Construction method and application of breast cancer model

Abstract

The invention provides a construction method and application of a breast cancer model.The spontaneous breast cancer model is constructed by means of FBXO22 specificity deficiency of the mammary gland of a mouse, and the breast cancer model can spontaneously form tumors, is high in tumor forming speed, large in tumor forming size and high in tumor forming weight, can be used for breast cancer drug screening and drug effect evaluation or diagnosis and has good application prospects. The FBXO22 gene can also be used for researching and revealing the pathogenesis of breast cancer caused by dysfunction of the FBXO22 gene. The invention has important significance for clinical research of breast cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular, relates to a method for constructing a breast cancer model and its application. Background Art

[0002] According to the latest global cancer burden data for 2020 released by the World Health Organization's International Agency for Research on Cancer (IARC), 60% of new cancer cases in 2020 came from the 10 most common tumors, of which female breast cancer accounted for 11.7%, surpassing lung cancer (11.4%) for the first time in terms of number, becoming the most diagnosed cancer in the world. For the first time, female breast cancer has become the most commonly diagnosed cancer, the number one killer threatening women's health, and has become one of the most pressing health issues today. With the continuous development of modern medicine, although there have been advances in cancer diagnosis, treatment and life expectancy, the improvement in mortality has not been significant.

[0003] The research on the occurrence, development and treatment of tumors needs to be conducted on the basis of corresponding animal models. The currently commonly used animal models are not ideal in terms of tumor formation mode, tumor formation speed, tumor load, etc. Therefore, more suitable animal models of breast cancer are still needed. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art. The present invention provides a method for constructing a breast cancer model and its application. The present invention constructs a spontaneous breast cancer model by specifically deleting FBXO22 in the mammary gland of mice. The breast cancer model can spontaneously form tumors, with a fast tumor formation speed, a large tumor volume, and a high tumor weight. It can be used for breast cancer drug screening, drug efficacy evaluation or diagnosis, and can also be used to study and reveal the pathogenesis of breast cancer caused by abnormal FBXO22 gene function. The present invention is of great significance for clinical research on breast cancer.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] A first aspect of the present invention provides a method for constructing a non-human animal model of breast cancer, wherein the non-human animal model of breast cancer lacks FBXO22 expression.

[0007] In the present invention, expression refers to the process of transcribing polynucleic acid into mRNA and translating into peptides, polypeptides or proteins. If the polynucleic acid is derived from genomic DNA and a suitable eukaryotic host cell or organism is selected, expression may include splicing of mRNA.

[0008] In the present invention, the lack of FBXO22 expression may refer to a reduction or loss of the expression level of FBXO22, or a reduction or loss of the activity function of FBXO22. The effect of lack of FBXO22 expression may be achieved by knocking out and / or knocking down FBXO22, or by administering an FBXO22 inhibitor. Regardless of the means used, as long as the effect of lack of FBXO22 expression is achieved, it falls within the scope of protection of the present invention. In one embodiment of the present invention, the lack of FBXO22 expression is the knocking out and / or knocking down of FBXO22 in non-human animals.

[0009] Knockout and / or knockdown refers to a gene sequence change that results in a loss of function and / or reduction of FBXO22, preferably such that FBXO22 expression is undetectable or insignificant. Knockout and / or knockdown may comprise a heterozygous knockout and / or knockdown or a homozygous knockout and / or knockdown of FBXO22. In the present invention, knockout and / or knockdown also includes conditional knockout and / or knockdown, wherein the change of FBXO22 may occur, for example, when the animal is exposed to a substrate that promotes the change of FBXO22, when a site recombinase (e.g., Cre in the Cre-LoxP system) is introduced to promote the change of FBXO22 at the site, or other methods for directing the change of FBXO22 after birth. Homozygous refers to a genetic condition that exists when identical alleles reside at corresponding loci on homologous chromosomes, and heterozygous refers to a genetic state that exists when different alleles reside at corresponding loci on homologous chromosomes.

[0010] Recombinase refers to an enzyme that performs site-specific recombination (SSR) to change the structure of DNA. This definition includes transposases, lambda (λ) integrase / excision enzymes, and site-specific recombinases. Recombinases come from, but are not limited to, Cre-lox, FLP / FRT, R / RS, Gin / gix, pSR1 system, cer system, and fim system. Recombinases can recognize wild-type or mutant recombinase sites, thereby promoting the occurrence of recombination events.

[0011] Furthermore, the knockout and / or knockdown can also be achieved by methods including but not limited to Cre-LoxP, FLP / FRT, R / RS, Gin / gix, Cin H / RS2, ParA / MRS, phiC31, CRISPR-Cas9, zinc finger nuclease technology, transcription activator-like effector nuclease technology, and interfering RNA methods.

[0012] In some embodiments of the present invention, the knockout and / or knockdown technique is selected from Cre-LoxP, interfering RNA. Further, the interfering RNA includes shRNA, siRNA, sgRNA. Further, the interfering RNA is selected from siRNA. In a specific embodiment of the present invention, the sequence of the siRNA is shown in SEQ ID NO: 1-4.

[0013] In the present invention, in order to achieve knockout and / or knockdown of FBXO22 using Cre-LoxP technology, it is necessary to insert LoxP sites with the same direction on both sides of the target gene (or some important fragments of the gene). Such transgenic animals carrying LoxP sites but in which the target gene can be expressed normally are called flox transgenic animals, and this process is called flox modification. To achieve knockout and / or knockdown, Cre recombinase is also required. Usually, a tool transgenic animal expressing Cre recombinase is prepared, and a specific Cre tool transgenic animal is mated with a flox transgenic animal to obtain a double-positive transgenic animal carrying both LoxP sites and Cre recombinase. In one embodiment of the present invention, the method for knockout and / or knockdown includes knocking out exon 1 and exon 2 of FBXO22 using Cre-LoxP technology.

[0014] Further, the knockout and / or knockdown method comprises the following steps:

[0015] (1) Flox modification was performed at both ends of the FBXO22 gene to construct Fbxo22 fl / fl Genotype animals.

[0016] (2) Fbxo22 fl / fl Genotyped animals were crossed with MMTV-Cre transgenic animals to obtain MMTV-Cre-Fbxo22 fl / fl Genotype animals.

[0017] (3) MMTV-Cre-Fbxo22 fl / fl The genotyped animals were crossed with the genotyped animals of MMTV-Cre-PyMT to obtain the F1 generation - MMTV-Cre-Fbxo22 + / fl -PyMT genotyped animals.

[0018] (4) Self-pollination of the F1 generation to produce MMTV-Cre-Fbxo22 fl / fl -PyMT genotyped animals.

[0019] Furthermore, overexpression of mammary gland-specific polyomavirus middle T antigen in animals with the MMTV-Cre-PyMT genotype can drive the expression of polyomavirus middle T antigen (PyMT) in mammary tissue, resulting in a mammary tumor phenotype.

[0020] Furthermore, the non-human animal model of breast cancer is a spontaneous breast cancer model.

[0021] Further, the knockout and / or knockdown is tissue specific. In certain embodiments, the non-human animal model of breast cancer lacks FBXO22 expression, ie, lacks FBXO22 specifically in the breast.

[0022] Furthermore, the tumor formation time of the non-human animal model of breast cancer is faster.

[0023] Furthermore, the non-human animal model of breast cancer can form obvious tumors at 12 weeks of age.

[0024] Furthermore, knocking out and / or knocking down FBXO22 in the present invention can accelerate the progression of "breast hyperplasia-breast intraepithelial neoplasia-breast cancer".

[0025] In the present invention, a non-human animal model refers to a non-human animal that has or displays characteristics of a disease or condition. Use as an animal model refers to any use of an animal for studying a disease or condition, such as for studying progression or development or response to new or existing therapies.

[0026] In the present invention, non-human animals include non-human vertebrates, more preferably mammals, which refer to all members of the class Mammalia, such as domesticated livestock (e.g., cattle, horses, pigs), pets (e.g., dogs, cats), or rodents. The term "rodent" refers to any and all members of the phylogenetic class Rodentia (e.g., mice, rats, squirrels, beavers, woodchucks, hamsters, guinea pigs, and agouti), including any descendants of all descendants derived therefrom. In a specific embodiment of the present invention, the mammal is selected from mice.

[0027] A second aspect of the present invention provides any of the following methods:

[0028] (1) A method for screening a drug candidate for treating breast cancer, the method comprising:

[0029] a) administering the drug candidate to a non-human animal with breast cancer prepared by the construction method described in the first aspect of the present invention;

[0030] b) detecting the therapeutic effect of the breast cancer on the drug candidate;

[0031] (2) A method for identifying an anticancer agent for treating drug-resistant breast cancer, the method comprising:

[0032] a) administering a pharmaceutical agent to a non-human animal with breast cancer prepared by the construction method described in the first aspect of the present invention;

[0033] b) performing one or more assays to determine whether the agent has a therapeutic effect on one or more abnormal symptoms associated with breast cancer;

[0034] c) identifying the agent as an anticancer agent for treating drug-resistant breast cancer when the agent has a therapeutic effect on one or more abnormal symptoms associated with breast cancer;

[0035] (3) A method for evaluating the therapeutic effect of a drug for treating breast cancer, the method comprising:

[0036] a) administering the drug to a non-human animal with breast cancer prepared by the construction method described in the first aspect of the present invention;

[0037] b) detecting the therapeutic effect of the drug on the breast cancer;

[0038] (4) A method for studying the pathogenesis of breast cancer, wherein the method comprises using a non-human animal with breast cancer prepared by the construction method described in the first aspect of the present invention to study the pathogenesis of breast cancer.

[0039] In the present invention, the term "treatment" refers to the administration of a compound or composition to control the progression of a disease. The control of disease progression is understood to be the achievement of beneficial or desired clinical results, including but not limited to alleviation of symptoms, reduction of disease duration, stabilization of pathological conditions (particularly avoiding additional deterioration), delay of disease progression, improvement and alleviation (partial and complete) of pathological conditions. The control of disease progression also involves an extension of survival compared to the expected survival without treatment. Further, the therapeutic effect includes but is not limited to reducing the number of tumor cells, reducing tumor size or tumor burden, inhibiting tumor cell infiltration into surrounding organs, inhibiting tumor metastasis or inhibiting tumor growth.

[0040] In the present invention, the method of screening drug candidates for treating breast cancer may involve administering different amounts of drug candidates (from no drug to an amount of drug close to the upper limit of the amount that can be successfully delivered to the animal, such as within the toxic limit), and may include delivering the drug in different formulations and routes. A single drug may be administered, or the drugs may be combined in a combination of two or more drugs, especially where the administration of a drug combination may result in a synergistic effect. The ability of a candidate agent to treat an existing cancer may be evaluated by administering the candidate drug to a transgenic animal and evaluating the regulation of the cancer phenotype. The regulation of the cancer phenotype may be evaluated, for example, by evaluating the presence or absence of an effect on, for example, tumor load, tumor number, tumor size, metabolic activity of tumor cells, progression-free survival (PFS), overall survival (OFS), etc. The candidate drug may be administered to the non-human animal model of breast cancer described in the first aspect of the present invention, followed by evaluating the ability of the candidate drug to promote cancer prevention. Cancer prevention refers to reducing the incidence and / or severity of tumors in an animal relative to the expected incidence and / or severity of cancer in the absence of intervention (e.g., without administering a drug with anticancer activity).

[0041] Drug candidates can be obtained from a wide variety of sources, including but not limited to synthetic, naturally occurring or recombinantly produced molecules, including small molecules, peptides, antibodies or other polypeptides. For example, random and directed synthesis of a variety of organic compounds and biomolecules, or natural compound libraries in the form of bacterial, fungal, plant or animal extracts, or natural or synthetic libraries and compounds modified by conventional chemical, physical or biochemical means, or directed or random chemical modification of known pharmacological agents, such as acylation, alkylation, esterification, amidation, etc. to generate structural analogs.

[0042] The drug of the present invention is preferably administered in a pharmaceutically acceptable vehicle. Suitable pharmaceutical carriers are known to those skilled in the art. For parenteral administration, the compound is usually dissolved or suspended in sterile water or saline. For enteral administration, the compound is incorporated into an inert carrier in the form of tablets, liquids or capsules. Suitable carriers can be starch or sugar, and include lubricants, flavoring agents, adhesives and other materials of the same nature. The compound can also be applied topically by topical application of solutions, creams, gels or polymeric materials (e.g., PluronicTM, BASF). Alternatively, the compound can be administered in liposomes or microspheres (or microparticles). Methods for preparing liposomes and microspheres for administration to patients are known to those skilled in the art. In essence, the material is dissolved in an aqueous solution, appropriate phospholipids and lipids are added together with surfactants if necessary, and the material is dialyzed or sonicated as needed. Microspheres formed by polymers or proteins are well known to those skilled in the art and can be customized to enter the bloodstream directly through the gastrointestinal tract. Alternatively, the compound can be incorporated and the microspheres or microsphere complexes can be implanted to slowly release over a period of days to months.

[0043] Furthermore, the anticancer agents include targeted small molecules, targeted macromolecules, proteasome inhibitors, immunosuppressants, tumor necrosis inhibitors, cytokines, activators of costimulatory molecules, and inhibitors of inhibitory molecules.

[0044] The drug of the present invention can also be used in combination with other drugs for treating breast cancer, and other therapeutic compounds can be administered simultaneously with the main active ingredient, or even administered simultaneously in the same composition. Other therapeutic compounds can also be administered separately in a separate composition or in a dosage form different from the main active ingredient. Partial doses of the main ingredient can be administered simultaneously with other therapeutic compounds, while other doses can be administered separately. During the course of treatment, the dosage of the pharmaceutical composition of the present invention can be adjusted according to the severity of the symptoms, the frequency of recurrence, and the physiological response of the treatment regimen.

[0045] Furthermore, the drugs include targeted small molecules, targeted macromolecules, proteasome inhibitors, immunosuppressants, tumor necrosis inhibitors, cytokines, activators of co-stimulatory molecules, and inhibitors of inhibitory molecules.

[0046] The third aspect of the present invention provides any of the following applications:

[0047] (1) Use of the non-human animal model of breast cancer prepared by the construction method described in the first aspect of the present invention in screening drug candidates for treating breast cancer;

[0048] (2) Use of the non-human animal model of breast cancer prepared by the construction method described in the first aspect of the present invention in identifying anticancer agents for treating drug-resistant breast cancer;

[0049] (3) Use of the non-human animal model of breast cancer prepared by the construction method described in the first aspect of the present invention in evaluating the therapeutic effect of drugs for treating breast cancer;

[0050] (4) Application of the non-human animal model of breast cancer prepared by the construction method described in the first aspect of the present invention in studying the pathogenesis of breast cancer.

[0051] Furthermore, the anticancer agents include targeted small molecules, targeted macromolecules, proteasome inhibitors, immunosuppressants, tumor necrosis inhibitors, cytokines, activators of costimulatory molecules, and inhibitors of inhibitory molecules.

[0052] Furthermore, the drugs include targeted small molecules, targeted macromolecules, proteasome inhibitors, immunosuppressants, tumor necrosis inhibitors, cytokines, activators of co-stimulatory molecules, and inhibitors of inhibitory molecules.

[0053] A fourth aspect of the present invention provides the use of FBXO22 in constructing a non-human animal model of breast cancer.

[0054] Furthermore, the non-human animal model refers to a non-human animal that has or exhibits characteristics of a disease or condition.

[0055] In the present invention, non-human animals include non-human vertebrates, more preferably mammals, which refer to all members of the class Mammalia, such as domesticated livestock (e.g., cattle, horses, pigs), pets (e.g., dogs, cats), or rodents. The term "rodent" refers to any and all members of the phylogenetic class Rodentia (e.g., mice, rats, squirrels, beavers, woodchucks, hamsters, guinea pigs, and agouti), including any descendants of all descendants derived therefrom. In a specific embodiment of the present invention, the mammal is selected from mice.

[0056] A fifth aspect of the present invention provides a method for regulating the expression level of PITRM1, the method comprising administering an FBXO22 regulator to regulate the expression level of FBXO22.

[0057] In the present invention, the term "regulate" includes regulation and control, and the regulation includes increasing the expression level of PITRM1 or decreasing the expression level of PITRM1.

[0058] Furthermore, the FBXO22 and the PITRM1 are negatively correlated and regulated, and a decrease in the expression of FBXO22 will lead to an increase in the expression of PITRM1, and an increase in the expression of FBXO22 will lead to a decrease in the expression of PITRM1.

[0059] Furthermore, the FBXO22 regulator includes a FBXO22 inhibitor and a FBXO22 promoter.

[0060] Furthermore, the expression level of FBXO22 can be reduced by FBXO22 inhibitors, or by knockout / knockdown. Regardless of the method, as long as the expression level of FBXO22 can be reduced, it falls within the protection scope of the present invention.

[0061] Furthermore, the FBXO22 inhibitors include nucleic acid inhibitors, protein inhibitors, and compounds.

[0062] Furthermore, the nucleic acid inhibitors include shRNA, siRNA, sgRNA, and antisense nucleic acids.

[0063] Furthermore, the nucleic acid inhibitor is selected from siRNA.

[0064] Furthermore, the sequence of the siRNA is shown in SEQ ID NO: 1-4.

[0065] Furthermore, the expression level of FBXO22 can be increased by using a FBXO22 promoter or by knock-in. Regardless of the method, as long as the expression level of FBXO22 can be increased, it falls within the protection scope of the present invention.

[0066] In the present invention, knock-in refers to the targeted insertion of a transgene into the host cell genome, resulting in expression of the transgene and / or altered expression of an endogenous target gene (e.g., elevated (including ectopic) or decreased expression), for example, by introducing an additional copy of the target gene or by operatively inserting a regulatory sequence that provides enhanced expression of the endogenous copy of the target gene. Knock-in transgenes may comprise heterozygous knock-ins of transgenes or homozygous knock-ins of transgenes. Knock-in also encompasses conditional knock-ins, in which expression of the transgene and / or altered expression of the endogenous target gene may occur, for example, by exposing the animal to a substance that promotes such expression, by introducing an enzyme that promotes recombination at the targeted insertion site, or by some other method for altering the targeted insertion site.

[0067] Further, the FBXO22 promoter includes nucleic acid molecules, carbohydrates, liposomes, small molecule chemicals, antibody drugs, polypeptides, proteins or interfering lentiviruses. In the present invention, the term nucleic acid refers to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). As equivalents, nucleic acids also include DNA or RNA analogs generated from nucleotide analogs and single-stranded (sense strand or antisense strand) and double-stranded polynucleotides when applicable. Liposomes refer to small vesicles composed of various types of lipids, phospholipids and / or surfactants that can be used to deliver drugs to mammals. Antibodies cover complete monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed by at least two complete antibodies, and antibody fragments, as long as they exhibit the desired antigen binding activity, they fall within the scope of protection of the present invention.

[0068] Furthermore, the FBXO22 promoter is selected from nucleic acid molecules and interfering lentiviruses.

[0069] In one embodiment of the present invention, the method for regulating the expression level of PITRM1 comprises knocking out and / or knocking down or overexpressing intracellular FBXO22.

[0070] Further, the cell includes cancer cells, and the cancer includes but is not limited to breast cancer, adrenocortical carcinoma, bladder urothelial carcinoma, cervical squamous cell carcinoma, endocervical adenocarcinoma, bile duct cancer, colon adenocarcinoma, lymphoid tumors, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe cell carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, leukemia, brain low-grade glioma, hepatocellular carcinoma, mesothelial cell carcinoma, ovarian cancer, pancreatic cancer, pheochromocytoma and paraganglioma, prostate cancer, rectal cancer, malignant sarcoma, melanoma, gastric cancer, testicular germ cell tumor, thyroid cancer, thymic carcinoma, endometrial carcinoma, uterine sarcoma, uveal melanoma, myeloma, lymphoma, lung cancer (lung adenocarcinoma, lung squamous cell carcinoma), sarcoma, anal cancer, melanoma, retinoblastoma. Further, the cancer is selected from breast cancer, and the cancer cell is a breast cancer cell.

[0071] In some embodiments, the breast cancer cells include but are not limited to 4T1, MDA-MB-231, MCF-7, HCC1395, HCC1937, MDA-MB-453, MDA-MB-468. In a specific embodiment of the present invention, the cells are MDA-MB-231 and MCF-7. In a specific embodiment of the present invention, after knocking out and / or knocking down FBXO22 of MDA-MB-231 and MCF-7 cells, the expression level of PITRM1 increases, and after overexpressing FBXO22 of MDA-MB-231 and MCF-7 cells, the expression level of PITRM1 decreases.

[0072] A sixth aspect of the present invention provides the use of FBXO22 in regulating the expression level of PITRM1.

[0073] A seventh aspect of the present invention provides the use of FBXO22 in the preparation of a drug for treating breast cancer.

[0074] Furthermore, the drug includes a FBXO22 promoter.

[0075] Furthermore, the FBXO22 promoter is a substance that can increase the expression level of the FBXO22 gene.

[0076] Furthermore, the FBXO22 promoter includes nucleic acid molecules, carbohydrates, liposomes, small molecule chemical drugs, antibody drugs, polypeptides, proteins or interfering lentiviruses.

[0077] Furthermore, the FBXO22 promoter is selected from nucleic acid molecules and interfering lentiviruses.

[0078] An eighth aspect of the present invention provides use of a reagent for detecting FBXO22 in diagnosing breast cancer.

[0079] Furthermore, the reagent is selected from the group consisting of primers that specifically amplify the FBXO22 gene, probes that specifically recognize the FBXO22 gene, or antibodies that specifically bind to FBXO22.

[0080] Furthermore, the reagent for detecting FBXO22 includes a chip, a kit or a test strip. The chip includes a solid phase carrier and an oligonucleotide probe fixed on the solid phase carrier, and the oligonucleotide probe includes an oligonucleotide probe for FBXO22 for detecting the expression level of FBXO22; the kit includes primers, probes or chips for detecting the expression level of FBXO22.

[0081] Furthermore, the kit also includes instructions or labels, positive controls, negative controls, buffers, adjuvants or solvents; the instructions or labels indicate that the kit is used to detect breast cancer.

[0082] Furthermore, the reagents include reagents for detecting the FBXO22 of the present invention by reverse transcription PCR, real-time quantitative PCR, in situ hybridization or gene chip.

[0083] Furthermore, the reagents for detecting the FBXO22 described in the present invention by reverse transcription PCR include at least a pair of primers for specifically amplifying the FBXO22; the reagents for detecting the FBXO22 described in the present invention by real-time quantitative PCR include at least a pair of primers for specifically amplifying the FBXO22; the reagents for detecting the FBXO22 described in the present invention by in situ hybridization include a probe that hybridizes with the nucleic acid sequence of the FBXO22; the reagents for detecting the FBXO22 described in the present invention by gene chip include a probe that hybridizes with the nucleic acid sequence of the FBXO22.

[0084] The present invention has the following advantages and beneficial effects:

[0085] The present invention provides a method for constructing a breast cancer model and its application. The present invention constructs a spontaneous breast cancer model by specifically deleting FBXO22 in the mammary gland of mice. The breast cancer model can spontaneously form tumors, has a fast tumor formation speed, a large tumor volume, and a high tumor weight. It can be used for breast cancer drug screening, drug efficacy evaluation or diagnosis, and can also be used to study and reveal the pathogenesis of breast cancer caused by abnormal FBXO22 gene function. The present invention has important significance for clinical research on breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 Figure 1 is a diagram of the construction of mammary gland-specific knockout FBXO22 mice, wherein Figure A is a diagram of the construction pattern of mammary gland-specific knockout FBXO22 mice; Figure B is a diagram of the PCR identification of mouse genotypes; Figure C is a diagram of the mammary gland tissue morphology of mice with different genotypes; Figure D is a statistical diagram of the body weight of mice with different genotypes; Figure E is a statistical diagram of the mammary gland weight of mice with different genotypes; Figure F is a statistical diagram of the mammary gland volume of mice with different genotypes; Figure G is a diagram of the Western blot result of FBXO22 after mammary gland-specific knockout; Figure H is a diagram of the H&E staining of mouse mammary gland tissue after mammary gland-specific knockout of FBXO22; Figure I is a statistical diagram of the Ki67 levels of mice with different genotypes.

[0087] Figure 2 Figure 1 is a diagram of the construction model of spontaneous breast cancer mice with mammary gland-specific deficiency of FBXO22, wherein Figure A is a diagram of the construction process of spontaneous breast cancer mice with mammary gland-specific deficiency of FBXO22; Figure B is a diagram of the mammary tumors formed in mice at 16 weeks of age; Figure C is a statistical graph of the weight of mammary tumors formed in mice at 16 weeks; Figure D is a statistical graph of the mammary gland volume formed in mice at 16 weeks; Figure E is a statistical graph of the proportions of different sizes of tumors formed in mice at 16 weeks; Figure F is an H&E staining image of mouse mammary tissue; and Figure G is a statistical graph of the Ki67 level in mouse breast cancer tissue. Figure 3These are the results of breast cancer progression caused by mammary gland-specific deletion of FBXO22, among which, Figure A is the tumor images of mice at 4, 9, 12 and 16 weeks of age; Figure B is the statistical results of mouse body weight, mammary gland weight and tumor volume after FBXO22 deletion; Figure C is the H&E staining and Sirius red staining of mouse fat pad / breast tumor tissue; Figure D is the Ki67, ErbB2, ER, EZH2 staining results of mouse fat pad / breast tumor tissue; Figure E is the statistical graph of Ki67, ErbB2, ER, EZH2 positive cells in mouse fat pad / breast tumor tissue.

[0088] Figure 4 Figure 1 is a graph showing the regulation of PITRM1 protein expression by FBXO22. Figure A shows the verification result of overexpressing mFBXO22 in 4T1 cells. Figure B shows the CO-IP detection result of FBXO22 and PITRM1. Figure C shows the result of protein immunoblotting detection of PITRM1 protein expression in MDA-MB-231 and MCF-7 cell lines, which is negatively regulated by FBXO22. Figure D shows the positive regulation of FBXO22 in MDA-MB-231 and MCF-7 cell lines by protein immunoblotting. Figure 1 shows the results of regulating mitochondrial autophagy-related markers; Figure E shows the results of treating MDA-MB-231 and MCF-7 cells with the proteasome inhibitor MG132, blocking the proteasome degradation pathway, and the expression level of PITRM1 is no longer regulated by FBXO22; Figure F shows the results of FBXO22 accelerating the degradation of PITRM1 after treating MDA-MB-231 and MCF-7 cells with the protein synthesis inhibitor CHX; Figure G is a schematic diagram of the results of FBXO22 promoting the ubiquitination modification of PITRMI.

[0089] Figure 5 The results verify that FBXO22 promotes mitochondrial autophagy and inhibits the occurrence of breast cancer by regulating PITRM1. Figure A shows that the protein levels of mitochondrial autophagy-related markers were significantly restored when PITRM1 was knocked down while FBXO22 expression was knocked down in MCF-7 and MDA-MB-231, compared with the single knockout of FBXO22. Figure B shows that after FBXO22 knockout mice were treated with the mitochondrial autophagy agonist urolithin A, the breast tumors were significantly smaller than those of untreated FBXO22 knockout mice. Figure C is a statistical graph of the volume of breast tumors of several mice. Figure D is a statistical graph of the weight of breast tumors of several mice. DETAILED DESCRIPTION

[0090] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0091] Example 1 Construction of mammary gland specific knockout FBXO22 mouse model

[0092] 1. Experimental methods

[0093] (1) First, the Cre / LoxP system was used to perform flox modification on the left side of exon 1 and the right side of exon 2 of FBXO22 ( Figure 1 A) We constructed conditional FBXO22 knockout mice and crossed them with mammary gland-specific recombinase MMTV-Cre transgenic mice to obtain MMTV-Cre + -Fbxo22+ / fl mice. Then MMTV-Cre + -Fbxo22 + / fl Female mice with MMTV-PyMT + Male transgenic mice were crossed to produce three genotypes of female mice: homozygous (MMTV-Cre + Fbxo22 fl / fl -PyMT + In this embodiment, it is referred to as Fbxo22 fl / fl , fl / fl), heterozygous (MMTV-Cre + Fbxo22 + / fl -PyMT + In this embodiment, it is referred to as Fbxo22 + / fl , + / fl), wild type (MMTV-Cre+Fbxo22 + / + -PyMT + In this embodiment, it is referred to as Fbxo22 + / + , + / +). Since the experimental model mice were required to be female from the same litter and to meet the three genotypes at the same time, the inventors conducted a large-scale breeding model.

[0094] (2) According to the mouse numbering method, the toes at the corresponding positions were cut and DNA was extracted. After amplification by PCR system, agarose gel electrophoresis was performed to identify the mouse genotype.

[0095] (3) In order to determine whether the loss of FBXO22 would affect the growth of mice and the development of mammary gland morphology and structure, we tested the MMTV-Cre + -Fbxo22 + / + Mouse, MMTV-Cre + -Fbxo22 + / fl Mice and MMTV-Cre + -Fbxo22 fl / fl Differences in mice. The mice were housed normally for 4 months, and the weight changes of female mice of the three genotypes were tracked.

[0096] (4) The liver, spleen, and mammary gland tissues of the three genotypes of mice were dissected and obtained, and the knockout effect of FBXO22 in the knockout mouse model was verified at the protein level by Western blot.

[0097] (5) Next, the mammary gland tissues of the three genotype mice were embedded and sectioned, and H&E staining and Ki67 staining were used to further observe the expression of MMTV-Cre + -Fbxo22 + / + , MMTV-Cre + -Fbxo22 + / fl , MMTV-Cre + -Fbxo22 fl / fl The morphology and structure of the mammary gland in female mice.

[0098] 2. Experimental results

[0099] (1) From the gel imaging, we can see that MMTV + A band with a molecular weight of about 556 bp appeared in mice, and the heterozygous Fbxo22 + / fl The genotype is two bands, Fbxo22 + / + (Wild type) 376bp and Fbxo22 fl / fl (homozygous) 410bp( Figure 1 B).

[0100] (2) Under the premise that the weight difference between the three genotypes of mice in the same litter is not large, the results ( Figure 1 C) shows that the appearance and morphology of the mammary glands of the three genotypes of mice are not significantly different, and the weight and volume of the mammary glands also support this finding ( Figure 1 DF). The results showed that the specific deletion of FBXO22 in mammary gland tissue did not affect the normal growth and development of mice, and the appearance and tissue morphology of the mammary gland of mice did not change.

[0101] (3) Tissue protein immunoblotting results showed that there was no significant difference in the levels of FBXO22 protein among the three genotypes of mice in the liver and spleen, but in the mammary gland, Fbxo22 fl / fl The FBXO22 protein level in mice was significantly reduced, successfully verifying that the knockout effect was specific ( Figure 1 G).

[0102] (4) Results Figure 1 H) found that the loss of FBXO22 also did not affect the structure of the mouse mammary gland. There was no significant difference in the mammary gland structure of the three genotypes of mice, and the levels of mammary gland proliferation of the three genotypes of mice were similar ( Figure 1 H), demonstrating that loss of FBXO22 does not affect mammary gland growth and development.

[0103] The above results indicate that mammary gland-specific knockout of FBXO22 has no significant effect on the normal development, morphology and tissue structure of the mammary gland in mice.

[0104] Note: *(p<0.05), **(p<0.01), ***(p<0.001), ****(p<0.0001)

[0105] Example 2 Mammary gland-specific deletion of FBXO22 promotes spontaneous breast cancer in PyMT mice

[0106] 1. Experimental methods

[0107] Make MMTV-Cre + -Fbxo22 fl / fl Mice with MMTV-Cre + -PyMT + Mice were crossed to obtain MMTV-Cre + -Fbxo22 + / fl -PyMT + Mice, then MMTV-Cre + -Fbxo22 + / fl -PyMT + MMTV-Cre + -Fbxo22 + / + -PyMT + (In this embodiment, it is referred to as Fbxo22 + / + , + / +) mice, MMTV-Cre + -Fbxo22 fl / fl -PyMT + (referred to as Fbxo22fl / fl, fl / fl in this example) mice ( Figure 2 A). The mice were fed with normal diet and the weight changes and spontaneous tumor formation were recorded. The earliest time when the tumor could be palpated in Fbxo22fl / fl mice was about 12 weeks. When the mammary glands of both genotype mice had tumors, the mammary gland tissues were collected. The time when both genotype mice had tumors was about 16 weeks. The tumor volume and weight of the mice were then statistically analyzed, and the breast cancer tissues were paraffin-sectioned and stained with H&E. At the same time, the breast cancer tissues of the two groups of mice were stained with Ki67.

[0108] 2. Experimental results

[0109] You can see Fbxo22 fl / fl The mammary tumors of mice were higher than those of Fbxo22 + / + The tumor tissue of the mice was larger in appearance ( Figure 2 B), Statistical analysis of tumor volume and weight of mice with two genotypes revealed that Fbxo22fl / fl The mammary tumor volume and weight of mice were compared with those of Fbxo22 + / + The difference was statistically significant (P<0.001) ( Figure 2 CE).

[0110] H&E staining results showed that in Fbxo22 + / + Mammary ducts and glands were found in the tumor tissues of mice, but in Fbxo22 fl / fl The mammary gland structure is basically not observed in the mouse mammary gland sections, and the entire field of view is basically filled with tumor foci ( Figure 2 F).

[0111] Ki67 staining results clearly show that Fbxo22 fl / fl Mouse breast cancer tissue showed more positive particles and higher immunohistochemical staining scores (IRS), suggesting that Fbxo22 fl / fl Mouse tissue has a stronger proliferation capacity ( Figure 2 G).

[0112] Note: *(p<0.05), **(p<0.01), ***(p<0.001), ****(p<0.0001)

[0113] Example 3 Mammary gland-specific deletion of FBXO22 accelerates the progression of "mammary hyperplasia-mammary intraepithelial neoplasia-breast cancer" in PyMT mice

[0114] 1. Experimental methods

[0115] (1) In order to more intuitively and in detail explore the function of FBXO22 in the development of breast cancer, mice aged 4, 9, 12, and 16 weeks were selected for observation and research. + -Fbxo22 + / + -PyMT + (In this embodiment, it is referred to as Fbxo22 + / + , + / +) and MMTV-Cre + -Fbxo22 fl / fl -PyMT + (In this embodiment, it is referred to as Fbxo22 fl / fl The weight and volume of fat pad tissue / breast cancer tissue of the two genotypes (fl / fl) were statistically analyzed separately.

[0116] (2) Next, H&E staining was performed on the mammary fat pad tissue / breast cancer tissue of spontaneous breast cancer mice at the above four time points to explore the effect of specific deletion of FBXO22 in the mammary gland on its occurrence and development. Since fibrosis characterized by increased collagen fibers is related to tumor progression, Sirius red staining was then performed to clarify the fibrosis during the development of the spontaneous breast cancer model. There are alkaline groups in collagen molecules, and Sirius red dye is a strong acidic dye. When the two are combined with each other, the birefringence phenomenon is enhanced. Under a general optical microscope, collagen fibers appear red and muscle fibers appear yellow.

[0117] (3) Fbxo22 + / + and Fbxo22 fl / fl Immunohistochemical staining of Ki67 was performed on mammary fat pad / breast cancer tissues of two genotype mice at four time periods. + / + and Fbxo22 fl / fl Female mice of the same littermate of the two genotypes were stained for ErbB2, ER, and EZH2 at the same time at 4, 9, 12, and 16 weeks of age.

[0118] 2. Experimental results

[0119] (1) Results Figure 3 A) shows that in the mammary gland tissue or mammary tumor tissue of mice collected at these four time periods, Fbxo22 + / + and Fbxo22 fl / fl The mammary fat pad tissue of these two genotypes of mice did not form tumors, but Fbxo22 fl / fl The fat pad tissue of mouse mammary gland is more + / + mice were enlarged; at 12 weeks of age, Fbxo22 fl / fl Mice with Fbxo22 + / + At this time, the mice still had no tumors; at 16 weeks of age, Fbxo22 fl / fl The tumors in the mice in the Fbxo22 group were already very serious. + / + The mice began to develop tumors, and the tumors were more severe than those of Fbxo22 fl / fl Mice are lighter.

[0120] For different time periods, Fbxo22 + / + and Fbxo22 fl / fl The weight and volume of fat pad tissue / breast cancer tissue of these two genotypes were statistically analyzed separately, and the differences were statistically significant ( Figure 3 B).

[0121] (2) From the results of H&E staining ( Figure 3C) As can be seen, at 4 weeks of age, Fbxo22 fl / fl Proliferation has begun in the mammary ducts of mice, and Fbxo22 + / + The structure of the mammary duct of mice is still normal; at 9 weeks of age, Fbxo22 fl / fl There was proliferation of mammary ducts in multiple locations in the mammary tissue of mice, and Fbxo22 + / + The proliferation of mice was relatively mild; at 12 weeks of age, Fbxo22 fl / fl Large tumor foci were clearly visible in the mouse mammary gland, and Fbxo22 + / + Mice are still in the slow proliferation stage of mammary ducts; by 16 weeks of age, Fbxo22 fl / fl The normal ductal morphology is basically invisible in the mammary gland structure of mice, and almost the entire field of view is filled with tumor lesions. + / + Some tumor foci begin to appear in the breast, and glands are partially visible.

[0122] Sirius red staining results ( Figure 3 C) Display, Fbxo22 fl / fl The mammary ducts of mice had obvious fibrosis at 4 weeks of age, which became more serious at 12 and 16 weeks of age. + / + Fibrosis progression in mice was higher than that in Fbxo22 fl / fl In mice, fibrosis itself is a simple and objective assessment indicator, which is consistent with the changes in H&E staining.

[0123] (3) Ki67 staining results ( Figure 3 D) shows that at 4 weeks of age, Fbxo22 + / + and Fbxo22 fl / fl Both genotypes of mice had Ki67 positive expression in fat pads, and Fbxo22 fl / fl Fbxo22 + / + More obviously, this suggests Fbxo22 fl / fl Mice began to enter the stage of mammary gland hyperplasia at 4 weeks of age. In the following three time periods, Fbxo22 fl / fl The Ki67 positive rate in the mammary gland tissue of mice was higher than that in Fbxo22 + / + The staining results were statistically analyzed by ImageJ ( Figure 3 E), the differences were statistically significant.

[0124] (4) ErbB2, ER, and EZH2 staining results ( Figure 3 D) shows that at 4 weeks of age, Fbxo22 + / + and Fbxo22 fl / fl ErbB2, ER, and EZH2 were positively stained in the mouse mammary fat pad tissue, but Fbxo22fl / fl The positive expression of Fbxo22 in mice was higher, and combined with the results of Ki67 staining, this suggests that fl / fl Fbxo22 + / + Mice entered the stage of mammary hyperplasia more quickly; at 9 weeks of age, Fbxo22 + / + and Fbxo22 fl / fl The mammary fat pad tissues of the two genotype mice showed more positive staining of ErbB2 and EZH2 than at 4 weeks of age. fl / fl The mouse mammary fat pad tissue showed strong positive staining of ER, suggesting that Fbxo22 fl / fl Mice may enter the stage of mammary intraepithelial neoplasia, and Fbxo22 + / + May still be in the stage of mammary hyperplasia; at 12 weeks of age, Fbxo22 + / + and Fbxo22 fl / fl In the mammary fat pad tissue / breast cancer tissue of both genotype mice, more positive staining of ErbB2 and EZH2 was observed than at 9 weeks of age, while at this time, a small amount of positive expression of ER was still observed in the mammary tissue of Fbxo22+ / + mice. fl / fl However, ER staining in mouse breast cancer tissue turned negative, indicating that Fbxo22 fl / fl It has entered the breast cancer stage and has a certain degree of invasiveness, and Fbxo22 + / + The mice were still in the mammary intraepithelial neoplasia stage; by 16 weeks of age, Fbxo22 + / + and Fbxo22 fl / fl The positive staining of ErbB2 and EZH2 in breast cancer tissues of both genotype mice continued to increase, while Fbxo22 + / + ER staining in mouse breast cancer tissue was initially negative, suggesting that Fbxo22 + / + The above results indicate that the mammary gland-specific deletion of FBXO22 can significantly accelerate the progression of breast cancer mice from mammary gland hyperplasia to mammary intraepithelial neoplasia to early breast cancer and then to late breast cancer. The staining results were statistically analyzed by ImageJ ( Figure 3 E), the differences were statistically significant.

[0125] Note: *(p<0.05), **(p<0.01), ***(p<0.001), ****(p<0.0001)

[0126] Example 4 Regulation of PITRM1 by FBXO22

[0127] 1. Experimental methods

[0128] (1) mFBXO22 (murine) was overexpressed in the mouse breast cancer cell line 4T1, followed by protein co-immunoprecipitation and proteomic analysis.

[0129] (2) MDA-MB-231 and MCF-7 cell lines were treated with the protease inhibitor MG132 and the protein synthesis inhibitor CHX, and then protein immunoprecipitation was performed to analyze whether FBXO22 regulated PITRM1 through the proteasome degradation pathway.

[0130] (3) Overexpression and knockdown of FBXO22 were performed on MDA-MB-231 and MCF-7, and Western Blot analysis was performed. FBXO22 was knocked down using siRNA, and the siRNA sequences are shown in Table 1.

[0131] Table 1 siRNA sequences

[0132] name siRNA Sequence number si-FBXO22-#1 GGUGGGAGCCAGUAAUUAUTT SEQ ID NO:1 si-FBXO22-#1 AUAAUUACUGGCUCCCACCTT SEQ ID NO:2 si-FBXO22-#2 GUUCGCAUCUUACCACAUATT SEQ ID NO:3 si-FBXO22-#2 UAUGUGGUAAGAUGCGAACTT SEQ ID NO:4 si-FBXO22-NC UUCUCCGAACGUGUCACGUTT SEQ ID NO:5 si-FBXO22-NC ACGUGACACGUUCGGAGAATT SEQ ID NO:6

[0133] (4) The binding relationship between FBXO22 and PITRM1 was verified by co-immunoprecipitation experiments, and the ubiquitination level of PITRM1 was detected.

[0134] 2. Experimental results

[0135] (1) Proteomic results Figure 4 A) PITRM1 is present in the gel filtration product, indicating that FBXO22 can specifically bind to PITRM1.

[0136] (2) Results Figure 4 B) It shows that in MDA-MB-231 cells, FBXO22 can pull down PITRM1, and PITRM1 can also pull down FBXO22, proving that the two can indeed bind to each other, which provides a premise for FBXO22 to ubiquitinate PITRM1.

[0137] (3) The results show that Figure 4 C), after overexpression of FBXO22 in MDA-MB-231 and MCF-7 cells, the protein expression level of PITRM1 decreased, while after knockdown of FBXO22 in MDA-MB-231 and MCF-7, the protein expression level of PITRM1 increased. This indicates that FBXO22 negatively regulates the protein expression of PITRM1. FBXO22 expression and degradation of RFC5 by ubiquitination regulate DNA damage repair, thereby delaying the occurrence and development of breast cancer.

[0138] (4) Results Figure 4D) showed that overexpression of FBXO22 in MDA-MB-231 and MCF-7 increased the expression levels of mitophagy markers Parkin and PINK proteins, while knockdown of FBXO22 decreased the expression levels of Parkin and PINK proteins.

[0139] (5) The results show that Figure 4 E), after treatment of MDA-MB-231 and MCF-7 with the proteasome inhibitor MG132, the expression of PITRM1 was no longer regulated by FBXO22. After treatment of MDA-MB-231 and MCF-7 with the protein synthesis inhibitor CHX, overexpression of FBXO22 accelerated the degradation of existing PITRM1 ( Figure 4 F), these results demonstrate that the regulation of PITRM1 by FBXO22 is dependent on the proteasome pathway.

[0140] (6) The ubiquitination level of PITRM1 was observed in the MDA-MB-231 cell line. It can be seen that after overexpression of FBXO22, the ubiquitination level of PITRM1 increased, and after knockdown of FBXO22, the ubiquitination level of PITRM1 decreased ( Figure 4 G).

[0141] Example 5 FBXO22 promotes mitochondrial autophagy and inhibits breast cancer by regulating PITRM1

[0142] 1. Experimental methods

[0143] (1) FBXO22 knockdown, PITRM1 knockdown, and simultaneous knockdown of FBXO22 and PITRM1 were performed in MDA-MB-231 and MCF-7, and Western Blot analysis was performed ( Figure 5 A).

[0144] (2) During spontaneous breast cancer in mice, MMTV-Cre was administered + -Fbxo22 fl / fl -PyMT + Mice (referred to as - / - in this example) were treated with urolithin A, a mitochondrial autophagy activator, by oral gavage (dose: 50 mg / kg, twice a week). After 16 weeks, breast tumor tissues were taken and photographed, and tumor volume and weight were measured ( Figure 5 BD).

[0145] 2. Experimental results

[0146] (1) The results showed that knocking down FBXO22 and interfering with PITRM1 expression in MDA-MB-231 and MCF-7 cells significantly restored the protein levels of mitochondrial autophagy-related markers compared with knocking down FBXO22 alone ( Figure 5A), indicating that FBXO22 promotes mitophagy by degrading PITRM1 through ubiquitination.

[0147] (2) The results showed that compared with untreated FBXO22 knockout mice, breast tumors in FBXO22 knockout mice treated with urea A were significantly reduced ( Figure 5 B), tumor volume decreased ( Figure 5 C), weight reduction ( Figure 5 D)

[0148] The above experimental results provide a possible explanation for the inhibitory effect of FBXO22 on tumor progression: because FBXO22 can specifically bind to PITRM1, ubiquitinate and degrade PITRM1, activate mitochondrial autophagy, and thus inhibit the occurrence and development of breast cancer.

[0149] Note: *(p<0.05), **(p<0.01), ***(p<0.001), ****(p<0.0001)

[0150] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for constructing a non-human animal model of breast cancer, characterized in that: The non-human animal model of breast cancer lacks FBXO22 expression; Preferably, the FBXO22 expression deficiency is knocking out and / or knocking down FBXO22 in a non-human animal; Preferably, the knockout and / or knockdown can be achieved by any of the following technologies: Cre-LoxP, FLP / FRT, R / RS, Gin / gix, Cin H / RS2, ParA / MRS, phiC31, CRISPR-Cas9, zinc finger nuclease technology, transcription activator-like effector nuclease technology, interfering RNA; Preferably, the knockout and / or knockdown technique used is selected from Cre-LoxP, interfering RNA; Preferably, the interfering RNA includes shRNA, siRNA, and sgRNA; Preferably, the interfering RNA is selected from siRNA; Preferably, the sequence of the siRNA is shown in SEQ ID NO: 1-4.

2. The construction method according to claim 1, characterized in that: The knockout and / or knockdown method includes knocking out exon 1 and exon 2 of FBXO22 using Cre-LoxP technology; Preferably, the knockout and / or knockdown method comprises the following steps: (1) Flox modification was performed at both ends of the FBXO22 gene to construct Fbxo22 fl / fl Genotype of animal; (2) Fbxo22 fl / fl Genotyped animals were crossed with MMTV-Cre transgenic animals to obtain MMTV-Cre-Fbxo22 fl / fl Genotype of animal; (3) MMTV-Cre-Fbxo22 fl / fl The genotyped animals were crossed with the genotyped animals of MMTV-Cre-PyMT to obtain the F1 generation - MMTV-Cre-Fbxo22 + / fl - Animals with the PyMT genotype; (4) Self-pollination of the F1 generation to produce MMTV-Cre-Fbxo22 fl / fl - Animals with the PyMT genotype; Preferably, the non-human animal model of breast cancer is a spontaneous breast cancer model; Preferably, the knockout and / or knockdown is tissue specific; Preferably, the knockout and / or knockdown is mammary gland specific; Preferably, the tumor formation time of the non-human animal model of breast cancer is faster.

3. The construction method according to claim 1, characterized in that: The non-human animal model refers to a non-human animal that has or displays characteristics of a disease or condition; Preferably, the non-human animal is a mammal; Preferably, the mammals include mice, rats, rabbits, dogs, pigs, monkeys, and sheep; Preferably, the mammal is selected from mice.

4. Any of the following methods: (1) A method for screening drug candidates for treating breast cancer, characterized in that: The method comprises: a) applying the agent to be screened to a non-human animal with breast cancer prepared by the construction method according to any one of claims 1 to 3; b) detecting the therapeutic effect of the agent to be screened on breast cancer; (2) A method for identifying an anticancer agent for treating drug-resistant breast cancer, characterized in that the method comprises: a) administering a drug to a non-human animal with breast cancer prepared by the construction method according to any one of claims 1 to 3; b) when drug resistance occurs, administering other drugs to the drug-resistant breast cancer non-human animal in a; c) performing one or more assays to determine whether the agent has a therapeutic effect on one or more abnormal symptoms associated with breast cancer; d) identifying the agent as an anticancer agent for treating drug-resistant breast cancer when the agent has a therapeutic effect on one or more abnormal symptoms associated with breast cancer; (3) A method for evaluating the therapeutic effect of a drug for treating breast cancer, characterized in that the method comprises: a) administering the drug to a non-human animal with breast cancer prepared by the construction method according to any one of claims 1 to 3; b) detecting the therapeutic effect of the drug on the breast cancer; (4) A method for studying the pathogenesis of breast cancer, characterized in that the method comprises using a non-human animal with breast cancer prepared by the construction method according to any one of claims 1 to 3 to study the pathogenesis of breast cancer; Preferably, the therapeutic effect includes reducing the number of tumor cells, reducing the size of the tumor or the tumor burden, inhibiting the infiltration of tumor cells into surrounding organs, inhibiting tumor metastasis or inhibiting tumor growth; Preferably, the anticancer agent includes targeted small molecules, targeted macromolecules, proteasome inhibitors, immunosuppressants, tumor necrosis inhibitors, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules; Preferably, the drug includes targeted small molecules, targeted macromolecules, proteasome inhibitors, immunosuppressants, tumor necrosis inhibitors, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules.

5. Any of the following applications: (1) Use of the non-human animal model of breast cancer prepared by the construction method according to any one of claims 1 to 3 in screening drug candidates for treating breast cancer; (2) Use of the non-human animal model of breast cancer prepared by the construction method according to any one of claims 1 to 3 in identifying anticancer agents for treating drug-resistant breast cancer; (3) Use of the non-human animal model of breast cancer prepared by the construction method according to any one of claims 1 to 3 in evaluating the therapeutic effect of a drug for treating breast cancer; (4) Use of the non-human animal model of breast cancer prepared by the construction method according to any one of claims 1 to 3 in studying the pathogenesis of breast cancer; Preferably, the anticancer agent includes targeted small molecules, targeted macromolecules, proteasome inhibitors, immunosuppressants, tumor necrosis inhibitors, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules; Preferably, the drug includes targeted small molecules, targeted macromolecules, proteasome inhibitors, immunosuppressants, tumor necrosis inhibitors, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules.

6. Application of FBXO22 in constructing non-human animal models of breast cancer; Preferably, the non-human animal model refers to a non-human animal that has or displays characteristics of a disease or condition; Preferably, the non-human animal is a mammal; Preferably, the mammals include mice, rats, rabbits, dogs, pigs, monkeys, and sheep; Preferably, the mammal is selected from mice.

7. A method for regulating the expression level of PITRM1, characterized in that: The method comprises administering an FBXO22 modulator to modulate the expression level of FBXO22; Preferably, the FBXO22 is negatively correlated with the PITRM1; Preferably, the FBXO22 regulator includes an FBXO22 inhibitor and an FBXO22 promoter; Preferably, the FBXO22 inhibitor includes nucleic acid inhibitors, protein inhibitors, and compounds; Preferably, the nucleic acid inhibitor includes shRNA, siRNA, sgRNA, and antisense nucleic acid; Preferably, the nucleic acid inhibitor is selected from siRNA; Preferably, the sequence of the siRNA is as shown in SEQ ID NO: 1-4; Preferably, the FBXO22 promoter can increase the expression level of the FBXO22 gene; Preferably, the FBXO22 promoter includes nucleic acid molecules, carbohydrates, liposomes, small molecule chemical drugs, antibody drugs, polypeptides, proteins or interfering lentiviruses; Preferably, the FBXO22 promoter is selected from nucleic acid molecules and interfering lentiviruses.

8. Application of FBXO22 in regulating PITRM1 expression level.

9. Application of FBXO22 in the preparation of drugs for treating breast cancer; Preferably, the drug comprises a FBXO22 promoter; Preferably, the FBXO22 promoter is a substance that can increase the expression level of the FBXO22 gene; Preferably, the FBXO22 promoter includes nucleic acid molecules, carbohydrates, liposomes, small molecule chemical drugs, antibody drugs, polypeptides, proteins or interfering lentiviruses; Preferably, the FBXO22 promoter is selected from nucleic acid molecules and interfering lentiviruses.

10. Application of reagents for detecting FBXO22 in the diagnosis of breast cancer; Preferably, the reagent is selected from the group consisting of primers that specifically amplify the FBXO22 gene, probes that specifically recognize the FBXO22 gene, or antibodies that specifically bind to FBXO22.

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