50kD.

[0073] (6) Incubation with primary antibodies: Incubate primary antibodies α-tubulin (internal reference protein), MTA1 (target protein), SOX2 and OLIG2 (dryness markers), and GFAP (differentiation markers) at 4°C overnight. Recover the primary antibodies and wash three times with TBST for 5 min each time.

[0074] (7) Incubate the secondary antibody (the secondary antibody is an antibody that binds to the primary antibody and carries an enzyme that can react with the substrate in the luminescent solution) for 90 min, recover the secondary antibody, and wash three times with TBST for 5 min each time.

[0075] (8) Add luminescent liquid and perform development and exposure.

[0076] like Figure 2 The results showed that in the three cell lines (GBM-1 GSC, GBM-2 GSC, and GBM-3 GSC), the expression of SOX2, a stem cell marker, was higher in glioma stem cells than in non-stem glioma cells, indicating that glioma stem cells possess stem cell characteristics. Conversely, the expression of GFAP, a differentiation marker, was higher in non-stem glioma cells than in glioma stem cells, indicating that non-stem glioma cells possess differentiation characteristics. Further quantitative analysis using Western blotting revealed that, considering the consistency of internal controls, and using normalized glioma stem cells as a control, the mean MTA1 expression level in non-stem glioma cells was only 19.5% of that in glioma stem cells. Therefore, MTA1 expression in glioma stem cells was significantly higher than that in non-stem glioma cells.

[0077] Example 3: Design of sgRNA and construction and validation of sgMTA1 plasmid

[0078] (1) sgRNA design: First, a highly specific sgRNA needs to be designed based on the target gene. This step includes: obtaining detailed information about the target gene from NCBI and selecting the editing region; opening the gene sequence using Snap-Gene software and finding the exon regions; designing sgRNA based on the exon sequences to ensure specificity and avoid off-target effects. The final designed sgRNA sequences are as follows: sgRNA-MTA1-1 5'-CTCTGCCCGCCACGCACATC-3' (SEQ ID No. 1); sgRNA-MTA1-25'-ATCGGGAGCTGTTCCTCTCC-3' (SEQ ID No. 2).

[0079] (2) Plasmid construction: The gene sgMTA1-1 was mutated and synthesized, and 5' (BsmBI) and 3' (BsmBI) were added. The gene was cloned into the vector LentiCRISPRv2.0 (Ampicillin) through 5' BsmBI and 3' BsmBI (using the recombination method) to construct the LentiCRISPRv2.0 vector of plasmid sgMTA1-1. One mini-scale recombinant plasmid DNA was prepared and divided into one tube and one tube of stab bacteria containing the recombinant plasmid. The sgMTA1-2 gene was mutated and synthesized by adding 5' (BsmBI) and 3' (BsmBI). The gene was cloned into the LentiCRISPR v2.0 (Ampicillin) vector via 5' BsmBI and 3' BsmBI (using recombination). The LentiCRISPR v2.0 vector containing plasmid sgMTA1-2 was constructed. One mini-scale recombinant plasmid DNA was prepared and divided into one tube and one tube containing the recombinant plasmid for skeletal sampling.

[0080] (3) Centrifuge the plasmid powder at 12000 rpm for 1 min, dissolve it in 10 μL ddH2O, and then perform the conversion.

[0081] The steps for the E. coli transformation experiment are as follows:

[0082] (1) Place competent cells on ice, add 100 ng / μL of plasmid, recombinant DNA, and 40-50 μL of competent cells (thaw on ice first).

[0083] (2) Place on ice for 40 minutes.

[0084] (3) Heat shock at 42℃ for 90s (drilling).

[0085] (4) Place on ice for 2 minutes, then add 100 μL of antibiotic-free LB (5 g of LB Broth (purchased by Sangon Biotech; catalog number: A507002-0250) dissolved in 200 mL of ddH2O, sterilized at high temperature and high pressure, and cooled to room temperature before use), and shake at 37℃ for 30-60 minutes (conversion).

[0086] (5) Add bacterial solution to the plate in the clean bench, spread the plate, and invert the incubator at 37°C overnight.

[0087] (6) Select a single clone, shake it slightly and add ampicillin (1 / 1000) for 6-8 hours.

[0088] (7) After the small-scale bacterial culture becomes turbid, transfer it to 200 mL of liquid LB medium with added ampicillin and culture for 12 h.

[0089] Plasmid extraction (purchased from Beijing Tiangen Biotech Co., Ltd.; catalog number: DP117), experimental steps are as follows:

[0090] (1) Add anhydrous ethanol to the rinsing solution PW before use.

[0091] (2) Take 200mL of bacterial culture (200mL is recommended for low copy number) and add it to a centrifuge tube. Collect bacteria at 8000rpm for 3min. Try to remove the supernatant. You can use clean absorbent paper to remove water droplets from the bottle wall.

[0092] (3) Add 12 mL of solution P1 to the centrifuge tube containing bacterial precipitate (first check if RNase A has been added), and vortex to completely suspend the bacterial cell precipitate.

[0093] (4) Add 12 mL of solution P2 to the centrifuge tube, and immediately gently invert it 6-8 times to fully lyse the bacteria. Let it stand at room temperature for 5 minutes.

[0094] (5) Add 12 mL of solution P4 to the centrifuge tube, and immediately gently invert it 6-8 times to mix thoroughly until a white precipitate appears. Let it stand at room temperature for about 10 minutes. Centrifuge at 8000 rpm for 5-10 minutes to allow the white precipitate to settle to the bottom of the tube. Carefully pour all the solution into filter CS1, and slowly push the push handle to filter. Collect the filtrate in a 50 mL tube.

[0095] (6) Column equilibration step: Add 2.5 mL of equilibration solution BL to the adsorption column CP6, centrifuge at 8000 rpm for 2 min, and discard the waste liquid in the collection tube.

[0096] (7) Add 0.3 times the volume of isopropanol to the filtrate, mix by inverting, and then transfer to the adsorption column CP6 (place the adsorption column in a 50mL collection tube).

[0097] (8) Centrifuge at 8000 rpm for 2 min at room temperature, discard the waste liquid in the collection tube, and put the adsorption column CP6 back into the collection tube.

[0098] (9) Add 10 mL of washing solution PW to the adsorption column CP6 (first check if anhydrous ethanol has been added), centrifuge at 8000 rpm for 2 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0099] (10) Repeat operation (9).

[0100] (11) Add 3 mL of anhydrous ethanol to the adsorption column CP6, centrifuge at 8000 rpm for 2 min at room temperature, and discard the waste liquid.

[0101] (12) Air-free 8000rpm, 5min.

[0102] (13) Open the CP6 cap and let it stand at room temperature for a few minutes to completely dry the rinsing solution.

[0103] (14) Place the adsorption column CP6 in a clean 50mL collection tube, add 1mL of ddH2O dropwise to the middle part of the adsorption membrane, and elute. Elute twice in total.

[0104] (15) DNA concentration was measured using a spectrophotometer.

[0105] The plasmid effect was verified by agarose gel electrophoresis. The experimental steps are as follows:

[0106] (1) 50mL TAE + 0.5g agar powder, cover with aluminum foil, microwave for a few minutes until boiling, the white solution becomes clear, indicating that it is completely dissolved (1% gel).

[0107] (2) During the cooling process, add 0.01% of EB dye into the conical flask.

[0108] (3) Turn the plate upside down, insert an 8-hole comb, and cool for 20 minutes.

[0109] (4) Take out the gel plate and place it in TAE buffer solution to remove the comb. The TAE needs to be completely submerged in the gel.

[0110] (5) Sample loading: Mix 2 μL of 6× sample loading solution (3 portions, corresponding to the plasmid) + 1 μL of plasmid evenly, then add ddH2O to make up to 12 μL. sgCTRL is used as the control group, and sgMTA1-1 and sgMTA1-2 are the experimental groups; 5 μL is loaded onto the marker.

[0111] (6) Electrophoresis at 100V for 1 hour;

[0112] (7) Development and exposure.

[0113] Figure 4 The results show that the sgMTA1-1 and sgMTA1-2 plasmids are in the correct positions and have normal supercoiled structures.

[0114] Example 4: Construction of glioma stem cells with MTA1 knocked out by sgRNA mediated by calcium phosphate co-precipitation transfection method

[0115] The experimental steps for calcium phosphate transfection are as follows:

[0116] (1) Seed cells 12 hours before transfection. For a 10 cm culture dish, seed 1.5 × 10⁶ cells. 6 293T cells (purchased from ATCC cell bank).

[0117] (2) Prepare transfection solution: ddH2O 360μL; CaCl2 (2.5M) 40μL; DNA 6μg (add 6μg to each of the four DNAs (PCDH-EF1-GFP, sgCTRL (purchased from Genewiz), sgMTA1-1, sgMTA1-2), and add 6μg to each tube of viral packaging plasmid (PAX2, VSVG); 2×HBS 400μL, for a total of 800μL.

[0118] (3) Slowly add DNA-CaCl2 solution dropwise into 2×HBS buffer.

[0119] (4) Let the transfection solution stand at room temperature for 10 minutes, then drop the mixture into DMEM medium (containing 500 mL DMEM; 50 mL fetal bovine serum FBS; and 1000 × 5 mL penicillin / streptomycin antibiotic solution). Gently shake the culture dish to mix; the medium should turn yellow.

[0120] (5) 12-16 hours after transfection, replace with NBM complete medium.

[0121] (6) After 36-48 hours of transfection, observe that the brightness of the PCDH-EF1-GFP group reaches more than 95%, and the virus can be collected (first centrifuge at 4500 rpm for 10 min, take the supernatant, and then filter it with a 0.22 μm filter to obtain the supernatant).

[0122] (7) Prepare GBM-1 GSC and GBM-2 GSC for passage, 500,000-800,000 per plate, and add virus solution for infection.

[0123] (8) Two days later, if the brightness of GSCs infected with PCDH-EF1-GFP is greater than 95%, Puromycin screening can be performed.

[0124] (9) GSC stable strains were obtained two days after Puromycin screening.

[0125] (10) Passage the GSCs transfected with the target plasmid.

[0126] (11) After culturing one generation of cells, the cells were harvested and the effect of MTA1 knockout in GSCs was verified by Western blotting (see Example 2 for specific experimental steps).

[0127] (12) Figure 5The results showed that the MTA1 protein expression levels in the sgMTA1-1 and sgMTA1-2 groups in GBM-1 GSC and GBM-2 GSC were much lower than those in the sgCTRL group (GBM-1 GSC: mean sgMTA1 knockout rate of 78.0%; GBM-2 GSC: mean sgMTA1 knockout rate of 87.6%), indicating that the MTA1 knockout GSC cell line was successfully constructed.

[0128] Example 5: Knocking out MTA1 significantly inhibited cell proliferation.

[0129] The CCK-8 (Cell Counting Kit-8) provides a simple and accurate analysis of cell proliferation and toxicity. Its basic principle is as follows: This reagent contains WST-8 [chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt], which is reduced by dehydrogenases in cells to a highly water-soluble yellow formazan dye under the action of the electron carrier 1-methoxy-5-methylphenazineonium sulfate (1-methoxyPMS). The amount of formazan dye produced is directly proportional to the number of viable cells. It offers rapid and sensitive detection with low toxicity, and the formazan dye produced is more soluble than MTT (kit purchased from Biosharp).

[0130] The specific experimental steps for CCK8 are as follows:

[0131] (1) Prepare cell suspension: Calculate the required number of cells based on the culture time. Taking GBM1-GSC as an example, use 100 μL of culture medium per well and 2,000 cells per well. Note that the suspension should be uniform and the number of cells per well should be consistent before inoculation.

[0132] (2) Experimental design: control group sgCTRL; experimental group sgMTA1-1, sgMTA1-2; leave one row around the top, bottom and left and right unused, PBS can be added, i.e., six replicates per group.

[0133] (3) Take the first day of inoculation as 0h, and add CCK-8 reagent at this time for each subsequent incubation.

[0134] (4) Note the treatment of 0h. After the cells have stabilized 2-4 hours after inoculation, add CCK-8 reagent for incubation and use it as the 0h data.

[0135] (5) When using CCK-8, be careful to avoid light; after adding 10 μL of CCK-8, shake it slightly left and right to mix it, being careful not to generate air bubbles, cover the well plate with tin foil to avoid light, and incubate it in an incubator at 37°C for 2 hours.

[0136] (6) After incubating CCK-8 for 2 hours, gently shake left and right to mix, being careful not to generate bubbles.

[0137] (7) Absorbance was measured using a 450nm microplate reader.

[0138] (8) If the OD value is not to be measured temporarily, 10 μL of 0.1M HCl solution or 1% w / v SDS solution can be added to each well, and the culture plate can be covered and stored at room temperature in the dark; the absorbance will not change if measured within 24 hours.

[0139] (9) Data processing: Cell viability = [(absorbance of experimental wells - absorbance of blank wells) / (absorbance of experimental wells - absorbance of control wells)] × 100%.

[0140] like Figure 6 The results showed that MTA1 knockout inhibited cell proliferation. In GBM-1 GSCs, sgCTRL showed a significant difference from the sgMTA1 treatment group starting from day two. At this point, the mean fold increase in the control group was 5.6435, while the mean fold increase in the treatment groups was 3.7319 for sgMTA1-1 and 2.4685 for sgMTA1-2, with inhibition rates of 66.1% and 43.7%, respectively. In GBM-2 GSCs, the control group also showed a significant difference from the sgMTA1 treatment group starting from day two. At this point, the mean fold increase in the control group was 6.6261, while the mean fold increase in the treatment groups was 4.2040 for sgMTA1-1 and 4.7591 for sgMTA1-2, with inhibition rates of 63.4% and 71.8%, respectively. The inhibition rate gradually increased with time, reaching its maximum on day four. At this point, the mean fold increase in the control group was 14.09 (GBM-1 GSC) and 14.7098 (GBM-2 GSC), while the mean fold increase in the treatment group was 7.6154 for sgMTA1-1, 6.0565 for sgMTA1-2 (GBM-1 GSC), 6.7382 for sgMTA1-1, and 8.7870 for sgMTA1-2 (GBM-2 GSC), with inhibition rates of 54.0% for sgMTA1-1, 43.0% for sgMTA1-2 (GBM-1 GSC), 45.8% for sgMTA1-1, and 59.7% for sgMTA1-2 (GBM-2 GSC). In conclusion, sgMTA1 has a significant inhibitory effect on the proliferation of glioma stem cells.

[0141] Example 6: Knocking out MTA1 significantly inhibited the spheroidization ability of glioma stem cells.

[0142] GBM-1 GSC and GBM-2 GSC cultured in NBM complete medium were transfected with plasmids (see Example 3 for specific experimental steps) to construct sgCTRL, sgMTA1-1, and sgMTA1-2 cell lines. Microscopic images were taken, and the number of cells was statistically analyzed and differentially analyzed.

[0143] like Figure 7A The results show representative images, demonstrating that knocking out MTA1 can significantly suppress the size and number of GSC spheres.

[0144] like Figure 7B and 7C The results show that the number and size of spheres were statistically analyzed in randomly selected fields of view. It can be seen that knocking out MTA1 can significantly suppress the number and size of spheres in GSC. Figure 7B This is a statistical analysis of the number of pellets formed. The control group is normalized to 1 (100%), and the pellet formation rates for the treatment groups are 43.3% and 41.0% (GBM-1 GSC) for sgMTA1-1, and 64.3% and 63.7% (GBM-2 GSC) for sgMTA1-2. In comparison, such as... Figure 7C Statistical analysis of globule size showed that MTA1 knockout had a greater inhibitory effect on globule size. After normalizing the control group to 1 (100%), the globule formation rates in the treatment groups were 40.0% and 35.1% (GBM-1 GSC) for sgMTA1-1, and 39.5% and 31.5% (GBM-2 GSC) for sgMTA1-1 and sgMTA1-2, respectively. In conclusion, MTA1 knockout significantly inhibits both the number and size of globules in GSCs.

[0145] In summary, this invention is the first to propose that MTA1 is a protein specifically highly expressed in glioma stem cells, and that high expression of MTA1 is associated with poor prognosis in GBM patients. This invention is also the first to construct a GSC cell line with MTA1 knockout, demonstrating the theory that targeting MTA1 can inhibit the proliferation and spheroidization ability of glioma stem cells. In conclusion, this invention provides a novel target and inhibitor for curing glioblastoma and improving its survival rate.

[0146] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of an MTA1 inhibitor in the preparation of a medicament or pharmaceutical composition for inhibiting the proliferation or spheroidization of glioma stem cells, wherein the MTA1 inhibitor is an sgRNA that reduces the level of MTA1 protein, and the sequence of the sgRNA is shown in SEQ ID No. 1 or SEQ ID No.

2.

2. Use of an MTA1 inhibitor in the preparation of a medicament or pharmaceutical composition for treating glioma stem cells, wherein the MTA1 inhibitor is an sgRNA that reduces the level of MTA1 protein, and the sequence of the sgRNA is shown in SEQ ID No. 1 or SEQ ID No.

2.

3. The use according to claim 1 or 2, characterized in that, The sgRNA was introduced into a lentiviral vector.

4. A drug or drug composition for treating glioma stem cells, characterized in that, The drug or drug composition includes an inhibitor of MTA1, wherein the inhibitor of MTA1 is an sgRNA that reduces the level of MTA1 protein, and the sequence of the sgRNA is shown in SEQ ID No. 1 or SEQ ID No.

2.

5. A drug or pharmaceutical composition that inhibits the proliferation or spheroidization ability of glioma stem cells, characterized in that, The drug or drug composition includes an inhibitor of MTA1, wherein the inhibitor of MTA1 is an sgRNA that reduces the level of MTA1 protein, and the sequence of the sgRNA is shown in SEQ ID No. 1 or SEQ ID No.

2.

6. The drug or drug composition according to claim 4 or 5, characterized in that, The sgRNA was introduced into a lentiviral vector.

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