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 application belongs to the field of biological medicine, and particularly relates to application of CCDC71L in diagnosis and treatment of radiation enteritis. BACKGROUND

[0002] Radiotherapy (RT) is a very effective cytotoxic therapy for local treatment of solid tumors by using high-energy ionizing radiation (IR) to cause DNA double-strand breaks, and then to cause cell cycle arrest, senescence and various cell death patterns. It is one of the three major treatment methods for tumors. In the process of clinical radiotherapy, it is inevitable to irradiate normal tissues, and the risk of damage to normal tissues by radiotherapy is still the most important factor limiting the dose of radiotherapy. Radiation enteritis is a common complication after radiotherapy for pelvic, abdominal and retroperitoneal malignant tumors. Patients may show symptoms such as abdominal pain, diarrhea and mucous blood stool, and even complications such as intestinal obstruction and intestinal perforation. So far, the antibiotics and anti-inflammatory agents used in the clinical treatment of radiation enteritis have poor effects, and the related basic research and drug development are not ideal. They cannot be effectively prevented and treated. With the increase in the number of patients receiving radiotherapy for malignant tumors, the incidence of radiation enteritis is also increasing. Therefore, it is crucial to explore the key role elements and analyze the molecular mechanisms of the occurrence and development of radiation enteritis, which helps to develop safe and effective therapeutic drugs or methods and improve the quality of life of patients. SUMMARY

[0003] In order to make up for the shortcomings of the prior art, the application provides application of CCDC71L in diagnosis and treatment of radiation enteritis.

[0004] In order to achieve the above-mentioned purpose, the application adopts the following technical solutions: The application provides the following application in any one aspect, and the application comprises: (1) application of a reagent for detecting the expression level of CCDC71L in preparation of a product for diagnosis of radiation enteritis; (2) application of an inhibitor of CCDC71L in preparation of a drug for treating radiation enteritis; (3) application of CCDC71L in screening of a candidate drug for treating radiation enteritis; (4) application of CCDC71L in construction of a computer model for diagnosis of radiation enteritis; (5) application of CCDC71L in construction of a system for diagnosis of radiation enteritis.

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

[0006] In the present application, treatment can refer to therapeutic treatment or prophylactic measures, wherein the object is to prevent or slow down (lessen) an undesired physiological condition, disorder or disease, or to obtain beneficial or desired clinical results. In the present application, treatment can refer to both treatment and prevention. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of extent of condition, disorder or disease; stabilized (i.e., not worsening) state of condition, disorder or disease; delay in onset or slowing of condition, disorder or disease progression; amelioration of the condition, disorder or disease state; and remission (whether partial or total), whether or not detectable to the patient or an observer, or improvement or amelioration of the condition, disorder or disease. Treatment can include eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival relative to expected survival if not receiving treatment.

[0007] Further, the reagent includes a probe specifically recognizing the CCDC71L gene, a primer specifically amplifying the CCDC71L gene, or a binding agent specifically binding to a protein encoded by the CCDC71L gene.

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

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

[0010] In the present invention, specific binding refers to a situation in which two or more molecules form a complex that can be measured under physiological or assay conditions and is selective. An antibody or antigen binding protein or other molecule is said to specifically bind to a protein, antigen, or epitope if such binding is not substantially inhibited while non-specific binding is inhibited under appropriately selected conditions. Specific binding is characterized by high affinity and selectivity for a compound, protein, epitope, or antigen. Non-specific binding typically has lower affinity.

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

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

[0013] In the present invention, a label refers to a composition that is capable of producing a detectable signal that indicates the presence of a target polynucleotide in an assay sample. Suitable labels include, but are not limited to, radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, bioluminescent moieties. Thus, a label is any composition that is capable of being detected by a device or method, including but not limited to spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical detection devices or any other suitable device. In some embodiments, a label can be detected visually without the aid of a device. Label is used to refer to any chemical group or moiety that has a detectable physical property or any compound that is capable of causing a chemical group or moiety to exhibit a detectable physical property, such as an enzyme that catalyzes the conversion of a substrate to a detectable product. A label also encompasses a compound that inhibits the exhibition of a particular physical property. A label can also be a compound that is a member of a binding pair, the other member of which has a detectable physical property.

[0014] wherein, the radioisotope includes, but is not limited to 3 H, 14 C, 35 S, 125 I, 131 I. The enzyme includes, but is not limited to, horseradish peroxidase, beta galactosidase, luciferase, alkaline phosphatase, acetylcholinesterase. The fluorescent molecule includes, but is not limited to, FITC, rhodamine, lanthanide phosphors.

[0015] Further, the product includes a kit, a chip, a test paper, a nucleic acid membrane strip.

[0016] In the present application, the kit also includes a buffer, a preservative, or a protein stabilizer. The kit can also contain the necessary ingredients for detecting the detectable agent (e.g., a substrate). The kit can also contain a control sample or a series of control samples, which can be assayed and compared to the test sample contained. Each ingredient of the kit is usually packaged in a separate container, and all of the different containers are packed in one package with instructions for observing whether the test subject has 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, a tool for collecting a biological sample, a tool for labeling a detection agent (a binding agent), a membrane for immobilizing a CCDC71L protein or a CCDC71L nucleic acid in a biological sample, a tool for applying a biological sample to the membrane, a tool for allowing a reagent to bind to CCDC71L in a biological sample of a subject, a secondary antibody, a tool for isolating total RNA from a biological fluid of a subject, a tool for performing gel electrophoresis, a tool for generating cDNA from the isolated total RNA, a tool for performing a hybridization assay, and a tool for performing PCR.

[0018] The kit can optionally include a set of instructions in printed or electronic (e.g., disk or disc) form regarding the components of the kit and / or how to perform the various assays (e.g., CCDC71L levels, comparison to control standards, etc.). The kit can also be marketed 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] Further, the inhibitor includes a nucleic acid inhibitor, a protein inhibitor, a compound.

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

[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] Further, the nucleic acid inhibitor includes an siRNA, an shRNA, a ribozyme, an antisense oligonucleotide.

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

[0025] In the present invention, an inhibitor refers to an agent that specifically binds to CCDC71L, preferably human CCDC71L, or to a polynucleotide of CCDC71L or a fragment thereof, and inhibits the activity and / or expression of the CCDC71L protein or polynucleotide. It includes nucleic acid inhibitors, protein inhibitors, compounds.

[0026] In which, the protein inhibitor includes an antibody, which is a monoclonal, chimeric, human or humanized antibody, or is 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 a compound, usually chemical, to add or remove functional groups to the chemical structure of the compound. Such modification usually involves the covalent addition of an agent or functional group to the compound. On the contrary, unmodified refers to the fact that the compound is not chemically changed from the most common form and / or 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 or prenylation, glypiation, glycosylphosphatidylinositol (GPI) anchor addition, fatty acylation, flavin moiety (e.g. FMN or FAD) addition, heme group addition, phosphopantetheinylation, retinoyl Schiff base, glycosylation, fucosylation, alkylation, amidation, amide bond formation, hydroxylation, phosphate (O-linked) or phosphoramidate (N-linked) formation, pegylation, biotinylation, carbamoylation, 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 extra bridge connecting the 2’ oxygen and the 4’ carbon. This bridge “locks” the ribose in the 3’-endo (north) conformation, which is the same conformation as often found in A-form duplexes. Whenever desired, the usually synthesized LNA nucleotides can be mixed with DNA or RNA residues in an oligonucleotide and hybridize to 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 a nucleic acid inhibitor, which includes siRNA, shRNA, ribozyme, antisense oligonucleotide.

[0031] 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 recognize a target gene or mRNA for degradation. The range of 19-25 nucleotides is the most preferred size for siRNA.

[0032] siRNA can include partially purified RNA, substantially pure RNA, synthetic RNA, or recombinantly produced RNA, as well as altered RNA that differs from natural RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. The alterations can include the addition of non-nucleotide material, such as to the ends of the siRNA or to one or more internal nucleotides of the siRNA; modifications that render the siRNA resistant to nuclease digestion (e.g., the use of 2'-substituted ribonucleotides or modifications to the sugar phosphate backbone); or the substitution of one or more nucleotides in the siRNA with deoxyribonucleotides. In addition, the siRNA can be modified to increase its stability, as described above for modified oligonucleotides, particularly by the introduction of one or more phosphorothioate linkages.

[0033] shRNA (small hairpin RNA) is a non-coding small RNA molecule that is capable of forming a hairpin structure, and the shRNA is capable of inhibiting the expression of a gene through the RNA interference pathway.

[0034] An antisense oligonucleotide (antisense nucleic acid sequence) can include a nucleotide sequence that is complementary to a sense nucleic acid encoding a protein (e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to a 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 in their entirety into the present application. The antisense oligonucleotide can be complementary to the entire coding strand of the target sequence, or only to a portion thereof. In another embodiment, the antisense oligonucleotide is antisense to a non-coding region of the coding strand of a nucleotide sequence in a CCDC71L mRNA. The 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 sequence-specific manner. Ribozymes and methods of their delivery 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, thus 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, ribozyme genes can be introduced into cells by gene delivery mechanisms known in the art.

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

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

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

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

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

[0041] Further, the product includes a kit, a chip, a test paper, a nucleic acid membrane strip.

[0042] Preferably, the kit further includes 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, immunoblotting method.

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

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

[0045] Preferably, the kit further includes an instruction manual.

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

[0047] Further, the pharmaceutical composition comprises 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 application, the pharmaceutical composition further comprises a pharmaceutically compatible carrier, which means a nontoxic material that is nontoxic to the subject at the dosages and concentrations employed. The pharmaceutically compatible carrier is nontoxic to the subject at the dosages and concentrations employed. The pharmaceutically compatible carrier is a natural or synthetic, organic or inorganic component that is nontoxic to the subject at the dosages and concentrations employed. In accordance with the present application, the pharmaceutically compatible carrier comprises one or more compatible solid or liquid filler diluents or encapsulating substances that are suitable for administration to the subject. The components of the pharmaceutical composition of the present application are typically free from significant interactive compatibility that negatively affect the desired pharmaceutical efficacy.

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

[0052] In the present application, the pharmaceutical composition further comprises a salt, which when used in medicine, should be a pharmaceutically compatible salt. However, pharmaceutically incompatible salts can be used to prepare a pharmaceutically compatible salt, and are included in the present application. 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 application, the pharmaceutical composition further comprises a suitable preservative, which includes but is not limited to benzalkonium chloride, chlorobutanol, paraben and thiomersal.

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

[0055] The composition suitable for parenteral administration generally includes sterile, aqueous or non-aqueous preparations of the active compound, which are 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 composition can be packaged in a container, package or dispenser with instructions for administration.

[0057] The fourth aspect of the present application provides a method for screening a drug for treating or preventing radiation enteritis.

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

[0059] Further, 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] The fifth aspect of the present application provides a method for inhibiting the proliferation and / or migration of rectal epithelial cells in vitro for non-therapeutic purposes.

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

[0062] The sixth aspect of the present application provides a method for inhibiting the expression of macrophage inflammatory factors in vitro for non-therapeutic purposes.

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

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

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

[0066] Further, the system comprises: (1) a detection unit: the detection unit is used to detect the expression amount of CCDC71L; (2) a result judging unit: the result judging unit is used to output the result of diagnosing radiation enteritis according to the result of the expression amount detected by the detection unit.

[0067] Further, the result judging unit comprises an input unit, an analysis unit and an output unit.

[0068] Further, the input unit is used to input the expression amount of CCDC71L.

[0069] Further, the analysis unit is used to analyze the result of diagnosing radiation enteritis according to the expression amount of CCDC71L.

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

[0071] Further, the expression amount of the CCDC71L includes the expression amount of CCDC71L mRNA and the expression amount of CCDC71L protein.

[0072] Advantages and beneficial effects of the present application: the CCDC71L marker provided by the present application can realize the diagnosis of radiation enteritis, and the siRNA sequence for targeting and knocking down CCDC71L is verified to be able to inhibit the proliferation and migration ability of rectal epithelial cells and to inhibit the expression of inflammatory factors. The CCDC71L marker provided by the present application provides a new idea for the diagnosis and treatment of radiation enteritis, and has a broad application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0073] Figure 1 showing time-specific changes in the transcriptome of ionizing radiation-induced mouse rectal tissue; wherein, Figures A-D show the volcano plot of differentially expressed genes in mouse rectal tissue at 4 hours (A), 3 days (B), 2 weeks (C) and 8 weeks (D) after irradiation with 25 Gy ionizing radiation; Figure E shows the GO function enrichment analysis of differentially expressed genes at different time points after irradiation, and the specific GO entries enriched at specific time points are listed in the figure; Figure F shows the KEGG pathway enrichment analysis of differentially expressed genes at different time points after irradiation, and the specific KEGG pathways enriched at specific time points are listed in the figure; Figure 2 showing time series clustering analysis of ionizing radiation-induced changes in the transcriptome of mouse rectal tissue; wherein, Figure A shows the time series clustering heat map of ionizing radiation-induced changes in the transcriptome of mouse rectal tissue; Figure B shows the GO function enrichment analysis of different Cluster characteristic genes, and the specific GO entries of different Clusters are listed in the figure; Figure C shows the KEGG pathway enrichment analysis of different Cluster characteristic genes, and the specific KEGG pathways of different Clusters are listed in the figure; Figure 3Figure 6 shows machine learning algorithm screens key genes in ionizing radiation-induced mouse rectal tissue; wherein, panel A shows the top 20 feature genes of Cluster 1 identified by Elastic Net machine learning and randomForest machine learning algorithms; panel B shows the top 20 feature genes of Cluster 2 identified by Elastic Net machine learning and randomForest machine learning algorithms; panel C shows the top 20 feature genes of Cluster 3 identified by Elastic Net machine learning and randomForest machine learning algorithms; panel D shows the top 20 feature genes of Cluster 4 identified by Elastic Net machine learning and randomForest machine learning algorithms; panel E shows the venn diagram of the intersection of the top 20 feature genes of Cluster 1 identified by Elastic Net machine learning and randomForest machine learning algorithms, resulting in three intersection genes Wdtcl, Mgat4a, and Entpd5; panel F shows the venn diagram of the intersection of the top 20 feature genes of Cluster 2 identified by Elastic Net machine learning and randomForest machine learning algorithms, resulting in two intersection genes CCDC71L and Nek2; panel G shows the venn diagram of the intersection of the top 20 feature genes of Cluster 3 identified by Elastic Net machine learning and randomForest machine learning algorithms, resulting in one intersection gene Cd68; panel H shows the venn diagram of the intersection of the top 20 feature genes of Cluster 4 identified by Elastic Net machine learning and randomForest machine learning algorithms, resulting in one intersection gene Scgb1b7; Figure 4 Figure 7 shows the verification of the role of the key gene CCDC71L screened by machine learning in ionizing radiation-induced mouse rectal tissue damage; wherein, panels A-D show that RT-qPCR verifies the expression changes of the 7 key genes (CCDC71L, Nek2, Scgb1b7, Wdtcl, Mgat4a, Entpd5, and Cd68) screened by machine learning algorithms at different time points (0 days, 1 day, 2 days, and 3 days) after irradiation; panel E shows that immunohistochemical staining shows that CCDC71L is significantly increased in the expression of mouse rectal tissue 3 days after irradiation; panel F shows that Western Blot analysis verifies the protein expression changes of CCDC71L at different time points (0 days, 1 day, 2 days, and 3 days) after irradiation; panel G shows that Western Blot verifies that a CCDC71L knockdown cell model is successfully constructed in a human rectal epithelial cell line; panel H shows that the colony formation experiment shows that after knocking down CCDC71L, the proliferation ability of human rectal epithelial cells is significantly reduced; panel 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 Display the AUC results of the filtered set; Figure 6 Display the AUC results of the validation set; Figure 7 The results show that CCDC71L participates in the ionizing radiation-induced intestinal inflammatory response by regulating macrophage infiltration and polarization. Figure A shows the changes in immune cells in mouse intestinal tissue before and after irradiation, as analyzed by seq-ImmuCCAI. M0 macrophages increased significantly, while M2 macrophages decreased significantly. Figures BC show the changes in the expression of MMP9, MMP2, VEGF, and ICAM-1 in bone marrow-derived macrophages from mice treated with CCDC71L knockdown, detected by qPCR (B) and Western Blot (C). Figure D shows the effect of CCDC71L knockdown on the migration ability of irradiated macrophages, as analyzed by Transwell migration assay. Figures EF show the mRNA expression and protein secretion levels of inflammatory factors IL-6, TNF-α, IL-1β, IFN-γ, and IL-12 in macrophages treated with CCDC71L knockdown, detected by RT-qPCR (E) and ELISA (F). Detailed Implementation

[0074] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0075] Example 1: Time-specific changes in the transcriptome of mouse rectal tissue induced by ionizing radiation RNA-seq sequencing was used to examine the transcriptomic changes in rectal tissue of mice at 4 hours, 3 days, 2 weeks, and 8 weeks after 25 Gy rectal irradiation (data from the GSE198331 dataset). Volcano plots were generated at the four time points using differential analysis with the control group. Figure 1 AD), and GO and KEGG functional enrichment analyses were performed on the differentially expressed genes. The results showed that the gene expression changes induced by ionizing radiation were time-specific (AD). Figure 1 E, F). Four hours after irradiation, pathways related to cell mitosis and chromosome separation (such as spindle fibers, sister chromatid separation, and chromosome separation) were enriched, as well as DNA replication and platinum-based drug resistance pathways, suggesting that ionizing radiation may primarily affect cell cycle progression and DNA damage response in the early stages. Figure 1E) After 3 days of irradiation, pathways related to glycolysis / gluconeogenesis and drug metabolism were enriched, suggesting that radiation might have disturbed the energy metabolism and detoxification function of cells. After 2 and 8 weeks of irradiation, the enriched pathways were mainly related to inflammatory responses (such as complement and coagulation cascades, phagocytosis, amoebiasis, chemokine signaling pathway, Staphylococcus aureus infection, etc.), indicating that the damage caused by ionizing radiation might have triggered a sustained inflammatory response in the later stage Figure 1 F) Overall, these findings revealed that the transcriptomic changes induced by ionizing radiation had different biological functions at different time points, providing important clues for understanding the molecular mechanisms of ionizing radiation damage.

[0076] Example 2 Time series clustering analysis of ionizing radiation-induced changes in mouse rectal tissue Time series clustering analysis was performed on the transcriptomic 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 using the Mfuzz R package, resulting in four characteristic gene sets Figure 2 A) GO and KEGG functional enrichment analysis of these characteristic gene sets found that the biological processes and signaling pathways enriched by different gene sets were different Figure 2 B, C) The characteristic genes of Cluster 1 were mainly enriched in GO biological processes related to cell structure and function, such as transmembrane transport and cytoskeletal reorganization Figure 2 B), as well as KEGG signaling pathways related to bile secretion, circadian rhythm, and steroid hormone biosynthesis Figure 2 C). The characteristic genes of Cluster 2 were enriched in GO biological processes related to cell cycle progression, such as spindle, chromosome segregation, and mitosis Figure 2 B), as well as KEGG signaling pathways related to cell cycle and DNA replication Figure 2 C). The characteristic genes of Cluster 3 were mainly enriched in biological processes and signaling pathways related to cell-cell communication and signal transduction, such as extracellular matrix, cytokine-cytokine receptor interaction, chemokine signaling pathway, and Rap1 signaling pathway Figure 2 B-C). The characteristic genes of Cluster 4 were closely related to epithelial cell differentiation, such as keratinization and keratinocyte differentiation Figure 2 B-C). Overall, these results suggest that the transcriptomic changes in mouse rectal tissue induced by ionizing radiation have time-specific and function-specific characteristics, and the 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 screening of key driver genes for radiation enteritis To further screen the most representative feature genes in each gene set, the importance of the feature genes in Clusters 1-4 was evaluated using two machine learning algorithms, Elastic Net and randomForest, resulting in the top 20 important feature genes in each gene set. Figure 3 To improve the reliability of feature gene screening, the project team conducted an intersection analysis on the top 20 feature genes selected by the two algorithms. The results showed that three genes in Cluster1—Wdtc1, Mgat4a, and Entpd5—were simultaneously selected by both algorithms. Figure 3 E); Cluster2 contains the intersection of the CCDC71L and Nek2 genes ( Figure 3 F); Cluster3 and Cluster4 each have only one intersection gene, Cd68 and Scgb1b7, respectively. Figure 3 These overlapping genes may play a key role in ionizing radiation-induced transcriptomic changes in mouse rectal tissue, with the CCDC71L gene ranking highly in both algorithms, warranting further investigation into its biological function and regulatory mechanism.

[0078] Example 4: Validation of the role of the key gene CCDC71L, screened by machine learning, in ionizing radiation-induced rectal tissue injury in mice. To validate the key genes screened by the machine learning algorithm, we performed real-time quantitative PCR (RT-qPCR) on the seven overlapping genes identified in Example 3. The results showed that the expression levels of these genes differed at different time points after mice (n=8) received 25 Gy irradiation, with CCDC71L showing a significant increase 3 days after irradiation. Figure 4 Immunohistochemical staining and Western blot analysis further confirmed changes in CCDC71L expression at the protein level, consistent with transcriptomic data. Figure 4 EF). We subsequently performed ROC analysis on the expression level of CCDC71L, and the AUC was 0.9013 in the selected set (GSE198331 dataset). Figure 5 In the validation set (ionizing radiation-induced mice), the AUC result was 0.9501. Figure 6 This indicates that CCDC71L is a biomarker for diagnosing radiation enteritis.

[0079] To investigate the biological function of CCDC71L, the research team knocked down the expression of CCDC71L in the human rectal epithelial cell line using siRNA. Figure 4 G), and phenotypic analysis of cell proliferation and migration was performed. The results showed that knockdown of CCDC71L significantly reduced both cell proliferation and migration abilities (G).Figure 4 The results suggest that CCDC71L may play an important role in regulating cell proliferation and migration. These experimental results preliminarily validate the reliability of transcriptome data analysis and lay the foundation for further research on ionizing radiation-induced rectal tissue damage.

[0080] Example 5: CCDC71L participates in ionizing radiation-induced intestinal inflammatory response by regulating macrophage infiltration and polarization. To investigate the role of CCDC71L in ionizing radiation-induced intestinal inflammatory responses, we first used the seq-ImmuCCAI algorithm to analyze the changes in immune cells in mouse intestinal tissue before and after irradiation. The results showed that irradiation significantly increased M0 macrophages while significantly decreasing M2 macrophages. Figure 7 A). To further verify the effect of CCDC71L on macrophage function, the research team collected the cell supernatant from intestinal epithelial cells treated with shRNA-CCDC71L. Mouse bone marrow-derived macrophages isolated and cultured in vitro were incubated with the irradiated cell supernatant. qPCR and Western Blot results showed that knockdown of CCDC71L significantly downregulated the expression of migration-related proteins such as MMP9, MMP2, VEGF, and ICAM-1. Figure 7 (BC) suggests that CCDC71L may promote macrophage infiltration into irradiated sites by positively regulating these proteins. This result was further validated by Transwell migration assays. Figure 7 D). Furthermore, in irradiated macrophages, knockdown of the CCDC71L group significantly inhibited the mRNA expression and protein secretion of inflammatory factors such as IL-6, TNF-α, IL-1β, IFN-γ, and IL-12. Figure 7 The results suggest that CCDC71L may also exacerbate the inflammatory response of radiation enteritis by promoting macrophage polarization to the M1 pro-inflammatory phenotype. In summary, these results indicate that CCDC71L may participate in ionizing radiation-induced intestinal inflammation by regulating cell proliferation and modulating macrophage infiltration and polarization, and that inhibiting CCDC71L expression can significantly suppress the expression of inflammatory factors in macrophages, thereby exerting a therapeutic effect on radiation enteritis.

[0081] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. The following application, characterized in that, The applications include: (1) Application of reagents for detecting CCDC71L expression levels in the preparation of products for the diagnosis of radiation enteritis; (2) Application of CCDC71L inhibitors in the preparation of drugs for treating 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 application according to claim 1, characterized in that, The reagents include probes that specifically recognize the CCDC71L gene, primers that specifically amplify the CCDC71L gene, or binding agents that specifically bind to the protein encoded by the CCDC71L gene. Preferably, the reagent further includes a detectable marker; Preferably, the label includes a radioactive isotope, an enzyme, a fluorescent molecule, or a magnetic particle; Preferably, the product includes a reagent kit, a chip, a test strip, and a nucleic acid membrane strip.

3. The application according to claim 1, characterized in that, The inhibitor reduces the expression level of CCDC71L; Preferably, the inhibitor includes nucleic acid inhibitors, protein inhibitors, and compounds; 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, thiophosphate, locked nucleic acid, and cholesterol; Preferably, the nucleic acid inhibitor includes siRNA, shRNA, ribozymes, and antisense oligonucleotides; Preferably, the nucleic acid inhibitor is selected from siRNA or shRNA.

4. The application according to claim 1, characterized in that, The medication used to treat 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-induced enteritis, characterized in that, The product includes reagents for detecting the expression level of CCDC71L; Preferably, the product includes a reagent kit, a chip, a test strip, and a nucleic acid membrane strip; Preferably, the kit further includes reagents required for detecting the expression level of the CCDC71L gene or protein by RT-PCR, qRT-PCR, microarray detection, DNA blotting, in situ hybridization, or immunoblotting. Preferably, the kit further includes a buffer, preservative, or protein stabilizer; Preferably, the kit further includes a control sample; Preferably, the kit also includes 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 includes a pharmaceutically compatible carrier; Preferably, the pharmaceutical composition further includes a buffer.

7. A method for screening treatments or preventing radiation enteritis, characterized in that, The method includes: (1) Contact the test drug with a system that expresses or contains CCDC71L; (2) Detect the expression level of CCDC71L in the system; (3) Select substances that can reduce the expression level of CCDC71L as candidate drugs for the treatment or prevention of 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 involves contacting rectal epithelial cells with an inhibitor of CCDC71L.

9. A method for inhibiting the expression of macrophage inflammatory factors in vitro for non-therapeutic purposes, characterized in that, The method includes contacting macrophages 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 includes: (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 diagnosis result of radiation enteritis based on the expression level 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 results of radiation enteritis diagnosis based on the expression level of CCDC71L; Preferably, the output unit is used to output the analysis results of the analysis unit; Preferably, the CCDC71L expression level includes the CCDC71L mRNA expression level and the CCDC71L protein expression level.

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