WO2020113877A1 - Function and use of e2f6 inhibitor - Google Patents

Abstract

Disclosed is a use of E2F6 as a target capable of increasing the sensitivity of temozolomide. Temozolomide is used for treating glioblastoma. E2F6 inhibitor and temozolomide treatment can increase the curability of glioblastoma. The invention provides scientific evidence for the pathogenesis and clinical treatment of glioblastoma from the clinical patient sample level, cell function level and molecular level.

Description

Functions and uses of E2F6 inhibitors Technical field

The invention belongs to the field of life science and technology, and specifically relates to the functions and uses of E2F6 inhibitors.

Background technique

Glioblastoma is the most common intracranial primary malignant tumor, which has strong proliferation and invasion ability and is prone to recurrence after surgery. The growth rate of glioblastoma is fast, 70% to 80% of patients have a disease course of 3 to 6 months, and only 10% of patients have a disease course of more than 1 year. Those with a longer course of disease may evolve from astrocytomas with a lower degree of malignancy. Due to the rapid tumor growth and extensive cerebral edema, the symptoms of increased intracranial pressure are obvious, and all patients have symptoms of headache and vomiting. Optic disc edema has headache, mental changes, limb weakness, vomiting, disturbance of consciousness and speech disorders. Tumor infiltration destroys brain tissue, causing a series of focal symptoms. Patients have varying degrees of hemiplegia, paraesthesia sensory disturbance, aphasia, and partial blindness. Nervous system examination can find hemiplegia, cerebral nerve damage, partial sensory disturbance and partial blindness. The incidence of epilepsy is rarer than astrocytoma and oligodendroglioma, and some patients have seizures. Some patients showed apathy, dementia, mental retardation and other mental symptoms.

Although many chemotherapeutic drugs, such as temozolomide, nitrosourea and cisplatin, have shown clinical benefit in patients with glioblastoma, the therapeutic effect of these drugs is still not very satisfactory, mainly because of internal or acquired resistance Medicinal. Temozolomide is an alkylating agent for standardized treatment of newly diagnosed glioblastoma patients. It has been shown to cause cell arrest in G2/M phase and mediate DNA damage and subsequent apoptosis. Although oral temozolomide increased the overall survival of glioblastoma patients, the drug resistance induced by cancer cells terminated further treatment. Therefore, overcoming drug resistance in glioma cells and completely treating glioblastoma are still major challenges in medicine.

Summary of the invention

In order to overcome the problems in the prior art, the object of the present invention is to provide the function and use of E2F6 inhibitors in glioblastoma.

In order to achieve the above objectives and other related objectives, the present invention adopts the following technical solutions:

The first aspect of the present invention provides the use of an E2F6 inhibitor for preparing a temozolomide synergistic drug for treating glioblastoma or for preparing a glioblastoma therapeutic drug in combination with temozolomide.

In the use for preparing a glioblastoma therapeutic drug in combination with temozolomide, the glioblastoma therapeutic drug has at least one of the following functions: it can inhibit the growth and proliferation of glioblastoma cells and promote glioblastoma Apoptosis of blastoma cells, inhibit the ability of glioblastoma cells to form tumors, and inhibit the growth of glioblastoma tissues.

Further, the E2F6 inhibitor refers to a molecule that has an inhibitory effect on E2F6 or NF-κB.

E2F6 is regulated by the EGFRvIII/PI3K/AKT/NF-κB pathway, and the activation of NF-κB phosphorylation after temozolomide-induced DNA damage is also an important component of increasing E2F6 expression. Therefore, the inhibition of E2F6 can be achieved by inhibiting NF-κB.

The Genbank accession number of the E2F6 gene is NM_198256.3. The nucleotide sequence is shown in SEQ ID NO: 16.

The Genbank accession number of the NF-κB gene is NM_021975.3. The nucleotide sequence is shown in SEQ ID NO: 17.

The inhibitory effect on E2F6 or NF-κB includes but is not limited to: the E2F6 inhibitor inhibits E2F6 activity, or inhibits E2F6 gene transcription or expression, or inhibits NF-κB activity, or inhibits NF-κB gene transcription or expression.

The E2F6 inhibitor may be siRNA, shRNA, antibody, or small molecule compound.

As listed in the embodiment of the present invention, the E2F6 inhibitor may be siRNA. The nucleotide sequence of the siRNA is shown in any one of SEQ ID NO: 1-3.

The therapeutic agent for glioblastoma necessarily contains an E2F6 inhibitor and temozolomide, and the E2F6 inhibitor and temozolomide are taken as the effective components of the aforementioned functions.

In the glioblastoma therapeutic drug, the active ingredients that exert the aforementioned functions may be only E2F6 inhibitors and temozolomide, and may also include other molecules that can perform similar functions.

The form of the drug for treating glioblastoma is not particularly limited, and may be in various forms such as solid, liquid, gel, semi-liquid, and aerosol.

The glioblastoma treatment drugs are mainly aimed at mammals, such as rodents and primates.

The second aspect of the present invention provides a method for treating tumors, or inhibiting tumor proliferation, invasion, and metastasis, which is to jointly administer an E2F6 inhibitor and temozolomide to a subject, or to administer a combination drug including an E2F6 inhibitor and temozolomide to a subject.

Further, the tumor is a glioblastoma.

Further, the E2F6 inhibitor refers to a molecule that has an inhibitory effect on E2F6 or NF-κB.

The E2F6 inhibitor inhibits E2F6 activity, or inhibits E2F6 gene transcription or expression, or inhibits NF-κB activity, or inhibits NF-κB gene transcription or expression.

Preferably, the E2F6 inhibitor is siRNA, shRNA, antibody, or small molecule compound.

More preferably, the nucleotide sequence of the siRNA is shown in any sequence of SEQ ID NO: 1 to 3.

The combination drug may be in any of the following forms: (1) E2F6 inhibitor and temozolomide are made into separate preparations, the dosage form of the preparation may be the same or different, and the administration route may also be the same or different; (2) The E2F6 inhibitor and temozolomide are formulated into a compound preparation.

The object is a mammal or a glioblastoma cell of the mammal. The mammal is preferably a rodent, an artiodactyl, an odd-hoofed animal, a rabbit-shaped animal, a primate, and the like. The primate animal is preferably a monkey, ape or sapiens. The glioblastoma cells may be glioblastoma cells in vitro, including but not limited to U87 or U87 EGFRvIII.

The subject may be a patient suffering from glioblastoma or an individual expecting to treat glioblastoma, or the subject may be isolated glioblastoma patients or individuals wishing to treat glioblastoma Blastoma cells.

The E2F6 inhibitor and temozolomide, or the combination drug of the E2F6 inhibitor and temozolomide can be administered to the subject before, during, and after receiving glioblastoma treatment.

The pharmaceutically effective dose of E2F6 inhibitor and the pharmaceutically effective dose of temozolomide can be administered in any combination of the following forms: (1) The pharmaceutically effective dose of E2F6 inhibitor and the pharmaceutically effective dose of temozolomide are made separately The formulation of the preparation is the same or different, and the route of administration is also the same or different; (2) A pharmaceutically effective dose of E2F6 inhibitor and a pharmaceutically effective dose of temozolomide are formulated into a compound preparation.

The pharmaceutically effective dose of the E2F6 inhibitor and the pharmaceutically effective dose of temozolomide are administered to patients in need of treatment via oral, injection, sublingual, rectal, vaginal, transdermal, or spray inhalation routes.

The dosage forms of the pharmaceutically effective dose of E2F6 inhibitor and the pharmaceutically effective dose of temozolomide are tablets, granules, capsules, pills, pills, powders, lotions, syrups, stomach plates, mixtures, medicinal liquor, tinctures, lozenges, liquid extraction Substances and extracts, ointments, gels, ointments, teas, lotions, paints, liniments, aerosols or sprays.

Further, the active ingredient of the pharmaceutical composition also includes at least one other glioblastoma treatment drug.

The combination drug may be in any of the following forms: (1) E2F6 inhibitor, temozolomide and other glioblastoma treatment drugs are made into separate preparations with the same or different dosage forms, and the route of administration is also Same or different; (2) E2F6 inhibitor, temozolomide and other glioblastoma treatment drugs are formulated into a compound preparation; (3) Temozolomide and other glioblastoma treatment drugs are formulated into a compound preparation, and E2F6 inhibitor It is formulated as an independent preparation, the dosage form of the preparation is the same or different, and the route of administration is also the same or different; (4) Temozolomide and E2F6 inhibitor are formulated as a compound preparation, and other glioblastoma therapeutic drugs are formulated as independent preparations. The dosage form is the same or different, and the route of administration is the same or different; (5) E2F6 inhibitor and other glioblastoma treatment drugs are formulated into a compound preparation, and temozolomide is formulated into an independent preparation. The dosage form of the preparation is the same or different. The approach is also the same or different.

In the third aspect of the present invention, there is provided a glioblastoma therapeutic drug combination, which includes an effective amount of an E2F6 inhibitor and an effective amount of temozolomide.

The glioblastoma therapeutic drug combination has at least one of the following functions: it can inhibit the growth and proliferation of glioblastoma cells, promote the apoptosis of glioblastoma cells, and inhibit the formation of glioblastoma cells Ability to inhibit glioblastoma tissue growth.

The glioblastoma therapeutic drug combination must contain an E2F6 inhibitor and temozolomide, and the E2F6 inhibitor and temozolomide are taken as the effective components of the aforementioned functions.

Further, the E2F6 inhibitor refers to a molecule that has an inhibitory effect on E2F6 or NF-κB.

E2F6 is regulated by the EGFRvIII/PI3K/AKT/NF-κB pathway, and the activation of NF-κB phosphorylation after temozolomide-induced DNA damage is also an important component of increasing E2F6 expression. Therefore, the inhibition of E2F6 can be achieved by inhibiting NF-κB.

The inhibitory effect on E2F6 or NF-κB includes but is not limited to: the E2F6 inhibitor inhibits E2F6 activity, or inhibits E2F6 gene transcription or expression, or inhibits NF-κB activity, or inhibits NF-κB gene transcription or expression. The E2F6 inhibitor may be siRNA, shRNA, antibody, or small molecule compound.

As listed in the embodiment of the present invention, the E2F6 inhibitor may be siRNA. The nucleotide sequence of the siRNA is shown in any one of SEQ ID NO: 1-3.

The therapeutic drug combination may be in any of the following forms: (1) The E2F6 inhibitor and temozolomide are made into separate preparations, the dosage forms of the preparations may be the same or different, and the route of administration may also be the same or different; (2 ) The E2F6 inhibitor and temozolomide are formulated into a compound preparation.

Further, the glioblastoma therapeutic drug combination further includes at least one other glioblastoma therapeutic drug as an effective component of the foregoing function.

The other glioblastoma therapeutic drugs refer to glioblastoma therapeutic drugs other than the E2F6 inhibitor and temozolomide.

Further, the therapeutic drug combination may be in any of the following forms: (1) E2F6 inhibitor, temozolomide and other glioblastoma therapeutic drugs are separately prepared into separate preparations, and the dosage forms of the preparations may be the same or different , The route of administration can also be the same or different; (2) E2F6 inhibitors, temozolomide and other glioblastoma treatment drugs are formulated into compound preparations; (3) temozolomide and other glioblastoma treatment drugs are formulated into compound preparations For the preparation, the E2F6 inhibitor is formulated as an independent preparation. The dosage form of the preparation may be the same or different, and the route of administration may also be the same or different; (4) The temozolomide and E2F6 inhibitor are formulated as a compound preparation, and other glioblastoma treatment The drug is formulated as an independent preparation, the dosage form of the preparation may be the same or different, and the route of administration may also be the same or different; (5) E2F6 inhibitor and other glioblastoma treatment drugs are formulated as a compound preparation, and temozolomide is formulated as an independent preparation The dosage form of the preparation is the same or different, and the administration route can also be the same or different.

According to a fourth aspect of the present invention, there is provided a method for treating glioblastoma, which is to administer an effective amount of an E2F6 inhibitor and temozolomide to a subject, and to administer an effective amount of other glioblastoma therapeutics to the subject and/or to The subject implemented other glioblastoma treatment methods.

An effective amount of an E2F6 inhibitor and temozolomide and at least one effective amount of other glioblastoma therapeutic drugs can be administered simultaneously or sequentially.

The other glioblastoma treatment drugs include, but are not limited to, anti-tumor antibodies, chemotherapy drugs, or targeted drugs.

Based on E2F6, which is the first gene discovered by the present invention that can enhance the resistance of temozolomide to treat glioblastoma, the fifth aspect of the present invention provides other new uses of E2F6. The E2F6 inhibitor has at least one of the following functions : Can increase temozolomide sensitivity.

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

After extensive and in-depth study of the present invention, it was found for the first time that E2F6 can be used as a target that can increase the sensitivity of temozolomide. Temozolomide is used to treat glioblastoma, and the combination of E2F6 inhibitor and temozolomide treatment can increase the incidence of glioblastoma. Curative. Therefore, the present invention provides strong scientific evidence for the pathogenesis of glioblastoma and the clinical treatment of glioblastoma from the clinical patient sample level, cell function level and molecular level.

BRIEF DESCRIPTION

Figure 1: TMZ acts on the IC50 of U87 cells, (LogIC50 is 2.528, Hillslope is 1.329, IC50 is 337.4).

Figure 2: CCK8 detects the TMZ concentration of U87 for tumor growth inhibition.

Figure 3: Relative counts distribution of sgRNA in each sample. The abscissa in the figure represents the sample, and the ordinate is Relative Log2 (CPM), where CPM = counts per million (similar to the common RPKM in sequencing results); the middle line represents the average value of Relative Log2 (CPM) for all samples; The upper and lower horizontal lines of each box represent the upper and lower 90% confidence intervals, the upper and lower edges of the box represent the upper and lower quartiles, and the black line in the middle represents the median.

Figure 4: RNAseq selects EGFRvIII-related resistance genes. (A) RNAseq showed that increasing the expression of EGFRvIII can increase the expression of E2F6mRNA. (B) Component analysis showed that the cells in each group after TMZ treatment were significantly different from those in the treatment group. (C) RNAseq showed that E2F6 increased after TMZ treatment. (D) RNAseq showed that the expression of E2F6 in TMZ treatment increased at 14 days compared to 7 days.

Figure 5: TCGA RNAseq and Rembrandt groups are used to show the level of E2F6 expression in WHOII-IV gliomas. E2F6 is positively correlated with WHO grade of glioma.

Figure 6A: Western blot analysis provided evidence about increased E2F6 expression by EGFRvIII and TMZ treatment, with GAPDH as a negative control.

Figure 6B: Using qRT-PCR to show increased E2F6 mRNA levels by EGFRvIII and TMZ treatment, GAPDH served as a negative control.

Figure 6C: A cell viability assay was performed to show that the gain or loss of E2F6 did not affect the proliferation of GBM cells, but E2F6 was treated by TMZ as TMZ resistance.

Figure 6D: Colony formation assay was used by TMZ treatment to show prolonged proliferation. Control cells overexpressing E2F6 and E2F6 silenced EGFRvIII cells were treated with TMZ for 14 days, indicating that E2F6 is a key factor in TMZ resistance.

Figure 6E: γ-H2AX is used to assess DNA damage caused by TMZ using immunofluorescence assays. The gain or loss of functional experiments indicates that E2F6 is resistant to DNA damage.

Figure 7A: Western blot analysis showed that E2F6 expression was positively correlated with NF-κB activation. GAPDH served as a negative control. Figure 7B: qRT-PCR shows that E2F6 mRNA levels are positively correlated with NF-κB activation.

Figure 7C: Western blot shows that EGFRvIII and TMZ treatment increased p-NF-κB expression. GAPDH served as a negative control.

Figure 7D: Using IGV to show ChIP-seq data (GSE46016), the E2F6 promoter in U87EGFRvIII cells is better enriched with H3K4me3 in U87 cells.

Figure 7E: ChIP-PCR analysis shows that H3K4me3 and p-NF-κB increase their binding to the E2F6 promoter through EGFRvIII.

Figure 7F: ChIp-PCR analysis showed that H3K4me3 and p-NF-κB treatment by MK-2206 reduced its binding to the E2F6 promoter.

Figure 7G: Schematic diagram of the regulatory mechanism of EGFRvIII/PI3K/AKT pathway on E2F6. EGFRvIII/PI3K/AKT and TMZ activate NF-κB through transcriptional regulation of E2F6 expression. And the binding on the E2F6 promoter region increased by H3K4me3 modification induced by the EGFRvIII/PI3K/AKT pathway further activates E2F6 transcription.

Figure 8A: Schematic diagram of identification of E2F6 as a TMZ resistance factor in vivo. Mouse isotope injection of U87, U87E2F6, U87EGFRvIII or U87EGFRvIII+E2F6KD cells. Each of these groups was injected intraperitoneally with 5 mg/kg/d of DMSO or TMZ, and rested for 2 days for 5 days. The bioluminescence was detected every 7 days, and the body weight of the mice was measured every 2 days.

Figure 8B: In vitro imaging fluorescence of mice implanted with intracranial tumors every 7 days in the U87, U87E2F6 and TMZ treatment groups.

Figure 8C: In vitro imaging fluorescence of mice implanted with intracranial tumors every 7 days in the U87EGFRvIII, U87EGFRvIII+E2F6KD and TMZ treatment groups.

Figure 8D: Measuring the body weight of U87, U87E2F6 and TMZ-treated mice every 2 days.

Figure 8E: The body weights of mice treated with U87EGFRvIII, U87EGFRvIII+E2F6KD and their TMZ were measured every 2 days. Figure 8F: Kaplan-Meier curve shows that when E2F6 expression is increased, the overall survival time of TMZ treatment decreases. Figure 8G: Kaplan-Meier curve shows that when E2F6 expression is silenced, overall survival time is increased by TMZ treatment.

Figure 8H: Representative immunostaining results of EGFRvIII, p-NF-κB and E2F6 in tumors of nude mice from the U87, U87E2F6 and TMZ treatment groups. Scale bar: 50 μm.

Figure 8I: Representative immunostaining results of EGFRvIII, p-NF-κB and E2F6 in tumors of nude mice from the U87EGFRvIII, U87EGFRvIII+E2F6KD and TMZ treatment groups. Scale bar: 50 μm.

In the drawings, * means p<0.05, *** means p<0.01, **** means p<0.001, and ns means not significant.

detailed description

The present invention found in research that E2F6 can be used as a target that can increase the sensitivity of temozolomide. Temozolomide is used to treat glioblastoma, and the combination of E2F6 inhibitor and temozolomide treatment can increase the curability of glioblastoma.

E2F6 inhibitor

The E2F6 inhibitor refers to a molecule that has an inhibitory effect on E2F6 or NF-κB.

E2F6 is regulated by the EGFRvIII/PI3K/AKT/NF-κB pathway, and the activation of NF-κB phosphorylation after temozolomide-induced DNA damage is also an important component of increasing E2F6 expression. Therefore, the inhibition of E2F6 can be achieved by inhibiting NF-κB.

The inhibitory effect on E2F6 or NF-κB includes but is not limited to: the E2F6 inhibitor inhibits E2F6 activity, or inhibits E2F6 gene transcription or expression, or inhibits NF-κB activity, or inhibits NF-κB gene transcription or expression.

The E2F6 inhibitor may be siRNA, shRNA, antibody, or small molecule compound.

Inhibiting E2F6 activity means reducing E2F6 activity. Preferably, the activity of E2F6 is reduced by at least 10%, preferably by at least 30%, better by at least 50%, more preferably by at least 70%, and best by at least 90% compared to before inhibition.

Inhibiting the transcription or expression of the E2F6 gene means that the E2F6 gene is not transcribed, or the transcription activity of the E2F6 gene is reduced, or the E2F6 gene is not expressed, or the expression activity of the E2F6 gene is reduced.

Those skilled in the art can use conventional methods to regulate E2F6 gene transcription or expression, such as gene knockout, homologous recombination, interference RNA, and the like.

The inhibition of E2F6 gene transcription or expression can be verified by PCR and Western Blot detection.

Preferably, the E2F6 gene transcription or expression is reduced by at least 10%, preferably by at least 30%, better by at least 50%, more preferably by at least 70%, and still better by at least 90% compared to the wild type At best, the E2F6 gene is not expressed at all.

Inhibiting the activity of NF-κB means decreasing the activity of NF-κB. Preferably, the activity of NF-κB is reduced by at least 10%, preferably by at least 30%, better by at least 50%, better by at least 70%, and best by at least 90% than before inhibition.

Inhibiting the transcription or expression of the NF-κB gene means that the NF-κB gene is not transcribed, or the transcription activity of the NF-κB gene is reduced, or the NF-κB gene is not expressed, or the expression of the NF-κB gene is reduced active.

Those skilled in the art can use conventional methods to regulate the transcription or expression of NF-κB genes, such as gene knockout, homologous recombination, and interference RNA.

The inhibition of NF-κB gene transcription or expression can be verified by PCR and Western Blot detection.

Preferably, the transcription or expression of the NF-κB gene is reduced by at least 10%, preferably by at least 30%, better by at least 50%, more preferably by at least 70%, and still better by at least 70% compared to the wild type 90%, optimally the NF-κB gene is not expressed at all.

Small molecule compound

In the present invention, it refers to a compound composed of several or dozens of atoms and having a molecular mass of less than 1,000.

E2F6 inhibitor preparation

The medicine is prepared by taking the E2F6 inhibitor as the main active ingredient or one of the main active ingredients. Generally, in addition to the active ingredients, according to the needs of different dosage forms, one or more pharmaceutically acceptable carriers or excipients are included in the medicine.

"Pharmaceutically acceptable" means that when the molecular body and composition are properly administered to an animal or human, they will not produce adverse, allergic, or other adverse reactions.

"Pharmaceutically acceptable carriers or excipients" should be compatible with E2F6 inhibitors, that is, they can be blended with them without significantly reducing the effectiveness of the pharmaceutical composition under normal circumstances. Specific examples of some substances that can be used as pharmaceutically acceptable carriers or excipients are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, Ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olives Oil, corn oil and cocoa butter; polyols such as malondiazine, glycerin, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; coloring Agents; flavoring agents; compressed tablets, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions; and phosphate buffers. These substances are used to help the stability of the formulation or to improve the activity or its bioavailability or to produce an acceptable mouthfeel or odor when taken orally as needed.

In the present invention, unless otherwise specified, the pharmaceutical dosage form is not particularly limited, and can be prepared as injections, oral liquids, tablets, capsules, pills, sprays and other dosage forms, and can be prepared by conventional methods. The choice of pharmaceutical dosage form should match the mode of administration.

Combination of therapeutic drugs and method of administration

When the therapeutic drug combination has only two effective components, an E2F6 inhibitor and temozolomide, the therapeutic drug combination may be any one of the following forms:

(1) The E2F6 inhibitor and temozolomide are made into separate preparations. The dosage form of the preparations can be the same or different, and the route of administration can also be the same or different;

(2) The E2F6 inhibitor and temozolomide are formulated into a compound preparation.

When the therapeutic drug combination has multiple active ingredients, the therapeutic drug combination may be in any of the following forms:

A) The E2F6 inhibitor, temozolomide, and other glioblastoma treatment drugs are made into separate preparations. The formulations of the preparations may be the same or different, and the route of administration may also be the same or different. During use, several drugs can be used at the same time, or several drugs can be used in succession. When administering drugs sequentially, other drugs should be applied to the body while the first drug is still effective for the body.

B) E2F6 inhibitors, temozolomide and other glioblastoma treatment drugs are formulated into compound preparations. When the E2F6 inhibitor, temozolomide medicine, and other glioblastoma treatment drugs are administered in the same route of administration and applied at the same time, the two can be formulated into a compound preparation.

(3) Partial distribution of active ingredients is made into compound preparations, and partial distribution is made into independent preparations. For example, temozolomide and other glioblastoma treatment drugs are formulated into a compound preparation, and E2F6 inhibitors are formulated as independent preparations; or temozolomide and E2F6 inhibitors are formulated into a compound preparation, and other glioblastoma treatment drugs are formulated. Independent preparations, the dosage forms of the preparations can be the same or different, and the route of administration can also be the same or different; (5) E2F6 inhibitors and other glioblastoma treatment drugs are formulated as compound preparations, and temozolomide is formulated as an independent preparation. The dosage forms are the same or different, and the route of administration can also be the same or different.

Commonly used methods of antibody administration are intravenous injection, intravenous drip or arterial infusion. The usage and dosage can refer to the existing technology.

Commonly used methods for small molecule compounds can be gastrointestinal administration or parenteral administration. siRNA, shRNA, and antibodies are generally administered parenterally. It can be administered locally or systemically.

An effective amount of an E2F6 inhibitor and temozolomide drugs and an effective amount of other glioblastoma therapeutic drugs can be administered simultaneously or sequentially. When in use, an effective amount of E2F6 inhibitor and temozolomide medicine and an effective amount of other glioblastoma treatment drugs can be used at the same time, an effective amount of E2F6 inhibitor and temozolomide medicine and an effective amount of other glioblastoma cells can also be used Tumor treatment drugs have been used one after another. When the drugs are administered one after another, other drugs should be applied to the organism within the period when the first medication is still effective for the organism.

Chemotherapy drugs include alkylating agents (such as nimustine, carmustine, lomustine, cyclophosphamide, ifosfamide, and glycosyl mustard, etc.), antimetabolites (such as deoxyfluoroguanosine, Nucleotide analogs such as doceferine, fluorouracil, mercaptopurine, and methotrexate), antitumor antibiotics (such as antibiotics such as actinomycin D, doxorubicin, and daunorubicin), antitumor animals and plants Ingredients (e.g. vinorelbine, paclitaxel, cephaloside, irinotecan, taxotere, vinblastine, etc.), anti-tumor hormone drugs (e.g. atamestane, anastrozole, amlutide, (Trazole, formestane, tamoxifen, etc.) as well as commonly used chemotherapeutic drugs such as cisplatin, dacarbazine, oxaliplatin, leroxadine, coplatin, mitoxantrone, and procarbazine.

Targeted drugs include EGFR blockers such as gefitinib (Gefitinib, Iressa and Iressa) and erlotinib (Erlotinib, Tarceva), monoclonal antibodies to specific cell markers such as cetuximab (Cetuximab) , Erbitux) and anti-HER-2 monoclonal antibodies (Herceptin, Trastuzumab, Herceptin), tyrosine kinase receptor inhibitors such as crizotinib (Crizotinib, Xalkori), anti-tumor angiogenesis drugs such as Bevacizumab, endostatin and Bevacizumab Etc., Bcr-Abl tyrosine kinase inhibitors such as Imatinib and Dasatinib, anti-CD20 monoclonal antibodies such as Rituximab, IGFR-1 kinase inhibitors such as NVP-AEW541, mTOR kinase inhibitors such as CCI-779, ubiquitin-proteasome inhibitors Such as Bortezomib and so on.

Other treatments for glioblastoma can be selected from surgical resection, radiofrequency ablation, argon-helium superconducting surgery, laser ablation, high-intensity focused ultrasound, and radiation therapy including X-knife, R-knife, 3D-CRT, and IMRT One or more.

Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments, It is not intended to limit the protection scope of the present invention. The test methods without specific conditions in the following examples generally follow the conventional conditions or the conditions recommended by the manufacturers.

When the numerical ranges are given in the embodiments, it should be understood that unless the present invention indicates otherwise, the two endpoints of each numerical range and any one value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, the methods, equipment, and materials described in the embodiments of the present invention can also be used according to the knowledge of the prior art by those skilled in the art and the description of the present invention. Any method, equipment and material of similar or equivalent prior art are used to implement the present invention.

Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in the technical field. Conventional technology.

TMZ refers to temozolomide. GBM refers to glioblastoma.

Example 1

1.1 Cell culture

Human glioma cells U87 were purchased from ATCC (American ATCC), and N5, N9, and N33 primary cells were cultured from glioma tissue surgically excised from glioma patients. Surgically removed gliomas are quickly contained in serum-free medium and kept in an ice bath. Cut the tissue into 0.5mm ³ pieces in a sterile operating table, and grow in serum-containing medium. After the tumor cells are shed and adhered, they are cultured as tumor cells. The cells were cultured in DMEM medium containing 10% heat-inactivated calf serum (FBS, Hyclone Corporation, USA). Each EGFRvIII cell line was derived from the transfection of a lentivirus constructed with a GV341 plasmid expressing EGFRvIII cDNA, followed by puromycin screening for 7 days. All these cells were grown in a humidified incubator with a constant temperature of 37°C and 5% CO ₂ . Overexpression of E2F6 and knockdown of E2F6 lentivirus were purchased from Jikai gene.

The GeCKO plasmid library is a genome-wide CRISPR knockout library that can knock out any coding genes and miRNAs within the human genome. The library contains 6 sgRNAs for each coding gene and 4 sgRNAs for each miRNA, and 1000 non-targeted control sgRNAs. HEK293T, a lentivirus packaging cell, is an adherent-dependent epithelial-like cell, and the growth medium is DMEM (containing 10% FBS). Adherent cells grow and proliferate in culture to form monolayer cells.

Table 1 E2F6 interfering RNA sequences

Interfering RNA	sequence	serial number
E2F6siRNA#1	5'-GAGGAACUUUCUGACUUAU-3'	SEQ ID NO: 1
E2F6siRNA#2	5'-AUGUCUAUUUGUGUGAAGU-3'	SEQ ID NO: 2
E2F6siRNA#3	5'-ACUUAGAUUACUGAGUAAU-3'	SEQ ID NO: 3

1.2 Plasmid transfection

1. 24h before transfection, 293T cells were trypsinized logarithmic growth phase, the culture medium containing 10% serum to adjust the cell density of about 5x10 ⁶ cells / 15mL, re-seeded in 10cm cell culture dishes, 37 ℃, 5% Cultivation in a CO ₂ incubator. It can be used for transfection when the cell density reaches 70%～80% in 24h;

2. Change to serum-free medium 2 hours before transfection;

3. Add the prepared DNA solutions (shuttle plasmid 20μg, pHelper1.0 vector plasmid 15μg, pHelper2.0 vector plasmid 10μg) to a sterilized centrifuge tube, mix with the corresponding volume of transfection reagent, adjust the total volume to 1mL, stand at room temperature for 15min;

4. The mixed solution was slowly added dropwise to the culture medium of 293T cells, mixed well, and cultured in a 37°C, 5% CO2 cell incubator;

5. After 6 hours of culture, discard the medium containing the transfection mixture, add 10 mL of PBS solution to wash it once, gently shake the petri dish to wash the residual transfection mixture and discard it;

6. Slowly add 20mL of cell culture medium containing 10% serum and continue culturing for 48-72h at 37°C in a 5% CO ₂ incubator.

1.3 Lentivirus concentration and purification

1. According to the state of the cells, collect the supernatant of 293T cells 24h and 48h after transfection (transfection can be counted from 0h) and store at 4℃;

2. Transfer the collected supernatant to a 0.22μm filter for suction filtration to remove cell debris, etc.;

3. Pass the sample collected in step 2 through the tangential flow filtration system, while concentrating the virus, removing DNA and protein residues to a great extent;

4. Use AKATA anion and cation chromatography system to further purify the sample, collect the sample, and store at 4℃;

5. Load the virus sample recovered in step 5 into the ultrafiltration concentration tube, centrifuge at 4°C and 5500 rpm, and adjust the time of each round of centrifugation according to the speed of venom concentration until it is concentrated to the target volume to prepare the sample for testing;

6. Collect the concentrated virus solution in a 1.5mL centrifuge tube and centrifuge at 11000rpm for 5min;

7. Aspirate the supernatant with a 2mL needle, purify it by 0.22μm (PVDF), and load it into the library according to certain specifications;

8. Prepare samples for testing.

1.4 Virus titer detection

1. 24 hours before infection, HEK293T adherent cells were passaged and counted, seeded in 96-well plates, the plating density was 4×10 ⁴ cells/well, and the volume was 100 μL;

2. According to the expected titer of the virus, prepare 7 to 10 sterile 1.5mL EP tubes, and add 90μL of serum-free medium to each tube;

3. Take 10 μL of the virus stock solution to be tested and add it to the first tube. After mixing, take 10 μL and add it to the second tube. Continue the same operation until the last tube;

4. Select the desired cell well, discard 90 μL of culture medium, add 90 μL of diluted virus solution, and culture in the cell incubator;

5. After 24 hours of cultivation, add 100 μL of complete medium;

6. 72h after infection, add the resistant drug puromycin, maintain the drug concentration of 5μg/mL, continue culturing for 24h, and observe the cell growth status.

7. Calculate the virus titer by the number of living cells after infection. For example, 3 cells survived in a well added with 1E-5μL virus stock solution, indicating that at least 3 virus particles in the well infected the cells. Virus titer = number of viable cells/ amount of virus stock solution, ie 3/(1E- 5)=3E+5 (TU/μL), which is 3E+8TU/mL.

Table 2 Reagents used to construct the virus

Reagent name	Reagent source	Cat.No.
Trypan blue	SIGMA	72-57-1
Fetal Bovine Serum FBS	Shanghai Weike Biochemical Reagent Co., Ltd.	A11-102
DMSO	Shanghai Trial Chemical Reagent Co., Ltd.	130701
DMEM	Hyclone	SH30022.01B

1.5 Use CCK-8 to detect IC50 of TMZ

1. After trypsinizing the cells of each experimental group in the logarithmic growth phase, resuspend the complete medium into a cell suspension and count.

2. Decide the density of the plated cells according to the growth rate of the cells (mostly 4000cell/well), 100μl per well, repeat for each group of 3-5 wells, and determine the number of plated according to the experimental design (if 5 days of detection, 5 96-well plates are plated) .

3. After uniformly spreading, after the cells are completely precipitated, observe the cell density of each experimental group under the microscope. If the density is not uniform, fix one group, fine-tune the amount of cells in other groups and plate again (eg: found Con group cells More, reduce the amount of cells and plate again), put it in a cell culture incubator.

4. Add medicine according to the experimental design the next day, no treatment is needed in the non-medicine group.

5. According to the experimental design time, add 10μLCCK-8 reagent to the well 2~4h before the end of the culture, without changing the liquid.

6. After 4h, the 96-well plate was placed on an oscillator and oscillated for 2-5min. The microplate reader was used to detect the OD value at 450nm.

7. Data analysis.

1.6 CCK8 detects the concentration of TMZ for U87 to inhibit tumor growth

1. After trypsinizing the cells of each experimental group in the logarithmic growth phase, resuspend the complete medium into a cell suspension and count;

2. Determine the plated cell density according to the killing effect of the drug (for IC70 drug concentration, the number of cell plating is about 500,000, the density is greater than 50%, for IC20 ~ IC30 drugs, the cell density is about 200,000, the density is about 20%), There is one well for each drug concentration, and a non-medicine group is set. The culture system is 2ml/well. During the plating process, it is necessary to ensure that the number of cells added to each well is the same. Incubate at 37°C and 5% CO ₂ incubator;

3. When the cells in the untreated group grow to about 80%, digest and count, and record the total amount of cells in each group;

4. According to the counting situation, pass the passage according to a certain proportion, and record the passage ratio;

5. The next day after passage, after the cells adhere to the wall, change the medium;

6. After the empty cells grow to about 80%, repeat the steps 3 to 5 until the culture is completed for 10 to 14 days;

7. Finally, the initial cell amount and the total number of cells counted for each count are aggregated to obtain the growth curve of each group of cells, and the inhibition rate of the drug-added group relative to the non-medicated group is calculated.

1.7 sgRNA sequencing

Amplify the target fragment from the target sequence through primers, and then use high-throughput sequencing technology to determine the sequence of the target fragment. Through sequence alignment, a large number of single nucleotide mutation sites (SNV) can be found and inserted into the deletion site (Insertion) /Deletion, InDel), Structure Variation (Structure Variation, SV), and through biological information means, analyze the mutation differences between different individual genomes, and complete the annotation at the same time.

1.8 Western blotting (Western blot)

After the cells are prepared, discard the culture medium, wash the cells 3 times with pre-chilled PBS at 4°C, discard the PBS, and place the petri dish on ice. The RIPA cell lysate was mixed with PMSF at 100:1 and pre-chilled on ice. Add 200μl of lysate to a 10cm dish, scrape the cells, transfer the cells and lysate to a 1.5ml EP tube, and lyse on ice for 30min. Centrifuge at 12000 rpm for 15 minutes at 4°C. Transfer the supernatant to another 1.5ml EP tube. BCA protein concentration detection: follow the instructions of BCA kit to make a standard curve. BCA and CuCl ₂ were mixed evenly at 50:1, and 200 μl of the mixed BCA solution was added to each auxiliary well. The collected protein was diluted to 1/10 with PBS, and 20 μl was added to each secondary well. The protein and BCA were mixed evenly and placed in a 37°C incubator for 30 min. The microplate reader detected the absorbance at 562 nm. The protein concentration was calculated using the standard curve. Add the remaining protein supernatant to protein loading buffer, boil at 100℃ for 10min, and store at -20℃. Polyacrylamide gel electrophoresis: formulating 10% polyacrylamide gel formulation as shown in Table 3:

Table 3 10% polyacrylamide gel ingredients

Separator	20ml	Compression rubber	5ml
ddH2O	8ml	ddH2O	6.89ml
30% acrylamide	6.66ml	30% acrylamide	1.7ml
Tris buffer (PH8.8)	5ml	Tris buffer (PH6.8)	1.25ml
10% ammonium persulfate (AP)	200μl	10% ammonium persulfate (AP)	100μl
10% SDS	100μl	10% SDS	50μl
TEMED	8μl	TEMED	10μl

Add 20-40μg protein to each well, electrophoresis at 80V for 40min, then change to 150V electrophoresis to the loading buffer to the bottom of the gel, stop the electrophoresis, transfer the protein to the PVDF membrane, and transfer to 100V for about 60min. 5% BSA blocked PVDF membrane for 1h, primary antibody blocked overnight (anti-p-NF-κB, p-AKT antibody (CST, 1:1000 dilution), anti-E2F6 (Gene, 1:1000 dilution), anti-GAPDH antibody (Millipore, 1:2000 dilution)). The next day, the PVDF membrane was rewarmed at room temperature for 1 hour, the antibody was discarded, and washed three times with PBST for 10 minutes each time. Incubate for 2h at room temperature with the corresponding anti-mouse or anti-rabbit combined with horseradish catalase secondary antibody, and wash again with PBST 3 times for 10min each time. G:BOXF3 gel imaging system (Syngene, UK) was used for immunoimaging.

1.9 Real-time quantitative PCR (qRT-PCR)

After the cells are prepared, discard the culture medium, wash the cells 3 times with pre-chilled PBS at 4°C, discard the PBS, and place the petri dish on ice. Follow the instructions of the TRIzol kit to extract total RNA from the cells using the TRIzol reagent. First, add 1ml of TRIzol to the six-well plate cells, pipet evenly, place in a 1.5ml RNase-free EP tube, place at room temperature for 15min, add 200μl of chloroform, mix upside down, and centrifuge at 12000g at 4°C for 15min. Transfer to another EP tube without RNase. Add equal volume of isopropanol, mix well, then centrifuge at 12000g at 4°C for 15 min. Discard the supernatant, and the bottom of the EP tube is visible. Wash with 75% ethanol 1ml, centrifuge at 12000g at 4°C for 15min. After discarding the supernatant, it was washed with 1ml of absolute ethanol and centrifuged at 12000g for 15 minutes at 4°C. After discarding the supernatant, the EP tube was placed on an ultra-clean table and opened to dry. The resulting precipitate was dissolved in 50 μl of DEPC-treated water and frozen at -80°C until use. Take 2μg total RNA as a template for reverse transcription. The reverse transcription system is shown in Table 4:

Table 4 RNA reverse transcription system

Total RNA	Xμl
OligodT	1μl
ddH 2 O	(10-X)μl
MgCl 2	5μl
5*buffer	2μl
RNase inhibitor	1μl
RNA reverse transcriptase	1μl
In total	20μl

The reverse transcription system obtained cDNA at 42°C for 1h and 70°C for 15min. 2μl of reverse transcribed cDNA was used for PCR amplification. The reaction system is shown in Table 5 and the primers are shown in Table 6:

Table 5 qPCR reaction system

Table 6 Primer sequences used

Primers	sequence	serial number
E2F6-Forward	5'-TCAGCAAAGTGAAGAATTGC-3'	SEQ ID NO: 4
E2F6-Reverse	5'-CGAGAGCACTTCATGGATAA-3'	SEQ ID NO: 5
GAPDH-Forward	5'-TTGGTATCGTGGAAGGACTCATG-3'	SEQ ID NO: 6
GAPDH-Reverse	5'-GTTGCTGTAGCCAAATTCGTTGT-3'	SEQ ID NO: 7

After the qPCR reaction system was prepared, it was run in the CFX96 _™ PCR instrument (Bio-Rad, USA). The PCR workflow is as follows: the first step is denaturation at 95°C for 5 minutes, the second step is 95°C for 15 seconds, the 53-58°C is 1 minute for 40 cycles, and the third step is 72°C for 10 minutes. The specificity of the qRT-PCR reaction was determined by the dissolution curve, and the resulting product was confirmed again using 1.2% agarose gel electrophoresis with the addition of GelRed fluorescent dye (Biotium, USA). mRNA is corrected using GAPDH, and microRNA is corrected using U6. The relative expression of genes is calculated using the formula 2^-ΔΔCt.

1.10 RNA sequencing

U87 cells and U87EGFRvIII cells were treated with temozolomide for 0, 7 and 14 days after transfection with sgRNA library. U87 cells and U87EGFRvIII cells were subjected to RNA sequencing to observe the mRNA changes. After the cells are ready, discard the medium, wash the cells 3 times with pre-chilled PBS at 4°C, discard the PBS, and place the petri dish on ice. Follow the instructions of the TRIzol kit to extract total RNA from the cells using the TRIzol reagent. First, add 1ml TRIzol to the 10cm Petri dish cells (1*10 ⁷ ), pipette evenly, place in a 1.5ml RNase-free EP tube, place at room temperature for 15min, add 200μl chloroform, mix upside down, centrifuge at 12000g at 4℃ 15min, transfer the clear supernatant to another RNase-free EP tube. Add equal volume of isopropanol, mix well, then centrifuge at 12000g at 4°C for 15 min. Discard the supernatant and see the sediment at the bottom of the EP tube. Wash with 75% ethanol 1ml, centrifuge at 12000g at 4°C for 15min. After discarding the supernatant, it was washed with 1ml of absolute ethanol and centrifuged at 12000g at 4°C for 15min. After discarding the supernatant, the EP tube was placed on an ultra-clean table and opened to dry. The resulting precipitate was dissolved in 50 μl of DEPC-treated water, and the total RNA was used for RNA sequencing. The total RNA was reverse transcribed to construct a cDNA library, which was then sequenced and tested using the BGI500 sequencing platform of Shenzhen Huada Gene. High-quality sequencing reads are matched using Bowtie2 according to the human reference genome (GRCh38), and the mRNA transcribed by the matched gene is normalized by mapping reads per million bases per million bases (FPKM) of the exon model . The sequencing results obtained can be analyzed and calculated in the next step.

1.11 Clinical sample collection and immunohistochemistry

Glioma specimens and corresponding clinical information were collected from the affiliated hospital of Hebei University. After surgical removal of the glioma, formalin was used for fixation, paraffin embedding and sectioning.

Immunohistochemical procedure

Dewaxing: Before dewaxing, the tissue sections are baked in a 60°C incubator for 1 hour, and then soaked in xylene (I) and (II) for 20 minutes each;

Hydration: 10 minutes each of anhydrous ethanol (I) and (II), then 95%, 10% each of 80% ethanol, and 5 minutes of distilled water;

Antigen repair: Place the slices in 0.01M citrate buffer (PH=7.4) and boil to 95°C for 15min, naturally cool to room temperature, rinse 3 times with PBS, 5min each time;

Blocking: 0.3% H ₂ O ₂ treatment for 10 min to extinguish endogenous peroxidase activity, PBS rinse 3 times, 5 min each time

Perforation: 1% tritonX-100 at room temperature for 10 minutes to lyse the cell membrane, rinsed with PBS for 3 times, 5 minutes each time;

Blocking: add normal goat serum blocking solution dropwise, incubate at 37℃ for 40min, and shake off excess liquid;

Primary antibody incubation: add 50 μl of primary antibody (1:100, diluted in antibody dilution) overnight at 4°C. The next day was rewarmed at 37°C for 45min, and rinsed with PBS 3 times, 5min each time;

Secondary antibody incubation: add 50μl of biotin-labeled secondary antibody (1:100, diluted in PBS), incubate at 37°C for 1h, rinse 3 times with PBS, 5min each time;

Third antibody incubation: add 50 μl of third antibody (horseradish peroxidase-labeled streptavidin 1:100, diluted in PBS), incubate at 37°C for 40min, rinse with PBS 3 times, 5min each time;

Color development: DAB color development 5-10min, master the degree of staining under the microscope;

Termination: Rinse with tap water to terminate the reaction;

Negative staining: Hematoxylin negative staining for 10min, hydrochloric acid and alcohol differentiation for 2 times, and ammonia water returning to blue for 40s;

Dehydration: 80%, 95% ethanol for 10 minutes each in order, absolute ethanol (I), (II) 10 minutes each, xylene (I) (II) 20 minutes each, resin sealing.

1.12 Patient glioma tissue chip data

691 specimens were selected from 3 independent human glioma databases: Chinese Glioma Genome Atlas Database (CGGA, http://www.cgcg.org.cn/), 248 cases, the National Cancer Institute Molecular Brain Tumor The database (Rembrandt, https://gdoc.georgetown, edu/gdoc) has 180 cases, and the Gene Expression Comprehensive Website (http://www.ncbi.nlm.nih.gov/geo/, GSE16011) has 263 cases. For the CGGA database, 248 frozen glioma specimens were collected from patients in the glioma center of Beijing Tiantan Hospital from 2006 to 2009. Patients who had received radiotherapy and chemotherapy before surgery were excluded from the study. The tumor histology of all patients was independently determined by two neuropathology experts based on the 2007 WHO Central Nervous System Tumor Classification Standards. The study was also approved by the Research Ethics Committee of Tiantan Hospital, and the informed consent of the patient or family members was obtained. Due to the low content of grade III glioma specimens in CGGA, only grade II and grade IV data were used for corresponding studies.

1.13 Cell proliferation experiment

The cell proliferation experiment was performed using the CCK8 kit (Dojindo Laboratories of Japan) according to the operating instructions. Briefly, in a 96-well plate, each cell is seeded with 2000 cells, and each treatment group has three auxiliary wells. The cells are grown in the corresponding medium for 0-96 hours. At the corresponding time, 10 μl of CCK8 is added to 100 μl of medium per well The solution was incubated for 2h, and then the absorbance of the resulting solution was detected using a 450nm microplate reader.

1.14 Chromatin immunoprecipitation (ChIP) and ChIP-qPCR experiments

The ChIP experiment used a commercially available ChIP kit (China Biyuntian). First, the large dish cells were washed twice with PBS, 1% formalin was added to cross-link the protein and chromatin at 37°C for 10 min, and then neutralized with glycine at room temperature for 5 min. All cells were washed with cold PBS, scraped off, collected and placed on ice. Next, the cells were sonicated using a Scientz-IID ultrasonic homogenizer (Scientz) for 10 s, with a total of 20 cycles of ultrasound lysis at 20 s intervals. Immunoprecipitation of equal amounts of chromatin and at least 1.5 μg antibody overnight. Antibodies are: H3K4me3, NF-κB (US CST) and normal mouse IgG (US Millipore). The immunoprecipitation product was incubated with protein A+G coated magnetic beads, the magnetic beads were washed, and the bound chromatin was eluted with ChIP elution buffer. The protein in the mixed solution was digested with proteinase K in a 45°C water bath for 4h. DNA was purified using a DNA purification kit (China Biyuntian). After co-immunoprecipitation was completed, qPCR was used to detect the E2F6 binding region and normalized with total chromatin (InPut). The normal mouse IgG group served as a negative control. The detection primer sequence is as follows:

Primer 1 forward: 5'-CGGTGTGTTGCCTTTTTATT-3' (SEQ ID NO: 8),

Reverse: 5'-AACAACGTCCAATTTCAGTG-3' (SEQ ID NO: 9);

Primer 2 forward: 5'-CACTGAAATTGGACGTTGTT-3' (SEQ ID NO: 10),

Reverse: 5'-AGGTCAGTGTTGATGCTTAG-3' (SEQ ID NO: 11);

Primer 3 forward: 5'-ATCTCTGCGGCTCAGAACTT-3' (SEQ ID NO: 12),

Reverse: 5'-AGGGAACAGGGGTGAGAGAA-3' (SEQ ID NO: 13);

Primer 4 forward: 5'-TTTCCTTTGCCAGCCTCTCC-3' (SEQ ID NO: 14),

Reverse: 5'-ACGCAGACGGAAAAAGAGGA-3' (SEQ ID NO: 15).

A three-step method was used to perform qPCR experiments. The reaction conditions were denaturation at 95°C for 3 min, followed by 40 cycles of annealing at 95°C for 15 s, 57°C for 30 s, and 72°C for 30 s, and finally extended at 72°C for 5 min. The PCR products were finally electrophoresed using GelRed containing 2% agarose gel.

1.15 Immunofluorescence experiment and confocal imaging

All cell lines used were planted on 10% collagen-coated coverslips. Cells were fixed with pre-chilled paraformaldehyde for 30 minutes, and washed with PBS three times for 5 minutes each time. 0.5% TritonX-100 permeable membrane, and then blocked with 1% BSA for 1h. The corresponding primary antibody (γ-H2AX (abcam) (1:200 dilution)) was used for binding overnight. The next day, the cells were re-warmed at 37°C for 1h, and washed with PBS three times for 10min each time. Incubate the cells with AlexaFluor488-conjugated secondary antibody (American Life Technologies, 1:100 dilution) and TRITC-labeled phalloidin for 2 hours at room temperature, and then stain the nuclei with DAPI and wash three times with PBS for 10 minutes each time. Covered with anti-quenching agent, placed under Olympus FluoView1200 system for imaging. In order to objectively compare different treatment groups with different immunofluorescence, all confocal scanning parameters remain unchanged, and the picture is almost unprocessed to maintain the authenticity of the data.

1.16 In situ tumor experiment

Five-week-old female nude mice (Institute of Oncology, Chinese Academy of Medical Sciences) were used to establish an intracranial glioma tumor model. Before intracranial injection of glioma cells, we transfected U87 cells with lentivirus overexpressing E2F6 and screened with puromycin to construct stable U87E2F6 cells; meanwhile, we transfected U87EGFRvIII cells with lentivirus overexpressing E2F6 siRNA Screening with puromycin to construct stable U87vIIIE2F6si cells. Then we used U87 cells, U87E2F6 cells, EGFRvIII cells, and EGFRvIIIE2F6si cells as cell models for making intracranial tumor experiments in mice. Discard the culture medium, wash the cells with PBS, add about 0.5ml of trypsin containing EDTA to each digestion for 2 minutes, observe that the cells are completely detached from the culture dish under the microscope, add the medium containing serum to stop the digestion, and count the cells. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, disperse the pelleted cells in an appropriate amount of PBS, and place on ice. Each nude mouse in each group was implanted intracranially with 500,000 cells under the guidance of a stereotaxic instrument. After one week of tumor growth, temozolomide was injected intraperitoneally at an amount of 5 mg/Kg/d for 5 days, and treatment was stopped for 2 days for a total of 2 weeks. . Tumor growth was performed on days 7, 14, 21, and 28 by in vitro imaging to detect tumor size, and the in vitro imaging fluorescence of each mouse was normalized with the initial fluorescence results. The body weight of the mice is measured and recorded every two days and recorded in a graph, and the error value in the figure represents the standard deviation (SD). The survival time of mice was recorded, and Kaplan-Meier survival curve was drawn. After temozolomide treatment and at the end of the experiment, the intracranial allogeneic tumors of the mice were taken out, washed with PBS, and soaked in formalin for 24 hours. The sections were embedded in paraffin for immunohistochemical detection. HE staining and anti-EGFRvIII, anti-p-NF-κB and anti-E2F6 antibodies were used to detect the corresponding protein expression.

1.17 Plate clone formation experiment

1. Take each group of cells in the logarithmic growth phase, digest with 0.25% trypsin and blow into individual cells, and suspend the cells in 10% fetal bovine serum in DMEM medium for use.

2. Dilute the cell suspension in multiples of gradients. Each group of cells is inoculated into a dish containing 10 mL of 37°C pre-warmed culture solution at a gradient density of 50, 100, and 200 cells per dish, and gently rotate to disperse the cells. Evenly. Incubate in a cell incubator at 37°C, 5% CO ₂ and saturated humidity for 2 to 3 weeks.

3. Observe frequently, when there are visible clones in the petri dish, terminate the culture. The supernatant was discarded and carefully rinsed twice with PBS. Add 4% paraformaldehyde to fix cells 5mL and fix for 15 minutes. Then remove the fixing solution, add an appropriate amount of GIMSA and apply the staining solution to stain for 10 to 30 minutes, then slowly wash off the staining solution with running water and air dry.

4. Invert the plate and overlay a transparent film with a grid, directly count the clones with the naked eye, or count the number of clones greater than 10 cells under a microscope (low magnification). Finally, the clonal formation rate was calculated. Clone formation rate = (number of clones/number of inoculated cells) × 100% The test method of plate clone formation is simple and suitable for adherent cells. Suitable substrates are glass and plastic bottles. The key to the success of the test is the preparation of the cell suspension and the seeding density. The cells must be well dispersed, there must be no cell mass, and the seeding density should not be too large.

1.19 Results analysis

1. E2F6 as a TMZ resistance gene in GBM cells

Comparison of EGFRvIII overexpression of mRNA in U87 cells. E2F6 is the only gene that increases after EGFRvIII overexpression and is resistant to TMZ (Figure 4A). Principal component analysis compared the cells before and after TMZ treatment and showed that the two groups were completely different (Figure 4B). Despite 7 or 14 days of treatment, the expression of E2F6 in TMZ-treated EGFRvIII cells increased (Figure 4C). TMZ treatment increased E2F6 expression at 14 days compared to 7 days (Figure 4D)

2. E2F6 expression is associated with classic glioma and WHO classification

We used TCGA RNAseq data and Rembrandt data to show whether E2F6 is related to glioma grade. Choose WHOII, III or IV glioma for our further study. As shown in Figure 5, E2F6 expression was significantly correlated with tumor grade (P<0.0001).

3. E2F6 as TMZ resistance gene in vitro

EGFRvIII enhances the killing effect against radiation by accelerating the repair of DNA double-strand breaks (DSBs). In order to identify the mechanism of chemotherapy resistance caused by EGFRvIII, GBM cells were exposed to TMZ for a long time and found that E2F6 levels in EGFRvIII cells increased, and TMZ stimulation also increased E2F6 expression, which is consistent with our previous observations, indicating that E2F6 plays a central role in TMZ resistance (Figure 6A and Figure 6B). Then, lentiviruses encoding E2F6 and E2F6 siRNAs were designed to gain or lose functional experiments. Calculating the amount of the three siRNAs we designed, siRNA#1 has a high efficiency in knocking down E2F6 (E2F6KD), so we use this siRNA as our tool to perform our subsequent experiments. The CCK8 viability assay confirmed the resistance of EGFRvIII cells to TMZ, and overexpression of E2F6 increased the resistance to TMZ, while silencing E2F6 reduced the resistance of EGFRvIII cells to TMZ (Figure 6C). In addition, we applied a colony formation test to prolong the exposure of GBM cells to TMZ within two weeks. Similar results indicate that E2F6 plays a major role in TMZ resistance (Figure 6D). Γ-H2AX (phosphorylated protein reflecting DNA damage) was used to measure the reflection of chromosome breaks caused by TMZ treatment. Under TMZ treatment, the increase or decrease of E2F6 molecules caused the fluorescence of γ-H2AX to decrease or increase, indicating that E2F6 inhibited TMZ-induced DNA damage (Figure 6E). In conclusion, by inhibiting the DNA damage response, E2F6 functions as a key factor in EGFRvIII cells in TMZ resistance.

4. E2F6 is regulated by H3K4me3 and NF-κB in the EGFR/PI3K/AKT pathway

After over-expression of NF-κB and JSH-23, respectively, the control group and EGFRvIII cells were treated for 48h, and the total protein and mRNA were collected by Western blot and qRT-PCR methods to detect the expression of E2F6. In protein (Figure 7A) and mRNA levels (Figure 7B), E2F6 was positively correlated with NF-κB activation in GBM cells, indicating that E2F6 may be regulated in the EGFRvIII/PI3K/AKT/NF-κB pathway. Through treatment with EGFRvIII and TMZ, NF-κB was found to be activated (Figure 7C), which means that E2F6 may be activated by EGFRvIII and TMZ-induced NF-κB activation.

To investigate how E2F6 is regulated by AKT, evaluate the chromatin state cell line of the E2F6 promoter regulated by EGFRvIII in GBM. ChIP-PCR analysis was performed using antibodies against H3K4me3 and p-NF-κB (p-p65) and 4 pairs of E2F6 promoter-specific genomic PCR primers. The amount of H3K4me3 and NF-κB binding to the promoter region increased (Fig. 7E). In contrast, the use of MK-2206 to block AKT produced completely different results (Figure 7F). These studies revealed that E2F6 transcription is regulated by H3K4me3 and NF-κB in the EGFRvIII/PI3K/AKT pathway (Figure 7G).

5. E2F6 is a therapeutic target that increases TMZ sensitivity

First, the lentivirus encoding E2F6 and U87EGFRvIII cells was transduced into U87 cells together with the lentivirus encoding E2F6siRNA#1 (E2F6KD). The in situ mouse model was constructed by intracranial injection of these four groups of cells. Each group was treated with 5 mg/kg/d of DMSO or TMZ, and the drug was discontinued for 5 days after 2 days for two weeks (Figure 8A). A bioluminescence imaging assay was performed to show that U87 cells showed high resistance to TMZ after E2F6 overexpression (Figure 8B), while U87EGFRvIII cells showed significant sensitivity to TMZ after knockdown of E2F6 (Figure 8C). Overexpression or silencing of E2F6 did not affect the body weight or overall survival time of these mice. However, mice in the E2F6 overexpression group treated with TMZ reduced body weight at a slower rate during their overall survival time (Figure 8D), while the E2F6 silence of TMZ-treated U87EGFRvIII survived at a faster rate during their overall survival Time (Figure 8E). It is worth noting that Kaplan-Meier survival curve analysis showed that the prognosis of patients with E2F6 overexpressing tumors is poor (Figure 8F), and the prognosis of E2F6 silent tumors after TMZ treatment is better (Figure 8G), indicating that E2F6 plays a key role in TMZ resistance. . Then we detected the expression of p-NF-κB and E2F6 by immunohistochemistry. p-NF-κB and E2F6 were increased in EGFRvIII or even treated by TMZ. Taken together, these data show that E2F6 is susceptible to TMZ-resistant GBM, and targeting E2F6 is a treatment strategy for TMZ-resistant GBM patients.

The above is only the preferred embodiment of the present invention, and does not limit any form and substance of the present invention. It should be noted that those of ordinary skill in the art, without departing from the method of the present invention, will also Several improvements and additions can be made, and these improvements and additions should also be regarded as the scope of protection of the present invention. Those skilled in the art, without departing from the spirit and scope of the present invention, can use the technical content disclosed above to make some alterations, modifications and evolutions of equivalent changes, all of the present invention Equivalent embodiments; meanwhile, any modification, modification and evolution of any equivalent changes made to the above embodiments according to the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims (53)

1. E2F6 inhibitors are used for preparing temozolomide synergistic drugs for treating glioblastoma or for preparing glioblastoma therapeutic drugs in combination with temozolomide.
2. The use according to claim 1, wherein the E2F6 inhibitor refers to a molecule having an inhibitory effect on E2F6 or NF-κB.
3. The use according to claim 1 or 2, wherein the E2F6 inhibitor inhibits E2F6 activity, or inhibits E2F6 gene transcription or expression, or inhibits NF-κB activity, or inhibits NF-κB gene transcription or expression.
4. The use according to any one of claims 1 to 3, wherein the E2F6 inhibitor is siRNA, shRNA, antibody, or small molecule compound.
5. The use according to claim 4, characterized in that the nucleotide sequence of the siRNA is shown in any sequence of SEQ ID NO: 1 to 3.
6. A glioblastoma therapeutic drug combination includes an effective amount of an E2F6 inhibitor and an effective amount of temozolomide.
7. The glioblastoma therapeutic drug combination according to claim 6, wherein the E2F6 inhibitor refers to a molecule having an inhibitory effect on E2F6 or NF-κB.
8. The glioblastoma therapeutic drug combination according to claim 6 or 7, wherein the E2F6 inhibitor inhibits E2F6 activity, or inhibits E2F6 gene transcription or expression, or inhibits NF-κB activity, or inhibits NF- κB gene transcription or expression.
9. The glioblastoma therapeutic drug combination according to any one of claims 6-8, wherein the E2F6 inhibitor is siRNA, shRNA, antibody, or small molecule compound.
10. The glioblastoma therapeutic drug combination according to claim 9, wherein the nucleotide sequence of the siRNA is shown in any one of SEQ ID NO: 1 to 3.
11. The therapeutic drug combination for glioblastoma according to any one of claims 6-10, characterized in that the therapeutic drug combination may be in any one of the following forms: (1) Separate preparation of an E2F6 inhibitor and temozolomide Into separate preparations, the dosage form of the preparations may be the same or different, and the route of administration may also be the same or different; (2) E2F6 inhibitor and temozolomide are formulated into a compound preparation.
12. The glioblastoma therapeutic drug combination according to any one of claims 6-10, wherein the active ingredient of the glioblastoma therapeutic drug combination further includes at least one other glioblastoma therapeutic drug .
13. The therapeutic drug combination for glioblastoma according to claim 12, wherein the therapeutic drug combination may be any one of the following forms: (1) E2F6 inhibitor, temozolomide and other glioblastoma cells Tumor therapy drugs are made into separate preparations, the dosage forms are the same or different, and the route of administration is also the same or different; (2) E2F6 inhibitors, temozolomide, and other glioblastoma treatment drugs are formulated into compound preparations; (3 )Temozolomide and other glioblastoma treatment drugs are formulated into a compound preparation, and the E2F6 inhibitor is formulated into an independent preparation. The dosage form of the preparation is the same or different, and the route of administration is also the same or different; (4) Temozolomide and E2F6 inhibitor Formulated into a compound preparation, and other glioblastoma treatment drugs are formulated into independent preparations, the dosage form of the preparation is the same or different, and the route of administration is also the same or different; (5) E2F6 inhibitor and other glioblastoma treatment drugs It is formulated into a compound preparation, and temozolomide is formulated into an independent preparation. The dosage form of the preparation is the same or different, and the route of administration is also the same or different.
14. A method for treating tumors includes the steps of: administering a pharmaceutical composition to a subject, the pharmaceutical composition containing a pharmaceutically effective dose of an E2F6 inhibitor and a pharmaceutically effective dose of temozolomide.
15. The method of claim 14, wherein the tumor is glioblastoma.
16. The method according to claim 14 or 15, wherein the E2F6 inhibitor refers to a molecule having an inhibitory effect on E2F6 or NF-κB.
17. The method according to any one of claims 14-16, wherein the E2F6 inhibitor inhibits E2F6 activity, or inhibits E2F6 gene transcription or expression, or inhibits NF-κB activity, or inhibits NF-κB gene transcription or expression.
18. The method according to any one of claims 14 to 17, wherein the E2F6 inhibitor is siRNA, shRNA, antibody, or small molecule compound.
19. The method according to any one of claims 14 to 18, wherein the nucleotide sequence of the siRNA is shown in any one of SEQ ID NO: 1 to 3.
20. The method according to any one of claims 14-19, wherein the therapeutic drug combination may be in any of the following forms: (1) E2F6 inhibitor and temozolomide are made into separate preparations, respectively The dosage form can be the same or different, and the route of administration can also be the same or different; (2) E2F6 inhibitor and temozolomide are formulated into a compound preparation.
21. The method according to any one of claims 14-20, wherein the subject is a mammal or a glioblastoma cell of the mammal.
22. The method according to claim 21, characterized in that the mammal is preferably a rodent, artiodactyla, odd-hoofed animal, rabbit-shaped animal, primate and the like.
23. The method of claim 22, wherein the primate is a monkey, ape, or sapiens.
24. The method according to claim 21, wherein the glioblastoma cells are glioblastoma cells in vitro.
25. The method of claim 24, wherein the isolated glioblastoma cell is U87 or U87 EGFRvIII.
26. The method according to any one of claims 14 to 25, wherein the subject is a patient suffering from glioblastoma or an individual expecting to treat glioblastoma.
27. The method according to any one of claims 14 to 25, wherein the subject is isolated glioblastoma cells of a glioblastoma patient or an individual who is expected to treat glioblastoma.
28. The method according to any one of claims 14 to 27, wherein the E2F6 inhibitor and temozolomide, or the combination of E2F6 inhibitor and temozolomide, can be treated before, during, or after glioblastoma treatment Subject administration.
29. The method according to any one of claims 14 to 28, wherein the pharmaceutically effective dose of the E2F6 inhibitor and the pharmaceutically effective dose of temozolomide can be administered in any combination of the following forms: (1) The pharmacologically effective dose of E2F6 inhibitor and the pharmacologically effective dose of temozolomide are made into separate preparations, the dosage forms of the preparations are the same or different, and the route of administration is also the same or different; (2) The pharmaceutically effective dose of E2F6 inhibitor and the pharmacologically effective A dose of temozolomide is formulated as a compound preparation.
30. The method according to any one of claims 14 to 29, wherein the pharmaceutically effective dose of the E2F6 inhibitor and the pharmaceutically effective dose of temozolomide are administered orally, by injection, by sublingual administration, by rectal administration, by vaginal administration The medicine, transdermal administration or spray inhalation route is used for patients in need of treatment.
31. The method according to any one of claims 14 to 30, wherein the dosage forms of the pharmaceutically effective dose of E2F6 inhibitor and the pharmaceutically effective dose of temozolomide are tablets, granules, capsules, pills, dropping pills, powders, washing Agents, syrups, stomach plates, mixtures, medicinal wine, tinctures, lozenges, liquid extracts and extracts, ointments, gels, ointments, teas, lotions, paints, liniments, aerosols or sprays.
32. The method according to any one of claims 14 to 31, wherein the active ingredient of the pharmaceutical composition further comprises at least one other glioblastoma treatment drug.
33. The method according to claim 32, wherein the drug combination may be any one of the following forms: (1) E2F6 inhibitor, temozolomide and other glioblastoma therapeutic drugs are made into separate Preparations, the dosage forms of the preparations are the same or different, and the route of administration is also the same or different; (2) E2F6 inhibitors, temozolomide and other glioblastoma treatment drugs are formulated into compound preparations; (3) temozolomide and other glioblastomas The cell tumor therapeutic drug is formulated into a compound preparation, and the E2F6 inhibitor is formulated into an independent preparation. The dosage form of the preparation is the same or different, and the route of administration is also the same or different; (4) The temozolomide and the E2F6 inhibitor are formulated into a compound preparation, and other gums Drugs for the treatment of glioblastoma are formulated into independent preparations. The dosage forms of the preparations are the same or different, and the route of administration is also the same or different; (5) E2F6 inhibitors and other glioblastoma treatment drugs are formulated into a compound preparation, and temozolomide is formulated. Into a separate preparation, the dosage form of the preparation is the same or different, and the route of administration is also the same or different.
34. A method for inhibiting tumor proliferation, invasion and metastasis includes the following steps: To administer a pharmaceutical composition to a subject, the pharmaceutical composition contains a pharmaceutically effective dose of an E2F6 inhibitor and a pharmaceutically effective dose of temozolomide.
35. The method of claim 34, wherein the tumor is a glioblastoma.
36. The method according to claim 34 or 35, wherein the E2F6 inhibitor refers to a molecule having an inhibitory effect on E2F6 or NF-κB.
37. The method according to any one of claims 34 to 36, wherein the E2F6 inhibitor inhibits E2F6 activity, or inhibits E2F6 gene transcription or expression, or inhibits NF-κB activity, or inhibits NF-κB gene transcription or expression.
38. The method according to any one of claims 34 to 37, wherein the E2F6 inhibitor is siRNA, shRNA, antibody, or small molecule compound.
39. The method according to any one of claims 34 to 38, wherein the nucleotide sequence of the siRNA is shown in any one of SEQ ID NO: 1 to 3.
40. The method according to any one of claims 34 to 39, wherein the therapeutic drug combination may be in any of the following forms: (1) E2F6 inhibitor and temozolomide are made into separate preparations, respectively The dosage form can be the same or different, and the route of administration can also be the same or different; (2) E2F6 inhibitor and temozolomide are formulated into a compound preparation.
41. The method according to any one of claims 34 to 40, wherein the object is a mammal or a glioblastoma cell of the mammal.
42. The method according to claim 41, characterized in that the mammal is preferably a rodent, artiodactyla, odd-footed animal, rabbit-shaped animal, primate and the like.
43. The method of claim 42, wherein the primate is a monkey, ape, or sapiens.
44. The method of claim 41, wherein the glioblastoma cells are glioblastoma cells in vitro.
45. The method of claim 44, wherein the isolated glioblastoma cell is U87 or U87 EGFRvIII.
46. The method according to any one of claims 34 to 45, wherein the subject is a patient suffering from glioblastoma or an individual who wishes to treat glioblastoma.
47. The method according to any one of claims 34 to 45, wherein the subject is glioblastoma cells isolated from a glioblastoma patient or an individual who is to be treated for glioblastoma.
48. The method according to any one of claims 34 to 47, wherein the E2F6 inhibitor and temozolomide, or the combination drug of the E2F6 inhibitor and temozolomide can be treated before, during, or after glioblastoma treatment Subject administration.
49. The method according to any one of claims 34 to 48, wherein the pharmaceutically effective dose of the E2F6 inhibitor and the pharmaceutically effective dose of temozolomide can be administered in any combination of the following forms: (1) The pharmacologically effective dose of E2F6 inhibitor and the pharmacologically effective dose of temozolomide are made into separate preparations, the dosage forms of the preparations are the same or different, and the route of administration is also the same or different; A dose of temozolomide is formulated as a compound preparation.
50. The method according to any one of claims 34 to 49, wherein the pharmaceutically effective dose of the E2F6 inhibitor and the pharmaceutically effective dose of temozolomide are administered orally, by injection, by sublingual administration, by rectal administration, by vaginal administration The medicine, transdermal administration or spray inhalation route is used for patients in need of treatment.
51. The method according to any one of claims 34-50, wherein the pharmaceutically effective dose of the E2F6 inhibitor and the pharmaceutically effective dose of temozolomide are in the form of tablets, granules, capsules, pills, drops, powders, and tablets Agents, syrups, stomach plates, mixtures, medicinal wine, tinctures, lozenges, liquid extracts and extracts, ointments, gels, ointments, teas, lotions, paints, liniments, aerosols or sprays.
52. The method according to any one of claims 34 to 51, wherein the active ingredient of the pharmaceutical composition further comprises at least one other glioblastoma treatment drug.
53. The method according to claim 52, characterized in that the drug combination can be in any of the following forms: (1) E2F6 inhibitor, temozolomide and other glioblastoma therapeutic drugs are made into independent Preparations, the dosage forms of the preparations are the same or different, and the route of administration is also the same or different; (2) E2F6 inhibitors, temozolomide and other glioblastoma treatment drugs are formulated into compound preparations; (3) temozolomide and other glioblastomas The cell tumor therapeutic drug is formulated into a compound preparation, and the E2F6 inhibitor is formulated into an independent preparation. The dosage form of the preparation is the same or different, and the route of administration is also the same or different; (4) The temozolomide and the E2F6 inhibitor are formulated into a compound preparation, and other gums Drugs for the treatment of glioblastoma are formulated as independent preparations. The dosage forms of the preparations are the same or different, and the route of administration is also the same or different; (5) E2F6 inhibitors and other glioblastoma treatment drugs are formulated as a compound preparation, and temozolomide is formulated. Into a separate preparation, the dosage form of the preparation is the same or different, and the route of administration is also the same or different.

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