CN115569198A

CN115569198A - Use of EFTUD2 inhibitors for treating medulloblastoma

Info

Publication number: CN115569198A
Application number: CN202211560235.7A
Authority: CN
Inventors: 吴海涛; 陈丽萍; 李莹
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2022-12-07
Filing date: 2022-12-07
Publication date: 2023-01-06
Anticipated expiration: 2042-12-07
Also published as: CN115569198B

Abstract

The invention belongs to the field of biomedicine, and particularly relates to an application of an EFTUD2 inhibitor in treating medulloblastoma. Also relates to the application of the reagent for detecting the expression level of EFTUD2 in diagnosing medulloblastoma and predicting prognosis of patients with medulloblastoma.

Description

Use of EFTUD2 inhibitors for treating medulloblastoma

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to an application of an EFTUD2 inhibitor in treating medulloblastoma.

Background

Medulloblastoma (MB) is the most common central nervous system malignancy in the posterior cranial fossa of children, accounts for 8% -10% of brain tumors of children, mainly occurs in the posterior cranial fossa, and can also spread to the whole brain and spinal cord through cerebrospinal fluid. Medulloblastoma grows extremely rapidly and belongs to an embryonic high-grade malignant tumor.

Medulloblastomas can appear throughout childhood and adulthood, occurring primarily in children under 14 years of age, with a total annual incidence of about 5/100 ten thousand in pediatric populations, with a minority of those over 20 years of age, with a male incidence of about 1.7 times that of women, with some medulloblastomas being genetically predisposed and positively correlated with the development of certain neuro-tumor syndromes (e.g., gorlin syndrome, turcot syndrome, curry-Jones syndrome, etc.).

Currently, surgical resection combined with postoperative chemoradiotherapy has become the standard treatment for patients with medulloblastoma, wherein surgical resection is the most important component of treatment, and the extent of tumor resection has a significant impact on patient prognosis. However, it has not been cured because of the difficulty of total surgical resection and the propensity of tumor cells to disseminate along the cerebrospinal fluid.

Currently, there is no very specific and effective drug for clinically treating medulloblastoma, so it is urgently needed to screen out an effective target for treating medulloblastoma.

Disclosure of Invention

In order to make up the current situation that no specific chemotherapy drugs for effectively treating medulloblastoma exist clinically, the invention aims to provide a new therapeutic target for medulloblastoma, thereby providing a new idea for developing drugs for preventing or treating medulloblastoma or inhibiting the proliferation of medulloblastoma cells.

The RNA spliceosome protein EFTUD2 described in the embodiments of the present invention is an important target molecule for regulating medulloblastoma, and siRNA that specifically knockdown EFTUD2 can significantly reduce the proliferation of medulloblastoma Daoy cells.

The specific technical scheme is as follows:

in one aspect, the invention provides the following uses of an EFTUD2 inhibitor:

1) Application in preparing products for treating Medulloblastoma (MB),

2) Use in inhibiting in vitro proliferation of medulloblastoma cells.

Preferably, the use is for non-therapeutic purposes, in vitro.

The EFTUD2 is called extension Factor Tu GTP Binding Domain Binding protein 2 (Elongation Factor Tu GTP Binding Domain protein 2) and can be called Snu, snrp116, U5-116KD and the like.

Preferably, the medulloblastoma comprises any type of medulloblastoma.

In particular, the world health organization classifies medulloblastoma into 3 types according to histopathology, namely classical, large cell/space variant, nodular and desmoplastic; alternatively, medulloblastomas are further classified into WNT type, SHH (sonic hedgehog) type, G3 (group 3) type and G4 (group 4) type according to genomics.

Preferably, the medulloblastoma of the present invention is an SHH (sonic hedgehog) type medulloblastoma.

Preferably, the product may also contain other drugs for treating cancer, more specifically drugs for treating medulloblastoma, such as cisplatin, carboplatin, cyclophosphamide, vincristine, semustine, fotemustine, mitomycin, paclitaxel, daunorubicin, camptothecin, or mabs specifically targeting medulloblastoma.

The term "EFTUD2 inhibitor" as used herein refers to a substance that targets, reduces or inhibits at least one activity of EFTUD 2.

Preferably, the EFTUD2 inhibitor comprises synthetic or naturally occurring.

Preferably, the EFTUD2 inhibitor includes a reagent used in a gene editing technology such as an RNA interference technology, an antisense oligonucleotide (ASO) technology, a CRISPR technology, a TALEN technology, a ZFN technology, a Cre-loxP gene recombination technology, and the like, and also includes a compound that can reduce the expression of EFTUD 2.

More specifically, the siRNA used according to the embodiment of the present invention, and more specifically, the target sequence thereof as shown in the embodiment of the present invention, may be referred to as an siRNA specifically targeting EFTUD 2.

Preferably, the product may further comprise reagents required for siRNA interference experiments, including transfection reagents; the transfection reagent may be a commercial product or a self-formulated reagent.

The siRNA of the present invention may also be referred to as Small interfering RNA (siRNA), short interfering RNA (short interfering RNA), or silencing RNA (silencing RNA). siRNA interference experiments are methods for effectively silencing or inhibiting the expression of a target gene.

Preferably, the product is a pharmaceutical composition.

Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or excipient.

Preferably, the pharmaceutically acceptable carrier, diluent or excipient includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye/colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, surfactant or emulsifier that has been approved by the U.S. food and drug administration or the national food and drug administration for use in humans or livestock.

The term "pharmaceutically acceptable" as used herein means that the EFTUD2 inhibitor or pharmaceutical composition thereof does not produce adverse, allergic or other untoward effects when properly administered to a subject.

The term "treating" as used herein includes the use of the EFTUD2 inhibitors provided herein to prevent or delay the onset of symptoms and complications of disease, particularly medulloblastoma. The treatment may also be prophylactic. Treatment of a tumor also refers to controlling the progression of the tumor in an individual, prolonging the life of a patient with the tumor, improving quality of life, alleviating symptoms, reducing or even eliminating the tumor, and inhibiting tumor metastasis. Anti-tumor effects include treatment of tumors and also prevention of tumorigenesis, recurrence and metastasis.

More specifically, the proliferation of the medulloblastoma cell is embodied by Ki167 ⁺ The amount of Ki167 when the proliferation of the medulloblastoma cell is inhibited ⁺ The expression level of (3) is decreased.

In another aspect, the invention provides a method of inhibiting proliferation of a medulloblastoma cell, the method comprising contacting or allowing an EFTUD2 inhibitor to enter a medulloblastoma cell.

Preferably, the contacting or entering may be by transfection, or other means of nucleic acid delivery.

Preferably, the method is performed in vitro and is for non-therapeutic purposes.

The term "contacting" as used herein when applied to a cell is used herein to describe the process of delivering an EFTUD2 inhibitor to a target cell. To achieve cell killing or stasis, the EFTUD2 inhibitor is delivered to the cells at a dose effective to kill the cells.

The term "transfection" as used herein describes a method of targeted delivery of DNA to eukaryotic cells using a delivery system such as adenovirus, AAV, retrovirus or plasmid-delivered gene transfer.

Preferably, the medulloblastoma cells include any type of cell, such as Daoy human medulloblastoma cells used in embodiments of the present invention; d341 Med human myeloblastoma, D283 Med human myeloblastoma are also acceptable.

Preferably, the medulloblastoma cells may also be ex vivo cancer cells (medulloblastoma cells) from a medulloblastoma patient.

In another aspect, the invention provides a method of treating medulloblastoma, the method comprising the step of administering an EFTUD2 inhibitor to a medulloblastoma patient.

Preferably, the mode of administration may be any clinically acceptable mode, such as oral, aerosol inhalation, rectal, nasal, buccal, parenteral, and the like.

In another aspect, the present invention provides a use of a reagent for detecting an expression level of EFTUD2 in the preparation of a product for predicting prognosis of a medulloblastoma patient.

Preferably, the indicators of prognosis include Objective Remission Rate (ORR), overall survival Rate (Overall survival Rate, OS), progression-free survival (PFS), time To Progression (TTP), disease-free survival (DFS), time to failure To Treat (TTF), response Rate (RR), complete Response (CR), partial Response (PR), and the like.

Preferably, the patient with low EFTUD2 expression will have a better prognosis, e.g., a longer overall survival rate.

In another aspect, the present invention provides a use of a reagent for detecting an expression level of EFTUD2 in the preparation of a product for diagnosing medulloblastoma.

In a specific embodiment, the EFTUD2 expression level of the present invention includes mRNA expression level and protein expression level.

Preferably, the EFTUD2 is expressed in the patient in an elevated amount in vivo.

Preferably, the reagent for measuring the expression level of mRNA of EFTUD2 comprises a reagent used in the following method: PCR-based detection method, southern hybridization method, northern hybridization method, dot hybridization method, fluorescence in situ hybridization method, DNA microarray method, ASO method, high throughput sequencing platform method. Such reagents are well known in the art and include, but are not limited to, specific nucleic acid probes that bind to the target sequence, specific primers that amplify the target sequence, non-specific fluorescent dyes, or combinations thereof.

Preferably, the reagent may further comprise a reagent for detecting the protein expression amount of EFTUD 2.

Preferably, the reagents at the protein level of the marker include reagents required for immunological detection.

Preferably, the immunological assay comprises an ELISA assay, an Elispot assay, a Western blot or a surface plasmon resonance method. Reagents required for immunological detection are well known in the art and include, but are not limited to, antibodies, targeting polypeptides capable of specifically binding to EFTUD 2.

Preferably, the expression level is measured in a sample taken from the subject.

Preferably, the sample comprises: peripheral blood, tissue, blood, serum, plasma, urine, saliva, semen, milk, cerebrospinal fluid, tears, sputum, mucus, lymph, cytosol, ascites, pleural effusion, amniotic fluid, bladder irrigation fluid and bronchoalveolar lavage fluid.

Preferably, the sample comprises a tissue, a cell line.

The terms "patient", "subject" and "subjects" as used herein interchangeably refer to an animal subject, particularly a vertebrate subject, more particularly a mammalian subject. Suitable vertebrates falling within the scope of the present invention include, but are not limited to, any member of the subphylum chordata, including primates, rodents (e.g., mice, rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cows), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, ducks, geese, companion birds such as canaries, budgerigars, etc.), marine mammals (e.g., dolphins, whales), reptiles (e.g., snakes, frogs, lizards, etc.), and fish.

Preferably, the subject is a primate (e.g., human, ape, monkey, chimpanzee).

Preferably, the subject is a human.

In another aspect, the present invention provides a method for confirming whether a test cell is a medulloblastoma cell or diagnosing medulloblastoma, the method comprising detecting the expression level of EFTUD2 in the test cell.

Preferably, the method is performed in vitro, for non-therapeutic purposes.

More specifically, when the expression level of EFTUD2 in the test cell is higher than a threshold value, the test cell can be judged as a medulloblastoma cell; or when the expression level of EFTUD2 in the test sample is higher than the threshold, the test sample is determined to be a patient with medulloblastoma.

In another aspect, the invention provides a method of predicting prognosis in a patient with medulloblastoma comprising detecting the amount of EFTUD2 expression in a sample from the patient with medulloblastoma.

Preferably, the sample is a cancer cell; more specifically, cells taken from cancer tissue.

Preferably, the method for detecting the expression level of the present invention includes a method for detecting the expression level of mRNA and/or a method for detecting the expression level of protein.

Preferably, the method for detecting the expression level of mRNA comprises: PCR-based detection method, southern hybridization method, northern hybridization method, dot hybridization method, fluorescence in situ hybridization method, DNA microarray method, ASO method, high throughput sequencing platform method.

Preferably, the method for detecting the expression level of the protein comprises the following steps: ELISA detection, elispot detection, western blotting or surface plasmon resonance.

More specifically, when the expression level of EFTUD2 in the test cell is higher than the threshold, the test sample can be determined to have a poor prognosis for the patient with medulloblastoma. The threshold value may vary according to different prognostic criteria.

As used herein, "threshold", the specific data for a threshold may depend on the particular measurement technique, e.g., western blot or qRT-PCR, used in the methods herein. If a different approach is used, the specific value of the threshold requires an analogic conversion, which is within the skill of the person skilled in the art.

The invention has the following advantages and beneficial effects:

the invention discovers that EFTUD2 can be used as a therapeutic target of medulloblastoma for the first time.

The invention also provides the use of an inhibitor specific for EFTUD2 in the manufacture of a product for treating medulloblastoma or inhibiting the proliferation of medulloblastoma cells.

Drawings

FIG. 1 is a graph showing the results of a comparison of the expression level of EFTUD2 between medulloblastoma and a control; a is the result of differential expression gene analysis, B is the signal path with significant enrichment difference, C is the result of measuring the EFTUD2 protein level in the two mouse cerebellums by western blot, D is the result of measuring the relative expression level of Eftud2 in the two mouse cerebellums by qRT-PCR, E is the result of measuring the medulloblastoma chip, and F is the result of measuring the expression level of EFTUD 2.

FIG. 2 is an experimental result of a comparison between mouse models expressing or not expressing EFTUD 2; a is the comparison of survival, B is the experimental result of Nissl staining, and C is the statistical result of the area on the B plot.

FIG. 3 is the results of an experiment in a cell line; a is the result of expression detection after siRNA knockdown of EFTUD2, B is the result of data statistics, C is the result of detection under fluorescence, and D is the result of detection of Ki 167.

Detailed Description

The present invention will be further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the invention in any way, and any person skilled in the art can modify the present invention by applying the teachings disclosed above and applying them to equivalent embodiments with equivalent modifications. Any simple modification or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.

Example 1 Presence of the spliceosome protein EFTUD2 Up-Regulation in RNA human and mouse medulloblastomas

1. Experimental materials

Mice: atoh1-Cre, smoM2, atoh1-Cre; smoM2 (the nomenclature used in the present invention for the hybrid mouse is ";" the genotype of the connecting parent and the female parent represents the genotype of the hybrid mouse).

Medulloblastoma chip (Avilabiio, DC-Bra 01022), SPN kit (China fir bridge, PV-9000), anti-EFTUD2 (Abcam, EPR 16055).

2. Experimental methods

1. Mouse hybridization: firstly, smoM2 and Atoh1-Cre are selected to be hybridized to obtain Atoh1-Cre, smoM2 (tumor model mouse-M-Smo) and Atoh1-Cre mouse as a control mouse.

2. Control mice and mice of the medulloblastoma tumor model (tumor model mouse-M-Smo) taken 30 days after birth (P30) were selected for cerebellar tissue collection and subjected to RNA-seq sequencing. And after the sequencing is finished, analyzing the differentially expressed genes and enriching the signal path.

3. According to the analysis of the second-generation RNA-seq sequencing result, the RNA spliceosome protein EFTUD2 is found to have an analysis result of obvious up-regulation, the cerebellum tissues of a control group and a tumor model mouse for P30 days are collected to prepare a protein sample and an RNA sample, and the expression of the protein and mRNA of the EFTUD2 in the cerebellum tissues of the control group and the model mouse is determined by western blot and qRT-PCR.

4. After deparaffinization and rehydration of the tissue chip containing normal cerebellar tissue and medulloblastoma samples, antigen retrieval was performed in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween20, ph 6.0) at 100 ℃ for 20 minutes. The chip was then neutralized with endogenous peroxidase (3%) for 15 min. The chip was then blocked with buffer (3% BSA) for 1 hour and incubated overnight with primary EFTUD2 antibody. The secondary antibody incubation and DAB visualization were performed according to the rabbit SPN kit (sequoia gold bridge, PV-9000). Subsequently, image J was used for analytical statistics.

3. Results of the experiment

As shown in FIG. 1, A and B are signal paths with significant differences in differentially expressed genes and enrichment found by RNA-seq sequencing analysis in cerebellar tissues of tumor mice compared with control mice. C-D protein and mRNA expression levels of EFTUD2 in the cerebellum of two mice were determined for western blot and qRT-PCR. In FIG. 1, E-F is an assay using a medulloblastoma chip (Avilabiio, DC-Bra 01022).

The data are presented as mean ± sd, with P values < 0.05 and < 0.001. The above results demonstrate a significant upregulation of EFTUD2 expression in human and mouse medulloblastoma tissues.

Example 2 EFTUD2 regulates the production of medulloblastoma

1. Experimental Material

Mice:

EFTUD2F/F，Atoh1-Cre，SmoM2，

Atoh1-Cre;SmoM2，Atoh1-Cre;SmoM2;EFTUD2F/F

2. experimental methods

1. Mouse hybridization: firstly, selecting EFTUD2F/F and Atoh1-Cre to make hybridization to obtain Atoh1-Cre, EFTUD 2F/-heterozygote, at the same time making SmoM2 and Atoh1-Cre undergo the process of hybridization to obtain Atoh1-Cre, smoM2 heterozygote, then further making hybridization of these two heterozygote mice to obtain Atoh1-creEFTUD2F/-, smoM2F/-, and making hybridization of Atoh1-Cre, EFTUD2F/-, smoM 2F/-and SmoM 2F/-to obtain Atoh1-Cre (control mouse-Ctrl), atoh1-Cre, smoM2 (tumor model mouse-M-KO) and Atoh1-Cre SmoM2, EFTUD2F/F (tumor-saving mouse-M-Smo knockout mouse-2-OTUD; EFD 2F/F).

2. Nissl staining: selecting three genotypes of Ctrl, M-Smo and EFTUD2 KO which are born in the same litter for 30 days, perfusing PFA, taking the whole brain tissue, fixing the whole brain tissue in the PFA for 24 hours, and performing gradient dehydration by using 10%,20% and 30% sucrose. Frozen sections with a thickness of 20 μm. Brain slices from similar sites were selected and washed three times with PBS. Fixing with 95% ethanol for 20min, washing with PBS for 3 times, each for 5min. Nissl dye liquor is used for dyeing for 2-3min, and washed by tap water. Drying brain slice or naturally drying, and sealing with neutral resin. And (6) sweeping the sheet.

3. Survival curve: the statistics of the death time of mice of three genotypes, at least 15 mice per genotype, were calculated using Prism software to derive survival curves.

4. Mouse cerebellum area statistics: mice of the above three genotypes at 30 days after birth were dissected to obtain cerebellum tissue and to count the area thereof, and the obtained data were counted with Prism software.

3. Results of the experiment

As shown in FIG. 2, A, M-Smo, EFTUD2 KO mice survived significantly longer than M-Smo mice. The weight of the cerebellum was significantly lower in the M-Smo, EFTUD2 KO mice than in the M-Smo mice, as indicated at BC in FIG. 2, and the data are presented as mean. + -. Standard error, where indicated by P < 0.05 and P < 0.001.

The above results demonstrate that EFTUD2 knockout inhibits the production of medulloblastoma.

Example 3 siRNA targeting EFTUD2 specifically inhibits proliferation of medulloblastoma DaoY cells

1. Experimental Material

Cell: daoY (ATCC)

Nucleic acid (A): EFTUD2 siRNA (Ruibo) RNAiMAX (Invitrogen)

Reagent: ki67 (BD, 550609), goat-anti-mouse 488 fluorescent secondary antibody (Biotum)

2. Experimental methods

After transfection of experimental group siRNA against EFTUD2 purchased from umbo and control group siRNA in the medulloblastoma cell line Daoy cells, western blot examined the expression of EFTUD2, and two groups of siRNA in which the EFTUD2 protein level was the lowest were picked after comparative analysis and named siRNA-1 and siRNA-2. Subsequent experiments non-specific siRNA was used as a control group, and siRNA-1 and siRNA-2 were used as two experimental groups for EFTUD2 knockdown. The three groups of cells are subjected to immunofluorescence detection of a cell proliferation marker Ki-67.

The specific operation method comprises the following steps:

1. siRNA purchased from Libo was dispensed and frozen at-20 ℃.

2. Seeded DaoY cells were seeded in six-well plates and siRNA transfected with the transfection reagent RNAimax for 48 hours.

3. And collecting DaoY cells after siRNA transfection, and preparing a total cell protein sample. Western blot identifies the protein level of EFTUD2, and two groups of siRNA with the most obvious EFTUD2 reduction are selected for subsequent experiments.

4. DaoY cells after siRNA transfection were labeled with cell proliferation marker Ki 67. Firstly, various groups of DaoY cells are planted in a confocal dish, siRNA transfection is carried out when the coverage rate is 20% -30, the cells are taken out of an incubator after 48 hours of transfection, the cells are fixed for 2 minutes by PFA at room temperature after culture medium is removed, and the cells are washed for three times of 5 minutes each time by 1xPBS. After 20 minutes of punching with 1% PBST at room temperature, 10% NGS (China fir gold bridge) was blocked for 1h at room temperature. Ki67 primary antibody (diluted in 5% ngs) was incubated overnight at 4 ℃. After rewarming the primary antibody at room temperature for 1h, the antibody was washed three times with PBS, 5 minutes each time. Secondary antibody (1.

3. Results of the experiment

As shown in fig. 3, EFTUD2 knockdown significantly inhibited the proliferation of medulloblastoma DaoY cells. The data are presented as mean ± sd, n =3, P < 0.05, and P < 0.001. The experimental results demonstrate that siRNA that specifically knockdown EFTUD2 can significantly inhibit proliferation of DaoY cells.

Claims

The following uses of eftud2 inhibitors:

1) Application of the medicine in preparing the medicine for treating medulloblastoma,

2) Use in inhibiting in vitro proliferation of medulloblastoma cells.
2. The use of claim 1, wherein the types of medulloblastoma include WNT type, SHH type, G3 type, and G4 type.
3. The use of claim 1, wherein the product further comprises cisplatin, carboplatin, a cyclic phosphoramide, a vincristine, semustine, fotemustine, mitomycin, paclitaxel, daunorubicin, camptothecin, or a monoclonal antibody that specifically targets medulloblastoma.
4. The use of claim 1, the EFTUD2 inhibitor comprises an agent used in RNA interference technology, antisense oligonucleotide technology, CRISPR technology, TALEN technology, ZFN technology, cre-loxP gene recombination technology, or a compound targeting EFTUD 2;

the agent used in the RNA interference technology is an siRNA that specifically targets EFTUD 2.
5. Application of reagent for detecting EFTUD2 expression level in preparation of products for predicting prognosis of medulloblastoma patients or diagnosing medulloblastoma.
6. The use according to claim 5, wherein the prognostic indicator comprises objective remission rate, overall survival rate, progression free survival, time to disease progression, disease free survival, time to treatment failure, response rate, complete response, partial response.
7. The use of claim 5, wherein the amount of EFTUD2 expression comprises mRNA expression and protein expression.
8. The use according to claim 7, wherein the reagent for measuring the expression level of mRNA of EFTUD2 comprises a reagent for use in the following method: PCR-based detection method, southern hybridization method, northern hybridization method, dot hybridization method, fluorescence in situ hybridization method, DNA microarray method, ASO method, high throughput sequencing platform method.
9. The use according to claim 7, wherein the reagent for detecting the protein expression level of EFTUD2 comprises a reagent required for an immunological assay comprising ELISA assay, elispot assay, western blotting or surface plasmon resonance assay.
10. A method comprising any one of:

1) A method of determining whether a test cell is a medulloblastoma cell, the method comprising detecting the amount of expression of EFTUD2 in the test cell, the method being of non-diagnostic interest;

2) A method of inhibiting proliferation of medulloblastoma cells in vitro, the method comprising contacting or allowing an EFTUD2 inhibitor to enter medulloblastoma cells.