Screen Of N6-methyladenosine RNA Modification As Biomarkers For Assessment Of Ionizing Radiation Dose

With the continuous development of nuclear technology,the application of ionizing radiation became more and more extensive.High dose of ionizing radiation(usually>1Gy)could lead to acute radiation syndrome(ARS),which involves multiple organ systems,with symptoms ranging from mild ones such as nausea and vomiting,to death.Although the body exposed to long-term low-dose radiation could not show obvious symptoms immediately,it might cause late effects,such as gene mutation or cell carcinogenesis.A rapid,accurate and high-throughput radiation dose assessment of exposed population is important to guide the classification diagnosis and the medical treatment of the injured after a radiation accident.Radiation biomarkers are biological changes that have a certain dependent relationship with radiation dose and time post irradiation in vivo.The biological dosimetry used for radiation biomarkers to estimate the absorbed dose truly reflects the exposure and physiological status of the body,and plays an important role in the rapid and accurate classification and accurate medical treatment of exposed population in the large-scale radiation accident rescue.The ideal radiation biological dosimetry should have the characteristics of fast,accurate,sensitive,minimally invasive and high throughput.Based on the current radiation biomarkers,researchers have not yet created a detection method or device that could be used for human diagnosis.Exploring new radiation biomarkers and their changing characteristics will help promote the development and application of radiation biological dosimetry.The study of new radiation biomarkers and its variation characteristics will be helpful to promote the development and application of radiation biological dosimetry.Among the various types of RNA modifications,the RNA N6-methyladenosine(m~6A)modification is one of the most abundant and has been shown to play an important role in regulating transcription,splicing,degradation,processing and translation in eukaryotic cells.The study of m~6A is an important topic in recent years.More studies have involved the relationships between RNA m~6A with external stimuli,such as ionizing radiation,but it is still unclear whether its modification level depends on the radiation dose and time post irradiation.At present,based on the m~6A chip and high-throughput sequencing technology,many studies have revealed the mechanism of RNA m~6A modification in pathological processes.The main aim of this work is to identify the candidate biomarkers by m~6A chip technology thereby to validate their characteristics in response to ionizing radiation,which will lay the foundation for the development and application of new radiation biodosimeters in the future.Methods:(1)Construction of RNA m~6A profiles after irradiation:Hematological analysis and body weight measurement after mice gamma-ray total-body irradiation(TBI)model;Transcriptome-wide quantification of m RNA expression and m~6A modification levels were performed using Epi-transcriptomic Microarray in peripheral blood mononuclear cells(PBMCs).Unsupervised clustering was performed by using timeclust function to show the distinguishable m~6A-modification pattern along different time points.(2)Dynamic changes of candidate RNA m~6A markers in mice TBI model:Sequence-based RNA adenosine methylation site predictor(SRAMP)was used to predict highly confident m~6A sites distributed across mice candidate markers transcripts.Specific primers for methylate RNA immunoprecipitation-quantitative real-time PCR(Me RIP-q PCR)assays were designed to target most of the predicted m~6A sites;To detect the dynamic changes of candidate markers in PBMCs from mice TBI model;Me RIP-q PCR was used to detect the dynamic changes of Ncoa4 RNA m~6A modification in mice X-ray TBI model;To validate the responsive specificity of Ncoa4 RNA m~6A modification in other mice irradiation models in response to radiation;Immunofluorescence and Western blotting(WB)were used to detect the dynamic changes ofγH2AX in mice TBI model.(3)Dynamic changes of NCOA4 RNA m~6A in monkeys gamma-ray TBI model,HUVECs and healthy human peripheral blood gamma-ray irradiation model in vitro:the homology of the RNA m~6A modification site of NCOA4 was analyzed by multiple sequence comparison among different species;SRAMP was used to predict highly confident m~6A sites distributed across monkey and human NCOA4 transcripts.Specific primers for Me RIP-q PCR assays were designed to target most of the predicted m~6A sites in monkey and human cell and blood samples;Dynamic changes of NCOA4 RNA m~6A were detected by Me RIP-q PCR in monkeys gamma-ray TBI model,HUVECs and healthy human peripheral blood gamma-ray irradiation model in vitro;Me RIP-q PCR was used to detect the dynamic changes of NCOA4 RNA m~6A modification in HUVECs exposed to X-ray and different other factors;(4)Dynamic changes of NCOA4 RNA m~6A in cancer patients undergoing radiation therapy:Me RIP-q PCR was used to detect dynamic changes of NCOA4 RNA m~6A modification in the peripheral blood of cancer patients receiving local radiotherapy;Me RIP-q PCR was used to assess the effect of long-term low-dose exposure on NCOA4RNA m~6A modification in the peripheral blood of long-term radiation exposure and healthy volunteers without known irradiation exposure.(5)Construction and accuracy evaluation of radiation dose estimation models:Algorithms were developed by fitting in the experimental data points using the goodness of fit model in mice and human after radiation.Receiver operating characteristic(ROC)analyses were then used to assess the performance of different model.Results:(1)The m~6A methylation of Ncoa4,Ate1 and Fgf22 m RNAs were identified as candidate biomarkers in mice gamma-ray TBI model.(2)The dose-effect and time-dependent relationships of candidate biomarkers were validated after irradiation in mice.Among these three candidate genes,the m~6A levels of Ncoa4 m RNA showed the longest duration post-irradiation.No significant confounding effect on Ncoa4 m~6A levels was observed due to differences in gender,age,immune or inflammatory status.The m~6A methylation of Ncoa4 m RNA outperformed the classical radiation biomarkerγ-H2AX in the long detectable time window after exposure.(3)The m~6A sites in Ncoa4m RNA are highly conserved across different species.The m~6A levels of NCOA4 m RNA increased significantly during longer time post-irradiation in monkey.Dose-response relationships of RNA m~6A methylation of NCOA4 could be detected in longer post-irradiation time in HUVECs and healthy human peripheral blood gamma-ray irradiation model in vitro.(4)Dose-response analysis showed that the NCOA4 m~6A levels increases gradually with the accumulated radiation dose in these patients receiving local physical radiotherapy.Those long-term low-dose exposed population had higher NCOA4 m~6A levels than the healthy volunteers.(5)The dose estimation model of candidate markers in mice gamma-ray TBI model were constructed,which could fit the observations sensitively and specifically.Based on NCOA4 RNA m~6A modification marker,the dose estimation model in humans receiving local radiotherapy model was constructed,which could detect the absorbed radiation doses well.Conclusion:In this work,it was the first attempt to find the RNA m~6A modification as radiation biomarkers and screen RNA m~6A modification sites of multiple genes as potential radiation biomarkers.We further validate the good dose-response relationship of NCOA4 RNA m~6A methylation and its temporal dynamics after irradiation exposure.Importantly,we constructed the novel biodosimetric models based on Ncoa4/NCOA4m~6A modification to estimate the radiation absorbed doses of mice with acute radiation exposure and cancer patients undergoing fractionated radiotherapy.Collectively,our study laid a foundation for the development of radiation biodosimeters based on RNA m~6A modification in the future.