Use of ILDR3 gene and / or ILDR3 protein in preparation of drugs for preventing and / or treating tumors, products for diagnosing tumors

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of ILDR3 gene and / or ILDR3 protein in preparation of medicines for preventing and / or treating tumors and products for diagnosing tumors. The application research finds that the ILDR3 gene and / or ILDR3 protein is lowly expressed in cancer tissues of colorectal cancer patients and colorectal cancer cells, and knocking out the ILDR3 gene promotes proliferation and migration and invasion of the colorectal cancer cells and tumor occurrence and development. Based on this, the ILDR3 gene and / or ILDR3 protein is overexpressed, so that good inhibition effect on proliferation and migration and invasion of the colorectal cancer cells and growth of subcutaneous tumors is achieved, and the purpose of inhibiting the colorectal cancer is achieved. The method of the application provides a new treatment target for prevention and / or treatment of the colorectal cancer, provides application of the ILDR3 gene as a target gene for preventing / treating / diagnosing the colorectal cancer, and provides a new strategy and direction for prevention and / or diagnosis and / or treatment of the colorectal cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of the ILDR3 gene and / or ILDR3 protein in the preparation of drugs for the prevention and / or treatment of tumors, and products for the diagnosis of tumors. Background Technology

[0002] Colorectal cancer (CC) is a common malignant tumor in the gastrointestinal tract. In the early stages, it may not have any symptoms. As the tumor grows, late-stage clinical manifestations include diarrhea, constipation, rectal bleeding, excessive gas, abdominal cramps, abdominal pain, and other symptoms. Its incidence and mortality rates are among the highest of all cancers, and the survival rate for patients diagnosed at an advanced stage is only 10%.

[0003] Currently, the conventional treatment methods for colorectal cancer in clinical practice mainly include surgery, radiotherapy, chemotherapy, and a combination of these. Surgical treatments such as lymph node dissection and colectomy not only carry certain surgical risks but also require adjuvant therapy after surgery, often accompanied by the risk of cancer metastasis. Chemotherapy, due to the toxic side effects of the drugs themselves, has limited efficacy, and during continuous drug treatment, cancer cells easily develop multidrug resistance, further reducing the effectiveness of anti-tumor therapy. Although using chemotherapy to assist surgery and radiotherapy can significantly improve the survival rate of colorectal cancer patients, the efficacy of chemotherapy is greatly limited due to the inherent low sensitivity of tumor cells to chemotherapy drugs and high dose-related toxicity. Furthermore, existing colorectal cancer treatments also suffer from problems such as non-specific targeting, poor solubility, low permeability, dose-dependent toxicity to normal tissues, and small tumor accumulation.

[0004] Therefore, developing new drugs for the treatment of colorectal cancer has become an important problem that urgently needs to be solved. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides the application of the ILDR3 gene and / or ILDR3 protein in the preparation of drugs for the prevention and / or treatment of tumors and products for the diagnosis of tumors, particularly relating to the application of the ILDR3 gene and / or ILDR3 protein in the preparation of drugs for the prevention and / or treatment of colorectal cancer and products for the diagnosis of colorectal cancer.

[0006] ILDR3 (Immunoglobulin-Like Domain Containing Receptor 3) is a type of tight junction protein between three cells, belonging to the Angulin family. ILDR3 is a single transmembrane protein, mainly expressed in the liver and intestine, but it is also expressed to varying degrees in other tissues.

[0007] The first aspect of the present invention is to provide the use of the ILDR3 gene and / or ILDR3 protein in the preparation of medicaments for the prevention and / or treatment of tumors.

[0008] The ILDR3 gene and / or ILDR3 protein described in this invention are widely present in humans and animals and have cross-species characteristics. Therefore, the application of the ILDR3 gene and / or ILDR3 protein described in this invention is not limited to humans, but also includes animals of other species that contain the ILDR3 gene and / or ILDR3 protein, such as cats, dogs, monkeys, and mice.

[0009] Preferably, the drug achieves the effect of preventing and / or treating tumors by inhibiting at least one of the clinical symptoms such as tumor cell proliferation, cell colony formation, cell invasion and metastasis, angiogenesis, and immune evasion.

[0010] Preferably, in the above-described applications provided by the present invention, the tumor includes at least one of rectal cancer, small bowel cancer, bladder cancer, skin cancer, and liver cancer.

[0011] Preferably, the tumor includes at least one of colorectal cancer and small bowel cancer.

[0012] A second aspect of the present invention is to provide an expression promoter for the ILDR3 gene and / or ILDR3 protein, wherein the expression promoter is a substance capable of enhancing the expression and / or activity and / or function of the ILDR3 gene and / or ILDR3 protein. Preferably, the expression promoter for the ILDR3 gene and / or ILDR3 protein comprises at least one of the following: a recombinant expression vector containing the ILDR3 gene or a gene fragment having more than 90% homology with the ILDR3 gene; a polypeptide, protein, nucleic acid and nucleic acid aptamer capable of upregulating the expression of the ILDR3 gene and / or ILDR3 protein; a naturally active substance; a biological agent; or a naturally / synthetically synthesized compound.

[0013] Preferably, the recombinant expression vector includes any one of the following: vector-based eukaryotic expression plasmids specifically targeting the ILDR3 gene and / or ILDR3 protein, adenovirus, adeno-associated virus, lentivirus, retrovirus, LNP liposome, microinjection technology, gene editing system elements, and homologous recombinant vectors.

[0014] As a further preferred embodiment, the ILDR3 gene and / or ILDR3 protein expression promoter targets and regulates the expression of the ILDR3 gene and / or ILDR3 protein by modulating signaling pathways.

[0015] A third aspect of the invention is the application of the expression promoter of the ILDR3 gene and / or ILDR3 protein, and more particularly, the application of the expression promoter of the ILDR3 gene and / or ILDR3 protein in the preparation of medicaments for the prevention and / or treatment of tumors.

[0016] Preferably, the application includes at least one of the following (A1)-(A3):

[0017] (A1) To prepare drugs for inhibiting tumor growth;

[0018] (A2) To prepare drugs for inhibiting the proliferation of tumor cells;

[0019] (A3) To prepare drugs for inhibiting tumor cell invasion and migration;

[0020] The tumor mentioned is either colorectal cancer or small bowel cancer.

[0021] This invention targets ILDR3 and found that overexpression of the ILDR3 gene and / or ILDR3 protein significantly inhibits tumor cell proliferation, particularly the enlargement of colorectal cancer tumors. In mice, overexpression of the ILDR3 gene and / or ILDR3 protein also significantly inhibits the subcutaneous tumor formation rate and growth rate of colorectal cancer cells, reducing the occurrence and development of orthotopic colorectal cancer in mice. Therefore, the ILDR3 gene and / or ILDR3 protein can be used as targets for the prevention and / or treatment and / or diagnosis of colorectal cancer, thus providing new targets for the prevention and / or treatment of colorectal cancer in humans and other animals, which has important theoretical and clinical significance.

[0022] A fourth aspect of the present invention is to provide a medicament comprising at least one of the above-mentioned ILDR3 gene and / or ILDR3 protein, and an ILDR3 gene and / or ILDR3 protein expression promoter, and using the latter as the main functional component of the medicament.

[0023] Preferably, the drug further includes pharmaceutically acceptable excipients, which include at least one of diluents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorbents, and lubricants. The drug is any dosage form selected from tablets, capsules, powders, pills, granules, solutions, suspensions, syrups, injections, suppositories, inhalers, and sprays.

[0024] As a further preferred embodiment, the content of the functional component in the drug is 1%-99%, that is, the main functional component added to the drug (at least one of ILDR3 gene, ILDR3 protein, ILDR3 gene and / or ILDR3 protein expression promoter) accounts for 1%-99% of the total weight of the drug.

[0025] A fifth aspect of the present invention is the use of a reagent for detecting the expression levels of the ILDR3 gene and / or ILDR3 protein in the preparation of a product for diagnosing colorectal cancer, said product comprising any one of a chip, a diagnostic reagent, or a diagnostic kit; wherein the test sample for said product is any one of cells, tissue, excrement, or serum.

[0026] Preferably, the product contains an antibody that specifically binds to the ILDR3 protein or a peptide of the ILDR3 protein, or primers that specifically amplify the ILDR3 gene, or probes that specifically detect the ILDR3 gene.

[0027] As a further preferred option, the product is used to detect the expression levels of the ILDR3 gene and / or ILDR3 protein. When the expression levels of the ILDR3 gene and / or ILDR3 protein are downregulated, colorectal cancer is diagnosed.

[0028] The beneficial effects of this invention are as follows:

[0029] (1) This invention provides the application of ILDR3 gene and / or ILDR3 protein in regulating the progression of tumors, especially colorectal cancer. Overexpression of ILDR3 gene and / or ILDR3 protein has a significant inhibitory effect on the growth of colorectal cancer tumor cells. Therefore, ILDR3 gene and / or ILDR3 protein can be used as a target for the treatment of colorectal cancer and applied to the prevention and / or treatment of colorectal cancer, thereby providing new strategies and directions for the prevention and treatment of colorectal cancer.

[0030] (2) This invention provides an overexpression virus that specifically targets the ILDR3 gene, which can efficiently enhance the expression of the ILDR3 gene in cells, thereby helping to inhibit the growth of colorectal cancer tumors;

[0031] (3) The present invention provides an application of a reagent for detecting the expression level of ILDR3 gene and / or ILDR3 protein in the preparation of products for diagnosing colorectal cancer. That is, the expression level of ILDR3 gene and / or ILDR3 protein is used as a biomolecular marker for the detection of colorectal cancer diagnostic products. The product is used to detect the expression level of ILDR3 gene and / or ILDR3 protein. When the expression level of ILDR3 gene and / or ILDR3 protein is downregulated, colorectal cancer is diagnosed. Attached Figure Description

[0032] Figure 1 The image shows the qRT-PCR results of ILDR3 gene expression in 24 pairs of colorectal cancer tissues and adjacent normal tissues.

[0033] Figure 2 The images show the immunohistochemical staining results of colorectal tumor tissue and corresponding adjacent normal tissue microarrays. In particular, 2a shows the immunohistochemical detection results of ILDR3 protein expression level in colorectal cancer tissue and adjacent normal tissue, and 2b shows the immunohistochemical analysis results of ILDR3 protein expression in colorectal cancer tissue and adjacent normal tissue.

[0034] Figure 3 Figure 3a shows the results of detecting the expression levels of ILDR3 protein and ILDR3 gene in normal human intestinal epithelial cells and colorectal cancer cell lines. 3b shows the results of detecting the expression level of ILDR3 protein and ILDR3 gene.

[0035] Figure 4 The figure shows the results of qRT-PCR detection of ILDR3 gene expression levels in Caco2 and HCT116 colorectal cancer cell lines after knockdown and overexpression. In the figure, 4a shows the ILDR3 gene expression level in Caco2 cells after knockdown and overexpression, and 4b shows the ILDR3 gene expression level in HCT116 cells after knockdown and overexpression.

[0036] Figure 5 The figure shows the results of the CCK-8 assay to detect the effect of ILDR3 expression level on the proliferation of colorectal cancer cell lines Caco2 and HCT116. In the figure, 5a shows the effect of ILDR3 expression level on the proliferation of Caco2 cells, and 5b shows the effect of ILDR3 expression level on the proliferation of HCT116 cells.

[0037] Figure 6 Figure 6a shows the effect of ILDR3 expression level on the clonogenic ability of colorectal cancer cell lines Caco2 and HCT116, and figure 6b shows the effect of ILDR3 expression level on the clonogenic ability of Caco2 cells and figure 6b on the effect of ILDR3 expression level on the clonogenic ability of HCT116 cells.

[0038] Figure 7 Figure showing the results of Transwell migration assays to detect the effect of ILDR3 expression levels on the migration ability of colorectal cancer cells;

[0039] Figure 8Figure showing the effect of ILDR3 expression level on the invasive ability of colorectal cancer cells as detected by Transwell invasion assay;

[0040] Figure 9 The procedure for constructing the AOM+DSS colon cancer model in ILDR3 gene knockout mice and the in situ tumor growth were presented. Among them, 9a shows the experimental results of weight change in the two groups of mice, 9b shows the size of the colon tumors in the two groups of mice, 9c shows the number of colon tumors in the two groups of mice, and 9d shows the HE staining results of the colon tissues in the two groups of mice.

[0041] Figure 10 Figure 10a shows the results of the spontaneous colorectal cancer tumor model experiment in ILDR3 double gene knockout mice. In this figure, 10a shows the size of the colonic tissue tumors in the two groups of mice, 10b shows the number of colonic tissue tumors in the mice, and 10c shows the HE staining results of the colonic tissues in the two groups of mice.

[0042] Figure 11 The growth of subcutaneous xenografts in nude mice in different groups is shown in Figure 11a, where 11b shows photographs of subcutaneous xenografts in different groups, and 11c shows the weight statistics of subcutaneous xenografts in different groups. Detailed Implementation

[0043] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0044] Example 1: Screening and discovery process of key colorectal cancer gene ILDR3 or protein ILDR3.

[0045] The 36 pairs of colorectal cancer tissue microarray samples and 24 pairs of colorectal cancer tissue cDNA microarray samples used in this embodiment were purchased from Shanghai Xinchao Biotechnology Co., Ltd. (product codes are HCollA060CS02 and cDNA-HColA060CS03, respectively).

[0046] (1) Expression of ILDR3 gene or ILDR3 protein in colon tissue of colorectal cancer patients

[0047] Immunohistochemical and real-time quantitative PCR experiments were performed on colorectal cancer tissue microarray samples and colorectal cancer tissue cDNA microarray samples. The specific procedures were performed according to the microarray instructions. The qRT-PCR results of cDNA microarrays from colorectal cancer patients' colon tumor tissues and corresponding adjacent normal tissues are attached. Figure 1 As shown in the attached figure, the immunohistochemical staining results of colon tumor tissue and corresponding adjacent normal tissue microarrays are as follows. Figure 2Among them, 2a shows the immunohistochemical detection results of ILDR3 protein expression level in colorectal cancer tissue and adjacent normal tissue, and 2b shows the immunohistochemical analysis results of ILDR3 protein expression in 36 pairs of colorectal cancer tissue and adjacent normal tissue.

[0048] Figure 1 The results showed that in all 24 patients, the expression of ILDR3 mRNA in tumor tissue was significantly lower than that in adjacent normal tissue.

[0049] Figure 2 It can also be seen that ILDR3 protein is mainly expressed on the cell membrane of normal intestinal tissue, while the epithelial cells of colon tumor tissue lose their columnar shape, become round, are arranged in a disordered manner, and have glandular hyperplasia, and the expression level of ILDR3 protein on the cell membrane is significantly reduced.

[0050] (2) Expression of ILDR3 protein in colorectal cancer cells and normal intestinal epithelial cells

[0051] Cell selection and culture: Human normal intestinal epithelial cell line HIEC-6, colorectal cancer cell lines Caco2 and HCT116 were cultured. HIEC-6 and HCT116 cells were routinely cultured in DMEM cell culture medium containing 10% fetal bovine serum, 1% penicillin and streptomycin. Caco2 cells were routinely cultured in DMEM / F12 cell culture medium containing 10% fetal bovine serum, 1% penicillin and streptomycin. The cells were cultured in an incubator at 37°C and 5% CO2. The medium was changed every 2 days and the cells were passaged at a ratio of 1:3.

[0052] The expression of ILDR3 protein in human colorectal cancer cells and normal intestinal epithelial cells was detected by Western blotting and qRT-PCR.

[0053] The expression levels of ILDR3 protein in human normal intestinal epithelial cell line (HIEC-6) and human colorectal cancer cell lines (Caco2, HCT116) are as follows: Figure 3 As shown.

[0054] As shown in 3a, the expression level of ILDR3 protein in human colorectal cancer cell lines Caco2 and HCT116 was generally lower than that in human normal intestinal epithelial cell line HIEC-6, and the difference was statistically significant.

[0055] In addition, the results of qRT-PCR detection of ILDR3 gene expression are shown in 3b. As can be seen from the figure, the expression level of ILDR3 gene in human colorectal cancer cell lines Caco2 and HCT116 is generally lower than that in human normal intestinal epithelial cell line HIEC-6, and the difference is statistically significant.

[0056] As can be seen above, the expression levels of the ILDR3 gene and / or ILDR3 protein are associated with the occurrence of colorectal cancer. The expression levels of both the ILDR3 gene and ILDR3 protein are significantly downregulated in colorectal cancer cells. Therefore, they can serve as molecular markers for colorectal cancer and be used for the clinical auxiliary diagnosis of colorectal cancer.

[0057] Example 2: Effects of ILDR3 on the proliferation, colony formation, migration, and invasion of colorectal cancer cells.

[0058] (1) Construction and identification of ILDR3 gene overexpression plasmid

[0059] Human colorectal cancer cell lines Caco2 and HCT116 were selected as ILDR3 overexpressing cell lines for research, and human embryonic kidney cells 293T were used as tool cells for lentiviral packaging.

[0060] The ILDR3 gene sequence was obtained from NCBI, synthesized by Sangon Biotech Inc., and then constructed into the mammalian cell overexpression vector PQCXIP.

[0061] Construction of ILDR3-overexpressing colorectal cancer cells: 293T cells were seeded into 15cm culture dishes and cultured overnight until the cell density reached approximately 70%-80% the next day. Before transfection, the culture medium in the dishes was replaced with 20 mL of fresh culture medium. The lentivirus overexpression packaging system was prepared in sequence. For one 15cm culture dish to be transfected, the following transfection reagents were prepared: Solution A: 500 μL DMEM medium, 10 μg PQCXIP-ILDR3 overexpression plasmid / control plasmid, 10 μg packaging plasmid pMDlg / pRRE; 10 μg packaging plasmid pRSV-Rev and 10 μg envelope plasmid pMD2.G; Solution B: 500 μL DMEM medium, 120 μL PEI transfection reagent; After incubating solutions A and B at room temperature for 5 min, solution B was mixed with solution A and incubated at room temperature for 20 min. The mixture was then evenly added to the corresponding culture dishes and cultured at 37℃ in a 5% CO2 incubator for 12 minutes. After h, replace with fresh culture medium and continue culturing in the incubator for 48 h. Then collect the culture supernatant and centrifuge to concentrate the virus, i.e., ILDR3 overexpressing lentivirus.

[0062] Lentiviral infection of colorectal cancer cells: Appropriate amounts of Caco2 and HCT116 cells were seeded into 6-well plates and cultured overnight until the cell density reached approximately 70%-80% the next day. The old culture medium was discarded, and 2 mL of fresh culture medium was added along with 0.1 mL of the previously packaged ILDR3 overexpressing lentivirus. The mixture was gently stirred and incubated at 37°C with 5% CO2 for 12 h. The old culture medium was discarded, and 2 mL of fresh culture medium was added. The cells were then incubated at 37°C with 5% CO2 for 36 h. 1 μg / mL puromycin was added to screen for positive cells. After 2-3 days, all uninfected cells were killed, leaving positive cells, which were the ILDR3 overexpressing cells. Following the same viral packaging procedure, the PQCXIP empty vector plasmid was transferred into the tool cells, and the cells were then infected and screened. The remaining positive cells were the control group cells.

[0063] (2) ILDR3 gene knockout

[0064] Human colorectal cancer cell lines Caco2 and HCT116 were selected as ILDR3 overexpressing cell lines for the study, and human embryonic kidney cells 293T were used as tool cells for lentivirus packaging. The cell culture method was the same as above.

[0065] shRNA design and vector construction: The shRNA targeting ILDR3 was synthesized by Sangon Biotech Co., Ltd., wherein the nucleotide sequence of the shRNA sense strand SEQ ID NO.1 is: TTTGAAGGAACACTGATGA; and the nucleotide sequence of the shRNA antisense strand SEQ ID NO.2 is: TCATCAGTGTTCCTTCAAA.

[0066] Take 5 μg of pSIREN-RetroQ vector, add 1 μL each of restriction endonucleases BamH1 and EcoR1, and incubate at 37℃ for 3 h. Mix the digested pSIREN-RetroQ vector with 6x DNA loading buffer and add it to a 1% agarose gel. After electrophoresis at 180V for 25 min, cut the gel and recover the 6.4 Kb of the cleaved vector. Mix the corresponding shRNA sense and antisense strands according to the following system. Set the annealing program in the PCR instrument: 37℃ for 30 min, 95℃ for 5 min, 90℃ for 1 min, and 85℃ for 1 min for annealing and recombination. The contents are: 2 μL sense oligonucleotide chain (F, 100 μM), 2 μL antisense oligonucleotide chain (R, 100 μM), 16 μL ddH2O, and a total volume of 20 μL.

[0067] The gel-cleaved fragments and annealed nucleotide recombinant fragments were ligated using T4 Ligase. The ligated shRNA was added to competent cells, incubated on ice for 30 min, heat-shocked at 42°C for 90 s, and then 500 μL of LB medium was added. The cells were cultured on a shaker at 37°C for 1 h. The culture was then spread onto ampicillin-resistant LB plates and incubated overnight at 37°C. Single clones were picked and added to 500 μL of ampicillin-resistant LB liquid medium and cultured on a shaker at 37°C for 12 h. A portion of the bacterial culture was taken for sequencing. The sequencing results were compared to identify the correct clones. The vector of the correct clones was extracted using the Tiangen Micrograft Medium Plasmid Extraction Kit, which is the ILDR3 gene knockout plasmid, for subsequent lentivirus packaging.

[0068] The construction and viral packaging of ILDR3 knockout colorectal cancer cells, followed by lentivirus infection of colorectal cancer cells, were carried out using the same steps as above.

[0069] The expression results of ILDR3 mRNA in Caco2 and HCT116 cells are shown in the appendix. Figure 4 As shown.

[0070] Among them, 4a shows the results of qRT-PCR detection of ILDR3 gene expression level after knockdown and overexpression in Caco2 colorectal cancer cell line, and 4b shows the results of qRT-PCR detection of ILDR3 gene expression level after knockdown and overexpression in HCT116 colorectal cancer cell line.

[0071] Figure 4 The results showed that the expression level of ILDR3 mRNA was significantly increased in both cell lines (Caco2 and HCT116) after infection with the overexpression virus and selection, while the expression level of ILDR3 mRNA was significantly decreased in both cell lines (Caco2 and HCT116) after infection with the knockdown virus and selection. It can be seen that ILDR3 overexpression cell lines and ILDR3 knockdown cell lines were successfully constructed in both cell lines.

[0072] The results of the CCK8 assay for cell proliferation are attached. Figure 5 As shown, 5a presents the results of the CCK-8 assay on the effect of ILDR3 expression level on the proliferation ability of colorectal cancer cell line Caco2; 5b presents the results of the CCK-8 assay on the effect of ILDR3 expression level on the proliferation ability of colorectal cancer cell line HCT116.

[0073] Figure 5 The results showed that, compared with the control group, the proliferation of the two types of colorectal cancer cells with ILDR3 knockdown was significantly enhanced, while the proliferation of colorectal cancer cells with ILDR3 overexpression was significantly inhibited.

[0074] The plate colony formation assay was used to detect the effect of ILDR3 on the clonogenic ability of colorectal cancer cells. The experimental results are as follows: Figure 6 As shown, Figure 6 In the table, 6a shows the results of the plate colony assay to detect the effect of ILDR3 expression level on the colony-forming ability of colorectal cancer cell line Caco2, and 6b shows the results of the plate colony assay to detect the effect of ILDR3 expression level on the colony-forming ability of colorectal cancer cell line HCT116.

[0075] Depend on Figure 6 It can be seen that, compared with the control group cells, the colony-forming ability of the two types of colorectal cancer cells with ILDR3 knockdown was significantly enhanced, while the colony-forming ability of colorectal cancer cells with ILDR3 overexpression was significantly inhibited.

[0076] Cell invasion assays were performed to detect the invasive ability of ILDR3 against colorectal cancer cells. The results are as follows: Figure 7 As shown.

[0077] Depend on Figure 7 It can be seen that, compared with the control group cells, the two types of colorectal cancer cells with ILDR3 knockdown had significantly enhanced invasive ability, while the invasive ability of colorectal cancer cells with ILDR3 overexpression was significantly inhibited.

[0078] Cell migration assays were used to detect the effect of ILDR3 on the cell migration ability of colorectal cancer cells. The experimental results are as follows: Figure 8 As shown. By Figure 8 It can be seen that, compared with the control group cells, the migration ability of the two types of colorectal cancer cells with ILDR3 knockdown was significantly enhanced, while the migration ability of colorectal cancer cells with ILDR3 overexpression was significantly inhibited.

[0079] Example 3: Effect of ILDR3 gene knockout on the ability of colorectal cancer to form in situ tumors in AOM-DSS model

[0080] To investigate the role of ILDR3 in the progression from colitis to colorectal cancer, a mouse model of colorectal cancer induced by the combined effects of AOM and DSS was constructed. The specific modeling process is as follows:

[0081] Male control mice aged 6-8 weeks and ILDR3 gene knockout mice (n=20 per group) were selected and injected intraperitoneally with AOM (10 mg / kg) at week 0.

[0082] Mice were fed sterilized water containing 1.75% DSS at weeks 1, 4, and 7, for one week each time, with a two-week interval between feedings and normal drinking water, to establish a colorectal cancer model induced by enteritis. Specific steps are as follows: Figure 9 As shown, the mice were weighed weekly, and the tumor growth was observed at the end of the week.

[0083] Figure 9 The procedure for constructing the AOM+DSS colon cancer model and the experimental results regarding mouse weight changes and tumor development are shown. Figure 9a shows that compared to the ILDR3 control group, the ILDR3 knockout group mice experienced significant weight loss during the modeling process, but their colorectal tumors grew more rapidly and were larger, as shown in Figure 9b. (The proportions of large, medium, and small tumors in the colon tissue of the ILDR3 knockout group mice were 40%, 33%, and 27%, respectively, while in the ILDR3 control group mice, these proportions were 28%, 20%, and 52%, respectively), and the number of tumors was also greater (as shown in Figure 9c). HE staining of the colon tissues of both groups of mice revealed more pronounced histological disorder in the ILDR3 knockout group mice, with destroyed crypt structures exhibiting loss of polarity and branching deformities, along with a large number of atypical cells (as shown in Figure 9d).

[0084] The above experimental results preliminarily indicate that the ILDR3 gene and / or protein can significantly inhibit the occurrence and development of colorectal cancer induced by "AOM+DSS".

[0085] Example 4: Effect of ILDR3 gene knockout on spontaneous colorectal cancer in situ tumor formation in mice

[0086] To further confirm the important role of ILDR3 in the progression of colorectal cancer, a spontaneous mouse model of colorectal cancer was constructed. The specific modeling process is as follows:

[0087] APC Min / + Mice were crossed with ILDR3 knockout mice to construct wild-type mice (ILDR3 knockout mice). Loxp / Loxp / APC Min / + (hereinafter referred to as control mice) and intestinal epithelial cell-specific ILDR3 knockout APCs Min / + Mice (ILDR3) Loxp / Loxp / Vil1-cre + / - / APC Min / + (Hereinafter referred to as ILDR3 double knockout mice). After feeding the two groups of mice with a normal diet for 24 weeks, the occurrence of tumors was detected.

[0088] Figure 10The results of experiments on tumor development and progression in a spontaneous mouse model of colon cancer are presented. Compared with the control group, the colorectal tumors in the ILDR3 double knockout group mice grew more rapidly and were larger. As shown in 10a, the proportions of large, medium, and small tumors in the colon tissue of the ILDR3 double knockout group mice were 53%, 31%, and 16%, respectively, while those in the control group mice were 24%, 16%, and 60%, respectively. The number of tumors was significantly increased (as shown in 10b). HE staining of the colon tissue of both groups of mice showed that the colon tissue of the ILDR3 double knockout group mice exhibited more obvious histological disorder, destruction and loss of polarity of crypt structure, branching deformity, and a large number of atypical cells (10c).

[0089] The above experimental results further confirm that the ILDR3 gene and / or protein can significantly inhibit the occurrence and development of spontaneous colorectal cancer.

[0090] Example 5: Effect of ILDR3 overexpression on the ability of subcutaneous tumor formation in colorectal cancer

[0091] Experimental Methods: Twenty 4-5 week old male C57BL / 6 mice were purchased from Jicui Pharmaceutical and housed in an SPF-grade animal facility for one week to ensure good mouse condition. Mc38 cells from the control group and the ILDR3 overexpression group, which were in good growth condition and in the logarithmic growth phase, were collected. Cells were digested with trypsin, counted, and resuspended in PBS to prepare a cell suspension at 1×10⁻⁶. 4 Cells / μL: 100 μL or more of the prepared cell suspension was injected into the forelimb axilla of C57BL / 6 mice, 0.3 cm from the back, using a 1 mL syringe. Ten mice were injected into each of the control and ILDR3 overexpression groups. Starting from day 3 after tumor formation, the length, width, and height of the tumor were measured every other day using calipers, and the weight of the tumor-bearing mice was also measured. The condition of the tumor-bearing mice was observed and recorded. Approximately 15 days after inoculation, tumor tissue was collected, and the volume and weight of the tumor were calculated and photographed, expressed as length × width. 2 This is the formula for calculating tumor volume; it is used to calculate tumor volume.

[0092] Results of subcutaneous xenograft tumor growth experiments in different groups of nude mice are as follows: Figure 11 As shown, Figure 11 Compared with the control group, the tumor formation ability of colorectal cancer cells in the ILDR3 overexpression group was significantly inhibited over time (11a), the tumor volume was significantly reduced (11b), and the weight was significantly reduced (11c).

[0093] This invention first identified ILDR3, a potential therapeutic target for colorectal cancer, through experiments using microarrays of tissue samples and cDNA microarrays from colorectal cancer patients. Gene knockout and overexpression techniques were then used to preliminarily verify the crucial role of ILDR3 in colorectal cancer progression. Experiments showed that in vitro overexpression of ILDR3 significantly inhibited the proliferation, colony formation, and migration / invasiveness of colorectal cancer cells. In vivo, ILDR3 knockout significantly enhanced the growth rate and number of in situ colonic tumors, while ILDR3 overexpression significantly inhibited tumor growth in tumor-bearing mice. Therefore, ILDR3 can serve as a novel therapeutic target for colorectal cancer, providing valuable evidence for the prediction, diagnosis, and treatment of clinical colorectal cancer patients.

[0094] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Use of an expression promoter of an ILDR3 gene and / or an ILDR3 protein for the manufacture of a medicament for treating a tumor, characterized in that, The tumor is colorectal cancer, and the expression promoter of the ILDR3 gene and / or ILDR3 protein is a recombinant expression vector containing the ILDR3 gene.

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