CN112011618B - Application of Rack1 as target in preparation of anti-SHH (single-stranded lymphoblastoma) medicament - Google Patents

Application of Rack1 as target in preparation of anti-SHH (single-stranded lymphoblastoma) medicament Download PDF

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CN112011618B
CN112011618B CN202010951238.8A CN202010951238A CN112011618B CN 112011618 B CN112011618 B CN 112011618B CN 202010951238 A CN202010951238 A CN 202010951238A CN 112011618 B CN112011618 B CN 112011618B
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吴海涛
刘凤姣
邵靖媛
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Academy of Military Medical Sciences AMMS of PLA
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Abstract

The invention discloses application of Rack1 as a target in preparation of an anti-SHH subtype medulloblastoma drug. According to the invention, firstly, an SHH subtype medulloblastoma mouse model is constructed, and then the Rack1 gene is knocked out in the particle neuron of the constructed mouse model at the same time, so that the result shows that the Rack1 gene knocking out can obviously inhibit the proliferation of tumor cells and obviously prolong the survival time of tumor mice, and the Rack1 gene plays a key role in the regulation and control of the SHH subtype medulloblastoma of the mouse. The invention discovers for the first time that the Rack1 gene can be used as a novel target of a medulloblastoma resistant medicament and has potential application value in the immunotherapy of medulloblastoma.

Description

Application of Rack1 as target in preparation of anti-SHH (single-stranded lymphoblastoma) medicament
Technical Field
The invention belongs to the technical field of tumor drug targeting research, and particularly relates to application of a Rack1 gene as a target point in preparation of an anti-SHH subtype medulloblastoma drug.
Background
Medulloblastoma (MB) is the most common neurological malignancy in children, the primary neuroectodermal tumor that occurs mainly in the cerebellum. Medulloblastoma occurs mostly in the posterior fossa, grows concealably, and is characterized by a lack of early symptoms. The first symptoms are headache, vomiting and unsteady gait, and then diplopia, ataxia, hypopsia, head position forcing, head enlargement and cough, and in severe cases subarachnoid hemorrhage and cerebellar crisis can occur. At present, the disease accounts for 25% of all children intracranial tumors, the prevalence rate of the disease in children under 15 years is 0.5/10 ten thousand, and the diseased age has the characteristics of double peaks of 3-4 years and 8-9 years. Medulloblastoma has high malignancy, strong invasiveness and transferability, is easy to spread through cerebrospinal fluid and affect lateral ventricles, also commonly affects spinal cords, especially cauda equina, and rarely affects lungs, bones and the like due to distant metastasis caused by blood spreading. In the 1970 s, the 5-year survival rate of medulloblastoma was only 30%, and with the continuous improvement of comprehensive diagnosis and treatment means such as microscopic operative resection, pathological typing, radiotherapy and chemotherapy, the average long-term survival rate of medulloblastoma can reach about 70% at present. Nevertheless, because the intellectual impairment of children patients caused by high-dose radiotherapy and the like seriously affects the quality of life of children patients and their families, targeted therapy and individualized therapy are the most interesting directions in the research field of medulloblastoma at present.
WHO (2007) central nervous system tumor guidelines classify medulloblastoma into 5 pathological subtypes: typical, fibroproliferative, with massive nodular, anaplastic and large cell types. However, histological subtypes and clinical manifestations are difficult to completely correspond, and more importantly, simple histopathology is difficult to predict clinical prognosis well. In a relatively long period, the medulloblastoma of children is divided into a critical type and a high-risk type internationally according to the age, metastasis, surgical resection degree and tissue morphology change degree of patients. Critical type means age>3 years old, no metastasis evidence, residual cancer focus after operation<1.5cm2. Those who do not meet one of the above conditions or who become metamorphic are of the high risk type. Age (age)<Age 3 also belongs to a high risk, but should be listed separately, regardless of medulloblastoma stage and surgical situation. With the development of molecular biology technology, the geneomics mutation spectrum of medulloblastoma is gradually analyzed, and the WHO divides medulloblastoma into 4 independent molecular subtypes according to different expression profiles: WNT type (Wnt subgroup), SHH type (Sonic Hedgehog subgroup), 3 type (group 3), 4 type (group 4), each subtype shows distinct characteristics in cytogenetics, epidemiology and clinical prognosis, and has a complex association with histology type, and is a currently internationally recognized molecular typing.
The SHH subtype medulloblastoma is a major subtype of medulloblastoma, accounting for about 30% of all molecular subtypes of medulloblastoma, and the SHH subtype medulloblastoma is formed due to a lot of reasons, any factor causing abnormal and continuous activation of SHH signaling pathway can cause the generation of SHH subtype medulloblastoma, and the research and development of finding new and specific medulloblastoma-related genes and taking the genes as targets are the key points of development and development of medulloblastoma treatment. In view of this, the present application finds, on the basis of preliminary experimental studies, that the specific knockout of the Rack1 gene in the mouse cerebellum development process can result in the significant inhibition of the proliferation of cerebellum granule neurons, and the inhibition of the SHH signal pathway, significantly inhibits the proliferation of tumor cells, and prolongs the survival time of tumor mice. Therefore, the discovery of the Rack1 gene provides a new action target and treatment for clinically treating the immunotherapy of the SHH subtype medulloblastoma
The Rack1 gene can be used as a target point to prepare an anti-SHH subtype medulloblastoma drug.
Disclosure of Invention
In view of the above problems in the prior art, the present invention aims to provide a novel therapeutic target for medulloblastoma, namely, a Rack1 gene, and an application of Rack1 gene as a target in preparing an anti-medulloblastoma drug. Aiming at the current situation that a plurality of treatment targets of medulloblastoma have drug resistance, the invention provides a new treatment target, and lays a foundation for the development of a subsequent anti-medulloblastoma drug and the research on the molecular mechanism of medulloblastoma.
The above object of the present invention is achieved by the following technical solutions:
the invention provides an application of a Rack1 gene in preparing an anti-medulloblastoma drug.
Preferably, the medulloblastoma is an SHH subtype medulloblastoma.
Further, the medicament is a medicament for inhibiting the growth of tumor cells and/or inhibiting the metastasis of tumor cells.
Further, the medicament comprises an agent for inhibiting the expression of the Rack1 gene.
Further, the reagent comprises a Rack1 antibody, a Rack1 ligand antibody, modified Rack1, a partial peptide of Rack1, siRNAs targeting a Rack1 gene sequence, shRNAs, antisense molecules, DNase, an expression vector containing the siRNAs, the shRNAs and the antisense molecules;
preferably, the Rack1 ligand comprises PKC (protein kinase C).
In a second aspect, the present invention provides a pharmaceutical composition against medulloblastoma.
Further, the pharmaceutical composition comprises an agent that inhibits the expression of the Rack1 gene.
Further, the reagent comprises a Rack1 antibody, a Rack1 ligand antibody, modified Rack1, a partial peptide of Rack1, siRNAs targeting a Rack1 gene sequence, shRNAs, antisense molecules, DNase, an expression vector containing the siRNAs, the shRNAs and the antisense molecules;
preferably, the Rack1 ligand comprises PKC (protein kinase C).
Further, the pharmaceutical composition also comprises a pharmaceutically acceptable carrier and/or an auxiliary material.
Further, the carrier and/or adjuvant includes pharmaceutically acceptable carriers, diluents, fillers, binders and other excipients, depending on the mode of administration and the designed dosage form. Therapeutically inert inorganic or organic carriers known to those skilled in the art include, but are not limited to, lactose, corn starch or derivatives thereof, talc, vegetable oils, waxes, fats, polyols (e.g., polyethylene glycol, water, sucrose, ethanol, glycerol), and the like, various preservatives, lubricants, dispersants, flavoring agents, wetting agents, sweeteners, fragrances, emulsifiers, suspending agents, preservatives, antioxidants, colorants, stabilizers, salts, buffers, and the like, to which suitable pharmaceutically acceptable carriers and formulations are also added, as well as those described in detail in Remington's Pharmaceutical Sciences (19th ed.,1995) for aiding the stability of the formulation or for improving the activity or its bioavailability or for producing an acceptable mouthfeel or odor in the case of oral administration, and those that can be used in such Pharmaceutical compositions can be in the form of their original compounds per se, or optionally in the form of a pharmaceutically acceptable salt thereof. The pharmaceutical composition thus formulated may be administered by any suitable means known to those skilled in the art, as desired, by administering a safe and effective amount of the drug of the present invention to a human.
The appropriate dose of the pharmaceutical composition of the present invention can be prescribed in various ways depending on factors such as the method of preparation, the mode of administration, the age, body weight, sex, disease state, diet, time of administration, route of administration, excretion rate and reaction sensitivity of the patient, and usually, a skilled physician can easily determine the prescription and the dose of administration effective for the desired treatment or prevention.
Further, the pharmaceutical composition also comprises an anti-tumor drug.
Further, the antitumor drugs comprise cisplatin, cyclophosphamide, lomustine, vincristine, procarbazine, hydroxyurea, doxorubicin, paclitaxel, imatinib, and adriamycin.
Further, the pharmaceutical composition can be prepared into various clinical pharmaceutical dosage forms as antitumor drugs according to needs, including but not limited to oral preparations or parenteral dosage forms, wherein the oral preparations include tablets, capsules, pills, granules, microcapsule tablets, suspensions, dripping pills and oral liquid preparations; the parenteral administration dosage form comprises an injection, an aerosol, a suppository or a subcutaneous administration dosage form.
Further, the route of administration of the pharmaceutical composition is not limited as long as it can exert the desired therapeutic or prophylactic effect, and includes, but is not limited to, intravenous, intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, intravesicular, intramuscular, intratracheal, subcutaneous, topical, inhalation, through the skin, through the pleura, through the mucosa, skin, gastrointestinal, intraarticular, intraventricular, rectal, vaginal, intracranial, intraurethral, intrahepatic. In some cases, the administration may be systemic. In some cases topical administration.
Further, the dosage of the pharmaceutical composition is not limited as long as the desired therapeutic effect or prophylactic effect is obtained, and may be appropriately determined depending on the symptoms, sex, age, and the like. The dose of the therapeutic or prophylactic pharmaceutical composition of the present invention can be determined using, for example, the therapeutic effect or prophylactic effect on a disease as an index.
In a third aspect, the invention provides a method of inhibiting proliferation of a medulloblastoma tumor cell.
Further, the method comprises reducing the expression level of the Rack1 gene.
Preferably, the method for reducing the expression level of the Rack1 gene comprises knocking out or knocking down the Rack1 gene.
The fourth aspect of the invention provides an application of the Rack1 gene in screening candidate drugs for treating medulloblastoma.
Further, the use includes a method of screening for a candidate agent for treating or preventing medulloblastoma or inhibiting the growth of medulloblastoma tumor cells.
In a fifth aspect, the invention provides a screening method for a candidate drug for treating medulloblastoma.
Further, the screening method comprises the following steps:
(1) treating a system expressing or containing a Rack1 gene with a test substance;
(2) detecting the expression of Rack1 gene in the system;
(3) selecting a test substance capable of inhibiting the expression level of the Rack1 gene.
Further, the system is selected from: a cell system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system.
Further, the test substances include, but are not limited to: interfering molecules, nucleic acid inhibitors, small molecule compounds and the like designed aiming at the Rack1 gene or the upstream or downstream gene thereof.
Further, the selected test agent in step (3) is a test agent that reduces the expression level of Rack1 as compared to the expression level detected in the absence of the test agent.
Further, the method for detecting the expression level of Rack1 includes but is not limited to: real-time fluorescent quantitative PCR, in-situ hybridization, gene chip, protein immunoblotting (Western blot), enzyme-linked immunosorbent assay (ELISA), and colloidal gold detection.
Furthermore, the real-time fluorescent quantitative PCR, in-situ hybridization and gene chip is used for detecting the expression level of the Rack1 gene on the gene level; the protein immunoblotting (Western blot), enzyme-linked immunosorbent assay (ELISA) and colloidal gold detection methods are used for detecting the expression level of the Rack1 gene on the protein level.
(1) Real-time fluorescent quantitative PCR (Real-time PCR, RT-PCR)
The real-time fluorescent quantitative PCR technology is characterized in that a fluorescent group is added into a PCR reaction system, the whole PCR process is detected in real time by utilizing fluorescent signal accumulation, and finally, an unknown template is qualitatively and quantitatively analyzed through a standard curve, and the technology has the following characteristics: the indicator which generates the fluorescence signal is used for displaying the amount of the amplified product, real-time dynamic continuous fluorescence monitoring is carried out, inaccurate terminal point quantification is avoided, the pollution of a sample and the product is eliminated, and no complex subsequent product processing process exists; the fluorescent signal is obtained by embedding fluorescent dye into double-stranded DNA or specifically combining fluorescent probe with target detection object, so that the sensitivity, specificity and accuracy of detection are greatly improved. The qRT-PCR has the advantages of high specificity, good sensitivity, rapidness and simplicity.
(2) In situ hybridization (In situ hybridization)
In situ hybridization chemistry is a mode of nucleic acid hybridization, and refers to a process of hybridizing a specific labeled nucleic acid with a known sequence as a probe with a nucleic acid in a cell or tissue section, so as to precisely and quantitatively locate a specific nucleic acid sequence, and in situ hybridization (LNA) based on Locked nucleic acid in situ hybridization (LNA-ISH)) is a currently applied probe mode. Has the advantages of safety, rapidness, high sensitivity, good specificity, long-term preservation of the probe and simultaneous display of various colors.
(3) Gene chip (Genechip)
A gene chip or DNA microarray (DNA Array) is prepared through in-situ photoetching or micro-spotting to fix a great number of DNA molecules on the surface of supporter (such as glass slide, silicon chip, polyacrylamide gel, nylon membrane, etc) to form dense two-dimensional molecular Array, hybridizing with the target molecules in biological sample, and quickly, parallelly and efficiently detecting and analyzing the intensity of hybridized signal by a special instrument such as laser confocal scan or charge Coupled Camera (CCD) to judge the number of target molecules in sample. Gene chip technology has become an effective basic and clinical medicine research method, which detects tens of thousands of gene expression levels or millions of DNA genetic markers such as Single Nucleotide Polymorphism (SNP), Copy Number Variation (CNV) and the like on a solid support at one time by the principle of nucleic acid hybridization, and has the incomparable high-throughput characteristic of the traditional method. Therefore, the gene chip technology provides a powerful technical platform for scientific research and clinical workers to understand a life complex system, and has the advantages of rapidness, high efficiency and automation.
(4) Protein immunoblotting (Western blot)
The western blotting method is a protein analysis method in which proteins are separated on polyacrylamide gel by protein gel electrophoresis (SDS-PAGE), then transferred from the gel to an NC membrane or a PVDF membrane, and then subjected to blotting detection by using an antibody, is a commonly used method for detecting protein characteristics, expression and distribution, and has the advantages of large analysis capacity, high sensitivity and strong specificity.
(5) Enzyme-linked immunosorbent assay (ELISA)
Enzyme-linked immunosorbent assay is the most widely used technique in enzyme immunoassay, and the principle of enzyme-linked immunosorbent assay is to combine antigen or antibody with substrate (enzyme) to keep the immunoreaction and enzyme activity. The marked antigen or antibody is combined with the ligand coated on the solid phase carrier, and then the ligand is reacted with the corresponding colorless substrate to display color, and the OD value is determined by visual inspection according to the color development depth or an enzyme-labeling instrument. The method mainly comprises a sandwich method, an indirect method, a competition method and a capture method, and has the advantages of simple and rapid operation, high sensitivity, strong specificity and wide application range.
(6) Colloidal gold detection method
The principle of the colloidal gold detection method is that a specific antibody is fixed on a membrane in a strip shape (T line), a colloidal gold labeled reagent is adsorbed on a binding body (gold pad), when an antigen to be detected is added on a sample pad at one end of a test strip, a sample moves forwards through capillary action, the colloidal gold labeled reagent on the binding pad is dissolved and then reacts with each other, and when the sample moves to a region of the fixed antigen or antibody, the binding body of the object to be detected and the gold labeled reagent is specifically bound with the binding body and is intercepted, and the binding body is gathered on a detection strip, so that a color development result can be observed through naked eyes. Has the advantages of rapidness, simple and convenient operation, low cost and good stability.
The invention has the advantages and beneficial effects that:
the invention firstly applies the Rack1 gene to the preparation of the medulloblastoma-resistant medicament, the application of the gene provides a new treatment target for medulloblastoma, lays a foundation for the development of the subsequent medulloblastoma-resistant medicament and the research of the molecular mechanism of medulloblastoma, and has potential application value in the immunotherapy of medulloblastoma.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 is a graph showing the results of histology and appearance morphology of the brains of P30 mice of four genotypes;
FIG. 2 is a graph showing the results of HE staining and immunofluorescence staining (NeuN staining) of the mouse cerebellum of P30 mice of four genotypes;
FIG. 3 shows a statistical plot of the sagittal sectional areas of the cerebellum of four genotype mice;
FIG. 4 shows survival plots for four genotype mice.
Detailed Description
The present invention will be described in further detail with reference to the following detailed description and accompanying drawings. In the following embodiments, numerous details are set forth in order to provide a better understanding of the present invention, and are set forth in order to illustrate, but not to limit the scope of the present invention. However, those skilled in the art will readily recognize that some of the features may be omitted or replaced with other elements, materials, methods in different instances. The experimental methods not specified in the examples are generally commercially available according to the conditions described in the conventional conditions or according to the conditions recommended by the manufacturers, and the materials, reagents and the like used in the examples, unless otherwise specified.
Example 1 establishment of SHH subtype medulloblastoma animal model
1. Experimental Material
Atoh1-cre mice were purchased from "The Jackson Laboratory" (No. 011104); SmoM2 mice were purchased from "The Jackson Laboratory" (No. 005130); rack1loxP/loxPThe mouse is provided by a military cognition and brain science research institute Zhang Ji teacher of military medical research institute, and the second exon 172bp of the mouse Rack1 gene is marked by loxP; NeuN is available from Milipore under the trade designation MAB 377; DAPI (H-1200; VECTASHIELD); goat anti-mouse IgG (H + L) Alexa Flour 568 was purchased from Biotium; 4% paraformaldehyde is available from Solebao.
2. Experimental methods
Atoh1-cre genotype mice were compared with SmoM2+/+The mice are hybridized, and an activated SmoM2 receptor is specifically expressed in cerebellar granule neurons of At oh1-Cre mice by using a Cre/loxp strategy, so that the excessive activation of an SHH signal channel is induced, and a typical cerebellum SHH subtype medulloblastoma animal model, namely Ato h1-Cre, is generated; SmoM2+/-。
3. Results of the experiment
The experimental result shows that the constructed Atoh 1-cre; SmoM2+/-Tumor mice developed distinct SHH subtype medulloblastoma (see fig. 1 and 2), Atoh 1-cre; SmoM2+/-The sagittal section area of the cerebellum of the tumor mouse is about 38mm2(see FIG. 3), and in addition, the results showed that Atoh1-cre was constructed; SmoM2+/-The tumor mouse model died around 40 days (see fig. 4), and the experimental result shows that the SHH subtype medulloblastoma animal model is successfully constructed, namely Atoh 1-cre; SmoM2+/-Tumor mouse model。
Example 2 establishment of a mouse model of tumor treated by a Gene knockout of Rack1 Gene
The experimental method comprises the following steps:
(1) establishing a Rack1 knockout tumor mouse model: firstly, utilizing Atoh 1-cre; SmoM2+/-And Atoh 1-cre; rack1F/FThe first filial generation of the mice gives heterozygote mice: atoh 1-cre; rack1F/WHeterozygote mice were then placed with SmoM2+/-；Rack1F/FHybridizing the mice to obtain the genotype Atoh 1-cre; SmoM2+/-；RackF/FThe gene of (3) treating a mouse.
(2) Four genotypes of P30 were taken: SmoM2+/-；Rack1F/F，Atoh1-cre；Rack1F/F，Atoh1-cre；SmoM2+/-，Atoh1-cre；SmoM2+/-；RackF/F. Four genotype mice were anesthetized with 1% pentobarbital, perfused with physiological saline and 4% paraformaldehyde, respectively, stripped of the whole brain, soaked in 4% paraformaldehyde, and fixed.
(3) After fixation in 4% paraformaldehyde for 24h, dehydration was carried out in 15% and 30% sucrose solution (in PBS) for 24h and 48h, respectively. Frozen sections were stored at-20 ℃.
(4) Selecting brain slices of the lumbricus cerebelli parts of four genotype mice close to the central position, firstly washing for 10min at room temperature by PBS, and repeating the washing step for three times. Then, the wells were punched with 0.3% PBST (Triton X-100) for 20 min. Blocking was then performed with 3% BSA (PBST) for 1h, and primary antibody (NeuN, 1: 400) was incubated at room temperature overnight at 4 ℃. The next day, washing with PBS for 10min, and this washing step was repeated three times. After incubation with fluorescent secondary antibody at room temperature for 1h, the washing was performed in PBS for 10min and the washing procedure was repeated three times. The slides were mounted with DAPI and photographed by confocal laser microscopy.
The experimental results are as follows: the results showed that Atoh 1-cre; SmoM2+/-；Rack1F/FMice showed dysmetallogenesis of SHH subtype medulloblastoma (see fig. 1 and fig. 2), Atoh 1-cre; SmoM2+/-；Rack1F/FThe mouse cerebellum sagittal section area is only 12mm2Left and right (see FIG. 3), in addition, the results show that Atoh1 was constructed-cre；SmoM2+/-(ii) a The survival time of a Rack1F/F mouse model can be prolonged to 89 days (see figure 4), and the successful construction of a Rack1 gene knockout gene therapy tumor mouse model, namely Atoh1-cre is proved; SmoM2+/-；Rack1F/FMouse model. The result shows that after the Rack1 gene is knocked out, the proliferation of tumor cells of a SHH subtype medulloblastoma mouse model can be obviously inhibited, the volume of tumor tissues is obviously reduced, and the survival time of the mouse is also obviously prolonged.
The above-described embodiments are only for illustrating the present invention and are not to be construed as limiting the present invention. As will be understood by those of ordinary skill in the art: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims (2)

1. Application of an agent for inhibiting the expression of the Rack1 gene in preparing an anti-SHH subtype medulloblastoma drug.
2. The use of claim 1, wherein the medicament is a medicament for inhibiting the growth of SHH subtype medulloblastoma cells.
CN202010951238.8A 2020-09-11 2020-09-11 Application of Rack1 as target in preparation of anti-SHH (single-stranded lymphoblastoma) medicament Active CN112011618B (en)

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