Application of CEBPD and FOS as ZFP36L2 transcriptional regulatory factors in development of radioactive injury treatment drug targets

Abstract

The invention belongs to the field of biomedicine, and particularly relates to application of CEBPD and FOS as ZFP36L2 transcriptional regulatory factors in development of radioactive injury treatment drug targets. Based on the fact that the inventor of the invention finds that transcription factors CEBPD and FOS can be combined with specific binding sites on the ZFP36L2 promoter and play a regulation role for the first time. The invention discloses a regulation and control mechanism of transcription factors CEBPD and FOS on ZFP36L2 expression of the transcription factors CEBPD and FOS, and a brand new molecular target is provided for development of radioactive injury treatment drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of CEBPD and FOS as ZFP36L2 transcriptional regulatory factors in the development of drug targets for treating radiation damage. Background Art

[0002] Radiotherapy is a local treatment method that uses radiation to treat tumors. However, while radiotherapy kills tumor cells, it also inevitably damages normal tissues, destroys the body's immune function, and produces a series of radiation injuries. With the increase of radiation dose or the continuation of radiotherapy, radiation damage gradually worsens. If intervention treatment is not carried out in time, it will not only reduce the treatment effect, but also aggravate the patient's condition and seriously affect the patient's quality of life.

[0003] ZFP36L2 (Zincfingerprotein36, C3Htype-like2) belongs to the ZFP36 protein family and undertakes the key task of regulating the post-transcription process of RNA. The protein accurately maintains the stability of gene expression through its unique zinc finger structure. In the inventor's previous research (see Chinese patent application number CN202411146999.0 for details), it was found that ZFP36L2 overexpression plays an important role in mitigating radiation / radioactive damage. By adjusting the expression level of ZFP36L2 in macrophages, radiation-induced mitochondrial dysfunction can be resisted. In vitro experimental verification showed that the supernatant of macrophages with overexpression of ZFP36L2 significantly improved the scratch healing ability of HUVEC under irradiation conditions. In addition, macrophages with overexpression of ZFP36L2 can significantly reduce the damage of abdominal tissues in transplanted mice under radiation environment. Therefore, studying the transcription factors and upstream pathways that directly act on ZFP36L2 will not only help to reveal its precise regulatory mechanism in radiation stress, but also provide a key theoretical basis for targeted intervention of radiation damage.

[0004] CEBPD (CCAAT / enhancer-binding proteindelta) belongs to the CCAAT enhancer-binding protein (C / EBP) family and is a transcription factor. CEBPD plays an important role in the regulation of genes related to immune and inflammatory responses. Some literature reports that it plays a role in radiation-induced intestinal damage by regulating the expression of inflammatory factors. In addition, CEBPD has an important effect on the invasiveness of glioblastoma (GBM) under hypoxic conditions, and it promotes the occurrence and development of tumors by activating the EGFR / PI3K signaling pathway. FOS (FBJosteosarcomaoncogene), as a member of the AP-1 transcription factor complex, has been shown to activate the FOS / JUN pathway through ionizing radiation and promote cell survival. However, it is still unclear whether CEBPD and FOS regulate the expression of the ZFP36L2 gene. Summary of the invention

[0005] The purpose of the present invention is to provide the application of CEBPD and FOS as ZFP36L2 transcriptional regulatory factors in the development of radiation injury therapeutic drug targets.

[0006] The inventors found through single-cell sequencing combined with SCENIC regulatory network analysis that CEBPD and FOS are the core transcription factors that regulate ZFP36L2. The GENIE3 algorithm shows that the two are the top two in terms of their contribution to the regulation of ZFP36L2 in monocytes after radiotherapy. The Regulon specificity score (RSS) and activity heat map show that CEBPD and / or FOS exhibit high specific regulatory activity and open state in classical monocytes, suggesting that they participate in the molecular mechanism of radiation damage repair by driving ZFP36L2 transcription, and verified through dual luciferase reporting that CEBPD and FOS can bind to specific sites of the ZFP36L2 promoter and exert a regulatory effect. Based on this, the inventors have completed the following invention.

[0007] The first aspect of the present invention is to provide the use of CEBPD and / or FOS as ZFP36L2 transcriptional regulatory factors in the development of drug targets for the treatment of radiation damage; CEBPD and / or FOS can positively regulate ZFP36L2 gene expression by binding to the binding site of the nucleotide sequence shown in SEQ ID NO: 1 and / or SEQ ID NO: 2 in the ZFP36L2 promoter region; the inventor's previous research (CN202411146999.0) has confirmed that ZFP36L2 overexpression has the effect of alleviating radiation-induced cell damage, so those skilled in the art can understand that CEBPD and / or FOS that have a positive regulatory effect on ZFP36L2 expression help to enhance its downstream repair function, and CEBPD and / or FOS, as transcription factors that regulate ZFP36L2, help to provide a new path for the target development of drugs for the treatment of radiation damage.

[0008] In this application, CEBPD and / or FOS regulate ZFP36L2 by binding to specific binding sites 3879bp-3856bp and / or 2854bp-2832bp upstream of ZFP36L2. The nucleotide sequence of the specific binding site is shown in SEQIDNO.1 and / or SEQIDNO.2, or has a nucleotide sequence that is at least 90% identical to SEQIDNO.1 and / or SEQIDNO.2.

[0009] In this application, CEBPD and / or FOS exerts a regulatory effect on ZFP36L2 by binding to the nucleotide sequence shown in SEQIDNO.3 and / or SEQIDNO.4 upstream of ZFP36L2; the nucleotide sequences shown in SEQIDNO.3 and SEQIDNO.4 are the core sequences of the nucleotide sequences shown in SEQIDNO.1 and SEQIDNO.2, respectively, or are the core sequences of nucleotide sequences having at least 90% identity with SEQIDNO.1 and / or SEQIDNO.2.

[0010] In this application, the drug target development method can be used to screen or verify compounds that can regulate the binding activity of CEBPD and / or FOS to the ZFP36L2 promoter, which may be achieved by including or at least partially following the steps: (a) constructing a reporter gene vector containing SEQ ID NO: 1 or SEQ ID NO: 2, for example, using the core regulatory sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2 in the promoter region of ZFP36L2 as a template to construct a reporter gene vector; transfecting the constructed vector into a suitable cell line, such as THP-1 cells, to establish a cell model that stably expresses the reporter gene for subsequent screening experiments; (b) detecting the effect of the candidate compound on the binding activity of CEBPD and / or FOS to the ZFP36L2 promoter, for example, adding the candidate compound to the above-mentioned transfected cells for incubation, which can use a luciferase reporter gene assay to reflect the change in the binding activity of CEBPD and / or FOS to the ZFP36L2 promoter, or using electrophoretic mobility shift analysis technology to determine whether the candidate compound affects the binding of CEBPD and / or FOS to the ZFP36L2 promoter fragment containing SEQ ID NO: 1 or SEQ ID NO: 2, or using chromatin immunoprecipitation technology to evaluate the effect of the candidate compound on the binding activity; (c) Screening out compounds that change the transcription level of ZFP36L2 by regulating the activity of CEBPD and / or FOS. For example, for candidate compounds that are shown to significantly affect the binding activity of CEBPD / FOS to the ZFP36L2 promoter in the above-mentioned test, further verifying the effect of the compound on the transcriptional regulation of ZFP36L2 by, for example, qPCR and Western blot, so as to determine the potential application impact of the compound in the treatment of radiation injury.

[0011] The second aspect of the present invention is to provide the use of CEBPD and / or FOS in the preparation of a product for regulating ZFP36L2 promoter activity and / or regulating ZFP36L2 expression; the regulation is positive regulation, and the product regulates ZFP36L2 transcription by overexpressing CEBPD and / or FOS proteins, overexpressing CEBPD and / or FOS genes, or activating endogenous CEBPD and / or FOS activity; for example, the product may include proteins expressing CEBPD and / or FOS, or nucleic acids encoding CEBPD and / or FOS, or drugs that activate ZFP36L2 gene transcription, as well as pharmaceutically acceptable excipients such as buffers, stabilizers, and the like.

[0012] The third aspect of the present invention is to provide a substance that promotes the expression of CEBPD and / or FOS for use in the preparation of a product that regulates the activity of the ZFP36L2 promoter and / or the expression of ZFP36L2, wherein the regulation is positive regulation; the substance may include a small molecule compound that activates the activity of CEBPD and / or FOS, or an mRNA or DNA nucleic acid sequence encoding CEBPD and / or FOS, or a cell that overexpresses CEBPD and / or FOS, and at least one of a functional antibody that enhances the activity of CEBPD and / or FOS.

[0013] The fourth aspect of the present invention is to provide a gene editing tool, comprising a nucleotide sequence as shown in SEQIDNO:1 and / or SEQIDNO:2 and / or SEQIDNO:3 and / or SEQIDNO:4 targeting the ZFP36L2 promoter, or a nucleotide sequence having at least 90% identity with SEQIDNO.1 and / or SEQIDNO.2; used to regulate the binding activity of CEBPD and / or FOS, and enhance the binding of CEBPD and / or FOS to ZFP36L2 by changing chromatin openness (such as demethylation) or recruiting auxiliary factors (such as co-activator proteins).

[0014] The significant advantage of the present invention is that the present invention reveals for the first time the regulatory effect of transcription factors CEBPD and FOS on the expression of ZFP36L2 through classical monocytes. The present invention uses single-cell analysis to screen out CEBPD and FOS as potential regulatory factors of ZFP36L2, and verifies through dual luciferase reporter that CEBPD and FOS can bind to specific sites in the promoter region of ZFP36L2; the inventor's previous research (CN202411146999.0) has confirmed that overexpression of ZFP36L2 has the effect of slowing down cell damage induced by radiation, thus revealing that CEBPD and FOS can bind to specific sites in the promoter region of ZFP36L2, which is expected to provide a new molecular target for the development of radiation injury treatment drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 This is a comparison diagram of single cell dimensionality reduction in the blood samples of patients undergoing chest radiotherapy before and after radiotherapy in the present invention; Figure 2 This is a group comparison diagram of the proportion of different cell types in whole blood before and after radiotherapy in the blood samples of patients undergoing chest radiotherapy according to the present invention; Figure 3 This is a graph showing changes in the number and proportion of different cell types in the blood samples of patients undergoing chest radiotherapy before and after radiotherapy in the present invention; Figure 4 This is a GOBP enrichment analysis diagram of the gene functions of different subtypes of monocytes in the blood samples of patients undergoing chest radiotherapy before and after radiotherapy in the present invention; Figure 5 The expression distribution diagram of the ZFP family genes provided by the present invention in the single cell dimensionality reduction diagram; Figure 6 A comparison diagram of the expression changes of the ZFP family genes provided by the present invention in different monocyte subtypes before and after radiotherapy; Figure 7 A heat map of ZFP36L2-related Regulon activity based on RSS calculation provided by the present invention; Figure 8 Provides a ranking diagram of the RSS values ​​of each cell group for the present invention; Fig. 9 Schematic diagram of the binding sites of CEBPD and FOS on the ZFP36L2 promoter of the present invention; Fig.10 This is a graph showing the results of the CEBPD dual luciferase reporter gene experiment of the present invention; Fig.11 This is a diagram showing the results of the FOS dual-luciferase reporter gene experiment of the present invention. DETAILED DESCRIPTION

[0017] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0018] The terms "first", "second" and "third" in the present invention are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Therefore, the features defined as "first", "second" and "third" can explicitly or implicitly include multiple features. In the description of this application, the meaning of "multiple" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined. All directional indications (such as up, down, left, right, front, back...) in the embodiments of the present application are only used to explain the relative position relationship, movement, etc. between the components under a certain specific posture (as shown in the accompanying drawings). If the specific posture changes, the directional indication also changes accordingly. In addition, the terms "including" and "having" and any of their variations are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device that includes a series of steps or modules is not limited to the listed steps or modules, but optionally also includes steps or modules that are not listed, or optionally also includes other steps or modules inherent to these processes, methods, products or devices.

[0019] Reference to "embodiments" herein means that the specific features, structures, or characteristics described in conjunction with the embodiments may be included in multiple embodiments of the present application. The appearance of the phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0020] The experimental methods used in the embodiments of the present invention are conventional methods unless otherwise specified, and the reagents and materials used are commercially available unless otherwise specified.

[0021] The research process of the transcriptional regulation mechanism of the present invention is as follows: 1) The inventor collected peripheral whole blood samples from patients undergoing chest radiotherapy in the Department of Radiotherapy of the Second Affiliated Hospital of Chengdu Medical College and the 416 Nuclear Industry Hospital. The samples were collected before the start of radiotherapy and after the entire course of treatment, and sent to Beijing Xunyin Biotechnology Co., Ltd. for single-cell omics analysis. By systematically mining single-cell omics data, all cell types were analyzed by dimensionality reduction and visualization, and the distribution results are as follows Figure 1 As shown, each point in the figure represents a single cell, the horizontal and vertical coordinates are the spatial coordinate values ​​after dimensionality reduction, and different colors represent different cell groups. Comparing the changes in cell distribution in samples before radiotherapy (right) and after radiotherapy (left), the overall impact of radiotherapy on the cell population is revealed. Given the low proportion of macrophages in the blood, monocytes, as precursor cells of macrophages, are closely related to their key role in immune regulation. Monocytes can be divided into three subgroups: classical monocytes, intermediate monocytes, and non-classical monocytes. Classical monocytes can further differentiate into macrophages. The inventors focused their research on the dynamic changes of the monocyte-macrophage axis.

[0022] See also Figure 2 , which is a grouped comparison of the proportion of different cell types in whole blood before and after radiotherapy. Statistical pie charts of the proportion of each cell type in whole blood before (right) and after (left) radiotherapy. Different colors represent the grouping information of different cell types, which intuitively shows the impact of radiotherapy on the proportion of different cell types. The inventors found that the proportion of classical monocytes in whole blood cells after radiotherapy was 20.23%, a significant increase compared to 14.23% before radiotherapy; the proportion of intermediate monocytes was 0.51% before radiotherapy, which increased to 1.23% after radiotherapy; the proportion of non-classical monocytes decreased from 6.17% before radiotherapy to 5.42% after radiotherapy.

[0023] See also Figure 3, which shows the changes in the proportion of different cell types before and after radiotherapy. The grouped bar graph shows the changing trend of the proportion of each cell type in the blood sample before and after radiotherapy. Different colors represent the groups of different cell types. The comparison results show the effect of radiotherapy on the number of each cell population. Further group comparison results showed that the number of classical monocytes and intermediate monocytes increased significantly after radiotherapy, while the number of non-classical monocytes decreased significantly. In addition, functional pathway analysis showed that the changes in gene expression pathways of different monocyte subtypes before and after radiotherapy were significantly different.

[0024] See also Figure 4 , which shows the GOBP enrichment analysis of gene functions of different subtypes of monocytes before and after radiotherapy. The results of GOBP (Gene Ontology Biological Process) enrichment analysis of gene changes in classical monocytes, intermediate monocytes and non-classical monocytes before and after radiotherapy reveal the differential effects of radiotherapy on the gene functions of the three types of monocytes. The genes that changed significantly in non-classical monocytes were enriched in protein transport-related pathways. GOBP (Gene Ontology Biological Process) enrichment analysis showed that the genes that changed significantly in classical monocytes after radiotherapy compared with before radiotherapy were enriched in endogenous immune response-related pathways; the genes that changed significantly in intermediate monocytes were mainly enriched in virus resistance pathways; and the genes that changed significantly in non-classical monocytes were enriched in protein transport-related pathways.

[0025] In summary, from Figure 1~Figure 4 The results showed that chest radiotherapy had a significant impact on the proportion of different monocyte subtypes in the patient's peripheral blood and their related functional pathways. These changes may be related to the regulatory mechanism of radiotherapy-induced immune response.

[0026] 2) To further explore the specific regulatory mechanism of ZFP36L2 in the monocyte-macrophage axis, the inventors further analyzed the expression characteristics of ZFP family genes in clinical samples in different subtypes of monocytes before and after radiotherapy.

[0027] See also Figure 5, which shows the expression distribution of ZFP family genes in the single-cell dimensionality reduction map. Each point in the dimensionality reduction analysis diagram represents a cell, and the horizontal and vertical coordinates are the spatial coordinates after dimensionality reduction. The color depth of the point represents the expression level of the ZFP family gene, and the darker the color, the higher the gene expression. Through the dimensionality reduction analysis diagram of the ZFP family genes at the single-cell level, it was found that the expression of ZFP36, ZFP36L2 and ZFP36L1 showed significant distribution differences in the overall cell map. In order to further explore its dynamic changes in different monocyte subtypes, the inventors took the three subtypes of classical monocytes, intermediate monocytes and non-classical monocytes as research objects for detailed analysis. For results, please see Figure 6 , which shows a comparison of the expression changes of ZFP family genes in different monocyte subtypes before and after radiotherapy. The horizontal axis is the grouping category (before and after radiotherapy), and the vertical axis is the standardized ZFP family gene expression. The width of the curve indicates the proportion of monocytes that meet the expression of the target gene: the wider the curve, the more cells that meet the conditions; the narrower the curve, the fewer cells that meet the conditions. After radiotherapy, the expression of ZFP36 was significantly reduced in both classical monocytes and non-classical monocytes compared to before radiotherapy, while no significant changes were observed in intermediate monocytes. The expression of ZFP36L2 also showed a significant downward trend in classical monocytes and non-classical monocytes; in contrast, the expression of ZFP36L1 was significantly increased in classical monocytes, while no significant differences were observed in other subtypes; Figure 5~Figure 6 These expression characteristics are highly consistent with the research results of the inventors in in vitro cell experiments and animal models (see CN202411146999.0 for details), indicating that ZFP36L2 may play a key role in radiation stress response by regulating the fate and function of monocytes and then regulating downstream macrophage function under radiation stress conditions.

[0028] Example 1, screening of ZFP36L2 as a key transcription factor in radiation stress response; based on single-cell sequencing (commissioned by Beijing Xunyin Biotechnology Co., Ltd.), systematic regulatory network analysis (Regulatory Network Analysis) was performed.

[0029] 1. Single-cell sequencing 1) Sample collection: Samples were collected from the Department of Radiotherapy of the Second Affiliated Hospital of Chengdu Medical College and the 416 Nuclear Industry Hospital. Peripheral blood samples were collected before radiotherapy and 24 hours after the end of the treatment. A total of 6 cases, 3 before radiotherapy and 3 after radiotherapy.

[0030] 2) Using the 10x Genomics Chromium system, single cells and barcoded gel beads were co-encapsulated in oil droplets to achieve single cell isolation, and cDNA with cell barcodes and UMIs was generated by reverse transcription.

[0031] 3) Library construction: cDNA was amplified and fragmented, and sequencing adapters were added to construct Illumina-compatible libraries. Paired-end sequencing was performed on the NovaSeq6000 platform, and an average of 50,000 reads were obtained per cell.

[0032] 4) Preprocessing: Use CellRanger software to align sequencing data to the reference genome and generate a gene expression matrix for each cell; remove cells with <200 genes and >20% mitochondrial gene ratio; perform LogNormalize on UMI counts; and obtain a single-cell gene expression matrix.

[0033] 2. Systemic regulatory network analysis 1) Transcription factor screening (GENIE3 algorithm) The single-cell gene expression matrix obtained by the above single-cell sequencing was input, and only highly variable genes (genes with the highest coefficient of variation in Top2000) were retained. The list of human transcription factors was obtained from the database (AnimalTFDB); the regulatory weight of each TF on ZFP36L2 was calculated by GENIE3, and the top 15 TFs were retained as candidate regulatory factors of ZFP36L2. The higher the weight, the stronger the co-expression correlation between the transcription factor and ZFP36L2, revealing its possible regulatory role. The top 15 TFs include CEBPD, FOS, CEBPB, JUNB, TFEC, EGRI, IRF2, KLF6, BACH1, SMAD3, FLI, KLF2, ATF6, TFE3 and RELB; among them, the top two transcription factors are CEBPD and FOS.

[0034] The top 15 candidate regulatory factors and weights of ZFP36L2 gene weight ranking are as follows:

[0035] 2) Regulatory factor (Regulon) specificity score (RSS,RegulonSpecificityScore) The Regulon specificity scores of the same regulatory factor (Regulon) in different monocyte subtypes were compared. The correlation between the regulatory factor and the specific cell subtype was evaluated by analysis based on the AUC value. The higher the RSS value, the more significant the regulatory effect of the transcription factor on the specific cell subtype. CEBPD and FOS showed the highest specificity in classical monocytes. CEBPD in classical monocytes had RSS=0.72 (P<0.001), and FOS in classical monocytes had RSS=0.711 (P<0.001), which had significant specificity.

[0036] Table of Regulon specificity scores (RSS) of CEBPD and FOS in different monocyte subtypes:

[0037] 3) Verify the subtype-specific regulation of the regulatory factor (Regulon) The AUC values ​​were binarized, and based on the RSS analysis results, a heat map of binary regulator activity of the top 5 regulators (Regulon) in different monocyte subtypes was drawn when radiotherapy was used as an exposure factor; the results can be found in Figure 7 , showing the binary heat map of the activity of the top 5 differential Regulons associated with ZFP36L2 in each single cell. Each row is a single cell, and each column is a Regulon. Black indicates active and white indicates inactive. CEBPD and FOS show a more open regulatory state in classical monocytes, suggesting that they may play a more active regulatory function in classical monocytes, thereby participating in the specific molecular response induced by radiotherapy. 4) Specific sorting of different monocyte subtypes See the results Figure 8 , Regulon is arranged in descending order according to RSS value for classical monocytes, intermediate monocytes and non-classical monocytes. The horizontal axis is Regulon and the vertical axis is RSS. It is clear that CEBPD and FOS have stronger regulatory specificity in classical monocytes and are the core participants in the regulation of ZFP36L2 gene expression in classical monocytes.

[0038] The above results indicate that CEBPD and FOS, as transcription factors of ZFP36L2, are significantly involved in the regulatory network of classical monocytes under radiotherapy exposure conditions and may play an important role.

[0039] Example 2, using a dual luciferase reporter gene system to detect the regulatory effects of CEBPD and FOS on ZFP36L2 gene expression; 1) Database search and positioning: The human ZFP36L2 gene was confirmed to be located in the p21 region of chromosome 2 through the NCBI database. Combined with the Ensembl database (gene ID: ENSG00000152518), its transcription start site (TSS) was identified in the promoter upstream region within the base pair range of 43,222,402 to 43,226,606 on chromosome 2; 2) Binding site prediction: The JASPAR database was used to predict the transcription factor binding sites in the promoter region of ZFP36L2 to obtain the target sequence; among them, the CEBPD binding site: sequence 5'-CGACTTCTTGTGCAAGGTAGGCC-3' (core region TTGTGCAA); the FOS binding site: sequence 5'-GCGCAGCCTGACTCGCCATCCG-3' (core region TGACTCG); 2) Construction of reporter plasmid: The ZFP36L2 promoter DNA and pcDNA3.1 plasmid were double-digested with XhoI and HindIII using the corresponding restriction endonucleases, respectively. After digestion at 37°C for 3 h, the digested ZFP36L2 promoter DNA and pcDNA3.1-Luciferase plasmid were subjected to 1% agarose gel electrophoresis, and the digested ZFP36L2 promoter DNA and pcDNA3.1-Luciferase plasmid were recovered respectively; the ZFP36L2 promoter double-digestion recovery product was ligated into the pcDNA3.1-Luciferase double-digestion recovery product to obtain a recombinant promoter reporter plasmid; the recombinant promoter reporter plasmid was transformed into Escherichia coli (DH5α) competent cells to obtain recombinant clones, 6 clones were selected for positive identification, and the selected positive clones were sequenced; after confirming that the nucleotide sequences were all correct, it was determined that the promoter reporter plasmid was obtained.

[0040] Effector plasmid construction: The empty plasmid was pcDNA3.1, and the CEBPD and FOS fragments were synthesized by a biological company according to NCBINM_005195.4 and NCBINM_005252.3, respectively, and the corresponding restriction endonuclease sites were added; the CEBPD, FOS fragments and pcDNA3.1 plasmid were double-digested with the corresponding restriction endonucleases, and after digestion at 37°C, they were subjected to 1% agarose gel electrophoresis, and the digested CEBPD, FOS and pcDNA3.1 plasmids were recovered respectively; the CEBPD and FOS double-digestion recovery products were connected to the corresponding pcDNA3.1 double-digestion recovery products, respectively, to obtain the effector plasmids (CEBPD overexpression plasmid and FOS plasmid); the effector plasmids were transformed into Escherichia coli (DH5α) competent cells to obtain recombinant clones, and 6 clones were picked for positive identification, and the selected positive clones were sequenced; after confirming that the nucleotide sequences were all correct, it was determined that the effector plasmid was obtained.

[0041] Among them, CEBPD promoter reporter plasmids include: ZFP36L2-CEBPD-WT plasmid: please refer to Fig. 9 , clone the ZFP36L2 promoter fragment containing the CEBPD binding site (SEQ ID NO: 1: 5'-CGACTTCTTGTGCAAGGTAGGCC-3'); ZFP36L2-CEBPD-MUT plasmid: see Fig. 9 , the CEBPD binding site core sequence SEQIDNO:3 "TTGTGCAA" was mutated to "CCACAAGG" (the sequence after mutation is 5'-CGACTTCCCACAAGGGGTAGGCC-3').

[0042] FOS promoter reporter plasmids include: ZFP36L2-FOS-WT plasmid: see Fig. 9 , clone the ZFP36L2 promoter fragment containing the FOS binding site (SEQ ID NO: 2: 5'-GCGCAGCCTGACTCGCCATCCG-3'); ZFP36L2-FOS-MUT plasmid: The FOS binding site core sequence SEQ ID NO: 4 "TGACTCG" was mutated to "CAGACAA" (the sequence after mutation is: 5'GCGCAGCCCAGACAACCATCCG-3').

[0043] 3) Grouping and cell transfection THP-1 cells were subcultured to passage 15 and 1×10^ 5 / well inoculated in 24-well plates; The CEBPD setting groups are as follows: pcDNA3.1-NC group: transfected with empty plasmid pcDNA3.1; pcDNA3.1-ZFP36L2 group: transfected with ZFP36L2-CEBPD-WT plasmid; pcDNA3.1-ZFP36L2+CEBPD-WT group; transfected with CEBPD overexpression plasmid and ZFP36L2-CEBPD-WT plasmid.

[0044] pcDNA3.1-ZFP36L2+CEBPD-MUT group: transfection of CEBPD overexpression plasmid and ZFP36L2-CEBPD-MUT plasmid.

[0045] The FOS set groups were as follows: pcDNA3.1-NC group: transfected with empty plasmid pcDNA3.1; pcDNA3.1-ZFP36L2 group: transfected with ZFP36L2-FOS-WT plasmid; pcDNA3.1-ZFP36L2+FOS-WT group: transfected with FOS overexpression plasmid and ZFP36L2-FOS-WT plasmid; pcDNA3.1-ZFP36L2+FOS-MUT group: transfection of FOS overexpression plasmid and ZFP36L2-FOS-MUT plasmid.

[0046] The CEBPD overexpression plasmid, FOS overexpression plasmid and pcDNA3.1 empty plasmid were transfected into THP-1 respectively, and the corresponding promoter reporter plasmid and Renilla luciferase plasmid (pRL-TK) were transferred according to the corresponding relationship of the above groups. For example, the pcDNA3.1-ZFP36L2+FOS-WT group needed to be transfected with FOS overexpression plasmid, ZFP36L2-FOS-WT plasmid and Renilla luciferase plasmid; after transfection, the transfected cells were collected, the cells were lysed with passive lysis buffer, and the supernatant was taken; firefly luciferase substrate and Renilla luciferase substrate were added in turn, and the fluorescence signal was detected by a microplate reader. The ratio of the expression amount of firefly luciferase to that of Renilla luciferase (relative activity = firefly fluorescence value / Renilla fluorescence value) was the relative luciferase signal (Relative Luciferase signals), and each group was repeated at least 3 times; Result analysis: See Fig.10 , the luciferase activity of the pcDNA3.1-ZFP36L2+CEBPD-WT group was significantly higher than that of the pcDNA3.1-ZFP36L2+CEBPD-MUT group (P<0.05), indicating that CEBPD can bind to specific sites in the promoter region of ZFP36L2 and play a regulatory role; See also Fig.11 The luciferase activity of the pcDNA3.1-ZFP36L2+FOS-WT group was significantly higher than that of the pcDNA3.1-ZFP36L2+FOS-MUT group, proving that FOS binds to specific sites in the ZFP36L2 promoter region and plays a regulatory role.

[0047] In summary, the present invention reveals for the first time the regulatory effects of transcription factors CEBPD and FOS on ZFP36L2 gene expression in classical monocytes. Through systematic analysis of single-cell sequencing data (SCENIC regulatory network construction and GENIE3 algorithm), CEBPD and FOS were screened as core regulatory factors of ZFP36L2, and their regulatory weights were significantly higher than other candidate factors (P<0.001). Further verification by dual luciferase reporter showed that CEBPD and FOS could bind to the SEQIDNO:1 (core binding site TTGTGCAA) and SEQIDNO:2 (core binding site TGACTCG) binding sites in the promoter region of ZFP36L2. Combined with the experimental results of the inventor's previous study (CN202411146999.0) that ZFP36L2 overexpression can significantly slow down radiation-induced cell damage, the present invention is expected to provide a new molecular target for the development of radiation injury therapeutic drugs by revealing that CEBPD and FOS activate ZFP36L2 transcription.

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of CEBPD and / or FOS as ZFP36L2 transcriptional regulators in the development of drug targets for the treatment of radiation injury.

2. The use according to claim 1, characterized in that: The CEBPD and / or FOS positively regulate the expression of ZFP36L2 by binding to the 3879bp-3856bp and / or 2854bp-2832bp binding sites upstream of ZFP36L2.

3. The use according to claim 2, characterized in that: The binding site includes the nucleotide sequence shown in SEQ ID NO: 1 and / or SEQ ID NO:

2.

4. The use according to claim 2, characterized in that: The binding site includes the nucleotide sequence shown in SEQ ID NO: 3 and / or SEQ ID NO:

4.

5. Use of CEBPD and / or FOS in any of the following: (a1) in the preparation of a product for promoting the expression of ZFP36L2; (a2) in the preparation of a product for enhancing the promoter activity of ZFP36L2.

6. The use according to claim 5, characterized in that: The CEBPD and / or FOS bind through the 3879bp-3856bp and / or 2854bp-2832bp binding sites upstream of ZFP36L2 to promote ZFP36L2 expression and / or enhance ZFP36L2 promoter activity; the binding site of ZFP36L2 includes the nucleotide sequence shown in SEQIDNO.1 and / or SEQIDNO.

2.

7. The use according to claim 6, characterized in that: The product promotes ZFP36L2 expression and / or enhances ZFP36L2 promoter activity by overexpressing CEBPD and / or FOS proteins, overexpressing CEBPD and / or FOS genes, or activating endogenous CEBPD and / or FOS activity.

8. Use of a substance that promotes the expression of CEBPD and / or FOS in any of the following: (b1) in the preparation of a product that promotes the expression of ZFP36L2; (b2) in the preparation of a product that enhances the promoter activity of ZFP36L2.

9. The use according to claim 8, characterized in that: The substance comprises at least one of a small molecule compound that activates the activity of CEBPD and / or FOS, an mRNA or DNA nucleic acid sequence encoding CEBPD and / or FOS, a cell that overexpresses CEBPD and / or FOS, and a functional antibody that enhances the activity of CEBPD and / or FOS.

10. A gene editing tool, characterized in that: The nucleotide sequence shown in SEQ ID NO: 1 and / or SEQ ID NO: 2 and / or SEQ ID NO: 3 and / or SEQ ID NO: 4 on the ZFP36L2 promoter is targeted to regulate the binding activity of CEBPD and / or FOS.

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