Application of CCDC71L in diagnosis and treatment of radiation enteritis

Abstract

The invention discloses an application of CCDC71L in diagnosis and treatment of radiation enteritis, and provides an application of a reagent for detecting the expression level of the CCDC71L in preparation of a product for diagnosing radiation enteritis and an application of an inhibitor of the CCDC71L in preparation of a medicine for treating radiation enteritis. The invention further provides a method for screening candidate drugs for treating or preventing radiation enteritis and a method for inhibiting the expression level of inflammatory factors in macrophages. Experiments prove that the CCDC71L can realize diagnosis of radiation enteritis, and discovers that inhibition of the CCDC71L can inhibit infiltration and polarization of macrophages so as to inhibit radiation-induced inflammatory reaction, and the CCDC71L marker provided by the invention provides a new idea for diagnosis and treatment of radiation enteritis, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the application of CCDC71L in diagnosing and treating radiation enteritis. Background Art

[0002] Radiotherapy (RT) is a very effective cytotoxic therapy for local treatment of solid tumors, and is one of the three major treatments for tumors. In the process of clinical radiotherapy, it is always inevitable to irradiate normal tissues, and the risk of damage to normal tissues by radiotherapy is still the most important factor limiting the radiotherapy dose during radiotherapy. Radiation enteritis is a common complication after radiotherapy for pelvic, abdominal and retroperitoneal malignancies. Patients may experience symptoms such as abdominal pain, diarrhea, and mucus and blood in the stool, and even complications such as intestinal obstruction and intestinal perforation. So far, antibiotics and anti-inflammatory agents used in clinical treatment of radiation enteritis have poor effects, and related basic research and drug development are not ideal, and effective prevention and treatment cannot be carried out. With the increase in the number of patients undergoing radiotherapy for malignant tumors, the incidence of radiation enteritis is also increasing. Therefore, it is crucial to explore the key elements and molecular mechanisms of the occurrence and development of radiation enteritis, which will help develop safe and effective therapeutic drugs or methods and improve the quality of life of patients. Summary of the invention

[0003] In order to make up for the deficiencies of the prior art, the present invention provides the use of CCDC71L in diagnosing and treating radiation enteritis.

[0004] In order to achieve the above object, the present invention adopts the following technical solution: The first aspect of the present invention provides any one of the following applications, comprising: (1) Application of reagents for detecting CCDC71L expression levels in the preparation of products for diagnosing radiation enteritis; (2) Application of CCDC71L inhibitors in the preparation of drugs for the treatment of radiation enteritis; (3) Application of CCDC71L in screening candidate drugs for the treatment of radiation enteritis; (4) Application of CCDC71L in constructing a computer model for the diagnosis of radiation enteritis; (5) Application of CCDC71L in constructing a system for diagnosing radiation enteritis.

[0005] In the present invention, CCDC71L includes wild type, mutant or fragment thereof. The term covers full-length, unprocessed CCDC71L, any form of CCDC71L derived from processing in cells, and naturally occurring variants (e.g., splice variants or allelic variants) of CCDC71L. The term covers, for example, human CCDC71L and CCDC71L from any other vertebrate source, including mammals, such as primates and rodents (e.g., mice and rats).

[0006] In the present invention, treatment may refer to therapeutic treatment or preventive measures, wherein the goal is to prevent or slow down (mitigate) an undesirable physiological condition, disorder or disease, or to obtain a beneficial or desired clinical result. In the present invention, treatment may refer to both treatment and prevention. Beneficial or desired clinical results include, but are not limited to, relief of symptoms; alleviation of the extent of a disorder, disorder or disease; stabilization (i.e., not worsening) of the state of a disorder, disorder or disease; delaying the occurrence of a disorder, disorder or disease or slowing its progression; improving a disorder, disorder or disease state; and relief (whether partial or complete) (whether detectable or undetectable) or improvement or improvement of a disorder, disorder or disease. Treatment may include causing a clinically significant response without excessive side effects. Treatment also includes a prolonged survival period compared to the expected survival period when not receiving treatment.

[0007] Furthermore, the reagent includes a probe that specifically recognizes the CCDC71L gene, a primer that specifically amplifies the CCDC71L gene, or a binding agent that specifically binds to a protein encoded by the CCDC71L gene.

[0008] In the present invention, the probe can be, for example, a full-length target gene nucleic acid or a portion thereof, such as an oligonucleotide having a length of at least 15, 30, 50, 100, 250 or 500 nucleotides and sufficient to specifically hybridize to the target gene mRNA or genomic DNA under stringent conditions.

[0009] In the present invention, primer refers to a short nucleic acid molecule, such as a DNA oligonucleotide, such as a sequence of at least 15 nucleotides, which can anneal with a complementary target nucleic acid molecule by nucleic acid hybridization to form a hybrid between the primer and the target nucleic acid chain. The primer can be extended along the target nucleic acid molecule by a polymerase. Therefore, the primer can be used to amplify the target nucleic acid molecule, wherein the sequence of the primer is specific for the target nucleic acid molecule, such as the primer will hybridize with the target nucleic acid molecule under very high stringency hybridization conditions.

[0010] In the present invention, specific binding refers to the situation where two or more molecules form a complex that can be measured under physiological or assay conditions and are selective. An antibody or antigen binding protein or other molecule is said to specifically bind to a protein, antigen or epitope when, under appropriately selected conditions, this binding is not substantially inhibited while nonspecific binding is inhibited. Specific binding is characterized by high affinity and selectivity for the compound, protein, epitope or antigen. Nonspecific binding generally has lower affinity.

[0011] Furthermore, the reagent also includes a detectable label.

[0012] Preferably, the label comprises a radioisotope, an enzyme, a fluorescent molecule, or a magnetic particle.

[0013] In the present invention, label refers to the composition that can produce the detectable signal indicating the presence of target polynucleotide in the assay sample. Suitable label includes but is not limited to radioisotope, nucleotide chromophore, enzyme, substrate, fluorescent molecule, chemiluminescent part, magnetic particle, bioluminescent part. Therefore, label is any composition that can be detected by device or method, including but not limited to spectrum, photochemistry, biochemistry, immunochemistry, electricity, optics, chemical detection device or any other suitable device. In some embodiments, label can be detected visually without the aid of device. Label is used to refer to any chemical group or part with detectable physical property or can cause chemical group or part to show any compound of detectable physical property, such as catalytic substrate conversion into detectable product enzyme. Label also encompasses the compound that suppresses the performance of specific physical property. Label can also be a compound as a member of a binding pair, and another member thereof has a detectable physical property.

[0014] Among them, radioactive isotopes include but are not limited to 3 H. 14 C. 35 S. 125 I. 131 I. Enzymes include, but are not limited to, horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, acetylcholinesterase. Fluorescent molecules include, but are not limited to, FITC, rhodamine, and lanthanide phosphors.

[0015] Furthermore, the products include test kits, chips, test papers, and nucleic acid membrane strips.

[0016] In the present invention, the kit also includes a buffer, a preservative or a protein stabilizer. The kit may also include an essential component (e.g., a substrate) for detecting a detectable agent. The kit may also contain a control sample or a series of control samples, which may be measured and compared with the test sample contained. Each component of the kit is usually packaged in a separate container, and all the different containers are packed into a package and carry instructions for observing whether the test subject suffers from a disease associated with abnormal expression of the target gene or is at risk of developing the disease.

[0017] Other components of the kit include, but are not limited to, tools for collecting biological samples, tools for labeling detection reagents (binding agents), membranes for fixing CCDC71L protein or CCDC71L nucleic acid in biological samples, tools for loading biological samples onto membranes, tools for binding reagents to CCDC71L in biological samples of subjects, secondary antibodies, tools for isolating total RNA from biological fluids of subjects, tools for performing gel electrophoresis, tools for generating cDNA from isolated total RNA, tools for performing hybridization assays, and tools for performing PCR.

[0018] The kit may optionally include a set of instructions in printed or electronic form (e.g., magnetic or optical disk) for information about the kit components and / or how to perform various assays (e.g., CCDC71L levels, comparisons with control standards, etc.). The kit may also be commercially available as part of a larger package that includes instruments for assaying other biochemical components.

[0019] Further, the inhibitor reduces the expression level of CCDC71L.

[0020] Furthermore, the inhibitors include nucleic acid inhibitors, protein inhibitors, and compounds.

[0021] Preferably, the inhibitor is selected from nucleic acid inhibitors.

[0022] Further, the nucleic acid inhibitor is unmodified or modified with one or more chemical groups selected from the group consisting of: 2'O-methoxy, phosphorothioate, locked nucleic acid and cholesterol.

[0023] Furthermore, the nucleic acid inhibitors include siRNA, shRNA, ribozymes, and antisense oligonucleotides.

[0024] Preferably, the nucleic acid inhibitor is selected from siRNA or shRNA.

[0025] In the present invention, an inhibitor refers to an inhibitor that can specifically bind to CCDC71L, preferably human CCDC71L, or bind to a polynucleotide or a fragment of CCDC71L, and inhibit the activity and / or expression of CCDC71L protein or polynucleotide, including nucleic acid inhibitors, protein inhibitors, and compounds.

[0026] Among them, the protein inhibitor includes an antibody, which is a monoclonal, chimeric, human or humanized antibody, or an antibody fragment or a synthetic antibody.

[0027] The nucleic acid inhibitor is unmodified or modified with one or more chemical groups selected from the group consisting of: 2'O-methoxy, phosphorothioate, locked nucleic acid and cholesterol.

[0028] In the present invention, modified refers to the change of the compound, usually chemical, to add or remove functional groups to the chemical structure of the compound. This modification generally relates to the covalent addition of reagents or functional groups to the compound. On the contrary, unmodified refers to the fact that the compound has not been chemically changed from the most common form and / or the form found in nature or under standard conditions. These modifications can be found in proteins, and these modifications include but are not limited to acylation (e.g., myristoylation, palmitoylation), isoprenylation (isoprenylation) or prenylation (prenylation), glycosylphosphatidylinositolization (glypiation), glycosylphosphatidylinositol (GPI) anchor addition, lipoylation, addition of flavin moieties (e.g., FMN or FAD), addition of heme groups, phosphopantetheinylation, retinyl Schiff bases, glycosylation, fucosylation, alkylation, amidation, amide bond formation, hydroxylation, phosphate (O- connection) or phosphoramidate (N- connection) formation, pegylation, biotinylation, carbamylation, oxidation and combinations thereof.

[0029] In the present invention, locked nucleic acid (LNA) refers to a modified RNA nucleotide. The ribose moiety of the LNA nucleotide is modified with an additional bridge connecting the 2' oxygen and the 4' carbon. The bridge "locks" the ribose in a 3'-endo (north) conformation, which is the same conformation often found in A-form duplexes. Whenever necessary, the commonly synthesized locked nucleic acid nucleotides can be mixed with DNA or RNA residues in oligonucleotides and hybridized with DNA or RNA according to the Watson-Crick base pairing rules. The locked ribose conformation enhances base stacking and backbone pre-organization.

[0030] In an embodiment of the present invention, the inhibitor is selected from nucleic acid inhibitors, including siRNA, shRNA, ribozyme, antisense oligonucleotide.

[0031] Among them, siRNA (small interfering RNA) refers to an isolated RNA molecule, preferably greater than 10 nucleotides in length, more preferably greater than 15 nucleotides in length, and most preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length, which is used to identify a target gene or mRNA to be degraded. The range of 19 to 25 nucleotides is the most preferred size for siRNA.

[0032] siRNA can include partially purified RNA, substantially pure RNA, synthetic RNA or recombinantly produced RNA and RNA that is modified from natural RNA by addition, deletion, substitution and / or change of one or more nucleotides. The changes may include the addition of non-nucleotide substances, such as addition to the end of the siRNA or one or more internal nucleotides of the siRNA; modifications that make the siRNA resistant to nuclease digestion (e.g., using 2'-substituted ribonucleotides or modifying the sugar-phosphate backbone); or replacing one or more nucleotides in the siRNA with deoxyribonucleotides. In addition, as described above for modified oligonucleotides, the siRNA can be modified to improve its stability, especially by introducing one or more phosphorothioate linkages.

[0033] shRNA (small hairpin RNA) is a non-coding small RNA molecule that can form a hairpin structure. shRNA can inhibit gene expression through the RNA interference pathway.

[0034] Antisense oligonucleotides (antisense nucleic acid sequences) may include nucleotide sequences complementary to sense nucleic acids encoding proteins (e.g., complementary to the coding strands of double-stranded cDNA molecules or complementary to CCDC71L mRNA). Antisense oligonucleotides and delivery methods are well known in the art (Goodchild, Curr. Opin. Mol. Ther., 6 (2): 120-128 (2004); Clawson et al., Gene Ther., 11 (17): 1331-1341 (2004)), which are incorporated by reference into the present invention in their entirety. Antisense oligonucleotides may be complementary to the complete coding strand of the target sequence, or only partially complementary to it. In another embodiment, the antisense oligonucleotide is antisense to the non-coding region of the coding strand of the nucleotide sequence in the CCDC71L mRNA. An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or more nucleotides in length.

[0035] Ribozymes are a class of RNAs that can be engineered to enzymatically cleave and inactivate other RNA targets in a specific, sequence-dependent manner. Ribozymes and methods of delivering them are well known in the art (Hendry et al., BMC Chem. Biol., 4(1):1 (2004); Grassi et al., Curr. Pharm. Biotechnol., 5(4):369-386 (2004); Bagheri et al., Curr. Mol. Med., 4(5):489-506 (2004); Kashani-Sabet M., Expert Opin. Biol. Ther., 4(11):1749-1755 (2004), each of which is incorporated herein by reference in its entirety. By cleaving the target RNA, ribozymes inhibit translation, thereby preventing expression of the target gene. Ribozymes can be chemically synthesized in the laboratory and structurally modified to increase their stability and catalytic activity using methods known in the art. Alternatively, the ribozyme gene can be introduced into the cell by gene delivery mechanisms known in the art.

[0036] In a specific embodiment of the present invention, the nucleic acid inhibitor is selected from siRNA and shRNA.

[0037] Furthermore, the drug for treating radiation enteritis reduces the expression of inflammatory factors.

[0038] Furthermore, the inflammatory factors include IL-6, TNF-α, IL-1β, IFN-γ, and IL-12.

[0039] A second aspect of the present invention provides a product for diagnosing radiation enteritis.

[0040] Furthermore, the product includes a reagent for detecting the expression level of CCDC71L.

[0041] Furthermore, the products include test kits, chips, test papers, and nucleic acid membrane strips.

[0042] Preferably, the kit further comprises reagents required for detecting the expression level of CCDC71L gene or protein by RT-PCR, qRT-PCR, biochip detection, Southern blotting, in situ hybridization, or immunoblotting.

[0043] Preferably, the kit further comprises a buffer, a preservative or a protein stabilizer.

[0044] Preferably, the kit further comprises a control sample.

[0045] Preferably, the kit further comprises instructions.

[0046] A third aspect of the present invention provides a pharmaceutical composition for treating radiation enteritis.

[0047] Furthermore, the pharmaceutical composition includes an inhibitor of CCDC71L.

[0048] Preferably, the pharmaceutical composition further comprises a pharmaceutically compatible carrier.

[0049] Preferably, the pharmaceutical composition further comprises a buffer.

[0050] In the present invention, the pharmaceutical composition also includes a pharmaceutically compatible carrier, and pharmaceutical compatibility refers to a non-toxic material that does not interact with the action of the active ingredient of the pharmaceutical composition. The pharmaceutically compatible carrier refers to a natural or synthetic, organic or inorganic component that is used in conjunction with the active ingredient to facilitate application. According to the present invention, the pharmaceutically compatible carrier includes one or more compatible solid or liquid fillers, diluents or encapsulating materials, and the carrier is suitable for administration to a patient. The components of the pharmaceutical composition of the present invention generally do not interact with each other that significantly affects the desired drug efficacy.

[0051] In the present invention, the pharmaceutical composition further comprises a buffer, which includes but is not limited to acetate, citrate, borate and phosphate.

[0052] In the present invention, the pharmaceutical composition also includes salts, which, when used in medicine, should be pharmaceutically compatible salts. However, pharmaceutically incompatible salts can also be used to prepare pharmaceutically compatible salts and are included in the present invention. Such pharmacologically and pharmaceutically compatible salts include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid. Pharmaceutically compatible salts can also be prepared as alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts or calcium salts.

[0053] In the present invention, the pharmaceutical composition may further include a suitable preservative as appropriate, including but not limited to benzalkonium chloride, chlorobutanol, paraben and thimerosal.

[0054] The pharmaceutical composition is usually provided in a uniform dosage form and can be prepared in a known manner. The pharmaceutical composition of the present invention can be in the form of, for example, capsules, tablets, lozenges, solutions, suspensions, syrups, elixirs, or in the form of emulsions.

[0055] Compositions suitable for parenteral administration generally include a sterile, aqueous or nonaqueous preparation of the active compound, which is preferably isotonic with the blood of the recipient. Examples of compatible carriers and solvents are Ringer's solution and isotonic sodium chloride solution.

[0056] The pharmaceutical compositions may be included in a container, pack, or dispenser together with instructions for administration.

[0057] A fourth aspect of the present invention provides a method for screening, treating or preventing radiation enteritis.

[0058] Further, the method comprises: (1) contacting the drug to be tested with a system expressing or containing CCDC71L; (2) detecting the expression level of CCDC71L in the system; (3) Select substances that can reduce the expression level of CCDC71L as candidate drugs for treating or preventing radiation enteritis.

[0059] Furthermore, the system is selected from: a cell system, a subcellular system, a solution system, a tissue system, an organ system or an animal system.

[0060] A fifth aspect of the present invention provides an in vitro method for inhibiting the proliferation and / or migration of rectal epithelial cells for non-therapeutic purposes.

[0061] Further, the method comprises contacting the rectal epithelial cells with an inhibitor of CCDC71L.

[0062] A sixth aspect of the present invention provides an in vitro method for inhibiting the expression of inflammatory factors in macrophages for non-therapeutic purposes.

[0063] Further, the method comprises contacting the macrophage with an inhibitor of CCDC71L.

[0064] Furthermore, the inflammatory factors include IL-6, TNF-α, IL-1β, IFN-γ, and IL-12.

[0065] A seventh aspect of the present invention provides a system for diagnosing radiation enteritis.

[0066] Further, the system comprises: (1) Detection unit: The detection unit is used to detect the expression level of CCDC71L; (2) Result judgment unit: The result judgment unit is used to output the result of radiation enteritis diagnosis based on the expression amount result detected by the detection unit.

[0067] Furthermore, the result judgment unit includes an input unit, an analysis unit and an output unit.

[0068] Furthermore, the input unit is used to input the expression level of CCDC71L.

[0069] Furthermore, the analysis unit is used to analyze the results of radiation enteritis diagnosis based on the expression level of CCDC71L.

[0070] Furthermore, the output unit is used to output the analysis result of the analysis unit.

[0071] Furthermore, the CCDC71L expression level includes CCDC71L mRNA expression level and CCDC71L protein expression level.

[0072] Advantages and beneficial effects of the present invention: The CCDC71L marker provided by the present invention can realize the diagnosis of radiation enteritis, and using the siRNA sequence for targeted knockdown of CCDC71L, it has been verified that inhibition of CCDC71L can inhibit the proliferation and migration ability of rectal epithelial cells and the expression of inflammatory factors. The CCDC71L marker provided by the present invention provides a new idea for the diagnosis and treatment of radiation enteritis and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 The graphs show the time-specific changes in the transcriptome of mouse rectal tissue induced by ionizing radiation; among them, Figures AD show the volcano plots of differentially expressed genes in mouse rectal tissue 4 hours (A), 3 days (B), 2 weeks (C) and 8 weeks (D) after irradiation with 25Gy ionizing radiation; Figure E shows the GO functional enrichment analysis of differentially expressed genes at different time points after irradiation, and the figure lists the specific GO entries enriched at specific time points; Figure F shows the KEGG pathway enrichment analysis of differentially expressed genes at different time points after irradiation, and the figure lists the specific KEGG pathways enriched at specific time points; Figure 2 The time series clustering analysis of the transcriptome changes of mouse rectal tissue induced by ionizing radiation is shown; Figure A shows the time series clustering heat map of the transcriptome changes of mouse rectal tissue induced by ionizing radiation; Figure B shows the GO functional enrichment analysis of the characteristic genes of different Clusters, and the figure lists the specific GO entries of different Clusters; Figure C shows the KEGG pathway enrichment analysis of the characteristic genes of different Clusters, and the figure lists the specific KEGG pathways of different Clusters; Figure 3The machine learning algorithm is used to screen the key genes of mouse rectal tissue induced by ionizing radiation; Figure A shows the top 20 characteristic genes of Cluster1 identified by Elastic Net machine learning and randomForest machine learning algorithms; Figure B shows the top 20 characteristic genes of Cluster2 identified by Elastic Net machine learning and randomForest machine learning algorithms; Figure C shows the top 20 characteristic genes of Cluster3 identified by Elastic Net machine learning and randomForest machine learning algorithms; Figure D shows the top 20 characteristic genes of Cluster4 identified by Elastic Net machine learning and randomForest machine learning algorithms; Figure E shows the intersection venn diagram of the top 20 characteristic genes of Cluster1 identified by Elastic Net machine learning and randomForest machine learning algorithms to obtain three intersection genes, Wdtc1, Mgat4a, and Entpd5; Figure F shows the intersection venn diagram of the top 20 characteristic genes of Cluster2 identified by Elastic Net machine learning and randomForest machine learning algorithms to obtain two intersection genes, CCDC71L and Nek2; Figure G shows the intersection venn diagram of the top 20 characteristic genes of Cluster2 identified by Elastic Net machine learning and randomForest machine learning algorithms The intersection venn diagram of the first 20 characteristic genes of Cluster3 identified by the Net machine learning algorithm and the randomForest machine learning algorithm was used to obtain an intersection gene Cd68; Figure H shows that the intersection venn diagram of the first 20 characteristic genes of Cluster4 identified by the Elastic Net machine learning algorithm and the randomForest machine learning algorithm was used to obtain an intersection gene Scgb1b7; Figure 4 Figure 4 shows the validation of the role of CCDC71L, a key gene screened by machine learning, in ionizing radiation-induced rectal tissue damage in mice; Figures AD show that RT-qPCR validated the 7 Figure 5 shows the expression changes of the key genes (CCDC71L, Nek2, Scgb1b7, Wdtc1, Mgat4a, Entpd5 and Cd68) at different time points (0 day, 1 day, 2 days and 3 days) after irradiation; Figure E shows that the expression of CCDC71L in the rectal tissue of mice increased significantly 3 days after irradiation by immunohistochemical staining; Figure F shows that Western Blot analysis verified the changes in protein expression of CCDC71L at different time points (0 day, 1 day, 2 days and 3 days) after irradiation; Figure G shows that Western Blot verified that the CCDC71L knockdown cell model was successfully constructed in the human rectal epithelial cell line; Figure H shows that the clone formation experiment shows that after knocking down CCDC71L, the proliferation ability of human rectal epithelial cells is significantly reduced; Figure I shows that the Transwell migration experiment shows that after knocking down CCDC71L, the migration ability of human rectal epithelial cells is significantly reduced; Figure 5 CCDC71L is involved in ionizing radiation-induced intestinal invasion by regulating macrophage infiltration and polarization Inflammatory response; Figure A shows the seq-ImmuCCAI analysis of immune cells in the intestinal tissue of mice before and after irradiation Changes, M0 macrophages increased significantly, and M2 macrophages decreased significantly; BC graph shows qPCR (B) Figure 4 and Western Blot (C) were used to detect the expression changes of MMP9, MMP2, VEGF and ICAM-1 in bone marrow-derived macrophages of irradiated mice treated with CCDC71L knockdown; Figure D shows the Transwell migration assay to analyze the effect of CCDC71L knockdown on the migration ability of irradiated macrophages; Figure 4 shows RT-qPCR (E) and ELISA (F) to detect the mRNA expression and protein secretion levels of inflammatory factors IL-6, TNF-α, IL-1β, IFN-γ and IL-12 in irradiated macrophages treated with CCDC71L knockdown. DETAILED DESCRIPTION

[0074] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0075] Example 1 Ionizing radiation induces time-specific changes in the transcriptome of mouse rectal tissue Using RNA-seq sequencing technology, the transcriptome changes of rectal tissues of mice were detected 4 hours, 3 days, 2 weeks and 8 weeks after receiving 25Gy rectal irradiation (data from GSE198331 dataset). By performing differential analysis with the control group, a volcano plot was drawn at the four time points ( Figure 1 AD), and GO and KEGG functional enrichment analysis was performed on the differentially expressed genes. The results showed that the changes in gene expression induced by ionizing radiation were time-specific ( Figure 1 E, F). After 4 hours of irradiation, pathways related to cell mitosis and chromosome separation (such as spindle, sister chromatid separation, chromosome separation, etc.), as well as DNA replication and platinum drug resistance pathways were enriched, suggesting that ionizing radiation may mainly affect cell cycle progression and DNA damage response in the early stage ( Figure 1 E). After 3 days of irradiation, pathways related to glycolysis / gluconeogenesis and drug metabolism were enriched, suggesting that radiation may disrupt the energy metabolism and detoxification function of cells. After 2 and 8 weeks of irradiation, the enriched pathways were mainly related to inflammatory response (such as complement and coagulation cascades, phagocytosis, amebiasis, chemokine signaling pathways, Staphylococcus aureus infection, etc.), indicating that the damage caused by ionizing radiation may have triggered a persistent inflammatory response in the later stage ( Figure 1 F). Collectively, these findings reveal that ionizing radiation-induced transcriptome changes have different biological functions at different time points, providing important clues for understanding the molecular mechanisms of ionizing radiation damage.

[0076] Example 2 Time series cluster analysis of ionizing radiation-induced changes in mouse rectal tissue The Mfuzz R package was used to perform time series cluster analysis on the transcriptome data of mouse rectal tissue at four time points: 4 hours, 3 days, 2 weeks, and 8 weeks after irradiation with 25 Gy of ionizing radiation, and four characteristic gene sets were obtained ( Figure 2 A). GO and KEGG functional enrichment analysis of these characteristic gene sets revealed that different gene sets were enriched in different biological processes and signaling pathways ( Figure 2 B, C). The characteristic genes of Cluster 1 are mainly enriched in GO biological processes related to cell structure and function, such as transmembrane transport and cytoskeleton remodeling ( Figure 2 B), and KEGG signaling pathways such as bile secretion, circadian rhythm, and steroid hormone biosynthesis ( Figure 2 C). The characteristic genes of Cluster 2 are enriched in GO biological processes related to cell cycle progression, such as spindle, chromosome separation, and mitosis ( Figure 2 B), and KEGG signaling pathways related to cell cycle and DNA replication ( Figure 2C). The characteristic genes of Cluster 3 are mainly enriched in biological processes and signaling pathways related to intercellular communication and signal transduction, such as extracellular matrix, cytokine-cytokine receptor interaction, chemokine signaling pathway, and Rap1 signaling pathway ( Figure 2 BC). The characteristic genes of Cluster 4 are closely related to epithelial cell differentiation, such as keratinization and keratinocyte differentiation ( Figure 2 BC). Collectively, these results suggest that ionizing radiation-induced transcriptome changes in mouse rectal tissue are temporally and functionally specific, and that biological processes and signaling pathways activated at different time points are different, reflecting the dynamic changes of ionizing radiation damage.

[0077] Example 3 Machine learning algorithm to screen key driver genes of radiation enteritis In order to further screen out the most representative characteristic genes in each gene set, the importance of the characteristic genes of Cluster 1-4 was evaluated using two machine learning algorithms, Elastic Net and randomForest, and the top 20 important characteristic genes in each gene set were obtained respectively ( Figure 3 AD). In order to improve the reliability of feature gene screening, the project team conducted an intersection analysis on the top 20 feature genes screened by the two algorithms. The results showed that three genes in Cluster 1, Wdtc1, Mgat4a, and Entpd5, were screened by both algorithms ( Figure 3 E); Cluster2 contains the intersection of two genes, CCDC71L and Nek2 ( Figure 3 F); Cluster3 and Cluster4 have only one overlapping gene, Cd68 and Scgb1b7, respectively ( Figure 3 GH). These intersection genes may play a key role in the transcriptome changes of mouse rectal tissue induced by ionizing radiation. Among them, the CCDC71L gene ranks very high in both algorithms, and its biological function and regulatory mechanism are worthy of further study.

[0078] Example 4 Verification of the role of the key gene CCDC71L screened by machine learning in ionizing radiation-induced rectal tissue damage in mice In order to verify the key genes screened by the machine learning algorithm, we performed real-time quantitative PCR (RT-qPCR) verification on the 7 intersection genes identified in Example 3. The results showed that the expression levels of these genes were different at different time points after mice received 25Gy irradiation, among which CCDC71L increased significantly after 3 days of irradiation ( Figure 4AD). Immunohistochemical staining and Western Blot analysis further confirmed the expression changes of CCDC71L at the protein level, which was consistent with the transcriptome data ( Figure 4 EF). We subsequently performed ROC analysis on the expression level of CCDC71L, and the AUC result was excellent, indicating that CCDC71L is a marker for diagnosing radiation enteritis. In order to explore the biological function of CCDC71L, the project team knocked down the expression of CCDC71L in human rectal epithelial cell lines by siRNA ( Figure 4 G), and performed phenotypic analysis of cell proliferation and migration. The results showed that after knocking down CCDC71L, the proliferation and migration abilities of cells were significantly reduced ( Figure 4 HI), suggesting that CCDC71L may play an important role in regulating cell proliferation and migration. These experimental results preliminarily verified the reliability of transcriptome data analysis and laid the foundation for in-depth research on rectal tissue damage induced by ionizing radiation.

[0079] Example 5 CCDC71L participates in ionizing radiation-induced intestinal inflammatory response by regulating macrophage infiltration and polarization To explore the role of CCDC71L in ionizing radiation-induced intestinal inflammatory response, we first used the seq-ImmuCCAI algorithm to analyze the changes in immune cells in the intestinal tissue of mice before and after irradiation. The results showed that M0 macrophages increased significantly after irradiation, while M2 macrophages decreased significantly ( Figure 5 A). To further verify the effect of CCDC71L on macrophage function, the project team collected the irradiated cell supernatant from intestinal epithelial cells treated with shRNA-CCDC71L. Mouse bone marrow-derived macrophages isolated and cultured in vitro were incubated with irradiated cell supernatant. qPCR and Western Blot results showed that knocking down CCDC71L could significantly downregulate the expression of migration-related proteins such as MMP9, MMP2, VEGF and ICAM-1 ( Figure 5 BC), suggesting that CCDC71L may promote the infiltration of macrophages into the irradiated injury site by positively regulating these proteins. This result was further verified by Transwell migration assay ( Figure 5 D). In addition, in irradiated macrophages, knockdown of CCDC71L can also significantly inhibit the mRNA expression and protein secretion of inflammatory factors such as IL-6, TNF-α, IL-1β, IFN-γ and IL-12 in macrophages ( Figure 5EF), suggesting that CCDC71L may also aggravate the inflammatory response of radiation enteritis by promoting the polarization of macrophages to the M1 proinflammatory phenotype. Taken together, these results indicate that CCDC71L may participate in the intestinal inflammatory response induced by ionizing radiation by regulating cell proliferation and regulating the infiltration and polarization of macrophages, and inhibiting the expression of CCDC71L can significantly inhibit the expression of inflammatory factors in macrophages, thereby playing a therapeutic effect on radiation enteritis.

[0080] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. Any of the following applications, characterized in that: The applications include: (1) Application of reagents for detecting CCDC71L expression levels in the preparation of products for diagnosing radiation enteritis; (2) Application of CCDC71L inhibitors in the preparation of drugs for the treatment of radiation enteritis; (3) Application of CCDC71L in screening candidate drugs for the treatment of radiation enteritis; (4) Application of CCDC71L in constructing a computer model for the diagnosis of radiation enteritis; (5) Application of CCDC71L in constructing a system for diagnosing radiation enteritis.

2. The use according to claim 1, characterized in that: The reagents include a probe that specifically recognizes the CCDC71L gene, a primer that specifically amplifies the CCDC71L gene, or a binding agent that specifically binds to a protein encoded by the CCDC71L gene; Preferably, the reagent further comprises a detectable label; Preferably, the label comprises a radioisotope, an enzyme, a fluorescent molecule, or a magnetic particle; Preferably, the products include a test kit, a chip, a test paper, and a nucleic acid membrane strip.

3. The use according to claim 1, characterized in that: The inhibitor reduces the expression level of CCDC71L; Preferably, the inhibitor includes a nucleic acid inhibitor, a protein inhibitor, or a compound; Preferably, the inhibitor is selected from nucleic acid inhibitors; Preferably, the nucleic acid inhibitor is unmodified or modified with one or more chemical groups selected from the group consisting of: 2'O-methoxy, phosphorothioate, locked nucleic acid and cholesterol; Preferably, the nucleic acid inhibitor comprises siRNA, shRNA, ribozyme, antisense oligonucleotide; Preferably, the nucleic acid inhibitor is selected from siRNA or shRNA.

4. The use according to claim 1, characterized in that: The drug for treating radiation enteritis reduces the expression of inflammatory factors; Preferably, the inflammatory factors include IL-6, TNF-α, IL-1β, IFN-γ, and IL-12.

5. A product for diagnosing radiation enteritis, characterized in that: The product includes a reagent for detecting the expression level of CCDC71L; Preferably, the product includes a test kit, a chip, a test paper, and a nucleic acid membrane strip; Preferably, the kit further comprises reagents required for detecting the expression level of CCDC71L gene or protein by RT-PCR method, qRT-PCR method, biochip detection method, Southern blotting method, in situ hybridization method, and immunoblotting method; Preferably, the kit further comprises a buffer, a preservative or a protein stabilizer; Preferably, the kit further comprises a control sample; Preferably, the kit further comprises instructions.

6. A pharmaceutical composition for treating radiation enteritis, characterized in that: The pharmaceutical composition includes an inhibitor of CCDC71L; Preferably, the pharmaceutical composition further comprises a pharmaceutically compatible carrier; Preferably, the pharmaceutical composition further comprises a buffer.

7. A method for screening, treating or preventing radiation enteritis, characterized in that: The method comprises: (1) contacting the drug to be tested with a system expressing or containing CCDC71L; (2) detecting the expression level of CCDC71L in the system; (3) Select substances that can reduce the expression level of CCDC71L as candidate drugs for treating or preventing radiation enteritis.

8. A method for inhibiting the proliferation and / or migration of rectal epithelial cells in vitro for non-therapeutic purposes, characterized in that: The method comprises contacting rectal epithelial cells with an inhibitor of CCDC71L.

9. A method for inhibiting the expression of macrophage inflammatory factors for non-therapeutic purposes in vitro, characterized in that: The method comprises contacting a macrophage with an inhibitor of CCDC71L; Preferably, the inflammatory factors include IL-6, TNF-α, IL-1β, IFN-γ, and IL-12.

10. A system for diagnosing radiation enteritis, characterized in that: The system comprises: (1) Detection unit: The detection unit is used to detect the expression level of CCDC71L; (2) Result judgment unit: The result judgment unit is used to output the result of radiation enteritis diagnosis according to the result of the expression amount detected by the detection unit; Preferably, the result judgment unit includes an input unit, an analysis unit and an output unit; Preferably, the input unit is used to input the expression level of CCDC71L; Preferably, the analysis unit is used to analyze the result of radiation enteritis diagnosis according to the expression level of CCDC71L; Preferably, the output unit is used to output the analysis result of the analysis unit; Preferably, the CCDC71L expression level includes CCDC71L mRNA expression level and CCDC71L protein expression level.

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