A method for constructing a spontaneous esophageal precancerous lesion or esophageal cancer non-human animal model and application thereof

Abstract

The application provides a method for constructing a spontaneous esophageal precancerous lesion or esophageal cancer non-human animal model and application thereof. The non-human animal model of spontaneous esophageal precancerous lesion or esophageal cancer is obtained by deleting the expression of Trp53 and Cdkn2a in the non-human animal. The non-human animal model prepared by the application can be used as an ideal animal model for screening drug candidates, evaluating the therapeutic effect of drugs, evaluating the toxicological effect of drugs and researching the pathogenesis of esophageal precancerous lesion or esophageal cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a method for constructing a non-human animal model of spontaneous esophageal precancerous lesions or esophageal cancer and its application. Background Technology

[0002] China accounts for approximately 53% of new esophageal cancer cases and 55% of esophageal cancer deaths globally. The high recurrence and metastasis rates of advanced esophageal cancer severely limit clinical treatment options, leading to poor prognosis and a five-year survival rate of only 20%. Common subtypes of esophageal cancer include esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC). ESCC is the most common histological subtype of esophageal cancer globally, with an annual incidence rate five times that of EAC. ESCC commonly occurs in the upper and middle two-thirds of the esophagus and often undergoes atypical squamous cell hyperplasia within the epithelial mucosa before developing into the final, typical invasive carcinoma. Depending on the proportion of atypical hyperplasia epithelial cells, this pathological stage is also named low-grade intraepithelial neoplasia (LGIN) and high-grade intraepithelial neoplasia (HGIN), which are currently recognized as the precancerous lesions of esophageal squamous cell carcinoma.

[0003] To investigate the mechanisms of esophageal cancer development and progression, researchers have created various tumor models, including organoid models, xenograft models, carcinogen- or diet-induced models, and genetically engineered mouse models. Compared to other models, genetically engineered mouse models can better provide the physiological, molecular, and histological characteristics of esophageal cancer, thereby determining the mechanisms of tumor development and treatment methods, and have enormous research potential.

[0004] However, existing genetically engineered animal models for esophageal cancer are time-consuming, and many fail to develop into esophageal cancer. They often require prolonged treatment with chemical carcinogens (mutagenic and tumor-inducing chemicals) in mice, eventually leading to esophageal cancer. Furthermore, the use of these chemical carcinogens damages systemic organs and the circulatory system of mice, such as bone marrow and immune cells. Therefore, existing models cannot simulate the multi-stage process of esophageal cancer development, posing difficulties and challenges to the exploration of mechanisms related to esophageal cancer development, drug screening, drug pharmacology and toxicology studies, and other research on esophageal cancer prevention strategies and treatments. This places new demands on animal models for esophageal cancer. Summary of the Invention

[0005] In view of this, in order to overcome the shortcomings of the prior art, the present invention is proposed.

[0006] The first aspect of the present invention provides a method for constructing a non-human animal model of esophageal precancerous lesions or esophageal cancer, the method comprising causing the non-human animal to lose expression of Trp53 and Cdkn2a.

[0007] In this invention, the esophageal cancer includes, but is not limited to, EAC and ESCC.

[0008] In some implementations, the esophageal cancer is selected from ESCC.

[0009] In this invention, the precancerous lesions of the esophagus include, but are not limited to, LGIN and HGIN.

[0010] In this invention, the LGIN exhibits atypical cell proliferation, but the atypical cells do not exceed half the thickness of the epithelial layer. Microscopically, the atypical cells show disordered basal cell arrangement, loss of cell polarity, slightly enlarged nuclei, slight irregular morphology, slightly darker staining than normal, and an increased nucleoplasmic ratio.

[0011] In this invention, HGIN exhibits atypical hyperplasia where cells occupy more than half the thickness of the epithelial layer, or even invade the entire epithelial layer. Invasion of the entire epithelial layer is termed carcinoma in situ. Microscopically, the atypical cells show disordered arrangement of basal cells with irregular margins, while the basement membrane remains continuous and intact. The epithelial cells exhibit loss of polarity, large, deeply stained nuclei, and irregular and diverse nuclear morphology, sometimes showing nuclear pyknosis and mitosis.

[0012] In this invention, Trp53 is a transformation-related protein (Trp53).

[0013] In this invention, Cdkn2a is a cyclin-dependent kinase inhibitor (Cdkn2a).

[0014] In this invention, expression loss can refer to a decrease or loss of Trp53 and Cdkn2a expression levels, or a decrease or loss of Trp53 and Cdkn2a activity. The effect of Trp53 and Cdkn2a expression loss can be achieved by knocking out or downregulating Trp53 and Cdkn2a, or by administering Trp53 and Cdkn2a inhibitors. Regardless of the method used, as long as the effect of Trp53 and Cdkn2a expression loss is achieved, it falls within the protection scope of this invention.

[0015] In some implementations, the construction method includes knocking out or knocking down Trp53 and Cdkn2a in non-human animals.

[0016] In some implementations, Trp53 and Cdkn2a in non-human animals are knocked out or knocked down using techniques including, but not limited to, site-specific recombination, gene editing, or gene expression regulation.

[0017] In some implementations, the site-specific recombination technology includes, but is not limited to, Cre-LoxP technology, FLP / FRT technology, Cin H / RS2 technology, Par A / MRS technology, and phiC31 technology.

[0018] In this invention, the Cre-loxP system in the Cre-loxP technology consists of two parts: Cre recombinase and loxP sites. Cre recombinase is a 38kDa DNA recombinase produced by the cyclization recombinase gene of bacteriophage P1. It can recognize a specific DNA fragment sequence at the loxP site and mediate a site-specific deletion of the DNA sequence between two loxP sites. The loxP site is a 34bp sequence composed of two 13bp inverted and palindromic repeat sequences and an 8bp core sequence.

[0019] In this invention, the FLP / FRT system in the FLP / FRT technology consists of a recombinase and a specific DNA sequence. The recombinase FLP is a monomeric protein composed of 423 amino acids found in yeast cells. Similar to Cre, FLP does not require any cofactors to function and exhibits good stability under various conditions. Another component of this system, the FLP recognition target (FRT), is very similar to the loxP site, also consisting of two 13 bp inverted repeat sequences and an 8 bp core sequence. When this system functions, the orientation of the FRT site determines whether the target fragment is deleted or inverted.

[0020] In this invention, the Cin H / RS2 technology and Par A / MRS technology are mediated by small serine recombinase.

[0021] In this invention, the integrase phiC31 in the phiC31 technology recognizes attP and attB and then cuts the double-stranded DNA starting from the base after TTG at the center of the two sequences and rotates it 180°, and then connects them to form 36bp attL and 37bp attR.

[0022] In some implementations, the gene editing technologies include, but are not limited to, CRISPR-Cas9 technology, zinc finger nuclease technology, and transcription activator-like effector nuclease technology.

[0023] In this invention, the zinc finger nuclease technology comprises two parts: zinc finger proteins and FokI endonuclease. Zinc finger proteins are a class of proteins abundant in eukaryotes, each containing approximately 30 amino acids, all controlled by a zinc ion. Zinc finger proteins are transcription factors possessing both DNA-binding and transcriptional activation domains, thus recognizing and binding to target DNA. FokI endonuclease, on the other hand, performs non-specific cleavage. The DNA-recognizing and DNA-cleaving portions of FokI are completely independent, and FokI operates as a dimer.

[0024] In this invention, the transcription activator-like effector nuclease (TALEN) technology consists of a TALE protein that recognizes and binds to DNA and a FokI nuclease that cuts the sequence. As a second-generation gene editing tool, TALEN technology is more flexible and efficient than ZFN technology. The DNA linker module in the TALE protein typically consists of 34 amino acids. Except for the two variable amino acid sites at positions 12 and 13, which can specifically recognize nucleotide bases, the other sequences are highly conserved. The TALE protein recognizes specific target sites through the two variable amino acids at positions 12 and 13. The FokI nuclease binds to the TALE sequence to form a TALEN protein. When a pair of TALEN proteins meet and recognize the target sequence together, the sequence is cut.

[0025] In this invention, Cas9 in CRISPR-Cas9 is a DNA endonuclease containing two domains: HNH and RuvC. The HNH domain cleaves the target strand, and the RuvC domain cleaves the non-target strand. Target recognition depends on the presence of a short (2-5 bp) protospacer adjacent motif (PAM) on one side of the target site; only sequences upstream or downstream of the PAM can be considered targets. Target-specific cleavage requires Cas9 to assemble with sgRNA. The assembled complex then searches for the target site within the genome. This target search requires finding a 20 nt complementary pair with the guide RNA in the genome, and also requires the presence of a conserved PAM region upstream or downstream of the target site. After PAM recognition, the Cas endonuclease is activated to cleave DNA. During Cas9-mediated interference, crRNA forms a unique dual-RNA structure that guides Cas9 to cleave the 20 bp complementary target sequence and the PAM sequence. By intentionally altering the guide RNA sequence in crRNA, DNA sequences at any PAM site in the genome can be modified. Once the guide RNA finds its target site, the DNA endonuclease Cas9, acting as an effector protein, specifically cuts the target site, causing a double-strand break in the DNA. This activates two repair mechanisms within the cell: non-homologous end joining and homologous recombination, leading to the deletion, insertion, or mutation of the target gene and ultimately modifying the gene.

[0026] In some implementations, the gene expression regulation technology includes, but is not limited to, RNA interference technology.

[0027] RNA interference (RNAi) is a highly conserved evolutionary phenomenon characterized by the efficient and specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA).

[0028] In some implementations, Trp53 and Cdkn2a can be knocked out or knocked down using different types of techniques (such as site-specific recombination, gene editing, or gene expression regulation).

[0029] In some implementations, Trp53 and Cdkn2a can be knocked out or knocked down using different types of techniques (such as Cre-LoxP, FLP / FRT, Cin H / RS2, Par A / MRS, and phiC31 techniques under site-specific recombination).

[0030] In some implementations, Trp53 and Cdkn2a can be knocked out or knocked down using the same technology (e.g., both are Cre-LoxP technology).

[0031] In some implementations, Trp53 can be knocked out or knocked down first, followed by Cdkn2a.

[0032] In some implementations, Cdkn2a can be knocked out or knocked down first, followed by Trp53.

[0033] In some implementations, Trp53 and Cdkn2a can be knocked out or knocked down simultaneously.

[0034] In some implementations, site-specific recombination techniques are used to knock out or knock down Trp53 and Cdkn2a in non-human animals.

[0035] In some implementations, the site-specific recombination technology is selected from Cre-LoxP.

[0036] In some implementations, the knockout or knockdown method includes knocking out exons 2-9 of the Trp53 gene and exon 2 of Cdkn2a using Cre-LoxP technology.

[0037] In some implementations, the construction method includes the following steps: Trp53 fl / fl Genotype non-human animals and Cdkn2a fl / fl Trp53 was obtained by hybridization of non-human genotype animals. fl / fl Cdkn2a fl / fl Genotype non-human animals, Trp53 fl / fl Cdkn2a fl / fl Crossing a non-human animal with the EDL2-iCre genotype with a non-human animal with the EDL2-iCre genotype yields EDL2-iCreTrp53. wt / fl Cdkn2a wt / fl Genotype non-human animals, EDL2-iCreTrp53 wt / fl Cdkn2a wt / fl Genotype of non-human animals and Trp53 fl / fl Cdkn2a fl / fl EDL2-iCreTrp53 was obtained by hybridization of non-human animals with different genotypes. fl / fl Cdkn2a fl / fl Genotype of non-human animal, abbreviated as Trp53 fl / fl Cdkn2a fl / fl Cre + Genotype of non-human animal. The Trp53 mentioned. fl / fl Genotype non-human animals refer to non-human animals in which the loxp site is inserted to the left of exon 2 and to the right of exon 9 of the Trp53 gene. The Cdkn2a... fl / flGenotype non-human animals refer to non-human animals in which the loxp site is inserted to the left of exon 2 and to the right of exon 2 of the Cdkn2a gene.

[0038] In some implementations, the construction method further includes constructing Trp53. fl / fl Genotypes of non-human animals and / or Cdkn2a fl / fl Steps for non-human genotype animals.

[0039] In some implementations, Trp53 is constructed by inserting loxp sites to the left of exon 2 and to the right of exon 9 of the Trp53 gene in non-human animals. fl / fl In non-human animals, loxp sites were inserted to the left of exon 2 and to the right of exon 2 of the Cdkn2a gene to construct Cdkn2a. fl / fl Genotypes of non-human animals.

[0040] In this invention, the EDL2-iCreTrp53 fl / fl Cdkn2a fl / fl Genotyped non-human animals exhibiting esophageal precancerous lesions or esophageal cancer, namely the aforementioned EDL2-iCreTrp53. fl / fl Cdkn2a fl / fl The genotype of non-human animals refers to the non-human animal models of esophageal precancerous lesions or esophageal cancer prepared in this invention.

[0041] In this invention, the EDL2-iCreTrp53 fl / fl Cdkn2a fl / fl Genotyped non-human animals exhibit spontaneous esophageal precancerous lesions or esophageal cancer.

[0042] In this invention, a non-human animal model refers to a non-human animal that has or displays characteristics of a disease or symptom.

[0043] In some implementations, the non-human animal is a non-human mammal.

[0044] In some implementations, the mammals include, but are not limited to, rodents, carnivores, chiropterans, hedgehogs, and insectivores.

[0045] In some embodiments, the mammal is selected from rodents.

[0046] In some embodiments, the rodents include, but are not limited to, hamsters, rats, dwarf rats, spiny rats, moles, thorny rats, and rock rats.

[0047] In some embodiments, the rodent is selected from the Muridae family.

[0048] In some implementations, the Muridae family includes mice and rats.

[0049] In some implementations, the murine species is selected from mice.

[0050] In some implementations, the mice include C57BL / 6 mice.

[0051] In some implementations, the knockout or knockdown is tissue- or cell-specific.

[0052] In some implementations, the knockout or knockdown is specific to esophageal squamous epithelial tissue or esophageal squamous epithelial cells.

[0053] In this invention, the EDL2 early cleavage cycle promoter, originally defined as one of the TATA boxes found in the EcoRI-BamHI fragment of the EB virus genome, is located upstream of the transcription start site of the BNLF2 short open reading frame and downstream of the BNLF1 (LMP1) open reading frame, and exhibits high promoter activity in squamous epithelial cells.

[0054] The second aspect of the present invention provides any of the following methods:

[0055] (1) A method for screening drug candidates for treating precancerous lesions or esophageal cancer, the method comprising:

[0056] a) Apply the screening reagent to non-human animals with esophageal precancerous lesions or esophageal cancer prepared by the construction method described in the first aspect of the present invention;

[0057] b) To detect the therapeutic effect of the reagent to be screened on precancerous lesions or esophageal cancer;

[0058] (2) A method for evaluating the therapeutic effect of a drug for treating precancerous lesions or esophageal cancer, the method comprising:

[0059] a) Applying the drug to non-human animals with esophageal precancerous lesions or esophageal cancer prepared by the construction method described in the first aspect of the present invention;

[0060] b) To test the therapeutic effect of the drug on the precancerous lesions or esophageal cancer of the esophagus;

[0061] (3) A method for evaluating the toxicological effects of a drug for treating precancerous lesions or esophageal cancer, the method comprising:

[0062] a) Applying the drug to non-human animals with esophageal precancerous lesions or esophageal cancer prepared by the construction method described in the first aspect of the present invention;

[0063] b) To detect the toxicological effects of the drug on the non-human animals;

[0064] (4) A method for studying the pathogenesis of esophageal precancerous lesions or esophageal cancer, wherein the method is to use non-human animals with esophageal precancerous lesions or esophageal cancer prepared by the construction method described in the first aspect of the present invention to study the pathogenesis of esophageal precancerous lesions or esophageal cancer.

[0065] In this invention, the method for screening drug candidates for treating precancerous lesions or esophageal cancer may involve administering varying amounts of the screening reagent (from none to near the upper limit of successful delivery to animals, e.g., within toxicity limits), and may include delivery of the screening reagent in different formulations and routes. A single screening reagent may be administered, or the screening reagent may be combined in combinations of two or more screening reagents, particularly where the administration of such combinations may result in a synergistic effect. The ability of the screening reagent to treat existing precancerous lesions or esophageal cancer can be evaluated by administering the screening reagent to non-human animals with precancerous lesions or esophageal cancer prepared by the construction method described in the first aspect of this invention, and by assessing the modulation of the precancerous lesion or esophageal cancer phenotype. The modulation of the precancerous lesion or esophageal cancer phenotype can be assessed, for example, by evaluating the presence or absence of effects on, for example, lesion severity, tumor burden, tumor number, tumor size, metabolic activity of tumor cells, progression-free survival (PFS), overall survival (OFS), etc. The ability of the screening reagent to promote the prevention of esophageal precancerous lesions or esophageal cancer can be evaluated by administering the screening reagent to a non-human animal model of esophageal precancerous lesions or esophageal cancer as described in the first aspect of the invention. Prevention of esophageal precancerous lesions or esophageal cancer refers to a reduction in the incidence and / or severity of esophageal precancerous lesions or esophageal cancer in animals relative to the expected incidence and / or severity of esophageal precancerous lesions or esophageal cancer without intervention.

[0066] In some implementations, the screening reagents include, but are not limited to, protein analogs, antibodies, DNA, RNA, and small molecule compounds.

[0067] In some embodiments, the small molecule compound is sourced from either newly synthesized or existing databases; wherein existing databases include, but are not limited to, general natural product databases (COCONUT, Super Natural II, NPASS), plant natural product databases (KNApSaCK, CMAUP, TriForC, Alkamid, NPACT DB, BioPhytMol), traditional Chinese medicine natural product databases (CEMTDD, CHDD, ETCM, TM-MC, TCMID, YaTCM), microbial natural product databases (StreptomeDB, NP Altas, ProCarDB, PAMDB, Lichen Database), marine natural product databases (MNPD, SWMD), natural product databases from different countries and regions (IMPPAT, NeMedPlant, MedPServer, TlPdb, AfroDB, ANPDB, BIOFACQUIM, NUBBEDB), food natural product databases (FooDB, BitterDB, Phenol-Explorer, PhytoHub, SuperSweet database), and toxic natural product databases (Exposome-Explorer, T3DB, Snake Neurotoxin). Databases such as TPPT, Natural Products Industry Catalog (Greenpharma, AnalytiConDiscovery, InterBioScreen, Indofine Chemical Company, Pi Chemicals Systems\Specs, TargetMol), databases for deduplicating MS data (MoNA, MassBank, METLIN, HMDB, YMDB, ReSpect, GNPS), and databases for deduplicating NMR data (NMRShiftDB, NAPROC-13) are available.

[0068] In this invention, the term "treatment" refers to administering a drug to control the progression of a disease. Control of disease progression should be understood as achieving beneficial or desired clinical outcomes, including but not limited to symptom relief, reduction of disease duration, stabilization of the pathological state (partially and completely), delay of disease progression, and improvement and remission of the pathological state. Control of disease progression also involves prolongation of survival compared to expected survival without treatment.

[0069] The third aspect of the present invention provides any of the following applications:

[0070] (1) The application of the non-human animal model of esophageal precancerous lesions or esophageal cancer prepared by the construction method described in the first aspect of the present invention in screening drug candidates for the treatment of esophageal precancerous lesions or esophageal cancer;

[0071] (2) The application of the non-human animal model of esophageal precancerous lesions or esophageal cancer prepared by the construction method described in the first aspect of the present invention in evaluating the therapeutic effect of drugs for treating esophageal precancerous lesions or esophageal cancer;

[0072] (3) The application of the non-human animal model of esophageal precancerous lesions or esophageal cancer prepared by the construction method described in the first aspect of the present invention in evaluating the toxicological effects of drugs for treating esophageal precancerous lesions or esophageal cancer;

[0073] (4) The application of the non-human animal model of esophageal precancerous lesion or esophageal cancer prepared by the construction method described in the first aspect of the present invention in the study of the pathogenesis of esophageal precancerous lesion or esophageal cancer.

[0074] The fourth aspect of this invention provides the use of Trp53 and / or Cdkn2a in constructing non-human animal models of esophageal precancerous lesions or esophageal cancer.

[0075] In some implementations, non-human animal models of esophageal precancerous lesions or esophageal cancer are constructed by knocking out or knocking down Trp53 and Cdkn2a in non-human animals.

[0076] The advantages and beneficial effects of this invention are as follows:

[0077] This invention provides a method for constructing a non-human animal model of spontaneous esophageal precancerous lesions or esophageal cancer and its application. This invention obtains a non-human animal model of esophageal precancerous lesions or esophageal cancer by deleting the expression of Trp53 and Cdkn2a in non-human animals. The method for constructing this non-human animal model of esophageal precancerous lesions or esophageal cancer provided by this invention eliminates the need for in vitro construction of cell cultures such as cells or organoids, followed by transplantation back into non-human animals, thus saving time and effort. The non-human animal model prepared using the method provided by this invention can progress to the HGIN stage within 12 months, allowing observation of the multi-stage pathological process of the esophagus from normal to precancerous lesions to cancer, and efficiently simulates the process of esophageal cancer development without the need for chemical carcinogen treatment. The non-human animal model prepared using the method provided by this invention can serve as an ideal animal model for screening drug candidates, evaluating drug treatment effects, assessing drug toxicology, and studying the pathogenesis of esophageal precancerous lesions or esophageal cancer. Attached Figure Description

[0078] Figure 1This is a waterfall chart of mutations in all microsamples of the 18 driver genes in the normal esophagus stage; the colors represent different types of mutations; the upper left bar chart represents the total number of mutations of the 18 driver genes in each microsample; the right half is the percentage of each mutation in all microsamples at each stage.

[0079] Figure 2 This is a waterfall chart of mutations in the 18 driver genes across all microsamples of esophageal LGIN; the colors represent different types of mutations; the upper left bar chart represents the total number of mutations in the 18 driver genes for each microsample; the right half shows the percentage of each mutation across all microsamples at each stage.

[0080] Figure 3 This is a waterfall chart of mutations in all microsamples of the 18 driver genes at the esophageal HGIN stage; the colors represent different types of mutations; the upper left bar chart represents the total number of mutations in the 18 driver genes for each microsample; the right half shows the percentage of each mutation in all microsamples at each stage.

[0081] Figure 4 This is a waterfall chart of mutations in all microsamples across the 18 driver genes at each ESCC stage; colors represent different mutation types; the upper left bar chart represents the total number of mutations in each of the 18 driver genes in each microsample. The right half shows the percentage of each mutation across all microsamples at each stage.

[0082] Figure 5 This is a bar chart of dN / dS for genes undergoing positive selection during the normal esophageal (NOR) stage; the colors represent different mutation types, and the q values ​​for the restriction hypothesis tests of the first four genes shown are all less than 0.05.

[0083] Figure 6 This is a bar chart of dN / dS for genes undergoing positive selection in esophageal LGIN; the colors represent different mutation types, and the q values ​​for the restriction hypothesis tests of the first four genes shown are all less than 0.05.

[0084] Figure 7 This is a bar chart of dN / dS of genes undergoing positive selection during the esophageal HGIN stage; the colors represent different mutation types, and the q values ​​for the restriction hypothesis tests of the first four genes shown are all less than 0.05.

[0085] Figure 8 This is a bar chart of dN / dS for genes undergoing positive selection during the ESCC stage; the colors represent different mutation types, and the q values ​​for the restriction hypothesis tests of the first four genes shown are all less than 0.05.

[0086] Figure 9This represents the co-occurrence rate (percentage) of TP53 gene mutation and CDKN2A gene mutation / deletion in multiple stages of esophageal cancer; the color represents the different types of the two gene mutation states and the percentage of mutations in all small samples at each stage.

[0087] Figure 10 It is EDL2-iCreTrp53 fl / fl Cdkn2a fl / fl Mouse construction strategy;

[0088] Figure 11 This is a graph showing the genotype PCR identification results for mice;

[0089] Figure 12 This is a graph showing the results of immunoblotting detection of p53 and p16 protein expression in esophageal epithelial tissues of mice of different genotypes;

[0090] Figure 13 This is a graph showing the results of immunohistochemical detection of the in situ expression of p53 and p16 proteins in the esophageal tissue of mice of different genotypes;

[0091] Figure 14 The images show the pathological results of precancerous lesions and cancer in the entire esophagus of mice with control genotypes (18 months old) and target genotypes (3, 6, 9, 12 and 18 months old).

[0092] Figure 15 This is a statistical graph showing the pathological status of precancerous lesions and cancers in the entire esophagus of mice with control genotypes (18 months old) and target genotypes (3, 6, 9, 12 and 18 months old). Detailed Implementation

[0093] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0094] Example 1: Screening for important gene deletion / mutation combinations based on precancerous lesion genomic data.

[0095] 1. Experimental Materials

[0096] Genomic data of normal human esophagus, precancerous lesions of esophagus, and esophageal cancer from the laboratory.

[0097] 2. Experimental Methods

[0098] Whole exome data quality control and somatic mutation identification

[0099] The 150bp paired-end sequences from whole-exome sequencing were converted from BCL to FASTQ format using the bcl2fastq software, and then processed using Cutadapt v.3.4. Sequencing adapters were removed from the reads, and then FastQC v.0.11.9 (http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ) software was used for data quality control to remove data with poor sequencing quality. Then, Sentieon Genomics workflow software was used. Reads were aligned to remove PCR amplification duplications, undergo base quality recalibration, and identify somatic mutations (including SNVs and indels). The main steps are as follows: First, the adapter-deadlocked reads were aligned to the hg19 reference genome using Sentieon's built-in Burrows–Wheeler Aligner (BWA-MEM) algorithm, resulting in a compressed BAM file. Next, the "Dedup" algorithm was applied to label and remove repetitive fragments generated by PCR amplification. Only reads with an alignment quality score greater than 20 were retained. The deduplicated BAM file was quality controlled using bamdst, with an average sequencing depth of 36.1x for 1275 samples. The base quality recalibration score matrix for each sample was calculated using Sentieon's GATK-like "QualCal" function. Based on this matrix information, the "TNhaplotyper" algorithm was further used to identify somatic mutations in each sample. The algorithm is essentially Mutect2 from GATK. This identification process uses paired peripheral blood DNA data from each sample as a germline reference and annotates each SNP site using the human single nucleotide polymorphism (SNP) data dbSNP138. For identified somatic mutations, SnpEff v.5.0e is further used for annotation.

[0100] To ensure the reliability of the results, a series of screening procedures were adopted for the obtained somatic cell mutations, as follows:

[0101] SNV:

[0102] Mutation sites with a coverage of more than 10-fold in both tissues and paired peripheral blood were preserved;

[0103] For sites in the tissue with 3 mutant reads, the total number of reads at that site must be at least 10.

[0104] Sites with a mutant allele frequency of 0.05 or higher were preserved;

[0105] Mutations with a maximum allele alignment quality score of 20 or higher were retained.

[0106] Mutation sites in the dbSNP database are removed unless the site also exists in the Catalog of Somatic Mutations in Cancer (COSMIC) database.

[0107] Indel:

[0108] Mutation sites with a coverage of more than 10-fold in both tissues and paired peripheral blood were preserved;

[0109] For sites in the tissue with 5 mutant reads, the total number of reads at that site must be at least 10.

[0110] Sites with a mutant allele frequency of 0.2 or higher were preserved;

[0111] Mutations with a maximum allele alignment quality score of 20 or higher were retained.

[0112] Mutation sites in the dbSNP database are removed unless the site also exists in the Catalog of Somatic Mutations in Cancer (COSMIC) database.

[0113] Mutation selectivity analysis

[0114] Extensive genomic studies of esophageal cancer have identified 27 frequently mutated genes, such as TP53, which has a mutation frequency of nearly 85% and can be detected in early stages, i.e., precancerous lesions. Other genes with alterations less than 20% include NOTCH1, KMT2D, CDKN2A, and NFE2L2. In addition, many other gene expression changes are associated with the development of esophageal cancer, such as EGFR, CyclinD1, KLF4, KLF5, and SOX2.

[0115] To further elucidate the role of driver gene mutations in ESCC development, the variation patterns of these 27 potential ESCC driver genes were analyzed in the data. Mutations were detected in at least one stage of the samples from 18 of the 27 genes: NOTCH1, TP53, KMT2C, FAT1, CREBBP, EP300, FAT2, PIK3CA, KMT2D, NOTCH3, CUL3, TP63, ZNF750, FBXW7, AJUBA, KDM6A, CDKN2A, and NFE2L2. NOTCH1 was found to be the most frequently mutated gene in the NOR and LGIN stages, while TP53 showed widespread mutations in both the HGIN and ESCC stages. The mutation frequency trends of both genes during carcinogenesis are opposite, but their overall mutation frequencies are relatively high, specifically 59.2% (NOR), 67.3% (LGIN), 9.3% (HGIN), and 10.6% (ESCC) for NOTCH1, and 29.2% (NOR), 58.4% (LGIN), 94.3% (HGIN), and 100% (ESCC) for TP53. However, for other driver genes, only CDKN2A showed a trend of gradually increasing mutation frequency with carcinogenesis.

[0116] To assess the selectivity of gene mutations in tissues, the dNdScv algorithm was used to calculate the dN / dS values ​​of 18 driver gene mutations mentioned in the paper. This algorithm, based on trinucleotide sequence information near the mutation site, uses maximum likelihood to assess whether the ratio of nonsynonymous mutations to synonymous mutations is statistically significant, thereby inferring whether selection of the mutated gene occurred during a biological process. To reduce false positives that might result from treating each small sample as a single study object, the tissue of each person at each stage was considered a whole, and the same repeated mutation within this whole was counted as a single mutation. Genes with a dN / dS value > 1 and a restriction hypothesis test q < 0.05 were considered to have undergone significant positive selection in the sample.

[0117] 3. Experimental Results

[0118] Experimental results are as follows Figures 1-9 As shown.

[0119] Example 2: Construction scheme and knockout effect detection of esophageal-specific knockout model of Trp53 and Cdkn2a dual genes

[0120] 1. Experimental Materials

[0121] Primers (Qingke Biotechnology), agarose, nucleic acid gel dye (Qingke Biotechnology, TSJ003), microwave oven, enzyme-free water, TAE.

[0122] 2. Experimental Methods

[0123] Construction scheme for esophageal-specific knockout model of Trp53 and Cdkn2a dual genes

[0124] (1) Loxp sites were inserted to the left of exon 2 and to the right of exon 9 of the Trp53 gene in C57BL / 6 mice to construct Trp53. fl / fl Genotype mice; loxp sites were inserted to the left of exon 2 and to the right of exon 2 of the Cdkn2a gene in C57BL / 6 mice to construct Cdkn2a. fl / fl Genotype mice;

[0125] (2) Trp53 fl / fl C57BL / 6 genotype mice and Cdkn2a fl / fl By crossing C57BL / 6 mice, homozygous Trp53 mice were obtained. fl / fl Cdkn2a fl / fl Genotype mice;

[0126] (3) Trp53 fl / fl Cdkn2a fl / fl Genotype mice were crossed with EDL2-iCre mice to obtain EDL2-iCreTrp53. wt / fl Cdkn2a wt / fl Genotype mice, abbreviated as Trp53 wt / fl Cdkn2a wt / fl Cre + Genotype mice;

[0127] (4) EDL2-iCreTrp53 wt / fl Cdkn2a wt / fl Genotype mice and Trp53 fl / fl Cdkn2a fl / fl Genotype mice were crossed to obtain EDL2-iCreTrp53. fl / fl Cdkn2a fl / fl Genotype mice, abbreviated as Trp53 fl / fl Cdkn2a fl / fl Cre + Genotype mice.

[0128] Simultaneously, a single-gene-specific knockout protocol was implemented for Trp53 and Cdkn2a.

[0129] Knockout effect test

[0130] (1) Cut off 3-5 mm of mouse tail tissue.

[0131] (2) DNA extraction: Add 1 μl proteinase K + 50 μl lysis buffer to each rat tail tissue sample, 55℃ for 90 minutes; 95℃ for 5 minutes.

[0132] (3) Design primers based on the gene and amplify the extracted DNA fragments by PCR.

[0133] (4) Determine the mouse genotype based on the agarose gel electrophoresis results.

[0134] 3. Experimental Results

[0135] Construction strategy such as Figure 10 As shown, exons 2-9 of Trp53 are cleaved by a specifically expressed Cre enzyme, resulting in the non-expression of p53 protein. Exon 2 of Cdkn2a is also cleaved by a specifically expressed Cre enzyme, leading to the loss of p16 protein expression. In EDL2-iCre-positive mice, it is specifically expressed in the esophageal squamous epithelium.

[0136] After construction, the mice were genotyped and identified. The experimental results are as follows: Figure 11 As shown, lanes 2, 3, and 8 represent the target mice (Trp53) with homozygous knockout of two genes. fl / fl Cdkn2a fl / fl Cre + ); Numbers 1 and 7 are littermate control mice (Trp53) fl / fl Cdkn2a fl / fl ); the others are partially knocked out or non-target mice.

[0137] Immunoblotting was used to detect the expression of p53 and p16 proteins in esophageal epithelial tissues of different mouse genotypes. The experimental results are as follows: Figure 12 As shown, the target mouse Trp53 fl / fl Cdkn2a fl / fl Cre + At the same time, p53 and p16 are not expressed.

[0138] Immunohistochemical staining was used to detect the in situ expression of p53 and p16 proteins in esophageal tissues of mice with different genotypes. The experimental results are as follows: Figure 13 As shown in Figure A, p53 and p16 are expressed in basal cells of the esophageal epithelium of control genotype mice; p53 and p16 are not expressed in the esophageal epithelium of target mice.

[0139] Example 3: Pathological diagnosis of precancerous lesions and cancer of the esophagus in mice at multiple stages.

[0140] 1. Experimental Materials

[0141] Three-week-old C57BL / 6 mice were housed under controlled environmental conditions, specifically: a temperature of 23±1℃, humidity of 50±10%, and a 12-hour light cycle.

[0142] Mice were euthanized by CO2 asphyxiation at weeks 18, 20, and 22. The esophagus of each mouse was removed, cleaned, and the number of tumors was counted. The length and width of each tumor were measured, and the tumor volume was calculated using the formula length × width^2 × 0.52. At different induction time points, five esophagus sections from each group were quickly placed in 4% paraformaldehyde fixative for paraffin sectioning and pathological assessment. During the experiment, the weight of each animal was measured every three days.

[0143] 2. Experimental Methods

[0144] Multiplex immunofluorescence (MIF) staining analysis was performed on sections of mouse tissue from multi-stage esophageal cancer (H&E contiguous) lesions. The TSA-RM seven-color multi-label kit provided by Beijing Bainuo Panoramic Biotechnology Co., Ltd. was used for the experiments. Opal Polaris 480, Opal 520, Opal 570, Opal 620, Opal 690, and Opal Polaris 780 correspond to antibodies targeting protein targets, generating different immunofluorescence signals. The basic principle is similar to traditional immunohistochemical staining methods, utilizing the specific interaction between antigen and antibody. High-density in situ labeling of target antigens can be achieved by using horseradish peroxidase-labeled secondary antibodies. Furthermore, tyramine signal amplification technology was used to activate the fluorescent dyes in the kit, stably covalently anchoring the fluorescent signal to specific antigens on the tissue sections. During staining, the previously bound primary and secondary antibodies were removed using microwave or high-pressure technology, but the labeled fluorescent signals were retained, thus achieving multiple labeling of multiple target antigens on the tissue sections. After these steps, the slides were stained with DAPI for nuclear staining and then mounted using VectaShield Hardset fixation medium. The slides were then imaged using the Vectra Polaris automated quantitative pathology imaging system (PerkinElmer). Autofluorescence was removed and multispectral images were analyzed using inForm software (Perkin Elmer).

[0145] (1) After obtaining the target genotype mice, the esophageal lesions were detected at multiple points at a time.

[0146] (2) Based on the test results, the design is updated and iterated to obtain a dynamic evolution map of esophageal cancer in multiple stages.

[0147] Under esophageal endoscopy, esophageal mucosa samples from suspected lesions are taken using biopsy forceps. After extraction, the mucosa is quickly placed on a sterile drape and spread out. To prevent epithelial drying and shrinkage, one sample from the same location should be immediately transferred to a 1.5ml cryovial in the same orientation for subsequent frozen sectioning and microsurgical cutting. The esophageal mucosa should be moved towards the lumen in a translational manner to simulate the sampling position as closely as possible for recording. The cryovial should be rapidly frozen on dry ice and then transferred to a -80°C freezer as soon as possible. The other sample is quickly placed in 4% paraformaldehyde fixative for paraffin sectioning and pathological interpretation.

[0148] Immediately after surgical resection, the excised specimen was rinsed with PBS buffer. The operating table was sterilized and draped. The esophagus was laid out longitudinally to expose the luminal surface, and photographs were taken before and after sampling. Throughout the process, the esophageal mucosa was kept moist with RPMI-1640 medium containing 30% FBS to prevent the epithelial cells from drying out. For each esophageal cancer gross specimen, three types of tissue samples were taken: the cancerous lesion, the proximal adjacent tissue (< 2 cm from the cancerous lesion), and the distal adjacent tissue (> 5 cm from the cancerous lesion). The muscularis propria and adventitia of the adjacent tissue were peeled off, leaving only the epithelium and submucosa. The separated tissue was approximately (0.5–2) cm × (0.5–2) cm in size. Two small pieces were cut from each tissue along the edge. One piece was kept on ice for later use, and the other piece was fixed in 4% paraformaldehyde fixative. These two pieces were used as frozen sections and paraffin-embedded tissue for subsequent pathological diagnosis.

[0149] Frozen or formaldehyde-fixed paraffin-embedded tissue sections were stained with H&E, and their morphology and pathological type were determined under a microscope. The results were photographed and recorded. The specific tissue morphology or pathological type and its diagnostic basis in this study are as follows:

[0150] (1) Normal stratified squamous epithelial cells (NOR): The cells are arranged neatly, the basal cell layer is 1–3 layers thick, polar, and the nuclei are normal in shape, size and staining. Mucosal immune cell infiltration and papillary structures are rare.

[0151] (2) Inflammation: The main changes are scattered or abundant infiltration of neutrophils and / or lymphocytes in the mucosa and submucosa. Basal cell morphology occasionally shows significant variations, such as cell enlargement and slightly irregular arrangement, but there are basically no atypia and the staining is normal. Or it may be accompanied by morphological changes such as increased number of papillae, increased height of penetration into the epithelium, and thickening of the epithelial layer.

[0152] (3) Low-grade intraepithelial neoplasia (LGIN): Atypical cell proliferation is present, but the atypical cells do not exceed 1 / 2 of the full thickness of the epithelial layer. Among them, the atypical cells are characterized by disordered basal cell arrangement, loss of cell polarity, slightly larger nuclei, slight irregular morphology, slightly darker staining than normal, and increased nucleocytoplasmic ratio.

[0153] (4) High-grade intraepithelial neoplasia (HGIN): Atypical cells occupy more than 1 / 2 of the full thickness of the epithelial layer or even invade the entire epithelial layer. When the entire layer is invaded, it is called carcinoma in situ. Under the microscope, the atypical cells are characterized by disordered arrangement of basal cells, irregular edges, and the basement membrane is still continuous and intact; the epithelial cells lose polarity, the nuclei are large and deeply stained, the nuclear morphology is irregular and diverse, and sometimes nuclear pyknosis and nuclear division can be seen.

[0154] (5) Invasive carcinoma: Atypical proliferative cells break through the basement membrane and grow into the submucosa, interrupting the basement membrane and blurring or eliminating the epithelial structure. The morphology of the epithelial cells is similar to that of high-grade intraepithelial neoplasia, and sometimes cancer cells can be seen in nests with keratin pearls in the center.

[0155] 3. Experimental Results

[0156] Mice with single-gene knockout of Trp53 and Cdkn2a cannot develop spontaneous pathological changes.

[0157] Experimental results are as follows Figure 14 As shown, 18-week-old control mice (Trp53) fl / fl Cdkn2a fl / fl The epithelium consisted of normal stratified squamous epithelium, without any precancerous lesions or cancer; the target mouse (Trp53) fl / fl Cdkn2a fl / fl Cre + From week 9 onwards, obvious LGIN and HGIN appeared, and the number of cancerous lesions increased in the 12-month-old and 18-month-old model mice. Figure 15 In the study, 18-week-old control mice exhibited normal stratified squamous epithelium with only about 1% inflammatory hyperplasia and no precancerous lesions. In contrast, the target mice showed significant LGIN and HGIN starting from week 9, and the number of cancerous lesions increased in the 12-month-old and 18-month-old model mice, eventually leading to cancerous lesions. Statistical results showed that cancerous lesions began to appear and enlarge in the esophagus of the target mice over time.

[0158] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A method for constructing a non-human animal model of esophageal squamous cell precancerous lesions or esophageal squamous cell carcinoma, characterized in that, The construction method involves deleting the expression of Trp53 and Cdkn2a in a non-human animal, namely a mouse.

2. The construction method according to claim 1, characterized in that, The construction method includes knocking out or knocking down Trp53 and Cdkn2a in non-human animals.

3. The construction method according to claim 2, characterized in that, Use site-specific recombination, gene editing, or gene expression regulation techniques to knock out or knock down Trp53 and Cdkn2a in non-human animals.

4. The construction method according to claim 3, characterized in that, The site-specific recombination technologies include Cre-LoxP technology, FLP / FRT technology, Cin H / RS2 technology, Par A / MRS technology, and phiC31 technology.

5. The construction method according to claim 3, characterized in that, The gene editing technologies include CRISPR-Cas9 technology, zinc finger nuclease technology, and transcription activator-like effector nuclease technology.

6. The construction method according to claim 3, characterized in that, The gene expression regulation technology includes RNA interference technology.

7. The construction method according to claim 3, characterized in that, Site-specific recombination techniques were used to knock out or knock down Trp53 and Cdkn2a in non-human animals.

8. The construction method according to claim 7, characterized in that, The site-specific recombination technology is selected from Cre-LoxP technology.

9. The construction method according to claim 8, characterized in that, Exons 2-9 of the Trp53 gene and exon 2 of Cdkn2a were knocked out using Cre-LoxP technology.

10. The construction method according to claim 9, characterized in that, The construction method includes the following steps: Trp53 fl / fl Genotype non-human animals and Cdkn2a fl / fl Trp53 was obtained by hybridization of non-human genotype animals. fl / fl Cdkn2a fl / fl Genotype non-human animals, Trp53 fl / fl Cdkn2a fl / fl Crossing a non-human animal with the EDL2-iCre genotype with a non-human animal with the EDL2-iCre genotype yields EDL2-iCreTrp53. wt / fl Cdkn2a wt / fl Genotype non-human animals, EDL2-iCreTrp53 wt / fl Cdkn2a wt / fl Genotype of non-human animals and Trp53 fl / fl Cdkn2a fl / fl EDL2-iCreTrp53 was obtained by hybridization of non-human animals with different genotypes. fl / fl Cdkn2a fl / fl Genotype of non-human animal, abbreviated as Trp53 fl / fl Cdkn2a fl / fl Cre + Genotypes of non-human animals.

11. The construction method according to claim 10, characterized in that, The construction method also includes constructing Trp53. fl / fl Genotypes of non-human animals and / or Cdkn2a fl / fl Steps for non-human genotype animals.

12. The construction method according to claim 10, characterized in that, Trp53 was constructed by inserting loxp sites to the left of exon 2 and to the right of exon 9 of the Trp53 gene in non-human animals. fl / fl In non-human animals, loxp sites were inserted to the left of exon 2 and to the right of exon 2 of the Cdkn2a gene to construct Cdkn2a. fl / fl Genotypes of non-human animals.

13. The construction method according to claim 1, characterized in that, The mice included C57BL / 6 mice.

14. A method for screening drug candidates for treating esophageal squamous cell precancerous lesions or esophageal squamous cell carcinoma, characterized in that, The method includes: a) Apply the screening reagent to non-human animals with esophageal squamous cell precancerous lesions or esophageal squamous cell carcinoma prepared by the construction method according to any one of claims 1-13; b) To detect the therapeutic effect of the reagent to be screened on esophageal squamous cell precancerous lesions or esophageal squamous cell carcinoma.

15. A method for evaluating the therapeutic efficacy of a drug for treating esophageal squamous cell carcinoma precancerous lesions or esophageal squamous cell carcinoma, characterized in that, The method includes: a) Applying the drug to non-human animals with esophageal squamous cell precancerous lesions or esophageal squamous cell carcinoma prepared by the construction method according to any one of claims 1-13; b) To test the therapeutic effect of the drug on the esophageal squamous cell precancerous lesion or esophageal squamous cell carcinoma.

16. A method for evaluating the toxicological effects of a drug for treating esophageal squamous cell carcinoma precancerous lesions or esophageal squamous cell carcinoma, characterized in that, The method includes: a) Applying the drug to non-human animals with esophageal squamous cell precancerous lesions or esophageal squamous cell carcinoma prepared by the construction method according to any one of claims 1-13; b) To detect the toxicological effects of the drug on the non-human animals.

17. A method for studying the pathogenesis of esophageal squamous cell precancerous lesions or esophageal squamous cell carcinoma, characterized in that, The method described herein is to study the pathogenesis of esophageal squamous cell precancerous lesions or esophageal squamous cell carcinoma in non-human animals prepared by the construction method according to any one of claims 1-13.

18. The use of a non-human animal model of esophageal squamous cell precancerous lesion or esophageal squamous cell carcinoma prepared by the construction method according to any one of claims 1-13 in screening drug candidates for the treatment of esophageal squamous cell precancerous lesions or esophageal squamous cell carcinoma.

19. The use of a non-human animal model of esophageal squamous cell precancerous lesion or esophageal squamous cell carcinoma prepared by the construction method according to any one of claims 1-13 in evaluating the therapeutic effect of a drug for treating esophageal squamous cell precancerous lesion or esophageal squamous cell carcinoma.

20. The use of a non-human animal model of esophageal squamous cell precancerous lesion or esophageal squamous cell carcinoma prepared by the construction method according to any one of claims 1-13 in evaluating the toxicological effects of a drug for treating esophageal squamous cell precancerous lesion or esophageal squamous cell carcinoma.

21. The use of the non-human animal model of esophageal squamous cell precancerous lesion or esophageal squamous cell carcinoma prepared by the construction method according to any one of claims 1-13 in studying the pathogenesis of esophageal squamous cell precancerous lesion or esophageal squamous cell carcinoma.

Images