Disease model construction method based on intestinal epithelium specific knockout Twist1 gene and application of disease model construction method in accurate treatment of inflammatory bowel disease

Abstract

The invention relates to the field of biomedicine, in particular to a disease model construction method based on intestinal epithelium specific knockout Twist1 gene and application of the disease model construction method in accurate treatment of inflammatory bowel disease, and the disease model construction method comprises the following steps: introducing a loxP site into the Twist1 gene through a CRISPR-Cas9 gene editing technology to generate a Twist1f / f mouse which can be conditionally knocked out; the method comprises the following steps: firstly, designing gRNA aiming at Twist1 and a donor vector containing loxP sites, and injecting the gRNA and the donor vector and Cas9 mRNA into a mouse fertilized egg together; mating the generated F0 generation with a wild type mouse to obtain an F1 generation Twist1f / f mouse; the F1 generation is mated with a transgenic mouse for expressing intestinal epithelium specific Cre recombinase, and finally a transgenic mouse (Twist1- / -) of which the intestinal epithelium specific knockout Twist1 is obtained; the knockout efficiency is verified through PCR, sequencing and protein expression analysis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to a method for constructing a disease model based on intestinal epithelium-specific knockout of the Twist1 gene and its application in the precision treatment of inflammatory bowel disease. Background Art

[0002] Inflammatory Bowel Disease (IBD) is a group of chronic and recurrent intestinal inflammatory diseases, mainly including Ulcerative Colitis (UC) and Crohn's Disease (CD). The global prevalence of IBD continues to rise. Its pathogenesis involves the interaction of multiple factors such as genetic susceptibility, environmental factors, intestinal flora imbalance, and immune system abnormalities, but the exact pathogenesis has not been fully elucidated. Long-term chronic inflammation significantly increases the risk of Colitis-Associated Colorectal Cancer (CAC). The risk of colorectal cancer in UC patients is 2-3 times that of the general population.

[0003] Twist1 is a highly conserved basic helix-loop-helix (bHLH) transcription factor that plays an important role in embryonic development, tissue injury repair, and tumor progression. Studies have shown that Twist1 is involved in various inflammatory responses and tumorigenesis processes and plays a role by regulating processes such as epithelial-mesenchymal transition (EMT), inflammatory factor expression, and cell proliferation. However, the specific functions and regulatory mechanisms of Twist1 in intestinal inflammation and intestinal tumorigenesis are still unclear.

[0004] Currently, the animal models for studying the function of Twist1 are mainly global knockout or specific organ knockout models, and a model specifically for studying the role of Twist1 in intestinal epithelium has not been established. Global knockout of Twist1 leads to embryonic lethality, making it impossible to study its role in adult intestinal diseases. In addition, existing disease models of inflammatory bowel disease are mostly based on chemical inducers or gene mutations, lacking research tools for the role of specific genes in intestinal epithelium, which limits the in-depth study of molecular mechanisms.

[0005] In view of the potential pro-inflammatory and pro-tumor effects of Twist1, establishing a disease model with intestinal epithelium-specific knockout of the Twist1 gene is of great significance for clarifying the role of Twist1 in the pathogenesis of inflammatory bowel disease and colorectal cancer and developing targeted treatment strategies. Summary of the Invention

[0006] The object of the present invention is to provide a method for constructing a disease model based on intestinal epithelial-specific knockout of the Twist1 gene and its application in the precision treatment of inflammatory bowel disease, for studying the mechanism of action of Twist1 in intestinal inflammation and tumorigenesis, and providing new technical means and theoretical basis for the precision diagnosis and innovative treatment of inflammatory bowel disease and colorectal cancer associated with colitis.

[0007] To achieve the above object, the present invention provides the following technical solutions: A method for constructing a disease model based on intestinal epithelial-specific knockout of the Twist1 gene, comprising the following steps: (1) Design a guide RNA (gRNA) targeting the mouse Twist1 gene and a donor vector containing loxP sites; (2) Co-inject the gRNA, the donor vector containing loxP sites, and Cas9 mRNA into mouse fertilized eggs to generate F0 generation mice with the Twist1 gene (Twist1f / f) marked with loxP sites; (3) Mate the F0 generation mice with wild-type mice to obtain F1 generation Twist1f / f mice; (4) Mate the F1 generation Twist1f / f mice with transgenic mice expressing Cre recombinase driven by an intestinal epithelial-specific promoter to obtain transgenic mice with intestinal epithelial-specific knockout of the Twist1 gene (Twist1- / -); (5) Verify the knockout efficiency of the Twist1 gene in the obtained mouse intestinal epithelial tissue by PCR, sequencing, and protein expression analysis.

[0008] In a preferred embodiment of the present invention, the gRNA in step (1) targets the exon boundary region of the mouse Twist1 gene, and its length is 18-22 nucleotides, and the GC content is 40-60%.

[0009] In a preferred embodiment of the present invention, the concentration of Cas9 mRNA for injection in step (2) is 50-150 ng / μL, the concentration of gRNA is 25-75 ng / μL, and the concentration of the donor vector is 5-20 ng / μL.

[0010] In a preferred embodiment of the present invention, the intestinal epithelial-specific promoter in step (4) is the villin promoter, and the transgenic mouse is a pVillin-Cre mouse.

[0011] The present invention also provides a method for inducing an enteritis model in Twist1- / - mice and control Twist1f / f mice obtained by using dextran sulfate sodium (DSS), wherein the concentration of DSS is 1.5-3.5%, and the treatment time is 5-10 days; then the differences in disease activity index, pathological changes of colon tissue, and levels of inflammatory factors between Twist1- / - mice and control Twist1f / f mice are compared.

[0012] In a preferred embodiment of the present invention, the concentration of DSS is 2.5%, the treatment time is 7 days, and then ordinary drinking water is given for 3-5 days as the recovery period.

[0013] The present invention also provides a method for inducing a colitis-associated colorectal cancer model in Twist1- / - mice and control Twist1f / f mice obtained by using azoxymethane (AOM) combined with dextran sulfate sodium (DSS), wherein the dose of AOM is 7.5-12.5 mg / kg body weight, and the concentration of DSS is 1.5-2.5%; then the differences in tumor incidence, tumor number, tumor size, and histopathological characteristics between Twist1- / - mice and control Twist1f / f mice are compared.

[0014] In a preferred embodiment of the present invention, the dose of AOM is 10 mg / kg body weight, the concentration of DSS is 2.0%, the DSS treatment cycle is to recover for 14 days after 7 days of treatment, and a total of 3 cycles are carried out, and the total observation time is 12 weeks.

[0015] The present invention also provides the application of the transgenic mice with intestinal epithelial-specific knockout of Twist1 gene in the preparation of therapeutic drugs for inflammatory bowel disease and colitis-associated colorectal cancer, including the following steps: (1) administering a candidate drug to be screened to the transgenic mice; (2) detecting and comparing the disease activity index, histopathological changes, levels of inflammatory factors, and / or tumor characteristics of the mice before and after drug administration; (3) evaluating the therapeutic effect of the candidate drug according to the comparison results.

[0016] The present invention also provides the application of the transgenic mice with intestinal epithelial-specific knockout of Twist1 gene in studying the association between intestinal microecology and the pathogenesis of inflammatory bowel disease, including the following steps: (1) collecting fecal samples and intestinal contents of the transgenic mice and control mice; (2) analyzing the differences in the composition and function of intestinal flora between the two groups of mice by 16S rRNA sequencing and / or metagenomic sequencing; (3) comparing the differences in intestinal microbial metabolites between the two groups of mice by metabolomics analysis; (4) verifying the causal relationship between Twist1 gene and changes in intestinal flora through fecal microbiota transplantation experiments.

[0017] The present invention has the following beneficial effects: 1. A transgenic mouse model with intestinal epithelium-specific knockout of the Twist1 gene was successfully constructed, overcoming the limitation of embryonic lethality caused by systemic knockout of Twist1, and providing an ideal tool for studying the role of Twist1 in adult intestinal diseases; 2. It was demonstrated that Twist1 plays a promoting role in the development of DSS-induced inflammatory bowel disease and AOM / DSS-induced colitis-associated colorectal cancer. Twist1 knockout significantly reduced the disease severity and tumor incidence; 3. The interaction between Twist1 and the intestinal microbiota was revealed, providing a new perspective for understanding the association between intestinal inflammation and the microbiome; 4. It provided a theoretical basis and experimental foundation for developing treatment strategies for inflammatory bowel disease and colorectal cancer targeting Twist1 and its signaling pathway; 5. The constructed disease model has the characteristics of high reliability, stable phenotype, and strong specificity, and can be widely applied to basic research, drug screening, and the development of individualized treatment strategies. Brief Description of the Drawings

[0018] Figure 1 This is a schematic diagram of the construction strategy of the transgenic mouse with intestinal epithelium-specific knockout of the Twist1 gene according to the present invention, as well as the PCR verification results, marking the DNA molecular weight marker, Twist1f / f-like and Twist1- / - samples, the Western Blot verification results, marking the protein molecular weight marker, Twist1f / f sample, Twist1- / - sample and internal reference control, and the experimental result diagram for verifying the Twist1 gene knockout efficiency by using PCR and Western Blot methods.

[0019] Figure 2 This is a comparison diagram of the phenotypes of Twist1- / - mice and Twist1f / f mice in the DSS-induced acute colitis model. From top to bottom, from left to right are the schematic diagram of the experimental design, the body weight change curve, the disease activity index change curve, the comparison of colon lengths, and the deletion of intestinal epithelial Twist1 alleviates inflammatory bowel disease.

[0020] Figure 3 This is a histological HE staining diagram, and the deletion of intestinal epithelial Twist1 can partially inhibit the inflammation of the colorectal region in mice.

[0021] Figure 4 This is a comparison diagram of the phenotypes of Twist1- / - mice and Twist1f / f mice in the AOM / DSS-induced colitis-associated colorectal cancer model. Figure 4 A is the schematic diagram of the experimental design, Figure 4 B is the macroscopic photograph and statistical chart of the tumor formation situation, Figure 4C is a statistical table of tumor incidence rates, Figure 4 D is a histopathological analysis diagram of tumors.

[0022] Figure 5 It is a comparative analysis diagram of the intestinal flora compositions of Twist1- / - mice and Twist1f / f mice, Figure 5 A is an alpha diversity analysis, a box plot of the Shannon index, marking the Twist1f / f group (5-1) and the Twist1- / - group (5-2); Figure 5 B is a beta diversity PCoA analysis diagram, marking the sample clustering of the Twist1f / f group (5-3) and the Twist1- / - group (5-4); Figure 5 C is a stacked bar chart of the flora compositions at the phylum level, marking the main phylum categories (5-5); Figure 5 D is a heat map of the differential flora at the genus level, marking the upregulated genera (5-6) and the downregulated genera (5-7).

[0023] Figure 6 It is the treatment effect diagram of the Twist1-targeted inhibitor, Figure 6 A is a bar chart comparing the disease activity indices of different treatment groups, marking the control group (6-2), the DSS group (6-3), the low-dose group (6-4), and the high-dose group (6-5); Figure 6 B is a comparison of the histopathological scores, marking the score data of each group (6-6); Figure 6 C is a comparison diagram of the co-treatment effects of TW-17 and probiotics, marking the single-treatment group (6-7) and the combined-treatment group (6-8).

[0024] Figure 7 It is metagenomic sequencing. Specific implementation manners

[0025] The following combines the attached Figure 1-7 A detailed description of the preferred implementation manners of the present invention will be given. It should be understood that the preferred implementation manners described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

[0026] Example 1: Construction of a transgenic mouse model with intestinal epithelium-specific knockout of the Twist1 gene This example provides a method for constructing a transgenic mouse model with intestinal epithelium-specific knockout of the Twist1 gene by using the CRISPR / Cas9 technology and the tissue-specific Cre-loxP system.

[0027] 1.1 Materials and reagents Experimental animals: C57BL / 6 mice, purchased from Cyagen Biosciences Inc.; pVillin-Cre transgenic mice, purchased from Cyagen Biosciences Inc.

[0028] Main reagents: Cas9 mRNA (source: Thermo Fisher Scientific); PCR primers (synthesized by Sangon Biotech Co., Ltd.); genomic DNA extraction kit (source: QIAGEN); Western Blot related antibodies and reagents (source: Cell Signaling Technology).

[0029] 1.2 gRNA design and donor vector construction According to the mouse Twist1 gene sequence (GenBank accession number: NM_011658.2), two pairs of gRNAs were designed, targeting the intron regions at the 5' end of exon 1 and the 3' end of exon 2 of the Twist1 gene respectively.

[0030] gRNA1 sequence: 5'-AGCTCGAGACCTAGATGTCANNGG-3' (targeting upstream of exon 1); gRNA2 sequence: 5'-TGCATTGCATAGCCGTAGTCNNGG-3' (targeting downstream of exon 2); Through in vitro cleavage efficiency testing, gRNA combinations with a cleavage efficiency > 80% were selected for subsequent experiments.

[0031] Donor vector construction: The homologous arm length was designed to be 1000 bp, and loxP sites were inserted upstream of exon 1 and downstream of exon 2 respectively, while introducing a resistance screening marker. The homology between the homologous arm and the target sequence in the donor vector was > 99.5% to ensure efficient homologous recombination.

[0032] 1.3 Microinjection and obtaining F0 generation mice A mixture of Cas9 mRNA (100 ng / μL), two gRNAs (50 ng / μL each), and the donor vector (10 ng / μL) was microinjected into the pronuclei of C57BL / 6 mouse fertilized eggs. The injection volume per time was controlled at 1 - 2 pL. The injected fertilized eggs were transplanted into the uterus of pseudopregnant mice, and F0 generation mice were obtained after normal delivery.

[0033] 1.4 Genotype identification and screening Genomic DNA was extracted from the tail tips of F0 generation mice, and PCR primers spanning the loxP sites were designed for genotype identification. The primer sequences are as follows: Upstream loxP detection primer pair: Forward: 5'-CTGAGACCTAGCTTGCAGTTCAGG-3'; Reverse: 5'-GACTTCCTGTGCATAGGCAATGC-3'; Downstream loxP detection primer pair: Forward: 5'-ACCTAGCCTGCAGTTGTAGACCT-3'; Reverse: 5'-GCTACGATCTAGCTGACTGCAGT-3'; The PCR products were analyzed by agarose gel electrophoresis and verified by sequencing to confirm the correct insertion of the loxP site. The obtained positive F0 generation founder mice were backcrossed with wild-type C57BL / 6 mice to obtain F1 generation Twist1f / f mice.

[0034] 1.5 Obtaining and verifying intestinal epithelial-specific knockout mice The F1 generation Twist1f / f mice were mated with pVillin-Cre transgenic mice (expressing Cre recombinase driven by the villin promoter, specifically expressed in intestinal epithelial cells) to obtain transgenic mice with intestinal epithelial-specific knockout of the Twist1 gene (Twist1- / -).

[0035] At the same time, three genotypes of mice were obtained as control and experimental groups: 1) Twist1f / f;pVillin-Cre- (control group, i.e., Twist1f / f); 2) Twist1f / f;pVillin-Cre+ (knockout group, i.e., Twist1- / -); 3) Wild-type mice (wild-type control) 1.6 Verification of gene knockout efficiency The knockout efficiency of the Twist1 gene in intestinal epithelium was verified by the following methods: 1) Verification at the DNA level: Genomic DNA was extracted from various segments of the mouse intestine (duodenum, jejunum, ileum, colon) and other tissues (liver, kidney, spleen, etc.), and PCR detection was performed using specific primers. For Twist1- / - mice, the Twist1 gene knockout fragment should be detected in intestinal tissues, while the intact Twist1f / f genotype should be maintained in other tissues.

[0036] 2) Verification at the RNA level: Total RNA was extracted from various tissues of the mice, and the expression level of Twist1 mRNA was detected by qRT-PCR. The primers were designed to span the knockout region to ensure specific detection. The results showed that compared with Twist1f / f mice, the expression level of Twist1 mRNA in the intestinal epithelial tissues of Twist1- / - mice decreased by >90%, while there was no significant difference in the expression levels of other tissues.

[0037] 3) Verification at the protein level: The expression level of Twist1 protein was detected by Western Blot. Total proteins were extracted from various tissues of mice and detected using anti-Twist1 specific antibody. The results showed that compared with Twist1f / f mice, the expression of Twist1 protein in the intestinal epithelial tissues of Twist1- / - mice was decreased by >85%, while there was no significant change in the expression levels of other tissues.

[0038] Meanwhile, the distribution and expression intensity of Twist1 protein in intestinal tissues were detected by immunohistochemistry to further confirm the tissue specificity of the knockout effect.

[0039] 1.7 Analysis of the basic characteristics of mouse phenotypes The basic phenotypes of the obtained Twist1- / - mice were analyzed, including: 1) Growth and development: Indices such as the weight change and survival rate of mice after birth were recorded. The results showed that there were no significant differences between Twist1- / - mice and Twist1f / f mice in terms of growth curve, weight growth rate, survival rate, etc., indicating that intestinal epithelial-specific knockout of Twist1 did not affect the normal growth and development of mice.

[0040] 2) Evaluation of the basic state of the intestine: Indices such as intestinal length, intestinal wall thickness, and tissue structure were analyzed. The results showed that under the basic state, there were no obvious abnormalities in the intestinal morphology and tissue structure of Twist1- / - mice and Twist1f / f mice. The crypt structure was intact, the epithelial cells were arranged regularly, and the number of goblet cells was normal.

[0041] 3) Evaluation of intestinal function: Indices such as intestinal permeability and ion transport function were tested. Under the basic state, there were no obvious differences in the intestinal function parameters between the two groups of mice.

[0042] The experimental results showed that we successfully constructed a transgenic mouse model with intestinal epithelial-specific knockout of the Twist1 gene, and this model had the following characteristics: 1) High knockout efficiency, with the expression of Twist1 in intestinal epithelial tissues decreased by >85%; 2) Strong tissue specificity, only knocked out in intestinal epithelial cells; 3) Stable phenotype under the basic state, without affecting the normal development of mice and the basic functions of the intestine.

[0043] Example 2: Protective effect of Twist1 knockout in a DSS-induced acute colitis model This example studied the effect of intestinal epithelial-specific knockout of the Twist1 gene on DSS-induced acute colitis, aiming to clarify the role of Twist1 in the occurrence and development of intestinal inflammation.

[0044] 2.1 Experimental design Experimental animals: Twist1f / f mice and Twist1- / - mice at 8-10 weeks of age, weighing 20-25 g, with half males and half females. There were 12 mice in each group.

[0045] Experimental grouping: 1) Twist1f / f control group (drinking normal water); 2) Twist1- / - control group (drinking normal water); 3) Twist1f / f + DSS group (treated with 2.5% DSS); 4) Twist1- / - + DSS group (treated with 2.5% DSS); Experimental protocol: 2.5% DSS (molecular weight 36,000 - 50,000 Da, MP Biomedicals) was dissolved in drinking water and freely available for 7 days, and then switched back to normal drinking water for 3 days as the recovery period. The control groups were given normal drinking water throughout the experiment.

[0046] 2.2 Evaluation of disease activity index The body weight, fecal characteristics, and fecal occult blood / bloody stools of the mice were recorded daily, and the disease activity index (DAI) was calculated: 1) Body weight loss: <5% (0 points), 5 - 10% (1 point), 10 - 15% (2 points), 15 - 20% (3 points), >20% (4 points); 2) Fecal consistency: normal (0 points), soft stools (2 points), diarrhea (4 points); 3) Anal bleeding: none (0 points), occult blood (2 points), visible bloody stools (4 points); The total score ranges from 0 to 12 points, and the higher the score, the more severe the disease.

[0047] The experimental results showed that after DSS treatment, the body weight of the mice in the Twist1f / f + DSS group decreased significantly, with the maximum decrease reaching 25.8 ± 3.2%, while the degree of body weight loss in the Twist1- / - + DSS group was significantly reduced, with the maximum decrease being 14.6 ± 2.5% (p < 0.01).

[0048] In terms of the disease activity index, the DAI score of the Twist1f / f + DSS group gradually increased and reached a peak of 9.2 ± 1.5 on the 7th day; while the DAI score of the Twist1- / - + DSS group was significantly lower, with a peak of 5.8 ± 1.2 (p < 0.01), indicating that knockout of intestinal epithelial Twist1 significantly alleviated the symptoms of DSS-induced colitis.

[0049] 2.3 Intestinal morphology and histopathological analysis Mice were sacrificed at the experimental endpoint (day 10), and colonic tissues were collected. The colonic length was measured, and histopathological analysis was performed.

[0050] The results of colonic length measurement showed that compared with the control group, the colonic length in the Twist1f / f + DSS group was shortened by 30.5 ± 3.8%, while that in the Twist1− / − + DSS group was shortened by 15.3 ± 2.4% (p < 0.01), indicating that Twist1 knockout alleviated DSS-induced colonic shortening.

[0051] Histopathological analysis: After fixation, paraffin embedding, and sectioning (5 μm thickness) of colonic tissues, HE staining was performed. Scoring was independently carried out by two pathologists who were unaware of the grouping, and the scoring criteria were as follows: 1) Inflammatory infiltration: 0 - 4 points (0 = normal, 4 = severe inflammation); 2) Epithelial damage: 0 - 4 points (0 = normal, 4 = extensive ulceration); 3) Crypt structure: 0 - 4 points (0 = normal, 4 = complete loss); The total score was 0 - 12 points, and a higher score indicated more severe damage.

[0052] The results showed that the histopathological score of the Twist1f / f + DSS group was 9.5 ± 1.3, showing extensive epithelial damage, loss of crypt structure, and infiltration of inflammatory cells; while the score of the Twist1− / − + DSS group was significantly reduced to 5.2 ± 1.1 (p < 0.01), showing mild to moderate epithelial damage and inflammatory infiltration, and better preservation of the crypt structure, indicating that Twist1 knockout significantly alleviated DSS-induced colonic tissue damage.

[0053] 2.4 Analysis of inflammatory factor expression The expression levels of inflammatory factors in colonic tissues and serum, including IL-1β, IL-6, TNF-α, IL-17, and IL-10, were detected by qRT-PCR and ELISA methods.

[0054] The qRT-PCR results showed that compared with the Twist1f / f + DSS group, the mRNA expression levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α in colonic tissues of the Twist1− / − + DSS group were decreased by 62.3 ± 8.5%, 58.7 ± 7.3%, and 45.2 ± 6.8% respectively (all p < 0.01), while the expression of the anti-inflammatory factor IL-10 was increased by 85.4 ± 12.6% (p < 0.01).

[0055] The ELISA results were consistent with the qRT-PCR results, indicating that Twist1 knockout regulated the expression profile of inflammatory factors, reduced the levels of pro-inflammatory factors, increased the expression of anti-inflammatory factors, and thus alleviated the colonic inflammatory response.

[0056] 2.5 Intestinal barrier function assessment The intestinal barrier function was evaluated by multiple methods, including: 1) FITC-dextran permeability test: FITC-dextran (molecular weight 4000 Da, 600 mg / kg body weight) was administered orally. Serum was collected 4 hours later, and the FITC fluorescence intensity in the serum was measured to reflect intestinal permeability. The results showed that the serum FITC level in the Twist1f / f + DSS group was 4.8 ± 0.6 times that of the control group, while that in the Twist1- / - + DSS group was 2.3 ± 0.4 times that of the control group (p < 0.01), indicating that Twist1 knockout improved DSS-induced intestinal barrier dysfunction.

[0057] 2) Analysis of tight junction protein expression: The expression and distribution of tight junction proteins (Claudin-1, Occludin, ZO-1, etc.) were detected by Western Blot and immunofluorescence staining. The results showed that the expression level and membrane localization integrity of tight junction proteins in the Twist1- / - + DSS group were significantly higher than those in the Twist1f / f + DSS group, indicating that Twist1 knockout contributed to maintaining the integrity of the intestinal epithelial barrier.

[0058] In summary, the results of Example 2 showed that intestinal epithelial-specific knockout of the Twist1 gene had a significant protective effect on DSS-induced acute colitis, manifested as reducing the disease activity index, decreasing the histopathological injury score, regulating the balance of inflammatory factor expression, and improving intestinal barrier function. This result confirmed the promoting role of Twist1 in the occurrence and development of intestinal inflammation.

[0059] Example 3: Anticancer effect of Twist1 knockout in an AOM / DSS-induced colitis-associated colorectal cancer model Please refer to Figure 4 A-4D. In this example, the effect of intestinal epithelial-specific knockout of the Twist1 gene on AOM / DSS-induced colitis-associated colorectal cancer (CAC) was studied to clarify the role of Twist1 in inflammation-related tumorigenesis.

[0060] 3.1 Experimental design Experimental animals: 8-10-week-old Twist1f / f mice and Twist1- / - mice, weighing 20-25 g, with an equal number of males and females. There were 20 mice in each group.

[0061] Experimental grouping: 1) Twist1f / f control group; 2) Twist1- / - control group; 3) Twist1f / f + AOM / DSS group; 4) Twist1- / - + AOM / DSS group; AOM / DSS treatment protocol: 1) On the 1st day: Intraperitoneal injection of AOM (10 mg / kg body weight, Sigma-Aldrich); 2) On the 8th - 14th days: 2.0% DSS in drinking water (first cycle); 3) On the 15th - 35th days: Normal drinking water for recovery (14 days); 4) On the 36th - 42nd days: 2.0% DSS in drinking water (second cycle); 5) On the 43rd - 63rd days: Normal drinking water for recovery (14 days); 6) On the 64th - 70th days: 2.0% DSS in drinking water (third cycle); 7) On the 71st - 84th days: Normal drinking water for recovery (14 days); 8) On the 85th day: Sacrifice the mice for analysis (total observation period: 12 weeks). During the experiment, the body weight changes and general conditions of the mice were monitored weekly.

[0062] 3.2 Tumor assessment At the end of the experiment (the 85th day), the mice were sacrificed, the colon tissues were collected, and the colon tumors were observed, counted, and measured under a dissecting microscope.

[0063] Macroscopic tumor assessment: 1) Tumor counting: Record the total number of tumors in the colon of each mouse; 2) Tumor size measurement: Measure the tumor diameter using a digital caliper, divided into four grades: <1 mm, 1 - 2 mm, 2 - 3 mm, >3 mm; 3) Tumor burden calculation: The sum of the volumes of all tumors, volume = length × width² × 0.5; 4) Tumor formation rate: Number of mice with tumors / total number of mice × 100%; The results showed that the tumor formation rate in the Twist1f / f + AOM / DSS group reached 40% (8 / 20), the average number of tumors was 4.5 ± 1.2 tumors / mouse, and the tumor diameters were mainly distributed between 2 - 3 mm, presenting obvious intestinal tumors; while the tumor formation rate in the Twist1- / - + AOM / DSS group was only 5% (1 / 20), the number of tumors was significantly reduced, being 1.2 ± 0.5 tumors / mouse (p < 0.01), and the tumor diameters were mainly < 2 mm, indicating that Twist1 knockout significantly inhibited AOM / DSS-induced colorectal tumorigenesis.

[0064] 3.3 Histopathological analysis The colon tissues were fixed, paraffin-embedded, sectioned, and then subjected to HE staining and immunohistochemical analysis.

[0065] Pathological analysis by HE staining: The tumors were graded by a pathologist according to WHO criteria: low-grade intraepithelial neoplasia, high-grade intraepithelial neoplasia, and invasive adenocarcinoma.

[0066] The results showed that the tumors in the Twist1f / f + AOM / DSS group mainly presented high-grade intraepithelial neoplasia and invasive adenocarcinoma, with disordered cell arrangement and obvious nuclear atypia, while the tumors in the Twist1- / - + AOM / DSS group mainly presented low-grade intraepithelial neoplasia, with relatively regular cell arrangement and lower nuclear atypia, indicating that Twist1 knockout not only reduced the number of tumors but also decreased the malignancy of the tumors.

[0067] Immunohistochemical analysis: The expressions of the proliferation marker Ki67, the apoptosis marker Cleaved-Caspase3, and the tumor-related molecule β-catenin were detected.

[0068] The results showed that in the tumor tissues of the Twist1- / - + AOM / DSS group compared with the Twist1f / f + AOM / DSS group: 1) The proportion of Ki67-positive cells decreased by 45.3 ± 6.8% (p < 0.01), indicating a decrease in cell proliferation activity; 2) The proportion of Cleaved-Caspase3-positive cells increased by 72.4 ± 9.5% (p < 0.01), indicating an increase in cell apoptosis; 3) The nuclear localization of β-catenin was significantly reduced, indicating a decrease in the activation of the Wnt signaling pathway; 3.4 Molecular mechanism study To explore the molecular mechanism by which Twist1 promotes colitis-associated colorectal cancer, the following experiments were conducted: 1) Analysis of the Wnt / β-catenin signaling pathway: The expressions of components of the Wnt signaling pathway (β-catenin, GSK-3β, APC, etc.) and downstream target genes (Cyclin D1, c-Myc, etc.) were detected by Western Blot and qRT-PCR.

[0069] The results showed that in the tumor tissues of the Twist1- / - + AOM / DSS group, the nuclear localization and total protein level of β-catenin decreased, the phosphorylation level of GSK-3β decreased, and the expressions of Wnt downstream target genes (Cyclin D1, c-Myc) were significantly decreased, indicating that Twist1 knockout inhibited the abnormal activation of the Wnt signaling pathway.

[0070] 2) Analysis of inflammation-related signaling pathways: The activation status of the NF-κB and STAT3 signaling pathways and the expressions of downstream inflammatory factors were detected.

[0071] The results showed that in the tumor tissues of the Twist1- / - + AOM / DSS group, the phosphorylation levels of NF-κB and STAT3 decreased, and the expressions of inflammatory factors (IL-6, TNF-α, IL-1β, etc.) decreased, indicating that Twist1 knockout inhibited the activation of inflammation-related signaling pathways.

[0072] 3) Analysis of epithelial-mesenchymal transition (EMT) markers: The expressions of EMT-related molecules (E-cadherin, Vimentin, N-cadherin, etc.) were detected.

[0073] The results showed that in the tumor tissues of the Twist1- / - + AOM / DSS group, the expression of the epithelial marker E-cadherin increased, and the expressions of the mesenchymal markers Vimentin and N-cadherin decreased, indicating that Twist1 knockout inhibited the EMT process, which might be one of the mechanisms for its inhibition of tumor progression.

[0074] Figure 4 A shows the schematic diagram of the construction plan for intestinal epithelial-specific knockout of Twist1 (Twist1- / -) mice. It was found by Western blot (WB) detection that the expression of Twist1 in the intestinal tissues of Twist1- / - transgenic mice was downregulated ( Figure 4 A). In contrast, AOM / DSS was used to induce CAC in Twist1f / f and Twist1- / - mice. Compared with Twist1f / f mice, the number of colorectal tumors in Twist1- / - mice was significantly reduced, and the tumor volume was also smaller (p < 0.05) ( Figure 4 B). The tumor formation rate of Twist1f / f mice reached 40%, while that of Twist1- / - mice was only 5% ( Figure 4C). Hematoxylin-eosin (HE) staining showed that compared with Twist1f / f mice, the colorectal glands formed in Twist1- / - mice were regularly arranged and had lower nuclear atypia ( Figure 4 D). The above experimental results indicated that Twist1 promoted tumorigenesis of CAC in mice.

[0075] In summary, the results of Example 3 showed that intestinal epithelial-specific knockout of the Twist1 gene significantly inhibited the occurrence and development of AOM / DSS-induced colitis-associated colorectal cancer, manifested as a decrease in the tumor incidence rate (from 40% to 5%), a reduction in the number and size of tumors, and a decrease in the malignancy of tumors. Mechanistic studies showed that Twist1 knockout exerted an anti-tumor effect by inhibiting the Wnt / β-catenin signaling pathway, weakening the activation of the inflammatory signaling pathway, and inhibiting the EMT process. This result confirmed the promoting role of Twist1 in the occurrence and development of inflammation-related colorectal cancer.

[0076] Example 4: Study on the effect of Twist1 knockout on the intestinal microecology Please refer to Figure 5 A-5D. In this example, the effects of intestinal epithelial-specific knockout of the Twist1 gene on the composition and function of the intestinal flora were studied to clarify the mechanism by which Twist1 participates in the pathogenesis of inflammatory bowel disease by regulating the intestinal microecology.

[0077] 4.1 Experimental design Experimental animals: 8-10-week-old Twist1f / f mice and Twist1- / - mice, with an equal number of males and females. There were 10 mice in each group.

[0078] Experimental grouping and sample collection: 1) Normal state group: Samples of Twist1f / f and Twist1- / - mice in the normal state; 2) DSS treatment group: Samples of Twist1f / f and Twist1- / - mice after treatment with 2.5% DSS for 7 days; Types of samples collected: 1) Fecal samples: Fresh feces were directly collected and quickly frozen and stored at -80°C; 2) Colonic contents: After sacrificing the mice, the colonic contents were aseptically collected and stored at -80°C; 3) Colonic tissues: Used for histological and gene expression analysis 4.2 16S rRNA sequencing analysis of the intestinal flora composition Total DNA was extracted from feces and colonic contents, and the V3-V4 region of 16S rRNA was amplified and sequenced by high-throughput sequencing using standard methods.

[0079] 1) Alpha diversity analysis: The Shannon index, Chao1 index, and Simpson index were used to evaluate the microbial diversity.

[0080] The results showed that under normal conditions, the alpha diversity of the microbiota in Twist1- / - mice (Shannon index: 4.8 ± 0.3) was higher than that in Twist1f / f mice (Shannon index: 4.2 ± 0.2) (p < 0.05); after DSS treatment, the degree of decrease in the microbial diversity of Twist1- / - mice (decrease of 20.8 ± 3.2%) was significantly less than that in Twist1f / f mice (decrease of 45.2 ± 5.6%) (p < 0.01), indicating that Twist1 knockout helps maintain the diversity of the gut microbiota, especially under inflammatory conditions.

[0081] 2) Beta diversity analysis: Principal coordinate analysis (PCoA) was performed based on UniFrac distance and Bray-Curtis distance.

[0082] The results showed that under normal conditions, there were significant differences in the microbial community structure between Twist1- / - mice and Twist1f / f mice (PERMANOVA, p < 0.01); after DSS treatment, the differences between the two groups were further enlarged, indicating that the Twist1 gene affects the overall composition of the gut microbiota and has a regulatory effect on the changes in the microbiota under inflammatory conditions.

[0083] 3) Microbial composition analysis: The dominant and differential microbiota were analyzed at the phylum level and genus level.

[0084] The results showed that at the phylum level, compared with Twist1f / f mice, the relative abundance of Bacteroidetes in Twist1- / - mice increased (35.2 ± 3.8% vs. 28.7 ± 3.2%, p < 0.05), and the relative abundance of Proteobacteria decreased (8.3 ± 1.2% vs. 15.6 ± 2.4%, p < 0.01).

[0085] At the genus level, the relative abundances of beneficial bacteria such as Lactobacillus and Bifidobacterium in Twist1- / - mice increased, while the relative abundances of potential pathogenic bacteria such as Bacteroides and Proteus decreased.

[0086] After DSS treatment, the Proteobacteria phylum in Twist1f / f mice increased significantly, while this change was significantly alleviated in Twist1- / - mice, indicating that Twist1 knockout alleviated the dysbiosis of the microbiota under inflammatory conditions.

[0087] 4.3 Metagenomic sequencing for analyzing the functions of the microbiota The functions of the microbiota were analyzed by metagenomic sequencing (sequencing depth ≥ 10 Gb per sample).

[0088] 1) Functional genomic analysis: The composition of functional genes in the microbiota was analyzed based on the KEGG and COG databases.

[0089] The results showed that compared with Twist1f / f mice, the gene abundances related to carbohydrate metabolism, amino acid metabolism, and short-chain fatty acid production in the microbiota of Twist1- / - mice were significantly increased, while the gene abundances related to inflammatory responses and virulence factors were decreased, indicating that Twist1 knockout promoted the enhancement of beneficial metabolic pathways and the attenuation of potentially harmful pathways.

[0090] 2) Analysis of microbiota metabolic pathways: HUMAnN2 was used to analyze the abundances of microbiota metabolic pathways.

[0091] The results showed that the abundances of the synthetic pathways of short-chain fatty acids (such as butyrate and propionate), tryptophan metabolism, and bile acid conversion-related pathways in the microbiota of Twist1- / - mice were increased, indicating that Twist1 knockout promoted the production of beneficial metabolites.

[0092] 4.4 Metabolomics for analyzing microbial metabolites Microbial metabolites in intestinal contents were analyzed based on liquid chromatography-mass spectrometry (LC-MS / MS).

[0093] 1) Analysis of short-chain fatty acids: The contents of short-chain fatty acids in colonic contents were measured.

[0094] The results showed that compared with Twist1f / f mice, the concentration of butyrate in the colonic contents of Twist1- / - mice increased by 45.2 ± 6.3%, and the concentration of propionate increased by 32.6 ± 5.1% (both p < 0.01), indicating that Twist1 knockout promoted the production of short-chain fatty acids.

[0095] 2) Analysis of bile acid metabolites: The contents of primary and secondary bile acids were detected.

[0096] The results showed that the contents of secondary bile acids (such as deoxycholic acid and lithocholic acid) in Twist1- / - mice increased, indicating that Twist1 knockout affected bile acid metabolism, which may be related to the changes in the intestinal microbiota.

[0097] 4.5 Microbiota transplantation experiments to verify causal relationships To verify the protective effect of the gut microbiota changes caused by Twist1 knockout on inflammation, a fecal microbiota transplantation experiment was conducted.

[0098] Experimental protocol: 1) Collect fresh feces from Twist1f / f and Twist1- / - mice; 2) Pretreat wild-type recipient mice with an antibiotic cocktail (vancomycin, neomycin, metronidazole, amphotericin B) for 5 days to eliminate the original microbiota; 3) Two days after stopping the antibiotics, orally gavage the donor fecal suspension (once a day for 5 consecutive days); 4) Two weeks after microbiota reconstruction, induce colitis with 2.5% DSS; 5) Evaluate the inflammation indexes and microbiota composition of the recipient mice; The results showed that: 1) Recipient mice that received the microbiota of Twist1- / - mice showed milder colitis symptoms after DSS induction, and the degree of weight loss, disease activity index, and histopathological score were all significantly lower than those of recipient mice that received the microbiota of Twist1f / f mice (p<0.01); 2) 16S rRNA sequencing analysis confirmed that the recipient mice successfully reconstructed the characteristic microbiota composition of the corresponding donor; 3) Intestinal gene expression analysis showed that recipient mice that received the microbiota of Twist1- / - mice had lower expression levels of inflammatory factors and higher expression levels of barrier function-related genes in the intestinal tissues; These results indicate that the gut microbiota changes caused by Twist1 knockout have a protective effect on DSS-induced colitis, confirming the causal relationship among Twist1 - microbiota - inflammation.

[0099] In summary, the results of Example 4 indicate that intestinal epithelial-specific knockout of the Twist1 gene significantly affects the gut microbiota composition and function, promotes the increase of beneficial microbiota, inhibits the growth of potential pathogenic bacteria, and enhances the production of beneficial metabolites (such as short-chain fatty acids). These changes may be one of the important mechanisms by which Twist1 knockout alleviates intestinal inflammation.

[0100] Example 5: Development of a Twist1-based therapeutic intervention strategy Please refer to Figure 6 A-6C. Based on the aforementioned research results, this example developed and preliminarily verified a therapeutic intervention strategy targeting Twist1, providing new ideas for the precision treatment of inflammatory bowel disease and colorectal cancer.

[0101] 5.1 Screening of Twist1-targeted inhibitors Based on the Twist1 protein structure, potential Twist1 inhibitors were screened from a compound library by molecular docking and virtual screening methods.

[0102] 1) Primary screening: 50 potential candidates were initially screened from 10,000 small molecule compounds by computer-aided drug design; 2) In vitro verification: 5 effective inhibitors were screened using in vitro transcriptional activation inhibition experiments and protein interaction analysis; 3) Cell experiments: The activity and specificity of the inhibitors were verified in colon cancer cell lines and intestinal epithelial cells; Finally, the compound TW-17 was identified as a lead compound with an IC50 of 1.5 μM, highly specific for Twist1, and significantly weaker inhibitory effects on other bHLH family proteins.

[0103] Among them, TW-17 is [4-(1H-benzo[d]imidazol-5-ylmethyl)piperazin-1-yl](pyridin-3-yl)methanone, and its chemical structural formula is as follows:

[0104] Its preparation method is as follows:

[0105] Potassium carbonate (1.49 g) was added to a solution of 5-(bromomethyl)-1H-benzo[d]imidazole (211 mg) and (piperazin-1-yl)(pyridin-3-yl)methanone (210 mg) in DMF (20 ml), and then the mixture was stirred at room temperature for 2 hours. Saturated aqueous sodium bicarbonate was added to the reaction mixture, and then it was extracted with EtOAc. The organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent; chloroform:methanol = 20:1 (V / V)) to obtain a white to slightly yellow solid (250.4 mg), [4-(1H-benzo[d]imidazol-5-ylmethyl)piperazin-1-yl](pyridin-3-yl)methanone, with a yield of 78%.

[0106] The in vitro inhibitory activity of W-17 against Twist1, IC 50 is 1.5 μM, highly specific for the dimer formed by Twist1 and E47, while the inhibitory effects on other bHLH family transcription factors such as MyoD and NeuroD are significantly weaker (IC 50 > 25 μM).

[0107] In the colon cancer cell line HCT116, TW-17 can significantly inhibit the expression of Twist1 target genes at a concentration of 5 μM, reduce the migration and invasion ability of tumor cells, and enhance the sensitivity of cells to chemotherapeutic drugs.

[0108] This structure has good drug chemical properties and can be used as a lead compound for the development of Twist1-targeted inhibitors for the treatment of inflammatory bowel disease and colitis-related colorectal cancer.

[0109] 5.2 Verification of the effect of TW-17 in in vitro models 1) Colon cancer cell lines: In human colon cancer cell lines HCT116 and SW480, after treatment with TW-17 (5 μM) for 48 hours, Twist1 activity decreased by 60 - 75%, while cell proliferation (decreased by 45 - 55%) and migration ability (decreased by 50 - 60%) were inhibited, and apoptosis increased (increased by 3 - 4 times).

[0110] 2) Intestinal organoid culture: Intestinal epithelial cells were isolated from wild-type mice and intestinal organoids were cultured. Treatment with TW-17 (3 μM, 72 hours) significantly reduced Twist1 expression and activity, and also reduced the expression of inflammatory factors under inflammatory conditions (IL-1β / TNF-α stimulation).

[0111] 3) Intestinal epithelial barrier function under inflammatory conditions: In the Caco-2 cell monolayer model, treatment with TW-17 significantly improved the barrier dysfunction caused by inflammatory factor stimulation, manifested as an increase in transepithelial electrical resistance and a decrease in permeability.

[0112] 5.3 Verification of the therapeutic effect of TW-17 in a DSS-induced colitis model Experimental design: 1) Wild-type C57BL / 6 mice at 8 - 10 weeks of age were divided into 4 groups (12 mice in each group): Control group (normal drinking water + solvent); DSS group (2.5% DSS + solvent); Low-dose TW-17 group (2.5% DSS + TW-17 10 mg / kg / day); High-dose TW-17 group (2.5% DSS + TW-17 20 mg / kg / day); 2) 2.5% DSS was given for 7 days, and TW-17 or solvent was injected intraperitoneally at the same time 3) Evaluate the changes in disease activity index, colon length, histopathology, and molecular markers The results showed: 1) Compared with the DSS + solvent group, the disease activity index of the TW-17 treatment group was significantly decreased (the high-dose group decreased by 40.5 ± 5.8%, p < 0.01); 2) The degree of colon shortening in the TW-17 treatment group was alleviated (the high-dose group was alleviated by 35.6 ± 4.7%, p < 0.01); 3) The histopathological score of the TW-17 treatment group was decreased (the high-dose group decreased by 45.2 ± 6.5%, p < 0.01); 4) The activity of Twist1 and the expression of downstream target genes in the intestinal tissue of the TW-17 treatment group were decreased; 5) The expressions of inflammatory factors (IL-1β, IL-6, TNF-α) in the TW-17 treatment group were decreased, and the expression of anti-inflammatory factor (IL-10) was increased; Dose-dependent analysis showed that the therapeutic effect of high-dose TW-17 (20 mg / kg / day) was better than that of low-dose (10 mg / kg / day), indicating that TW-17 has a dose-dependent therapeutic effect on DSS-induced colitis.

[0113] 5.4 Verification of the preventive effect of TW-17 in the AOM / DSS colorectal cancer model Experimental design: 1) Wild-type C57BL / 6 mice at 8 - 10 weeks of age were divided into 3 groups (15 mice in each group): Control group (AOM / DSS + solvent); Prevention group (AOM / DSS + prophylactic administration of TW-17); Treatment group (AOM / DSS + therapeutic administration of TW-17); 2) Prevention group: From the start of AOM injection until the end of the experiment, TW-17 (20 mg / kg) was intraperitoneally injected daily; 3) Treatment group: TW-17 (20 mg / kg) injection was started after the end of the first DSS cycle; 4) The tumorigenesis and related indicators were evaluated according to the method in Example 3; The results showed: 1) Compared with the control group, the tumor incidence rate in the prevention group was significantly decreased (20% vs. 45%, p < 0.01); 2) The average number of tumors in the prevention group was decreased (1.8 ± 0.5 vs. 4.6 ± 1.3, p < 0.01); 3) The size of tumors in the prevention group was decreased and the malignancy was reduced; 4) The tumor indicators in the treatment group were improved, but the effect was not as good as that of the prevention group; 5) Molecular mechanism analysis showed that TW-17 exerted its anti-tumor effect by inhibiting Twist1 activity, downregulating the Wnt / β-catenin signaling pathway, attenuating the inflammatory response, and inhibiting the EMT process; These results indicated that targeted inhibition of Twist1 had the potential to prevent colitis-associated colorectal cancer, especially with better efficacy in early intervention.

[0114] 5.5 Synergistic therapeutic effect of TW-17 combined with probiotics Based on the interaction between Twist1 and the gut microbiota, the effect of combined treatment with TW-17 and probiotics was investigated.

[0115] Experimental design: 1) Mice with DSS-induced colitis were divided into 4 groups (10 mice per group): Control group (DSS + solvent); TW-17 group (DSS + TW-17 20 mg / kg / day); Probiotic group (DSS + probiotic mixture containing Lactobacillus and Bifidobacterium, 1×10 9 CFU / day); Combined treatment group (DSS + TW-17 + probiotics); 2) The treatment lasted for 14 days (7 days of DSS treatment + 7-day recovery period); 3) The disease activity index, colon histology, and changes in the gut microbiota were evaluated; The results showed that: 1) The use of TW-17 or probiotics alone significantly improved the symptoms of colitis; 2) The effect of the combined treatment group was significantly better than that of the single-drug treatment group, manifested as: The disease activity index decreased by 60.5 ± 7.2% (vs. single use of TW-17: 40.3 ± 5.6%; single use of probiotics: 35.2 ± 4.8%); The histopathological score decreased by 65.3 ± 8.1% (vs. single use of TW-17: 44.5 ± 6.3%; single use of probiotics: 38.6 ± 5.5%); The reduction in the expression of inflammatory factors and the improvement of barrier function were more obvious; 3) 16S rRNA sequencing analysis showed that the microbial community structure in the combined treatment group was closer to the healthy state, and the proportion of beneficial bacteria was higher; These results indicated that the combined strategy of targeting Twist1 and regulating the gut microbiota had a synergistic therapeutic effect, providing a new idea for the comprehensive treatment of inflammatory bowel disease.

[0116] In summary, the results of Example 5 indicate that the Twist1-based therapeutic intervention strategy shows good therapeutic effects in inflammatory bowel disease and colitis-associated colorectal cancer models, confirming the application value and translational potential of the present invention.

[0117] The method for constructing a disease model with intestinal epithelial-specific knockout of the Twist1 gene and its application provided by the present invention have the following remarkable advantages and beneficial effects: 1. Ideal research tool: A transgenic mouse model with intestinal epithelial-specific knockout of the Twist1 gene was successfully constructed, overcoming the limitation of embryonic lethality caused by systemic knockout of Twist1, and providing an ideal tool for studying the role of Twist1 in adult intestinal diseases. Under basal conditions, the mice grow and develop normally, and there are no obvious abnormalities in intestinal morphology and function, ensuring the reliability of the research results.

[0118] 2. Confirmation of the pro-inflammatory effect of Twist1: In the DSS-induced inflammatory bowel disease model, Twist1- / - mice showed significantly reduced inflammatory responses, with lower body weight loss, disease activity index, and histopathological scores than the control group, and the inflammatory cytokine expression profile shifted towards the anti-inflammatory direction, confirming the promoting effect of Twist1 in the occurrence and development of intestinal inflammation.

[0119] 3. Revelation of the pro-cancer effect of Twist1: In the AOM / DSS-induced colitis-associated colorectal cancer model, the tumor incidence of Twist1- / - mice was significantly reduced (5% vs. 40%), the number of tumors decreased, and the malignancy degree decreased, confirming the promoting effect of Twist1 in inflammation-related tumorigenesis.

[0120] 4. Elucidation of the interaction between Twist1 and the intestinal microbiota: Through 16S rRNA sequencing, metagenomic sequencing, and metabolomic analysis, it was revealed that Twist1 knockout promoted the increase of beneficial bacteria, the decrease of harmful bacteria, and the increase of beneficial metabolites such as short-chain fatty acids, and the causal role of this change in disease protection was confirmed through fecal microbiota transplantation experiments.

[0121] 5. Provision of new therapeutic targets: Based on the research results of Twist1, a small molecule inhibitor TW-17 targeting Twist1 was developed, and its therapeutic effects on inflammatory bowel disease and prevention of colorectal cancer were verified in vitro and in vivo models, providing new targets and ideas for precision treatment of diseases.

[0122] 6. New comprehensive treatment strategy: It was found that the combination of Twist1 inhibition and probiotics has a synergistic therapeutic effect, providing a scientific basis for multi-target and multi-level comprehensive treatment of inflammatory bowel disease and colorectal cancer.

[0123] 7. Wide application value: The technology and model of the present invention can be widely applied to: ① the field of basic research to study the mechanism of action of Twist1 and related molecules in intestinal diseases; ② the field of drug research and development to screen therapeutic drugs targeting Twist1 and its signaling pathway; ③ the field of personalized medicine to guide disease typing and precision treatment based on Twist1 expression and activity.

[0124] In summary, the disease model of intestinal epithelial-specific knockout of Twist1 gene and its application provided by the present invention not only clarify the role of Twist1 in intestinal inflammation and tumors at the molecular mechanism level, but also provide new technical means and theoretical basis for the precision diagnosis and innovative treatment of inflammatory bowel disease and colorectal cancer, with important scientific value and application prospects.

[0125] It should be noted that the above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for constructing a disease model based on intestinal epithelial-specific knockout of the Twist1 gene, characterized in that: The following steps are involved: (1) Designing guide RNA (gRNA) targeting the mouse Twist1 gene and a donor vector containing loxP sites; (2) co-injecting the gRNA, the donor vector containing the loxP site, and the Cas9 mRNA into mouse fertilized eggs to produce F0 generation mice containing the Twist1 gene marked with the loxP site (Twist1f / f); (3) F0 mice were mated with wild-type mice to obtain F1 Twist1f / f mice; (4) F1 generation Twist1f / f mice were mated with transgenic mice expressing Cre recombinase driven by an intestinal epithelium-specific promoter to obtain transgenic mice with intestinal epithelium-specific knockout of the Twist1 gene (Twist1- / -); (5) The knockout efficiency of the Twist1 gene in the mouse intestinal epithelial tissue was verified by PCR, sequencing, and protein expression analysis.

2. The method according to claim 1, characterized in that The gRNA in step (1) targets the exon boundary region of the mouse Twist1 gene, which has a length of 18-22 nucleotides and a GC content of 40-60%.

3. The method according to claim 1, characterized in that In step (2), the concentration of Cas9 mRNA for injection is 50-150 ng / μL, the concentration of gRNA is 25-75 ng / μL, and the concentration of donor vector is 5-20 ng / μL.

4. The method according to claim 1, characterized in that: In the step (4), the intestinal epithelial-specific promoter is the villin promoter, and the transgenic mice are pVillin-Cre mice.

5. The method according to claim 1, characterized in that The following steps are also included: (6) Dextran sulfate sodium (DSS) was used to induce enteritis models in Twist1- / - mice and control Twist1f / f mice, where the concentration of DSS was 1.5-3.5% and the treatment time was 5-10 days; (7) Compare the differences in disease activity index, colon tissue pathological changes, and inflammatory factor levels between Twist1- / - mice and the control group Twist1f / f mice.

6. The method according to claim 5, characterized in that The DSS concentration was 2.5%, and the treatment time was 7 days, followed by 3-5 days of normal drinking water as a recovery period.

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