Product for detecting radiation exposure based on IFIT3 marker and application

Abstract

The invention belongs to the field of biomedicine, and particularly relates to a product for detecting radiation exposure based on an IFIT3 marker and application. Through detection, the expression of the IFIT3 in a blood sample of a subject after radiotherapy is obviously reduced, the IFIT3 is verified to have the characteristics of high sensitivity and high specificity, whether an individual is subjected to the risk of radiation exposure or injury or not can be detected by detecting the expression of the IFIT3, and the IFIT3 can be used as a basis for supporting the individual to be subjected to radiation exposure or early radiation injury and has a wide clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and more particularly, to products and applications for detecting radiation exposure based on the IFIT3 marker. Background Art

[0002] Ionizing radiation is widely used in medical radiotherapy, nuclear industry, aerospace and other fields. With the widespread application of nuclear technology and the increase of regional security risks, emergencies such as nuclear accidents and radiation leaks also pose potential threats to public health. After an individual is exposed to radiation, it is not certain that obvious radiation damage will occur. Whether it occurs and its severity are affected by multiple factors such as radiation dose, irradiation technique, tissue tolerance and individual repair ability. Some individuals may only show reversible mild reactions, or even may be in a state without obvious clinical manifestations. Such situations are difficult to detect through traditional clinical observations or imaging examinations, but may become potential causes of long-term health risks. Therefore, the research and development of products for detecting radiation exposure or early radiation damage have important social significance and wide application value. Currently, technologies such as RNA-seq and whole-genome DNA microarrays for identifying early radiation response genes are the main directions of radiation biology research. Relevant research mostly screens biomarkers for detecting radiation exposure or damage through means such as gene expression analysis and protein concentration detection.

[0003] As the role of the interferon signaling pathway in radiation response has been gradually revealed, interferon-stimulated genes (ISGs) have become a research hotspot due to their clustered differential expression. However, existing research mostly focuses on exploring the mechanisms of ISGs in radiation-induced immune activation, apoptosis or tumor drug resistance, and the expression changes of some ISGs overlap in non-radiation scenarios such as viral infections and autoimmune diseases. No gene markers with high sensitivity and good specificity have been screened out, especially the application in the convenient detection scenario of peripheral blood is still blank. As a core member of the IFIT family, IFIT3 encodes a protein that inhibits viral translation by recognizing the 5'-triphosphate structure of viral RNA and participates in cell cycle regulation and innate immune response. Existing research shows that IFIT3 is significantly upregulated through the JAK-STAT pathway in viral infections, and shows bidirectional regulation in oxidative stress, but its expression pattern under radiation exposure and its potential as a marker for detecting exposure or damage have not been reported. Summary of the Invention

[0004] The object of the present invention is to provide a product and application for detecting radiation exposure based on the IFIT3 marker.

[0005] Based on the single-cell sequencing data of peripheral blood samples from radiotherapy patients, the inventors ranked the top 40 genes at the overall level and found that some interferon-induced genes were significantly downregulated in clusters after irradiation, including the IFIT3 gene, which indicates that IFIT3 plays an important role in the radiation response. Through experiments, it was verified that IFIT3 meets the characteristics of high sensitivity and good specificity in the diagnosis of radiation exposure in radiotherapy patients and is expected to be applied to products for detecting individual radiation exposure or early radiation damage. Based on this, the inventors completed the following invention of the present invention.

[0006] The first aspect of the present invention is to provide the application of IFIT3 as a biomarker in the preparation of products for diagnosing or assisting in the diagnosis of radiation exposure.

[0007] In this application, the product includes a reagent for detecting the expression level of IFIT3 in a sample; for example, the reagent is used to detect the expression level of the IFIT3 gene and / or the expression level of the IFIT3 protein in the sample.

[0008] In this application, the reagent can rely on methods such as real-time quantitative qRT-PCR, RT-PCR, or gene chips to perform quantitative or semi-quantitative analysis of the mRNA transcript of IFIT3; or rely on methods such as enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), immunoturbidimetry, etc. to measure the concentration of the IFIT3 protein.

[0009] In this application, the reagent for detecting the expression level of IFIT3 in a sample includes: oligonucleotide probes targeting the IFIT3 mRNA sequence, such as designed for specific fragments of IFIT3 mRNA, which can detect mRNA through hybridization techniques such as Northern hybridization and in situ hybridization; PCR primers targeting IFIT3 mRNA, used for PCR amplification techniques such as ordinary PCR, real-time quantitative PCR, and nested PCR to analyze the mRNA level; monoclonal or polyclonal antibodies targeting IFIT3, detecting protein expression through immunological techniques such as Western Blot, immunohistochemistry, and immunofluorescence; specific nucleic acid aptamers of IFIT3, using their specific binding ability to recognize IFIT3 molecules; IFIT3-targeted molecularly imprinted polymers, used for specific recognition in affinity chromatography or sensor construction.

[0010] In this application, the above-mentioned reagents such as probes, primers, antibodies, or nucleic acid aptamers can be prepared or obtained by conventional methods in the art such as chemical synthesis, genetic engineering preparation, and hybridoma technology. For example, the oligonucleotide probe can be directly prepared by chemical synthesis according to the known IFIT3 mRNA sequence.

[0011] In this application, although the samples described in the embodiments of the present application are derived from the peripheral blood samples of chest radiotherapy patients, their applications are not limited to this scenario and can also be extended to other radiation exposures such as peripheral blood or other body fluid samples of head and neck radiotherapy patients, irradiated populations in nuclear accidents, etc.

[0012] In this application, although the samples in this embodiment are derived from the radiotherapy population, the expression change of IFIT3 is directly related to ionizing radiation. Therefore, the markers described in the present invention are expected to be applicable to most types of individuals exposed to ionizing radiation, including but not limited to radiotherapy patients, irradiated individuals in nuclear accidents, radiation workers, etc.

[0013] In this application, the reagent may contain auxiliary components such as buffer, reaction substrate, standard product, etc. in addition to detection components such as probes, primers, and antibodies, all of which are within the protection scope of the present invention.

[0014] In this application, the products include but are not limited to detection tools such as chips, test strips, and reagent kits; the detection can be realized relying on high-throughput sequencing platforms such as Illumina sequencers, enzyme labelers, fluorescence quantitative PCR instruments, etc., but the protection of the present invention is based on the reagent combination for IFIT3 detection rather than specific equipment.

[0015] The second aspect of the present invention is to provide a reagent kit, which realizes the detection of radiation exposure or injury by measuring the expression level of IFIT3 in a sample; the reagent kit may include: reagents for detecting the expression of the IFIT3 gene such as primers and probes; reagents for detecting the expression of the IFIT3 protein such as antibodies and markers; optional auxiliary materials such as positive controls, negative controls, and calibrators. Those skilled in the art can understand that the above components and auxiliary materials included in the reagent kit are all within the protection scope of the present invention.

[0016] The remarkable advantage of the present invention is that: the present invention provides a biomarker with high sensitivity and good specificity for diagnosing radiation exposure. The expression of IFIT3 in the blood samples of subjects after radiotherapy (radiation) is significantly down-regulated, which can accurately judge whether the subjects are at risk of radiation exposure or injury. By detecting the expression level of IFIT3 in the peripheral blood samples of subjects, it can be used for diagnosing or assisting in diagnosing radiation exposure or early radiation injury, and can be used as a basis to support that an individual has suffered radiation exposure or early radiation injury, and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0018] Figure 1 This is a grouped comparison chart of the proportions of different cell types in the blood samples of chest radiotherapy patients before and after radiotherapy provided by the present invention; Figure 2 This is a comparison chart of single-cell dimensionality reduction of the blood samples of chest radiotherapy patients before and after radiotherapy provided by the present invention; Figure 3 This is an expression distribution chart of the IFIT3 gene in the single-cell dimensionality reduction chart provided by the present invention; Figure 4 This is a comparison chart of the expression changes of the IFIT3 gene before and after radiotherapy provided by the present invention; Figure 5 This is a scatter plot of the IFIT3 expression levels in the control group and the radiotherapy group provided in Example 1 of the present invention; Figure 6 This is an ROC curve diagram drawn according to the IFIT3 expression levels in the control group and the radiotherapy group in Example 1 of the present invention; Figure 7 This is a scatter plot of the IFIT3 expression levels in the control group and the radiotherapy group provided in Example 2 of the present invention; Figure 8 This is an ROC curve diagram drawn according to the IFIT3 expression levels in the control group and the radiotherapy group in Example 2 of the present invention; Detailed implementation manners

[0019] Next, the technical solutions of the present invention will be described clearly and completely in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0020] The terms "first", "second", and "third" in the present invention are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first", "second", and "third" may explicitly or implicitly include multiple such features. In the description of this application, the meaning of "multiple" is at least two, such as two, three, etc., unless otherwise specifically defined. All directional indications (such as up, down, left, right, front, back...) in the embodiments of this application are only used to explain the relative positional relationship and movement conditions between components in a specific posture (as shown in the drawings). If the specific posture changes, the directional indications will change accordingly. In addition, the terms "comprising" and "having" and any variations thereof are intended to cover non-exclusive inclusion. 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 further includes steps or modules not listed, or optionally further includes other steps or modules inherent to these processes, methods, products, or devices.

[0021] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in connection with the embodiments can be included in multiple embodiments of this application. The phrase appears in various places in the specification and does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment mutually exclusive with other embodiments. Those skilled in the art will explicitly and implicitly understand that the embodiments described herein can be combined with other embodiments.

[0022] The design process of the present invention: The inventor collected a total of 6 peripheral blood whole blood samples from patients receiving chest radiotherapy in the Radiotherapy Department of the Second Affiliated Hospital of Chengdu Medical College · 416 Nuclear Industry Hospital. The total dose range of the course of radiotherapy was 40 - 60 Gy. The samples were collected from the whole peripheral blood before the start of radiotherapy and within 24 hours after the end of the entire course of radiotherapy. 3 samples were taken before radiotherapy and 3 samples were taken after radiotherapy, and they were sent to Beijing Xunyin Biotechnology Co., Ltd. for single-cell omics analysis. By systematically mining single-cell omics data, dimensionality reduction analysis and visualization processing were performed on all cell types, and the distribution results are as Figure 1 and Figure 2 shown, Figure 1 which is a grouped comparison of the proportions of different cell types in whole blood before and after radiotherapy. The statistical pie charts of the proportions of each cell type in whole blood before (right) and after (left) radiotherapy, with different colors representing the grouped information of different cell types, intuitively show the impact of radiotherapy (radiation) on the proportions of different cell types. Figure 2 This is a comparison diagram of single-cell dimensionality reduction before and after radiotherapy in the blood samples of radiotherapy patients, Figure 2Each point in it represents a single cell, with the horizontal and vertical coordinates being the space coordinate values after dimensionality reduction, and different colors representing different cell groups. By comparing the changes in cell distribution in the samples before radiotherapy (right) and after radiotherapy (left), the overall impact of radiotherapy (radiation) on the cell population is revealed.

[0023] The inventors ranked the top 40 genes at the overall level and found that interferon-stimulated genes (ISGs) were significantly downregulated in clusters after irradiation. Among these differentially expressed genes, the expression level of the IFIT3 gene was significantly reduced, suggesting that it may be a potential molecular indicator for radiation exposure or early radiation damage diagnosis. See Figure 3 The expression distribution of the IFIT3 gene in the single-cell dimensionality reduction map is shown. Each point in the dimensionality reduction analysis map represents a cell, and the horizontal and vertical coordinates are the space coordinates after dimensionality reduction. The color depth of the point represents the expression level of the IFIT3 gene, and the darker the color, the higher the gene expression. Through the dimensionality reduction analysis map of the IFIT3 gene at the single-cell level, it was found that the IFIT3 expression showed significant distribution differences in the cell atlas. See Figure 4 It is a comparison chart of the expression changes of the IFIT3 gene before and after radiotherapy. The horizontal axis is the grouping category (before radiotherapy and after radiotherapy), and the vertical axis is the normalized expression level of the IFIT3 gene. The wider the curve, the more cells meet the conditions; the narrower the curve, the fewer cells meet the conditions. The expression of IFIT3 was significantly reduced after radiotherapy compared with before radiotherapy.

[0024] Based on the above findings, the inventors further compared the samples before and after radiotherapy and detected the expression level of IFIT3 in peripheral blood. It was found that IFIT3 showed good specificity and sensitivity when used to detect radiation exposure. Based on these research results, the inventors completed the products and applications for detecting radiation exposure based on the IFIT3 biomarker. The following will detail this process through specific examples.

[0025] Example 1, screening of the differentially expressed gene IFIT3, was carried out by RNA (RNA-seq) sequencing, specifically including the following steps: 1) Sample collection: A total of 82 peripheral blood whole blood samples were collected from the radiotherapy department of the Second Affiliated Hospital of Chengdu Medical College · 416 Nuclear Industry Hospital from patients receiving chest radiotherapy. The total dose range of the course of irradiation was 40 - 60 Gy. The peripheral blood of the patients was collected before radiotherapy and within 24 hours after the last radiotherapy. The samples included a control group and a radiotherapy group. The control group included 41 pre-radiotherapy samples, and the radiotherapy group included 41 post-radiotherapy samples.

[0026] 2) Blood cell separation and RNA extraction: Blood cells were separated by conventional methods to obtain relatively pure white blood cell precipitates, and RNA was extracted from the white blood cell precipitates. After the RNA extraction was completed, a spectrophotometer was used to measure the concentration and purity of the RNA to ensure that the OD260 / OD280 ratio was between 1.8 and 2.2. Then, agarose gel electrophoresis was used to detect the integrity of the RNA, and the clarity and brightness ratio of the 28S and 18S rRNA bands were observed to ensure that the RNA quality met the requirements of subsequent experiments. 3) Library construction: Total RNA was fragmented by conventional methods and used as a template to synthesize cDNA by reaction with reverse transcriptase and random primers. Adapters containing PCR primer binding sites and sequencing recognition tags were ligated to both ends of the cDNA, and then multiple rounds of PCR amplification were performed with primers complementary to the adapters to increase the cDNA concentration to meet the sequencing requirements.

[0027] 4) RNA sequencing: Sequencing was performed using the Illumina series of high-throughput sequencing platforms. Before sequencing, the sequencing platform was calibrated and debugged to ensure that the instrument was in the best working condition. Reagents, consumables, etc. required for sequencing were prepared, the constructed library was appropriately diluted to make its concentration meet the requirements for loading onto the machine, and then sequencing was performed to obtain a large number of sequencing reads.

[0028] 5) Data processing and analysis: The number of sequencing reads of the IFIT3 gene in each sample before and after radiotherapy was calculated separately. The results are shown in Figure 5 , in the samples of the radiotherapy group compared with the control group, the expression level of IFIT3 was significantly down-regulated, and the Mann-Whitney test p < 0.05, indicating a statistically significant difference.

[0029] Taking whether radiotherapy was received as the binary classification result and the expression level of the IFIT3 gene as the predictor, by changing the threshold for judging the disease, the sensitivity and specificity at different thresholds were calculated, and an ROC curve was plotted. The results are shown in Figure 6 As shown, the area under the ROC curve AUC = 0.9917, the sensitivity was 95.12%, and the specificity was 95.12%, indicating that using the expression level of the IFIT3 gene as a diagnostic indicator can better judge whether the subject has been radiation-exposed and has good diagnostic efficacy.

[0030] Example 2, the diagnostic effect of IFIT3 was further verified by enzyme-linked immunosorbent assay (ELISA), including the following steps: 1) Sample collection: A total of 70 peripheral blood samples were collected from patients receiving chest radiotherapy in the radiotherapy department of the Second Affiliated Hospital of Chengdu Medical College - Nuclear Industry 416 Hospital. The total dose range of the course of radiotherapy was 40 - 60 Gy. Peripheral blood of the patients was collected before radiotherapy and within 24 hours after the last radiotherapy. The samples included a control group and a radiotherapy group. The control group included 35 pre - radiotherapy samples, and the radiotherapy group included 35 post - radiotherapy samples. Among them, all patients were completely independent of the patients in the screening level of Example 1, with no intersection.

[0031] 2) Antibody and sample treatment: Antibodies specific to the IFIT3 protein were coated on microtiter plates and incubated overnight at 4 °C to allow the antibodies to adsorb on the surface of the wells. The microtiter plates were filled with a blocking solution containing BSA and incubated at room temperature for 1 - 2 h. Protein samples from the control group and the radiotherapy group were added to the coated microtiter plates, and standards and blank controls were set simultaneously. Incubation was carried out at room temperature for 1 - 2 h to allow the IFIT3 protein in the samples to bind to the coated antibodies.

[0032] 3) Incubation: Primary antibody incubation: The liquid in the wells was discarded, and after washing, a primary antibody specific to the IFIT3 protein was added and incubated at room temperature for 1 - 2 h to allow the primary antibody to bind to the IFIT3 protein bound to the coated antibody. Secondary antibody incubation: After washing, a secondary antibody labeled with an enzyme was added and incubated at room temperature for 1 - 2 h, and the secondary antibody bound to the primary antibody.

[0033] 4) Adding substrate for color development and measurement: The substrate of the enzyme was added, and the enzyme catalyzed the substrate to develop color. The depth of the color was proportional to the content of the IFIT3 protein in the sample. The optical density values (OD values) of each well were measured using an enzyme - linked immunosorbent assay (ELISA) reader at a specific wavelength, and the relative content of the IFIT3 protein in the sample, that is, the relative optical density value, was calculated according to the standard curve drawn with the standards.

[0034] 5) Data processing and analysis: The relative optical density values of the IFIT3 protein in patients before and after radiotherapy were compared and statistically analyzed. For the results, please refer to Figure 7 Among the patients after radiotherapy, the expression level of IFIT3 was significantly down - regulated, and the Mann - Whitney test p < 0.05, which was statistically significant.

[0035] Taking whether having received radiotherapy as the result of binary classification and using the expression level of the IFIT3 protein as a predictor, by changing the threshold for judging the disease, the sensitivity and specificity at different thresholds were calculated, and an ROC curve was plotted. For the results, please refer to Figure 8 As shown in

[0036] In summary, the present invention verifies the application of IFIT3 as a biomarker in detecting radiation-exposed products through radiotherapy individual cases, and this IFIT3 biomarker has two characteristics of high sensitivity and good specificity; while the biological effects of ionizing radiation, including medical radiotherapy, nuclear accident radiation, industrial rays, etc., have commonalities, and their core mechanisms are all to trigger DNA damage, oxidative stress, and activation of the interferon signaling pathway through the action of ionizing radiation. Radiotherapy, as a typical scenario of ionizing radiation under controlled conditions, has a high degree of consistency in the induced radiation response mechanism with other non-medical radiation exposures. Therefore, the biomarker screened for the radiotherapy population in this application can also reflect the overall response of the body to ionizing radiation, has a scientific basis for generalization to other radiation exposure scenarios, can provide a basis for the clinical diagnosis of radiation exposure, provide an early warning for the diagnosis of early radiation damage, and has broad clinical application prospects.

[0037] The foregoing is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of IFIT3 as a biomarker in the preparation of a product for detecting radiation exposure.

2. The application according to claim 1, wherein The product includes a reagent for detecting the expression level of IFIT3 in a test sample.

3. The application according to claim 2, characterized in that The expression level of IFIT3 in the test sample includes detecting the expression level of the IFIT3 gene.

4. The application according to claim 3, characterized in that, The product detects the expression level of the IFIT3 gene in the sample by any one of qRT-PCR, RT-PCR or gene chip.

5. The application according to claim 2, wherein The expression level of IFIT3 in the test sample includes detecting the expression level of IFIT3 protein in the sample.

6. The application according to claim 5, wherein The product detects the expression level of IFIT3 protein in the sample by any one of enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), immunoturbidimetry.

7. The application according to claim 2, wherein the reagent is at least one of an oligonucleic acid probe targeting the IFIT3-encoding DNA sequence, a PCR primer targeting the IFIT3-encoding DNA sequence, an IFIT3-specific nucleic acid aptamer, or an IFIT3-targeted molecularly imprinted polymer.

8. The application according to claim 2, wherein the sample is a peripheral blood sample.

9. Use of a reagent for detecting IFIT3 in the preparation of a kit for diagnosing local ionizing radiation exposure, characterized in that, The reagent for detecting IFIT3 is a reagent for detecting IFIT3 in a peripheral blood sample.

10. The application according to claim 9, characterized in that The reagent for detecting IFIT3 is an RNA-seq analysis reagent or an enzyme-linked immunoassay reagent.

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