Products and applications for detecting radiation exposure based on the IFIT3 marker

Abstract

This invention belongs to the field of biomedicine, and particularly relates to products and applications for detecting radiation exposure based on the IFIT3 marker. Testing has shown that IFIT3 expression is significantly downregulated in blood samples from subjects after radiotherapy, confirming the high sensitivity and specificity of IFIT3. IFIT3 expression can be used to assess an individual's risk of radiation exposure or injury, providing support for predicting radiation exposure or early radiation damage, and thus has broad clinical application prospects.

Description

Technical Field

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

[0002] Ionizing radiation is widely used in medical radiotherapy, the nuclear industry, aerospace, and other fields. With the widespread application of nuclear technology and increasing regional safety risks, emergencies such as nuclear accidents and radiation leaks pose a potential threat to public health. Individuals exposed to radiation do not necessarily experience overt manifestations of radiation damage. The occurrence and severity of radiation damage are influenced by multiple factors, including radiation dose, irradiation technique, tissue tolerance, and individual repair capacity. Some individuals may only experience mild, reversible reactions or even no obvious clinical manifestations. These conditions are difficult to detect through traditional clinical observation or imaging studies, but they may pose a potential risk to long-term health. Therefore, research and development of products that can detect radiation exposure or early radiation damage has significant social significance and broad application value. Currently, the identification of early radiation response genes using technologies such as RNA-seq and whole-genome DNA microarrays is a major focus of radiation biology research. Related studies often screen and detect biomarkers of radiation exposure or damage through methods such as gene expression analysis and protein concentration measurements.

[0003] As the role of the interferon signaling pathway in radiation response has been gradually revealed, interferon-induced genes (ISGs) have become a research hotspot due to their clustered differential expression. However, existing research has primarily focused on the mechanisms of ISGs in radiation-induced immune activation, apoptosis, or tumor resistance. Furthermore, the expression changes of some ISGs overlap in non-radiation settings, such as viral infection and autoimmune diseases. Genetic markers with high sensitivity and specificity have yet to be identified, especially for convenient peripheral blood testing. IFIT3, a core member of the IFIT family, 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 responses. Existing studies have shown that IFIT3 is significantly upregulated in viral infection through the JAK-STAT pathway and exhibits bidirectional regulation in response to oxidative stress. However, its expression pattern in response to radiation exposure and its potential as a biomarker for detecting exposure or damage have not been reported. Summary of the Invention

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

[0005] Based on single-cell sequencing data from peripheral blood samples of radiotherapy patients, the inventors ranked the top 40 genes at a global level and found that some interferon-induced genes were significantly downregulated in clusters after irradiation, including the IFIT3 gene, suggesting that IFIT3 plays an important role in radiation response. Experiments also verified that IFIT3 meets the two characteristics of high sensitivity and good specificity in diagnosing 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.

[0006] The first aspect of the present invention is to provide the use of IFIT3 as a biomarker in the preparation of a product 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 a sample.

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

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

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

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

[0012] In this application, although the samples in this example were derived from a population undergoing radiotherapy, changes in IFIT3 expression are directly correlated with ionizing radiation. Therefore, the marker described in this invention is expected to be applicable to a wide range of individuals exposed to ionizing radiation, including but not limited to radiotherapy patients, nuclear accident victims, and radiation workers.

[0013] In this application, the reagent may contain not only detection components such as probes, primers, and antibodies, but also auxiliary components such as buffers, reaction substrates, and standards, all of which fall within the scope of protection of the present invention.

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

[0015] A second aspect of the present invention provides a kit for detecting radiation exposure or damage by measuring the expression level of IFIT3 in a sample. The kit may include reagents for detecting IFIT3 gene expression, such as primers and probes; reagents for detecting IFIT3 protein expression, such as antibodies and markers; and optional auxiliary materials, such as positive controls, negative controls, and calibrators. Those skilled in the art will appreciate that all of the aforementioned components and auxiliary materials included in the kit fall within the scope of the present invention.

[0016] The significant advantages of the present invention are that it provides a biomarker with high sensitivity and good specificity for diagnosing radiation exposure. The expression of IFIT3 in the blood samples of the subjects after radiotherapy (irradiation) is significantly downregulated, which can accurately determine whether the subjects are exposed to radiation or at risk of damage. By detecting the expression level of IFIT3 in the peripheral blood samples of the subjects, it can be used to diagnose or assist in the diagnosis of radiation exposure or early radiation damage, and can serve as a basis for supporting individuals suffering from radiation exposure or early radiation damage, 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 is a brief introduction to the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 A group comparison chart of the proportion of different cell types in whole blood in blood samples of chest radiotherapy patients before and after radiotherapy provided by the present invention;

[0019] Figure 2 A comparison diagram of single-cell dimensionality reduction in blood samples of chest radiotherapy patients before and after radiotherapy provided by the present invention;

[0020] Figure 3 This is the expression distribution diagram of the IFIT3 gene in the single-cell dimensionality reduction map provided by the present invention;

[0021] Figure 4 This is a comparison chart of the expression changes of the IFIT3 gene before and after radiotherapy provided by the present invention;

[0022] Figure 5 This is a scatter plot of IFIT3 expression levels in the control group and radiotherapy group provided in Example 1 of the present invention;

[0023] Figure 6 This is a ROC curve diagram drawn based on the IFIT3 expression levels in the control group and the radiotherapy group in Example 1 of the present invention;

[0024] Figure 7 This is a scatter plot of IFIT3 expression levels in the control group and radiotherapy group provided in Example 2 of the present invention;

[0025] Figure 8 This is a ROC curve diagram drawn based on the IFIT3 expression levels in the control group and the radiotherapy group in Example 2 of the present invention; DETAILED DESCRIPTION

[0026] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0027] The terms "first," "second," and "third" in this application are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of the technical features indicated. Therefore, features specified as "first," "second," or "third" may explicitly or implicitly include multiple such features. In the description of this application, "multiple" means at least two, for example, two, three, etc., unless otherwise specifically defined. All directional indications (such as up, down, left, right, front, back, etc.) in the embodiments of this application are intended only to illustrate the relative positional relationships and movement of components in a specific posture (as shown in the accompanying drawings). If the specific posture changes, the directional indications will also change accordingly. Furthermore, the terms "including," "having," and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product, or device comprising a series of steps or modules is not limited to the listed steps or modules and may optionally include steps or modules not listed, or may optionally include other steps or modules inherent to such process, method, product, or device.

[0028] References to "embodiments" herein mean that a particular feature, structure, or characteristic described in connection with the embodiments may be included in multiple embodiments of the present application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor does it constitute an independent or alternative embodiment that is mutually exclusive of other embodiments. It is understood, both explicitly and implicitly, by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0029] The design process of this invention: The inventor collected peripheral blood samples from a total of 6 patients undergoing chest radiotherapy at the Department of Radiation Oncology of the Second Affiliated Hospital of Chengdu Medical College and the 416 Nuclear Industry Hospital. The total irradiation dose of the treatment course ranged from 40-60Gy. The samples were collected before the start of radiotherapy and within 24 hours after the end of the entire treatment course. The whole peripheral blood samples of 3 patients before radiotherapy and 3 patients after radiotherapy were sent to Beijing Xunyin Biotechnology Co., Ltd. for single-cell omics analysis. By systematically mining the single-cell omics data, dimensionality reduction analysis and visualization processing were performed on all cell types, and the distribution results are shown as follows: Figure 1 and Figure 2 As shown, Figure 1 A pie chart comparing the proportions of different cell types in whole blood before and after radiotherapy. Different colors represent different cell type groups, visually demonstrating the effects of radiotherapy (radiation) on the proportions of different cell types. Figure 2 This is a comparison diagram of single cell dimensionality reduction in blood samples of radiotherapy patients before and after radiotherapy. Figure 2Each dot represents a single cell, and the horizontal and vertical axes represent the spatial coordinates after dimensionality reduction. Different colors represent different cell groups. Comparing the changes in cell distribution in samples before (right) and after (left) radiotherapy reveals the overall impact of radiotherapy (radiation) on cell populations.

[0030] 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 IFIT3 was significantly reduced, suggesting that it may be a potential molecular marker for the diagnosis of radiation exposure or early radiation damage. Figure 3 The expression distribution of the IFIT3 gene in the single-cell dimensionality reduction plot is shown. Each point in the dimensionality reduction analysis plot represents a cell, and the horizontal and vertical axes are the spatial coordinates after dimensionality reduction. The color 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 plot of the IFIT3 gene at the single-cell level, it was found that IFIT3 expression showed significant distribution differences in the cell map. See Figure 4 This figure compares changes in IFIT3 gene expression before and after radiotherapy. The horizontal axis represents grouping (before and after radiotherapy), and the vertical axis represents normalized IFIT3 gene expression. Wider curves indicate a greater number of cells meeting the criteria; narrower curves indicate a lower number of cells meeting the criteria. IFIT3 expression was significantly decreased after radiotherapy compared to before.

[0031] Based on these findings, the inventors further compared pre- and post-radiotherapy samples to measure IFIT3 expression levels in peripheral blood. They found that IFIT3 exhibited good specificity and sensitivity when used to detect radiation exposure. Based on these research findings, the inventors developed products and applications for detecting radiation exposure based on the IFIT3 marker. This process will be described in detail below through specific examples.

[0032] Example 1, screening of the differentially expressed gene IFIT3 by RNA (RNA-seq) sequencing, specifically comprising the following steps:

[0033] 1) Sample Collection: Peripheral blood samples were collected from 82 patients undergoing thoracic radiotherapy at the Second Affiliated Hospital of Chengdu Medical College and the 416th Nuclear Industry Hospital. Total treatment doses ranged from 40 to 60 Gy. Peripheral blood samples were collected before and within 24 hours after the last dose of radiotherapy. Samples included both a control group (41 pre-radiotherapy samples) and a radiotherapy group (41 post-radiotherapy samples).

[0034] 2) Blood Cell Isolation and RNA Extraction: Blood cells were separated using conventional methods to obtain a relatively pure white blood cell pellet, from which RNA was extracted. After RNA extraction, the concentration and purity of the RNA were determined using a spectrophotometer, ensuring that the OD260 / OD280 ratio was between 1.8 and 2.2. The integrity of the RNA was then tested by agarose gel electrophoresis, observing the clarity and brightness ratio of the 28S and 18S rRNA bands to ensure that the RNA quality met the requirements of subsequent experiments.

[0035] 3) Library Construction: Total RNA is fragmented using conventional methods and used as a template to synthesize cDNA using reverse transcriptase and random primers. Adapters containing PCR primer binding sites and sequencing tags are ligated to both ends of the cDNA. Multiple rounds of PCR amplification using primers complementary to the adapters are then performed to increase the cDNA concentration to meet sequencing requirements.

[0036] 4) RNA sequencing: Sequencing is performed using the Illumina series of high-throughput sequencing platforms. Before sequencing, the sequencing platform is calibrated and debugged to ensure optimal instrument performance. The necessary reagents and consumables are prepared. The constructed library is appropriately diluted to a concentration that meets the requirements of the instrument. Sequencing is then performed to generate a large number of sequence fragments.

[0037] 5) Data processing and analysis: The number of sequencing reads of the IFIT3 gene in each sample before and after radiotherapy was calculated. Figure 5 Compared with the control group, the expression level of IFIT3 in the samples of the radiotherapy group was significantly downregulated, and the Mann-Whitney test p < 0.05, the difference was statistically significant.

[0038] Whether or not radiotherapy had been performed was used as the binary result, and the expression level of the IFIT3 gene was used as the predictor. By changing the threshold for judging the disease, the sensitivity and specificity at different thresholds were calculated, and the ROC curve was drawn. Figure 6 As shown, the area under the ROC curve AUC = 0.9917, the sensitivity is 95.12%, and the specificity is 95.12%, indicating that using the expression level of the IFIT3 gene as a diagnostic indicator can better determine whether the subject has been exposed to radiation, and has good diagnostic efficacy.

[0039] Example 2, further verifying the diagnostic effect of IFIT3 by enzyme-linked immunosorbent assay (ELISA), comprising the following steps:

[0040] 1) Sample Collection: Peripheral blood samples were collected from 70 patients undergoing thoracic radiotherapy at the Second Affiliated Hospital of Chengdu Medical College and the 416 Nuclear Industry Hospital. Total treatment doses ranged from 40 to 60 Gy. Peripheral blood samples were collected before and within 24 hours after the last radiotherapy session. The samples included a control group (35 pre-radiotherapy samples) and a radiotherapy group (35 post-radiotherapy samples). All patients were completely independent of the patients in the screening phase of Example 1 and did not overlap with each other.

[0041] 2) Antibody and Sample Processing: Coat a microplate with an antibody specific for IFIT3 protein and incubate at 4°C overnight to allow the antibody to adsorb to the plate surface. Fill the microplate with a blocking solution containing BSA and incubate at room temperature for 1-2 hours. Add protein samples from the control and radiotherapy groups to the coated microplate, along with standard and blank controls, and incubate at room temperature for 1-2 hours to allow the IFIT3 protein in the samples to bind to the coated antibody.

[0042] 3) Incubation: Primary Antibody Incubation: Discard the liquid in the wells, wash, and then add a primary antibody specific for IFIT3 protein. Incubate at room temperature for 1-2 hours to allow the primary antibody to bind to the IFIT3 protein bound to the coated antibody. Secondary Antibody Incubation: After washing, add an enzyme-labeled secondary antibody and incubate at room temperature for 1-2 hours to allow the secondary antibody to bind to the primary antibody.

[0043] 4) Add substrate for color development and measurement: Add enzyme substrate, which catalyzes the enzyme to develop color. The intensity of the color is proportional to the IFIT3 protein content in the sample. Use a microplate reader to measure the optical density (OD) of each well at a specific wavelength. Calculate the relative IFIT3 protein content (relative optical density) based on the standard curve drawn using the standard.

[0044] 5) Data processing and analysis: The relative optical density values ​​of IFIT3 protein in patients before and after radiotherapy were compared and statistically analyzed. Figure 7 In patients after radiotherapy, the expression level of IFIT3 was significantly downregulated, and the Mann-Whitney test p < 0.05, which was statistically significant.

[0045] Whether or not radiotherapy had been performed was used as a binary result, and the expression level of IFIT3 protein was used as a predictor. By changing the threshold for judging the disease, the sensitivity and specificity at different thresholds were calculated, and the ROC curve was drawn. Figure 8 As shown, the area under the ROC curve AUC = 0.9118, the sensitivity is 71.43%, and the specificity is 97.14%, indicating that the IFIT3 protein detection result is basically consistent with the IFIT3 gene detection result in Example 2, further verifying that IFIT3 can be used as a predictive indicator for detecting radiation exposure.

[0046] In summary, the present invention has verified the application of IFIT3 as a biomarker in detecting radiation exposure products through the examples of radiotherapy individuals, and the IFIT3 marker has the two characteristics of high sensitivity and good specificity; and the biological effects of ionizing radiation, including medical radiotherapy, nuclear accident radiation, industrial radiation, etc., have commonalities, and their core mechanisms are all to induce DNA damage, oxidative stress and interferon signaling pathway activation through the action of ionizing radiation. Radiotherapy is a typical scenario of ionizing radiation under controlled conditions, and the radiation response mechanism it induces is highly consistent with other non-medical radiation exposures. Therefore, the biomarkers screened for radiotherapy populations in this application can also reflect the body's overall response to ionizing radiation, have a scientific basis for extension to other radiation exposure scenarios, can provide a basis for the clinical diagnosis of radiation exposure, and provide early warning for the diagnosis of early radiation damage, and have broad clinical application prospects.

[0047] 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 principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of IFIT3 as a biomarker in the preparation of products for detecting radiation exposure, characterized in that: The product includes a reagent for detecting the expression level of IFIT3 in a sample, wherein the expression of IFIT3 is downregulated in radiation-exposed patients, and the sample is a peripheral blood sample.

2. The use according to claim 1, characterized in that The detecting the expression level of IFIT3 in the sample includes detecting the expression level of the IFIT3 gene.

3. The use according to claim 2, characterized in that The product detects the expression level of the IFIT3 gene in a sample by any one of qRT-PCR, RT-PCR or gene chip.

4. The use according to claim 1, characterized in that The detecting the expression level of IFIT3 in the sample includes detecting the expression level of IFIT3 protein in the sample.

5. The use according to claim 4, characterized in that The product detects the expression level of IFIT3 protein in a sample by any one of enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA), and immunoturbidimetry.

6. The use according to claim 1, wherein the reagent is at least one of an oligonucleic acid probe targeting an IFIT3 encoding DNA sequence, a PCR primer targeting an IFIT3 encoding DNA sequence, an IFIT3-specific nucleic acid aptamer, or an IFIT3-targeting molecularly imprinted polymer.

7. 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.

8. The application according to claim 7, characterized in that: The reagent for detecting IFIT3 is an RNA-seq analysis reagent or an enzyme-linked immunosorbent assay reagent.

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