Application of MYRF gene in preparation of composition for treating glioma

Abstract

The invention discloses application of an MYRF gene in preparation of a composition for treating glioma, overexpression of the MYRF gene in glioma cells enables the glioma cells to be transdifferentiated into glial cells, MBP and MAG signals are up-regulated, and PDGFR alpha signals are down-regulated. The invention also provides an application of the MYRF gene in preparation of a glioma cell transdifferentiation reagent, and a method and a composition for transdifferentiating glioma cells into glioma cells. The invention has great significance on treatment of glioma, provides a new strategy for treatment of glioma, possibly contributes to reducing the malignant degree of tumor and overcoming the drug resistance of tumor cells, and possibly improves the effect of immunotherapy by changing the tumor microenvironment. In addition, the invention also helps to deeply understand the pathological mechanism of glioma, and provides a theoretical basis for developing new therapeutic targets and drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, relates to the application of MYRF gene in preparing a composition for treating glioma, and generally relates to a composition and method for treating glioma, wherein the composition comprises a combination of exogenous nucleic acids encoding MYRF polypeptide or biologically active fragments thereof packaged by a lentivirus. Background Art

[0002] Brain glioma refers to a tumor that originates from glial cells and is the most common primary intracranial tumor. Glioblastoma (GBM) is a high-grade glioma of grade IV in the World Health Organization (WHO) classification, which is mainly formed in astrocytes that support nerve cells. Statistics from the U.S. Central Brain Tumor Registry and the U.S. Centers for Disease Control and Prevention show that glioblastoma is the most common malignant central nervous system tumor, accounting for 50.1% of all primary central nervous system malignant tumors, with the highest incidence rate of about 3.26 cases per 100,000 people. GBM is one of the most difficult tumors to treat and has a poor prognosis among systemic tumors. It is highly invasive and grows in an infiltrative manner. It is not easy to completely remove it surgically, and it is prone to recurrence after surgery. The median survival period is only 12 to 15 months, and the five-year survival rate does not exceed 5%. At the same time, neurological dysfunction caused by tumors, such as worsening headaches, nausea and vomiting, blurred vision or diplopia, and epileptic seizures, will seriously affect the patient's quality of life. Despite major advances in cancer treatment, the overall 5-year survival rate for glioblastoma multiforme has stagnated at just under 5%.

[0003] The current standard treatment for GBM includes surgical resection of the tumor, adjuvant chemotherapy and radiotherapy with the DNA alkylating agent temozolomide (TMZ). In addition, the presence of a small number of glioma stem cells (GSCs) in glioblastoma is associated with GBM chemoresistance, heterogeneity, and recurrence. In addition, although temozolomide has been shown to target GSCs, it is also more tumorigenic. Therefore, other therapeutic strategies against GBM are needed. Summary of the invention

[0004] The first objective of the present invention is to address the deficiencies in the prior art and to provide an application of a MYRF gene in the preparation of a reagent for treating glioma.

[0005] Furthermore, the MYRF gene was overexpressed in glioma cells, causing them to transdifferentiate into glial cells.

[0006] The second objective of the present invention is to provide the use of MYRF gene in preparing a glioma cell transdifferentiation reagent, wherein the transdifferentiation specifically comprises: transdifferentiation of glioma cells into glial cells.

[0007] The third object of the present invention is to provide a method for transdifferentiating glioma cells into glial cells, wherein the method specifically comprises contacting the glioma cells with an exogenous nucleic acid encoding a MYRF polypeptide or a biologically active fragment thereof.

[0008] Alternatively, the method specifically involves delivering an expression vector comprising an exogenous nucleic acid encoding a MYRF polypeptide or a biologically active fragment thereof to glioma cells.

[0009] Furthermore, the expression vector is a recombinant lentiviral expression vector.

[0010] Furthermore, the recombinant lentiviral expression vector is a psPAX2 or pMD2.G expression vector.

[0011] Furthermore, the glioma cells include human glioma cells (U87) and human glioma cell line (U251).

[0012] Further, the stages of glioma cells include grade I, grade II, grade III, and grade IV.

[0013] The fourth object of the present invention is to provide a composition for treating glioma, comprising one or more of the following ingredients:

[0014] (a) MYRF polypeptide or a biologically active fragment thereof;

[0015] (b) an exogenous nucleic acid encoding a MYRF polypeptide or a biologically active fragment thereof;

[0016] (c) an expression vector of an exogenous nucleic acid encoding a MYRF polypeptide or a biologically active fragment thereof.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention provides a composition and method for transdifferentiating malignant glial cells (e.g., gliomas) into glial cells, and an exogenous nucleic acid encoding a MYRF polypeptide or a biologically active fragment thereof mediates the effective transdifferentiation of glioma cells into glial cells. The present invention is of great significance for the treatment of gliomas, which not only provides a new strategy for the treatment of gliomas, but also may help reduce the malignancy of tumors, overcome the drug resistance of tumor cells, and may improve the effect of immunotherapy by changing the tumor microenvironment. In addition, the present invention also helps to deeply understand the pathological mechanism of gliomas and provide a theoretical basis for the development of new therapeutic targets and drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Bright-field images from an optical microscope.

[0020] Figure 2 Immunohistochemistry images and qPCR statistical graphs, where A is the immunofluorescence image of U251 cells, and B is the qPCR statistical graph of U251 cells and U87 cells.

[0021] Figure 3 These are immunohistochemical images and bar graphs, where A is the immunofluorescence image and B is the statistical graph of the fluorescence signal.

[0022] Figure 4 In situ hybridization images.

[0023] Figure 5 This is a western blot image of U251 cells.

[0024] Figure 6 These are western blot images of U251 and U87. DETAILED DESCRIPTION

[0025] Unless otherwise defined herein, all technical and scientific terms used in the present disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs.

[0026] The present disclosure includes methods designed for transdifferentiation of glioma cells into glial cells. In this regard, the present disclosure includes data proving that after U251 cells and U87 cells overexpress the MYRF gene, effective transdifferentiation of glioma cells into glial cells is provided, such as upregulation of MBP and MAG signals, downregulation of PDGFRα signals, etc.

[0027] U251 cells, also known in the art as U-251 cells, and formerly as U-373 cells, are commercially available, such as product number 09063001 from MILLIPORE-SIGMA. The cells are derived from malignant glioblastoma tumors, and in particular human astrocytic glioblastoma cells. U87 cells, also known in the art as U-87, U87-mg cells, are commercially available, such as product number 89081402 from MILLIPORE-SIGMA, and the cells are derived from human glioma cells, and in particular human brain astrocytic glioblastoma cells. Therefore, certain embodiments of the present disclosure are confirmed in vitro, and it is expected that the data can be extended to in vivo applications, including but not limited to animal models of gliomas and related glioma cell cancers, and for the treatment of humans. The present disclosure includes treatment methods using overexpressed MYRF genes.

[0028] In embodiments, contacting the glioma cells with an exogenous nucleic acid encoding a MYRF polypeptide or a biologically active fragment thereof comprises administering the composition to a subject in need thereof.

[0029] In an embodiment, the subject in need of the method suffers from a CNS malignancy (e.g., cancer), including but not necessarily limited to glioma cells or glioma cell-related disorders. In an embodiment, cancer comprises only a single type of glioma. In an embodiment, cancer comprises more than one type of glioma cells, such as a mixed glioma. In an embodiment, the glioma is a high-grade glioma or a low-grade glioma. It is well known in the art to grade gliomas. In general, low-grade gliomas are considered to be grade I or grade II. A non-limiting example of a grade I glioma is a pediatric pilocytic astrocytoma. Grade I gliomas may have mutations in human BRAF genes. The most common grade II gliomas are astrocytomas and oligodendrogliomas. Grade II gliomas often have mutations in human IDH1 or IDH2 genes. High-grade gliomas are grade III or grade IV tumors. Grade III gliomas include anaplastic astrocytomas and anaplastic oligodendrogliomas. Grade IV gliomas are referred to as glioblastomas. Without intending to be bound by any particular theory, it is believed that the compounds and methods are suitable for treating any cancer comprising one or more of the described types of gliomas. In an embodiment, the cancer thus comprises one or more astrocytomas, including but not necessarily limited to astrocytomas, anaplastic astrocytomas, and glioblastomas. In an embodiment, the cancer comprises ependymomas, including but not necessarily limited to anaplastic ependymomas, myxopapillary ependymomas, and subependymomas. In an embodiment, the cancer comprises oligodendrogliomas, including but not limited to oligodendrogliomas, anaplastic oligodendrogliomas, and anaplastic oligoastrocytomas. In an embodiment, the cancer comprises low-grade oligodendrogliomas. In an embodiment, the cancer comprises optic nerve gliomas. In an embodiment, the cancer comprises subependymomas. In an embodiment, the cancer comprises glioma cell stem gliomas. In an embodiment, the method reduces or eliminates glioma stem cells. In an embodiment, the method is therapeutic to glioblastomas. In an embodiment, the method is therapeutic to glioblastoma multiforme.

[0030] Glioma cells are contacted with exogenous nucleic acids encoding MYRF polypeptides or their biologically active fragments, and it is expected that at least some glioma cells in the subject will be transdifferentiated into glial cells. In an embodiment, transdifferentiation into glial cells occurs in a period of about 7 to 14 days. In an embodiment, transdifferentiation into glial cells occurs in 7 to 8 days, 7 to 9 days, 7 to 10 days, 8 to 9 days, 8 to 10 days, 8 to 11 days, 9 to 10 days, 9 to 11 days, 9 to 12 days, 10 to 11 days, 10 to 12 days, 10 to 13 days, 11 to 12 days, 11 to 13 days, 11 to 14 days, 12 to 13 days, 12 to 14 days or 13 to 14 days. In embodiments, the time of transdifferentiation into glial cells is no more than 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 days.

[0031] In addition to transdifferentiating glioma cells into glial cells, the present disclosure further encompasses generating new glial cells from endogenous glial cells, and may include generating new glial cells from glial-like cells generated due to damage or disease conditions of the central or peripheral nervous system (such as but not limited to glioma).

[0032] The glial cells produced using the method are typically functional glial cells, and therefore can have at least one of the following non-limiting example functions: synapse formation, axon formation, dendrite formation or neurotransmitter release. Non-limiting examples of the glial cells produced by the method and composition are monopolar, bipolar or multipolar glial cells. Non-limiting examples of neurons include any type of glial cells, such as basket cells, Lugaro cells, medium-sized spiny glial cells, Purkinje cells or spindle cells. In an embodiment, the glial cells are selected from the group consisting of cholinergic glial cells, GABAergic glial cells, glutamatergic glial cells, dopaminergic glial cells, adrenergic glial cells, motor glial cells, peptidergic glial cells and serotonergic glial cells.

[0033] Functional glial cells can exhibit properties that can include, but are not necessarily limited to, firing repetitive action potentials, developing multiple dendritic branches, and releasing neurotransmitters including, but not necessarily limited to, glutamate.

[0034] (Glutamate / glutamicacid), dopamine, acetylcholine, serotonin, norepinephrine (Norepinephrine / noradrenaline) and gamma-aminobutyric acid (GABA). Therefore, it is expected that the present disclosure will promote the development of new cortical pre-glioma cell glial cells, middle glioma cell glial cells, post-glioma cell glial cells, spinal cord glial cells or peripheral glial cells or a combination thereof by using the methods described herein, and those skilled in the art can adjust the method as needed in a manner that is obvious in view of the benefits of the present disclosure. In an embodiment, glioma cells are transdifferentiated into glial cells expressing one or both of the OL (Oligodendrocyte) differentiation markers myelin associated glycoprotein (myelin associated glycoprotein, MAG) or myelin basic protein (Myelin basic protein, MBP). In an embodiment, the glial cells produced express myelin associated glycoprotein (MAG). In an embodiment, the glial cells produced express reduced levels of platelet-derived growth factor receptor alpha polypeptide (Platelet-derived growth factor receptoralpha, PDGFRα). In the embodiment, the glial cells are post-mitotic glial cells. The glial cells produced in the embodiment have a reduced cell proliferation ability relative to other groups. The data provided in the present disclosure prove that overexpression of the MYRF gene promotes the appearance of early glial cell marker MAG in the U251 glioblastoma cell line.

[0035] As used herein, the term "subject" refers to any animal subject. Non-limiting examples of animal subjects include experimental animals (eg, non-human primates, rats, mice), etc.

[0036] In embodiments, the subject is in need of glial cell generation. In embodiments, the subject is in need of glial cell generation having a glioma.

[0037] Generally, the method disclosed herein comprises administering to a subject a composition of an exogenous nucleic acid overexpressing a MYRF gene, so that the number of glial cells in the subject increases. In an embodiment, an effective amount (which may include a therapeutically acceptable amount) is an amount that can achieve a desired effect, such as reducing glioma cells and / or glioma cells and / or glioma tumor growth rate, inhibiting glioma tumor formation, eradicating glioma cells, preventing glioma recurrence, and / or inhibiting extension along white matter bundles and / or infiltration into adjacent glioma cell tissues. In an embodiment, the extraneural metastasis of gliomas is suppressed. In an embodiment, the effective amount of a compound is a value adjusted at least in part based on the concentration of the compound described in the following examples, and the value in the embodiment reflects the local concentration of the compound in the glioma cell environment.

[0038] "Active ingredient" refers to a substance that acts on glioma cells to transdifferentiate the cells into glial cells. Therefore, "compositions of exogenous nucleic acids that overexpress MYRF polypeptides" do not include reagents added to, for example, pharmaceutical preparations to contain active ingredients or otherwise facilitate the delivery of active ingredients to glial cells, such reagents including but not necessarily limited to buffers, salts, pharmaceutically suitable excipients, carriers, etc. Therefore, in an embodiment, the present disclosure includes viral preparations and methods of using the preparations, wherein the only active ingredient in the viral preparation consists of a composition of exogenous nucleic acids that overexpress MYRF polypeptides in 293t cells.

[0039] Non-limiting examples of tests to assess the amount of atrophy in glioma cells in a subject in need thereof include Nissle staining, MRI, functional magnetic resonance imaging (fMRI), and PET scans.

[0040] In embodiments, the method is used to treat any type of glioma.Non-limiting examples of glioma symptoms include headaches, seizures, nausea and vomiting, confusion, weakness, numbness, and loss of balance.

[0041] The following examples are provided to illustrate the invention but are not intended to be limiting in any way.

[0042] Example 1: Overexpression of the MYRF gene significantly induced morphological changes in U251 cells and U87 cells

[0043] method

[0044] The specific steps for packaging and producing lentivirus in HEK293T cells are as follows: HEK293T cells are inoculated in a T-75 cell culture dish and cultured in a high-glucose medium (DMEM) supplemented with 10% fetal bovine serum. When the cell density is 80%-90%, the medium is changed. Prepare the plasmid mixed solution and the 293Fectin transfection reagent dilution solution separately: Solution A: Add the virus packaging plasmid psPAX2, pMD2.G and the overexpression plasmid of the MYRF gene in a certain mass ratio to 90μL DMEM culture medium, with a total mass of 10μg (overexpression plasmid: psPAX2: pMD2.G = 4:3:

[0045] 1) Mix evenly by blowing with the tip of the gun; Solution B: Mix DMEM and 293Fectin transfection reagent at a ratio of 1:1 with 45μL of each reagent. Let the prepared solution A and solution B stand at room temperature for 5 minutes each. Mix solution A and solution B thoroughly and let stand at room temperature for 20 minutes. Add the mixed solution after standing evenly to the supernatant of HEK293T cell culture medium. After mixing the mixed solution and the culture medium evenly, put the cells back into the constant temperature incubator. Replace with fresh complete culture medium 8 hours after transfection. Take the cell supernatant culture medium once at 48 and 72 hours after plasmid transfection, and then filter it with a 0.22μm filter. Add the filtered viral supernatant directly to the cells to be infected, or place it in a 4°C refrigerator for short-term storage.

[0046] Lentivirus infection of U251 cells and U87 cells, the specific steps are as follows: U251 cells and U87 cells are inoculated in a six-well plate, cultured in a high-glucose medium (DMEM) supplemented with 10% fetal bovine serum, and lentiviral infection is performed when the cell density is about 60%. The old culture medium is discarded, the above-filtered virus supernatant is added, and the cells are returned to the incubator for culture. After 48 hours of infection, the cell growth state and the expression intensity of green fluorescent protein (GFP) are observed under a microscope, and fresh complete culture medium is replaced. After overnight culture, the old culture medium is discarded and replaced with screening culture medium to screen positive cells. After the cells are fully grown, they are subcultured and expanded, and after continued anti-biological screening, stable knockdown or overexpression cell stable transfection lines are obtained, and the cells are collected for subsequent experiments or frozen for standby use.

[0047] result

[0048] The results are as follows Figure 1 As shown in the figure, after U251 cells and U87 cells were overexpressed with MYRF gene, their growth state was observed under the bright field of optical microscope, which showed that overexpression of MYRF gene significantly induced morphological changes of U251 cells and U87 cells. After overexpression of MYRF gene, the morphology of glioma cells was bipolarized and tended to OPC.

[0049] Example 2: After overexpressing the MYRF gene, U251 and U87 cells express early OL protein

[0050] Cell slides were spread in 24-well plates. U251, U87 cells and U251 and U87 cells overexpressing MYRF were digested and centrifuged, resuspended and inoculated, and the old culture medium was discarded after attachment. Washed with PBS three times for 5 minutes each time. PBS was discarded and cross-linked with 4% paraformaldehyde at room temperature for 15 minutes. 4% PFA was discarded and washed with PBS three times for 5 minutes each time. PBS was discarded and incubated with PBS blocking solution containing 5% goat serum for 1 hour at room temperature. Then the blocking solution was discarded, and the primary antibody (MYRF antibody, MAG antibody) was diluted with the blocking solution. After adding the primary antibody, the cell slides were placed in a dark room and incubated overnight at 4°C. On the second day, the primary antibody was discarded and washed with PBS three times for 5 minutes each time. PBS was discarded and the secondary antibody was added and incubated at room temperature for 1 hour in the dark. The secondary antibody was discarded and washed with PBS three times for 5 minutes each time. PBS was discarded and the cell nuclei were counterstained with DAPI stain for 5 minutes and sealed with 3M sealing agent. Observe and photograph using an inverted fluorescence microscope.

[0051] result

[0052] The results are as follows Figure 2 As shown in the immunohistochemistry images of U251 cells overexpressing the MYRF gene, U251 differentiated into early OL after overexpressing the MYRF gene. The bar charts of U251 cells and U87 cells overexpressing the MYRF gene showed that after overexpressing the MYRF gene, U251 and U87 cells expressed early OL protein, were MAG positive, and MBP was significantly upregulated, PDGFRα was significantly downregulated, and glioma cells showed transdifferentiation.

[0053] Example 3: Changes in proliferation ability of U251 after overexpression of MYRF gene and SOX-10 gene

[0054] Cell slides were spread in 24-well plates. U251 cells and U251 cells overexpressing MYRF gene, SOX10 gene, and co-overexpressing the above genes were digested and centrifuged, resuspended and inoculated, BRDU was added to the culture medium, and the old culture medium was discarded after attachment. Wash with PBS 3 times, 5 minutes each time. Discard PBS and crosslink with 4% paraformaldehyde at room temperature for 15 minutes. Discard 4% PFA and wash with PBS 3 times, 5 minutes each time. Discard PBS and incubate with PBS blocking solution containing 5% goat serum at room temperature for 1 hour. Then discard the blocking solution, dilute the primary antibody (KI67 antibody) with the blocking solution, add the primary antibody, and incubate the cell slide in a dark room at 4°C overnight. The next day, discard the primary antibody, wash with PBS 3 times, 5 minutes each time. Discard PBS, add the secondary antibody and incubate at room temperature in the dark for 1 hour. Discard the secondary antibody and wash with PBS 3 times, 5 minutes each time. PBS was discarded, the cell nuclei were counterstained with DAPI for 5 minutes, and the sections were sealed with 3M sealing medium. The sections were observed and photographed using an inverted fluorescence microscope.

[0055] U251 and U87 cells were plated in one well of a 6-well plate, and the reverse transcription process was performed according to PrimeScript TM The real-time quantitative PCR was performed according to the following system and reaction conditions:

[0056] Table 1 qPCR system

[0057]

[0058] Table 2 qPCR reaction conditions

[0059]

[0060] The relative expression was calculated according to 2-ΔΔCT.

[0061] result

[0062] The results are as follows Figure 3 As shown in the immunostaining images, the changes in the proliferation ability of U251 after overexpression of the MYRF gene and overexpression of the SOX-10 gene were shown. The BRDU and KI67 proliferation experiments showed that the proliferation ability of glioma cells after overexpression of the MYRF gene was weakened compared with other groups.

[0063] Example 4: In situ hybridization

[0064] The preparation of ISH probe is as follows: Find the coding sequence of MBP gene, design primers, Forward Primer: CGAGGCCTGGATGTGATGG, Reverse primer: GGGGAACAAGTCAGGGCT. Obtain the target gene fragment by PCR and clone it on pBluescript II KS plasmid and send it for sequencing; after the sequence alignment is correct, Escherichia coli is expanded and the plasmid is extracted. After the plasmid is linearized by restriction endonuclease, it is transcribed to obtain a digoxigenin-labeled probe that recognizes the mRNA of the gene to be detected. The specific operation steps are as follows: Single enzyme digestion to achieve plasmid linearization. The enzyme digestion system is as follows:

[0065]

[0066] After thorough mixing, place in a water bath at 37°C overnight. To recover the digestion product, add 5 μL sodium acetate buffer (pH 5.2) and 120 μL anhydrous ethanol to 50 μL digestion system, mix thoroughly and place in a -20°C refrigerator for about 20 hours; centrifuge at 14000 rpm for 15 minutes at 4°C the next day; wash the precipitate with 75% ethanol, then centrifuge at 14000 rpm for 5 minutes at 4°C, repeat twice; pour off the supernatant ethanol, dry the precipitate, and dissolve it with 30-50 μL DEPC-H2O; reverse transcription in vitro, the reverse transcription system is as follows:

[0067]

[0068]

[0069] Place in a 37℃ water bath for 2h, take 1μL of reverse transcription product for 0.8% agarose electrophoresis (1μL product in the electrophoresis system, add 1μL10 Loading Buffer and 8μL DEPC-H2O). Add 2μL DNaseI&Buffer, digest for 15-30min; add 2μL 0.2M EDTA, place on ice for 5min to terminate the reaction; add 74μL DEPC-H2O to dilute; add 1 / 10 volume of pH 5.2 sodium acetate solution, mix thoroughly, add 1 volume of isopropanol, place in a -20℃ refrigerator overnight to precipitate nucleic acids; centrifuge at 4℃, 13000rpm for 10min; remove the supernatant, wash the precipitate with 75% ethanol, centrifuge for 10min under the same conditions, repeat twice; remove the supernatant, dry the residual ethanol, add 30-50μL DEPC-H2O according to the volume of the precipitate to dissolve, measure the concentration; add an equal volume of deionized formamide, and freeze at -80℃.

[0070] The in situ hybridization experiment process is as follows:

[0071] Day 1: Ensure that the entire process is RNase-Free

[0072] The required reagents must be prepared in advance and treated with 0.1% DEPC overnight before wet heat sterilization. The instruments required for the experiment (hybridization glass jar, scissors, tweezers, blades, 500mL measuring cylinder, etc.) were placed in a 180°C oven for more than 2 hours to inactivate possible RNase; cell slides were spread in 24-well plates, U251 and U25 cells overexpressing MYRF gene and SOX10 gene were digested and centrifuged, resuspended and inoculated, and the old culture medium was discarded after adhesion, and the cell slides were fixed in 4% PFA for 20 minutes; PBS was divided into three parts and placed in glass jars, labeled ①②③, and the fixed cell slides were washed once in ①PBS and ②PBS, each for 5 minutes; treated in a solution containing 1mg / L proteinase K for 10 minutes; washed once in ①PBS and ②PBS, each for 5 minutes; and washed again in 4 % PFA for 10 min; wash once in ①PBS and ②PBS, each for 5 min; soak in TEA solution containing 0.25% acetic anhydride for 10 min; wash once in ②PBS and ③PBS, each for 5 min; block with hybridization solution at room temperature for 1 h: carefully cover the slide with hybridization solution using a pipette, about 50 μL per slide, and place the slide in an SSC box; process the probe: dilute the probe with hybridization solution according to the dilution ratio (final concentration is about 0.5 ng / μL); heat the probe mixture in an 85℃ metal bath for 5 min and then quickly insert it on ice; pour off the hybridization solution on the slice, cover the slide with the folded boat, and add the probe mixture; put the tissue slice with the probe back into the SSC box and hybridize in a 65℃ oven overnight;

[0073] the next day:

[0074] Discard the probe mixture and soak the slices in 0.2x SSC solution in a 65°C water bath for 3 times, 20 minutes each time. Cool to room temperature and then transfer to room temperature 0.2x SSC solution for 5 minutes. Equilibrate in 0.1M pH 7.4 Tris / 150mM NaCl solution twice, 10 minutes each time. Block the slices with 10% sheep serum at room temperature for 1 hour; add anti-digoxigenin antibody coupled to alkaline phosphatase AP (Roche, 11093274910; 1:5000 dilution) and cover the slices at 4°C overnight. Day 3: Wash the slides three times in 0.1M pH 7.4Tris / 150mM NaCl solution for 20 min each time; equilibrate in 0.1M pH 7.4Tris / 150mM NaCl / 50mM MgCl2 solution for 10 min; prepare the color developer NBT / BCIP (Roche, 11681451001, 1:50 dilution) with the solution in the previous step, cover the slides, and develop color for 1 to 10 h in the dark, and observe the progress of color development at any time under a microscope; place the sections in ddH2O to terminate the color development reaction, decolorize with methanol for 20-30 min, rinse twice with ddH2O, and seal the sections for storage.

[0075] result

[0076] The results are as follows Figure 4 As shown, the in situ hybridization images showed that the expression level of MBP protein increased after overexpression of SOX-10 gene and overexpression of MYRF gene.

[0077] Example 5: Western blot test for MAG protein expression

[0078] U251 cells overexpressing the MYRF gene were plated in a 10 cm cell culture dish, and the cells were harvested on days 14, 21, and 28, and western blot was performed together with the U251 cells and Glioma tissue.

[0079] After washing the cells twice with PBS, add an appropriate amount of RIPA lysis buffer mixed with protease inhibitors in advance to the dish and place it on ice for 5 minutes. After mixing the lysis buffer and cells, transfer them to the EP tube, use an ultrasonic disruptor to sonicate at 40% power for 8 times, centrifuge at 12000rpm, and 4℃ for 15 minutes, and use the BCA kit to determine the protein content in the collected supernatant. Mix ReagentA and ReagentB in a volume ratio of 50:1 in advance for use. Take 1uL of the cell protein supernatant and dilute it 25 times with water. Add 200uL of the mixed solution + 25uL of protein diluent to the ELISA plate and incubate at 37℃ for 30min; read the OD value at a wavelength of 595nm; calculate the sample protein concentration according to the standard curve to prepare for the next follow-up experiment. Configure WB electrophoresis gel: fix the glass plate; prepare separation gel (4.5mL); seal the separation gel with ddH2O or isopropanol and place at room temperature for 20-30min; pour out H2O and rinse with water; prepare concentrated gel (2.5mL); insert comb (comb, 10well, 1.5mm, Bio-Rad, #65339) and place at room temperature for 10-20min. Mix the protein sample supernatant with 5×SDS-Loading Buffer and heat denature in a 95℃ metal bath for 5min. Remove the gel plate and fix it on the electrophoresis tank bracket; add electrophoresis buffer to the electrophoresis tank, and start spotting after confirming that the gel does not leak; load the sample with 30ng protein per well according to the needs, and perform 80v constant voltage electrophoresis in the concentrated gel first. After 30min of constant voltage 80V electrophoresis, when the sample enters the separation gel, increase it to 120V and run for another 60min. When the marker is obviously separated and the sample is close to the lower edge of the separation gel, stop the electrophoresis. The specific electrophoresis time can be adjusted according to actual conditions.

[0080] Transfer: PVDF membrane was first activated by soaking in methanol for 1 min, and the sponge and filter paper were soaked with transfer solution; the gel plate was taken out, the concentrated gel was cut off, and the separation gel was taken out and placed in transfer buffer; a "sandwich" was made in the transfer buffer, and black foam pad, filter paper, separation gel, PVDF membrane, filter paper and black foam pad were placed in sequence, and the bubbles between them were driven out, and the black gel pad and the white gel pad were sandwiched in the order, and the positive and negative electrodes were placed in the electrode slot; the transfer buffer was poured in; the electrophoresis tank was placed in an ice water bath; the membrane was transferred in an ice water bath at a constant current of 300 mA for 90 min.

[0081] Place the PVDF membrane facing the gel upwards, soak it in 5% skim milk prepared in TBST and block it on a shaker at room temperature for 1 hour. After pouring out the milk, incubate it with the diluted primary antibody (MAG antibody) prepared in the blocking solution on a shaker at 4°C overnight. Recover the primary antibody and freeze it, wash it 3 times with TBST, 10 minutes each time; incubate it with the secondary antibody at room temperature for 1 hour, wash it 3 times with TBST, 10 minutes each time; add ECL evenly to the membrane and expose it in the machine.

[0082] result

[0083] The results are as follows Figure 5 As shown, the western blot image shows that the expression level of MAG protein in U251 increases with time after overexpressing the MYRF gene.

[0084] Example 6: Western blot to test other gene expressions

[0085] U251 cells, U87 cells, and U251 cells and U87 cells overexpressing the MYRF gene were plated in 10 cm cell culture dishes. After 14 days of culture, the cells were collected for western blot.

[0086] After washing the cells twice with PBS, add an appropriate amount of RIPA lysis buffer mixed with protease inhibitors in advance to the dish and place it on ice for 5 minutes. After mixing the lysis buffer and cells, transfer them to the EP tube, use an ultrasonic disruptor to sonicate at 40% power for 8 times, centrifuge at 12000rpm, and 4℃ for 15 minutes, and use the BCA kit to determine the protein content in the collected supernatant. Mix ReagentA and ReagentB in a volume ratio of 50:1 in advance for use. Take 1uL of the cell protein supernatant and dilute it 25 times with water. Add 200uL of the mixed solution + 25uL of protein diluent to the ELISA plate and incubate at 37℃ for 30min; read the OD value at a wavelength of 595nm; calculate the sample protein concentration according to the standard curve to prepare for the next follow-up experiment. Configure WB electrophoresis gel: fix the glass plate; prepare separation gel (4.5mL); seal the separation gel with ddH2O or isopropanol and place at room temperature for 20-30min; pour out H2O and rinse with water; prepare concentrated gel (2.5mL); insert comb (comb, 10well, 1.5mm, Bio-Rad, #65339) and place at room temperature for 10-20min. Mix the protein sample supernatant with 5×SDS-Loading Buffer and heat denature in a 95℃ metal bath for 5min. Remove the gel plate and fix it on the electrophoresis tank bracket; add electrophoresis buffer to the electrophoresis tank, and start spotting after confirming that the gel does not leak; load the sample with 30ng protein per well according to the needs, and perform 80v constant voltage electrophoresis in the concentrated gel first. After 30min of constant voltage 80V electrophoresis, when the sample enters the separation gel, increase it to 120V and run for another 60min. When the marker is obviously separated and the sample is close to the lower edge of the separation gel, stop the electrophoresis. The specific electrophoresis time can be adjusted according to actual conditions.

[0087] Transfer: PVDF membrane was first activated by soaking in methanol for 1 min, and the sponge and filter paper were soaked with transfer solution; the gel plate was taken out, the concentrated gel was cut off, and the separation gel was taken out and placed in transfer buffer; a "sandwich" was made in the transfer buffer, and black foam pad, filter paper, separation gel, PVDF membrane, filter paper and black foam pad were placed in sequence, and the bubbles between them were driven out, and the black gel pad and the white gel pad were sandwiched in the order, and the positive and negative electrodes were placed in the electrode slot; the transfer buffer was poured in; the electrophoresis tank was placed in an ice water bath; the membrane was transferred in an ice water bath at a constant current of 300 mA for 90 min.

[0088] Place the PVDF membrane facing the gel upwards, soak it in 5% skim milk prepared in TBST and block it on a shaker at room temperature for 1 hour. Pour out the milk and cut the membrane, incubate it with diluted primary antibodies (Nestin antibody, SOX2 antibody, PDGFRa antibody) prepared in blocking solution on a shaker at 4℃ overnight. Recover the primary antibody and freeze it, wash it 3 times with TBST, 10 minutes each time; incubate the secondary antibody at room temperature for 1 hour, wash it 3 times with TBST, 10 minutes each time; add ECL evenly to the membrane and expose it in the machine.

[0089] result

[0090] The results are as follows Figure 6 As shown in the western blot image, it shows that the stemness of U251 and U87 is weakened after overexpressing the MYRF gene. In U251, Nestin, SOX2, and PDGFRa expression are weakened, and in U87, PDGFRa expression is weakened.

[0091] While the invention has been described by way of particular embodiments, routine modifications will be apparent to those skilled in the art, and such modifications are intended to fall within the scope of the invention.

Claims

1. The use of MYRF gene in the preparation of a reagent for treating glioma, characterized in that: Overexpression of the MYRF gene in glioma cells caused them to transdifferentiate into glial cells.

2. The use of MYRF gene in the preparation of glioma cell transdifferentiation reagents, characterized in that: Overexpression of the MYRF gene in glioma cells caused them to transdifferentiate into glial cells.

3. The use according to claim 1 or 2, characterized in that: After overexpression of the MYRF gene in glioma cells, MBP and MAG signals were upregulated, and PDGFRα signal was downregulated.

4. A method for transdifferentiating glioma cells into glial cells, characterized in that: The method specifically involves contacting glioma cells with exogenous nucleic acid encoding a MYRF polypeptide or a biologically active fragment thereof.

5. A method for transdifferentiating glioma cells into glial cells, characterized in that: The method specifically involves delivering an expression vector comprising an exogenous nucleic acid encoding a MYRF polypeptide or a biologically active fragment thereof to glioma cells.

6. The method according to claim 5, characterized in that The expression vector is a recombinant lentiviral expression vector.

7. The method according to claim 6, characterized in that The recombinant lentiviral expression vector is a psPAX2 or pMD2.G expression vector.

8. The method according to claim 4 or 5, characterized in that: The glioma cells include human glioma cells U87 and human glioma cell line U251.

9. The method according to claim 8, characterized in that The stages of the glioma cells include grade I, grade II, grade III and grade IV.

10. A composition for treating glioma, characterized in that: The composition includes one or more of the following ingredients: (a) MYRF polypeptide or a biologically active fragment thereof; (b) an exogenous nucleic acid encoding a MYRF polypeptide or a biologically active fragment thereof; (c) an expression vector of an exogenous nucleic acid encoding a MYRF polypeptide or a biologically active fragment thereof.

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