Application of high-expression MTA1 in targeted glioma stem cells in treatment of glioblastoma

Abstract

The invention provides an application of high-expression MTA1 in targeted glioma stem cells (GSC) in treatment of glioblastoma (GBM). According to the invention, the high-level expression of MTA1 protein molecules by the GSC in the GBM is proposed for the first time, the high-expression MTA1 is positively correlated with the poor prognosis of a GBM patient, and the dryness, cell proliferation and balling ability of the GSC are also promoted. The MTA1 highly expressed in the targeted GSC can inhibit the malignant progress of GBM. The invention provides a new target and an inhibitor for targeted therapy of GBM.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of targeting MTA1 highly expressed in glioma stem cells (GSC) in the treatment of glioblastoma. More specifically, the present invention relates to the application of targeting MTA1 highly expressed in glioma stem cells (GSC) to inhibit the proliferation and spheroidization ability of glioma stem cells in the treatment of glioblastoma. Background Art

[0002] Glioblastoma (GBM) is a primary malignant tumor that is most common in the central nervous system, accounting for about 16% of all primary brain tumors. It has a special growth location, extremely strong invasiveness, and common postoperative recurrence characteristics, resulting 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 supplemented with surgical chemotherapy and radiotherapy. However, the overall prognosis of patients is still poor, with a median survival of less than 2 years and a low long-term survival rate. Therefore, there is an urgent need to develop new therapies to improve the prognosis of GBM patients.

[0003] There is a group of GSCs with self-renewal ability in GBM. GSCs are a type of cell population with strong self-renewal ability existing in gliomas. They are involved in many pathological processes such as tumor invasion, immune escape, and tumor angiogenesis, and are a key target for the treatment of GBM.

[0004] Previous studies have shown that MTA1 is upregulated in a variety of malignant tumors, including breast cancer, prostate cancer, cervical cancer, liver cancer, and esophageal cancer. As the malignancy of the tumor increases, the expression level of MTA1 also increases accordingly. MTA1 promotes the proliferation of tumor cells by regulating the expression of cell cycle and proliferation-related genes.

[0005] However, the mechanism of how MTA1 mediates the malignant phenotype of glioblastoma is still unclear. There is an urgent need for in-depth research on how MTA1 mediates the malignant phenotype of glioblastoma in order to develop drugs targeting MTA1, a key protein in glioblastoma, for clinical application. Summary of the invention

[0006] The present invention proposes for the first time that compared with non-stem glioma cells, there is a high MTA1 protein expression level in glioma stem cells.

[0007] The present invention also proposes that high expression of MTA1 is associated with poor prognosis of GBM patients. Therefore, MTA1 is a potential target for treating glioblastoma.

[0008] The present invention also constructs a GSC cell line with MTA1 knockout for the first time, and proposes for the first time that MTA1 plays a key role in the maintenance of 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] In one aspect, the present invention provides use of a reagent for detecting MTA1 protein levels in preparing a diagnostic and / or prognostic composition or kit for glioblastoma, especially glioma stem cells.

[0011] On the other hand, the present invention provides a composition or kit for diagnosing and / or prognosing glioblastoma, especially glioma stem cells, characterized in that the composition or kit comprises a reagent for detecting the level of MTA1 protein.

[0012] In some embodiments, the reagent for detecting the level of MTA1 protein is an antibody.

[0013] In another aspect, the present invention provides use of an MTA1 inhibitor in the preparation of a medicament or a pharmaceutical composition for treating glioblastoma, especially glioma stem cells.

[0014] In another aspect, the present invention provides use of an MTA1 inhibitor in the preparation of a drug or a pharmaceutical composition for inhibiting the proliferation and spheroidization ability of glioblastoma, especially glioma stem cells.

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

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

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

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

[0019] Preferably, the MTA1 inhibitor treats cancer by inhibiting the proliferation and sphere-forming ability of glioma stem cells, thereby killing glioma stem cells.

[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 comprises detecting the MTA1 protein level of the cells to be tested and comparing it with the MTA1 protein level of reference non-stem glioma cells; if the MTA1 protein level of the cells is higher than that of the reference non-stem glioma cells, the cells are determined to be glioma stem cells.

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

[0022] definition

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

[0024] CCK-8: A reagent used to easily and accurately measure the number of viable cells.

[0025] Sphere-forming ability: includes the number of spheres and the size of spheres. The number of spheres refers to the number of stem cell spheres formed by tumor stem cells, which is one of the criteria for measuring the stemness of tumor cells. The size of spheres refers to the size of stem cell spheres formed by tumor stem cells, which is one of the criteria for measuring the stemness of tumor cells.

[0026] Co-IP technology: Based on the specific interaction between antibodies and antigens, target protein-specific antibodies are used to indirectly capture proteins bound to specific target proteins, thereby identifying relevant protein-protein interactions in vivo.

[0027] Beneficial Effects

[0028] Previous studies have shown that MTA1 is highly expressed in a variety of human malignant tumors, including breast cancer, prostate cancer, cervical cancer, liver cancer, and esophageal cancer, and is involved in the occurrence, development, and metastasis of tumors. Specifically, MTA1 may affect the invasiveness and metastasis of tumors by regulating cell functions and participating in signal transduction pathways. In addition, MTA1 is closely related to high tumor recurrence and poor prognosis. MTA1 also promotes the proliferation of tumor cells by regulating the expression of cell cycle and proliferation-related genes.

[0029] The present invention firstly finds that MTA1 has a high protein expression level in glioma stem cells compared with non-stem glioma cells.

[0030] The present invention discovered for the first time that high expression of MTA1 in glioma stem cells is positively correlated with poor prognosis of GBM patients. Therefore, high expression of MTA1 is a very promising marker for evaluating the prognosis of glioblastoma patients.

[0031] The present invention first discovered that MTA1 plays a key role in the proliferation and spheroidization of glioma stem cells. By constructing a GSC cell line with stable knockout of MTA1, it was found that after MTA1 was knocked out, the proliferation and spheroidization of glioma stem cells were inhibited. Therefore, the present invention first discovered that MTA1 plays a key role in promoting the proliferation and spheroidization of glioma stem cells. Therefore, MTA1 is a very promising target for the treatment of glioblastoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This figure shows the results of using immunofluorescence to detect whether MTA1 is highly expressed in glioma stem cells at the same location as the stemness markers SOX2 and OLIG2, and to verify the correlation between MTA1 and the stemness of glioma stem cells.

[0033] Figure 2 This is a graph showing the results of a western blotting experiment verifying that MTA1 is highly expressed in glioma stem cells (GSCs) compared to non-stem glioma cells (NSTCs).

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

[0035] Figure 4 This is the result of agarose gel electrophoresis to verify that the sizes of sgMTA1-1 and sgMTA1-2 plasmids are in the correct position and have normal supercoiled structures.

[0036] Figure 5 This figure shows the results of using Western blotting experiments to verify whether the GSC cell lines of sgCTRL and sgMTA1 were successfully constructed.

[0037] Figure 6 This is a result diagram of using CCK-8 reagent to explore the effect of MTA1 knockout on the proliferation of glioma stem cells. The experiment was performed using biological replicates of two cell lines.

[0038] Fig. 7A , Figure 7B and Figure 7C To investigate the effect of MTA1 on the spheroid formation ability of glioma stem cells using sgMTA1 cell lines and sgCTRL cell lines, biological replicates of two cell lines were used for the experiment. Fig. 7AThe following are representative pictures selected; Figure 7B and 7C This is a statistical chart after counting the images. DETAILED DESCRIPTION

[0039] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

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

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

[0042] The lysis buffer formula is as follows:

[0043] 20mM beat-GPA (Tianjin Ai (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, A500850); 1MDTT (BioFROXX, 111GR005); 1:50 Cocktails (RocheDiagnostics GmbH, 11873580001).

[0044] The statistical analysis involved in the present invention was performed using GraphPad Prism statistical software, the mean values ​​of two groups were compared using t-test, and the mean values ​​of multiple groups were compared using one-way analysis of variance. A P value less than 0.05 was considered to be statistically significant.

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

[0046] 1. The steps of the cell slide experiment are as follows:

[0047] (1) Prepare culture dishes and round glass slides: Add one coverslip to a 12-well plate (add the corresponding number of coverslips according to the staining requirements. The number of stainings can be changed. You can also use a 6-well plate or a 6-cm culture dish; NEST, catalog number 801011). Repeat 3 times for each cell line.

[0048] (2) Diluting the matrix gel: Add 500 μL of matrix gel stock solution (thawed on ice; purchased from Corning, Cat. No. 354234) to 14.5 mL of ice-cold PBS (pre-cooled at 4°C) and mix well, i.e. diluting the solution to 30 times (20, 30, or 40 times is acceptable).

[0049] (3) Solidifying the matrix gel: Add 1 mL of the diluted matrix gel to the 12-well plate prepared in step 1, evenly spread over the coverslip and the bottom of the culture dish, and place in a 37°C incubator to solidify for half an hour.

[0050] (4) Prepare cells: Digest cells and prepare 4.5×10 5 3 mL NBM complete medium (including 500 ml NBM; 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 double antibody solution (1000×) 50 µl) to suspend cells. After the gel has solidified under the microscope, add the cell suspension along the side wall to the culture dish. After the cell suspension covers the bottom, place it in a 37℃ incubator and culture for 24 hours. It can be seen that the cells sink to the bottom of the dish and extend polarly.

[0051] 2. Immunofluorescence experimental steps are as follows:

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

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

[0054] (3) MTA1 and SOX2 antibodies were mixed with 1% BSA in PBS solution, incubated at 4°C overnight, and washed with PBS three times at the end of incubation, each time for 5 minutes.

[0055] (4) Incubate with fluorescent secondary antibody (secondary antibody is an antibody that binds to the primary antibody and has a fluorescent group that can be excited by laser) at room temperature for 2 hours. Hoechst and secondary antibody can be incubated together (diluted to 1 / 10,000). Wash three times with PBS. Keep away from light. After incubation, wash three times with PBS, 5 minutes each time.

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

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

[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: MTA1 protein is specifically highly expressed in glioma stem cells compared to non-stem glioma cells

[0060] In order to find proteins that are specifically highly expressed in GSCs, three different glioma stem cell lines (GBM-1 GSC, GBM-2 GSC and GBM-3 GSC) and corresponding non-stem glioma cell lines (GBM-1 NSTC, GBM-2 NSTC and GBM-3 NSTC) (for cell line preparation methods, see Zhou K, Yao YL, Ping YF, et al. VDAC2 interacts with PFKP to regulate glucose metabolism and phenotypic reprogramming 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. After several days, glioma stem cells and non-stem glioma cells were collected, and 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 steps of Western blotting experiment are as follows:

[0061] Preparation: Pre-cool in a 4℃ centrifuge; take out the RIPA lysis buffer from a 20℃ refrigerator and place it on ice to melt; prepare the spectrophotometer measuring cup: wash the inner wall with alcohol first and then ddH2O, respectively, and dry; dilute with 5x concentration Coomassie Brilliant Blue: 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 RIPA protein lysis buffer (70 μL for <1 million cells, 90 to 120 μL for 2 to 3 million cells, and 120 μL for 3 million cells) according to the cell pellet, and lyse on ice for 15 min.

[0064] (3) Centrifuge at 4°C, 12,000 rpm for 15 min. Place the supernatant in a new EP tube.

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

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

[0067] Preparation: electrophoresis buffer system (Tris base 250mM, Glycine 1.92mM, SDS 35mM); transfer buffer system (Glycine 1.92M, Tris base 250mM, 20% volume of methanol), add HCl to adjust the pH to 7.6, can be reused 3 to 4 times; TBST system (NaCl 2.74M, KCl 54mM, Tris base 380mM, 1 / 1000 volume of Tween-20); 5% skim milk powder (40mL TBST, 2g milk powder); antibody dilution (primary antibody 1:1000, secondary antibody 1:5000, use primary antibody diluent or 1% BSA).

[0068] (1) Prepare 8% separation gel (prepare according to the instructions) and aspirate the residual gel in the well before loading the sample.

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

[0070] (3) Transfer: Cut a PVDF membrane of 4 cm × 8 cm, activate it in methanol in advance, install the electroporation clamp in the order of black clamp-sponge-filter paper-gel-PVDF membrane-filter paper-sponge-white clamp, and transfer in an ice bath at a constant current of 300 mA for 120 min; (reference value: one gel: 1 h; two gels: 1.5 h; three gels: 2 h; four gels: 2.5 h).

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

[0072] (5) Membrane cutting: Cut the PVDF membrane according to the location of the target protein: α-tubulin 55kD, MTA1 80kD, SOX234kD, OLIG2 32kD, GFAP 50kD.

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

[0074] (7) Incubate with secondary antibody (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, each time for 5 min.

[0075] (8) Add luminescent liquid and carry out 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 stemness marker of glioma stem cells, was higher than that of non-stem glioma cells, indicating that glioma stem cells have stem cell characteristics; the expression of GFAP, a differentiation marker of non-stem glioma cells, was higher than that of glioma stem cells, indicating that non-stem glioma cells have differentiation characteristics. Further quantitative results of protein immunoblotting showed that, considering the consistency of internal references, with normalized glioma stem cells as the control, the average expression of MTA1 in non-stem glioma cells was only 19.5% of that in glioma stem cells. Therefore, the expression of MTA1 in glioma stem cells is significantly higher than that in non-stem glioma cells.

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

[0078] (1) sgRNA design: First, it is necessary to design a highly specific sgRNA based on the target gene. This step includes: obtaining detailed information of the target gene on NCBI and selecting the editing region; using Snap-Gene software to open the gene sequence and find the exon region; designing sgRNA based on the exon sequence 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-MTA1-2 5'-ATCGGGAGCTGTTCCTCTCC-3' (SEQ ID No.2).

[0079] (2) Plasmid construction: The synthetic gene sgMTA1-1 was mutated, 5' (BsmBI) and 3' (BsmBI) were added, and the gene was cloned into the vector LentiCRISPRv2.0 (Ampicillin) through 5' BsmBI and 3' BsmBI (using a recombination method), and the LentiCRISPRv2.0 vector of plasmid sgMTA1-1 was constructed. A mini-scale recombinant plasmid DNA was prepared and divided into 1 tube and 1 tube containing the recombinant plasmid. The synthetic gene sgMTA1-2 was mutated, 5' (BsmBI) and 3' (BsmBI) were added, the gene was cloned into the vector LentiCRISPR v2.0 (Ampicillin) by 5' BsmBI and 3' BsmBI (using a recombinant method), and the LentiCRISPR v2.0 vector of plasmid sgMTA1-2 was constructed. A mini-scale recombinant plasmid DNA was prepared and divided into 1 tube and 1 tube of puncture bacteria containing the recombinant plasmid.

[0080] (3) The plasmid powder was centrifuged at 12000 rpm for 1 min, dissolved in 10 μL ddH2O, and then transformed.

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

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

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

[0084] (3) Heat shock at 42°C for 90 s (punching).

[0085] (4) Place on ice for 2 min, take out and add 100 μL of antibody-free LB (5 g of LB Broth (purchased from Sangon; catalog number: A507002-0250) dissolved in 200 mL of ddH2O, sterilized by high temperature and high pressure, and cooled to room temperature before use), and shake at 37°C for 30-60 min (transformation).

[0086] (5) Add bacterial solution to the plate in the clean bench and place the plate upside down in an incubator at 37°C overnight.

[0087] (6) Select a single clone and shake the bacteria with ampicillin (1 / 1000) for 6-8 hours.

[0088] (7) When the microbial culture solution becomes turbid, transfer it to 200 mL of liquid LB medium supplemented with ampicillin and culture the solution for 12 h.

[0089] Extract plasmid (purchased from Beijing Tiangen Biochemical Technology Co., Ltd.; product number: DP117), the experimental steps are as follows:

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

[0091] (2) Take 200 mL of bacterial solution (200 mL is recommended for low copy number) and add it to a centrifuge tube at 8000 rpm for 3 min to collect bacteria. Remove the supernatant as much as possible. Use clean absorbent paper to absorb water droplets on the bottle wall.

[0092] (3) Add 12 mL of solution P1 (check whether RNase A has been added) to the centrifuge tube containing the bacterial cell pellet and vortex to thoroughly suspend the bacterial cell pellet.

[0093] (4) Add 12 mL of solution P2 to the centrifuge tube and immediately and gently invert the tube upside down 6-8 times to fully lyse the bacteria. Place the tube at room temperature for 5 min.

[0094] (5) Add 12 mL of solution P4 to the centrifuge tube, and immediately and gently invert the tube 6-8 times to mix thoroughly until a white precipitate appears in the solution. Place the tube at room temperature for about 10 minutes. Centrifuge at 8000 rpm for 5-10 minutes to allow the white precipitate to centrifuge to the bottom of the tube. Pour all the solution carefully into the filter CS1, and slowly push the 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 inversion, and transfer to the adsorption column CP6 (the adsorption column is placed in a 50 mL collection tube).

[0097] (8) Centrifuge at 8000 rpm at room temperature for 2 min, 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 rinse solution PW to the adsorption column CP6 (first check whether 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 step (9).

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

[0101] (12) Idle at 8000 rpm for 5 min.

[0102] (13) Open the lid of CP6 and let it stand at room temperature for several minutes to allow the rinse solution to dry completely.

[0103] (14) Place the adsorption column CP6 in a clean 50-mL collection tube and drip 1 mL of ddH2O onto the middle of the adsorption film to elute it. Repeat the elution process twice.

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

[0105] Agarose gel electrophoresis experiment verifies the plasmid effect. The experimental steps are as follows:

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

[0107] (2) During the cooling process, add 1 part per ten-thousandth 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 sheet and place it in TAE buffer to remove the comb. The TAE should cover the gel.

[0110] (5) Loading: 2 μL 6× loading solution (3 portions, corresponding to the plasmid) + 1 μL plasmid, mix evenly - make up to 12 μL with ddH2O, sgCTRL as the control group, sgMTA1-1 and sgMTA1-2 as the experimental groups; 5 μL on the marker.

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

[0112] (7) Development and exposure.

[0113] Figure 4 The results showed that the sizes of sgMTA1-1 and sgMTA1-2 plasmids were in the correct position and had normal supercoiled structures.

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

[0115] Calcium phosphate transfection, the experimental steps are as follows:

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

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

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

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

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

[0121] (6) 36-48 hours after transfection, when the brightness of the PCDH-EF1-GFP group reaches above 95%, the virus can be collected (first centrifuge at 4500 rpm for 10 min, take the supernatant, and then filter the supernatant through a 0.22 μm filter).

[0122] (7) Prepare GBM-1 GSCs and GBM-2 GSCs for subculturing, with 500,000 to 800,000 per dish, and add virus solution for infection.

[0123] (8) After 2 days, if the brightness of GSCs infected with PCDH-EF1-GFP group is greater than 95%, Puromycin screening can be performed.

[0124] (9) After two days of Puromycin selection, a stable GSC strain was obtained.

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

[0126] (11) After one generation of culture, the cells were harvested and western blotting was performed to verify the knockout effect of MTA1 in GSCs (see Example 2 for the specific experimental steps).

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

[0128] Example 5: Knockout of MTA1 can significantly inhibit cell proliferation

[0129] CCK-8 (Cell Counting Kit-8) can be used for simple and accurate cell proliferation and toxicity analysis. Its basic principle is: the reagent contains WST-8 [chemical name: 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfonic acid benzene) -2H-tetrazolyl monosodium salt], which is reduced to a highly water-soluble yellow formazan product (Formazan dye) by dehydrogenase in the cell under the action of the electron carrier 1-methoxy-5-methylphenazinium dimethyl sulfate (1-methoxy PMS). The amount of formazan generated is proportional to the number of living cells. Its detection is rapid and sensitive, with low toxicity, and the generated formazan product is more soluble than MTT (Kit purchase company: biosharp).

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

[0131] (1) Prepare cell suspension: Calculate the required number of cells based on the culture time. For GSC1, 100 μL of culture medium and 2,000 cells per well are used. Make sure the suspension is uniform and the number of cells per well is consistent before inoculation.

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

[0133] (3) The inoculation time on the first day is 0 h, and CCK-8 reagent should be added at this time for incubation each time thereafter.

[0134] (4) Pay attention to the 0h treatment. After the cells are stable 2-4h after inoculation, add CCK-8 reagent for incubation and use this as the 0h data.

[0135] (5) When using CCK-8, be careful to avoid light. After adding 10 μL of CCK-8, shake slightly from side to side to mix well. Be careful not to generate bubbles. Cover the well plate with tin foil to avoid light and incubate in a 37°C incubator for 2 h.

[0136] (6) After incubating CCK-8 for 2 h, gently shake the tube left and right to mix well, taking care not to create bubbles.

[0137] (7) Measure the absorbance at 450 nm using an enzyme-labeled instrument.

[0138] (8) If the OD value is not to be measured temporarily, 10 μL of 0.1 M 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 away from light. If measured within 24 hours, the absorbance will not change.

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

[0140] like Figure 6 The results showed that knocking out MTA1 inhibited cell proliferation. In GBM-1 GSCs, sgCTRL showed significant differences from the sgMTA1 treatment group from the second day. At this time, the mean proliferation fold of the control group was 5.6435, while the mean proliferation fold of the treatment group was sgMTA1-1: 3.7319; sgMTA1-2: 2.4685, and the inhibition rates were 66.1% and 43.7%, respectively; in GBM-2 GSCs, the control group showed significant differences from the sgMTA1 treatment group from the second day. At this time, the mean proliferation fold of the control group was 6.6261, while the mean proliferation fold of the treatment group was sgMTA1-1: 4.2040; sgMTA1-2: 4.7591, and the inhibition rates were 63.4% and 71.8%. The inhibition rate gradually increased with time and reached the maximum on the fourth day. At this time, the mean proliferation times of the control group were 14.09 (GBM-1 GSC) and 14.7098 (GBM-2 GSC), and the mean proliferation times of the treatment group were sgMTA1-1: 7.6154; sgMTA1-2: 6.0565 (GBM-1 GSC) and sgMTA1-1: 6.7382; sgMTA1-2: 8.7870 (GBM-2 GSC), and the inhibition rates were sgMTA1-1: 54.0%; sgMTA1-2: 43.0% (GBM-1 GSC) and sgMTA1-1: 45.8%; sgMTA1-2: 59.7% (GBM-2 GSC). In summary, sgMTA1 has a significant inhibitory effect on the proliferation of glioma stem cells.

[0141] Example 6: Knockout of MTA1 can significantly inhibit the spheroidization ability of glioma stem cells

[0142] GBM-1 GSC and GBM-2 GSC cultured in NBM complete medium were subjected to plasmid transfection treatment (see Example 3 for specific experimental steps) to construct sgCTRL, sgMTA1-1, and sgMTA1-2 cell lines, and images were taken using a microscope to perform statistical and differential analysis on the number of spheres.

[0143] like Fig. 7A The results show representative images selected, which show that knocking out MTA1 can significantly inhibit the size and number of GSC spheres.

[0144] like Figure 7B and 7C The results show that the number and size of spheres in randomly selected fields were counted. It can be seen that knocking out MTA1 can significantly inhibit the number and size of GSC spheres. Figure 7B The sphering rate of the control group was normalized to 1, i.e., 100%, and the sphering rates of the treatment groups were sgMTA1-1: 43.3% and sgMTA1-2: 41.0% (GSC1) and sgMTA1-1: 64.3% and sgMTA1-2: 63.7% (GSC2). Figure 7C Statistics on the size of spheres showed that knocking out MTA1 had a higher inhibitory effect on the size of spheres. After normalizing the control group to 1, i.e., 100%, the sphere rates of the treatment groups were sgMTA1-1: 40.0% and sgMTA1-2: 35.1% (GSC1) and sgMTA1-1: 39.5% and sgMTA1-2: 31.5% (GSC2). In summary, knocking out MTA1 had a significant inhibitory effect on the number and size of GSC spheres.

[0145] In summary, the present invention proposes for the first time that MTA1 is a protein that is specifically highly expressed in glioma stem cells, and that high expression of MTA1 is associated with poor prognosis in GBM patients. The present invention constructs a GSC cell line with MTA1 knockout for the first time, and the theory that targeting MTA1 can inhibit the proliferation and spheroidization ability of glioma stem cells. In short, the present invention provides new targets and inhibitors for curing glioblastoma and improving the survival rate of glioblastoma.

[0146] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is 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 in the scope of protection of the present invention.

Claims

1. Use of a reagent for detecting MTA1 protein levels in the preparation of a composition or kit for the diagnosis and / or prognosis of glioblastoma, especially glioma stem cells.

2. A diagnostic and / or prognostic composition or kit for glioblastoma, especially glioma stem cells, characterized in that: The composition or kit includes a reagent for detecting the level of MTA1 protein.

3. The use according to claim 1 or the composition or kit according to claim 2, characterized in that: The reagent for detecting the MTA1 protein level is an antibody.

4. Use of an MTA1 inhibitor in the preparation of a drug or a pharmaceutical composition for inhibiting the proliferation or spheroidization ability of glioblastoma, especially glioma stem cells.

5. Use of MTA1 inhibitors in the preparation of drugs or pharmaceutical compositions for treating glioblastoma, especially glioma stem cells.

6. A drug or pharmaceutical composition for treating glioblastoma, especially glioma stem cells, characterized in that: The medicament or pharmaceutical composition comprises an inhibitor of MTA1.

7. A drug or pharmaceutical composition for inhibiting the proliferation or spheroidization ability of glioblastoma, especially glioma stem cells, characterized in that: The medicament or pharmaceutical composition comprises an inhibitor of MTA1.

8. The use according to claim 4 or 5 or the medicament or pharmaceutical composition according to claim 6 or 7, characterized in that The MTA1 inhibitor is a sgRNA that reduces the level of MTA1 protein. Preferably, the sequence of the sgRNA is shown as SEQ ID No.1 or SEQ ID No.

2.

9. The use, medicine or pharmaceutical composition according to claim 8, characterized in that: The sgRNA was introduced into a lentiviral vector.

10. A method for distinguishing glioma stem cells from non-stem glioma cells, characterized in that: The method comprises detecting the MTA1 protein level of the cells to be tested and comparing it with the MTA1 protein level of reference non-stem glioma cells. If the MTA1 protein level of the cells is higher than that of the reference non-stem glioma cells, the cells are determined to be glioma stem cells.

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