An Exploratory Study Of MiRNA As A Biological Marker Of Ionizing Radiation

ObjectiveNuclear terrorist attacks,nuclear power plant accidents,medical nuclear accidents and other types of nuclear accidents seriously damage national defense construction and the safety of people's lives and property.It is necessary and urgent to build and improve the nuclear emergency response system.After a radiation accident,the first problem we have to face is how to assess the radiation dose accurately.There are great limitations in traditional ionizing radiation dose detection methods and it is difficult for them to be applied to the radiation dose assessment of a large number of people after nuclear accidents.Therefore,there is an urgent need for a rapid,accurate and simple marker to detect radiation dose in the field of radiation dosimetry.Studies have shown that ionizing radiation can alter the expression of specific micro RNA(miRNA)in various cells,tissues and body fluids.The changes in the content of some specific miRNAs were related to the radiation dose.This indicates that miRNA has the possibility of being a biological indicator of ionizing radiation.When nuclear radiation and nuclear contamination occur,human beings are usually exposed to ionizing radiation all over their bodies,therefore,patients who receive total body irradiation(TBI)are more representative of the victims of nuclear radiation.However,in the previous studies on miRNA as a biological marker of ionizing radiation dose,the research objects were animals(mainly mice)and human bodies receiving local radiotherapy.The research field of miRNA as a biological marker of ionizing radiation dose is still in the blank stage,with people receiving total body radiotherapy as the research object.Therefore,this study selected patients who received total body irradiation as the study object,simulating the victims after nuclear radiation,to test the possibility of miRNA as a new biological indicator of ionizing radiation.Method1.Sixteen patients with acute leukemic leukemia who underwent total body irradiation were selected as the research subjects.The general information,examination results and detailed therapeutic regimen of the patients were collected.Blood samples were collected for each patient before TBI(0Gy),after 5Gy radiotherapy,and after cumulative radiotherapy of 10 Gy.2.The preliminary screening of specific miRNA was performed by two methods.The first method is the gene chip method.A total of 6 plasma samples were analyzed,respectively from 3 patients collected before and after radiotherapy.The second method was to perform a meta-analysis using miRNA as a biological indicator of ionizing radiation dose.This meta-analysis can not only provide preliminary screening results for the following studies,but also help us find out the shortcomings of previous studies,and help us have a more comprehensive understanding of the influence of ionizing radiation on human miRNA.The specific steps of the meta-analysis was carried out in accordance with the PRISMA guidelines.R software specialized in Meta analysis was used to accomplish data analysis,heterogeneity analysis,heterogeneity treatment,subgroup analysis,bias evaluation and bias correction.3.A total of 48 plasma samples of 16 patients were quantitatively verified by q PCR of miRNA,plasma samples from before radiotherapy,after 5Gy radiotherapy and after 10 Gy radiotherapy.Preliminary screening of miRNAs was performed by gene microarray and meta-analysis.Paired sample t-test was used to further screen out specific miRNAs with statistically significant differences in content changes before and after radiotherapy(5Gy,10Gy).4.Linear regression was used to analyze the correlation between the content changes of specific miRNA and radiotherapy dose.According to the analysis results,the unitary linear regression model and multiple linear regression model of specific miRNA content and radiotherapy dose were established.Receiver operating characteristic curve(ROC)and the area under the ROC curve(AUC)were used to evaluate the effectiveness of the regression models.Results1.After 10 Gy total body radiotherapy,the patients had no obvious discomfort;peripheral blood cells decreased to varying degrees;most of the adverse reactions were mild,mainly oral mucosal reaction and radioactive enteritis,which could be improved after symptomatic treatment.In this study,16 patients with acute leukemic leukemia after hematopoietic stem cell transplantation were selected.The 2-year disease-free survival rate after transplantation was 75% and the 3-year disease-free survival rate after transplantation was 66.7%.Patients with matched sibling allogeneic transplantation have a longer disease-free survival.2.Meta-analysis of 37 articles found that there were statistically significant changes in the content of 21 miRNA after receiving ionizing radiation,thereinto,the expression of 11 miRNA increasing,and the expression of 10 miRNA decreasing.A total of 6 specific miRNA content changes were found to be correlated with ionizing radiation dose in the Meta-analysis,which were miR-142,miR-21,miR-34,miR-320,miR-200 b and miR-339.The 6 specific miRNA have the potential to be dose markers of ionizing radiation,and the results need to be verified in the following q PCR experiments.3.Gene microarray analysis found that there were statistically significant differences in the content of 11 specific miRNA after receiving ionizing radiation,which were miR-1225-5p,miR-1273G-3p,let-7a-5p,miR-7107-5p,miR-548 d,miR-590-5p,miR-21-5p,miR-200 b,miR-150-5p,miR-34a-5p and miR-199 a.Among them,6 were up-regulated and 5 were down-regulated after ionizing radiation.The minimum absolute value of content change was 1.52 and the maximum was 7.15.The p value of each t-test was less than 0.05.4.11 miRNA were screened out by gene microarray,and 6 specific miRNA were screened out by meta-analysis,and 3 miRNA were screened out by both methods.Therefore,a total of 14 miRNA were included in subsequent experiments.5.As verified by q PCR,11 specific miRNA showed stable and consistent change trend(up-regulation/down-regulation)after 5Gy and 10 Gy ionizing radiation,suggesting that the 11 specific miRNA have the potential to become biological markers of ionizing radiation.6.Using a linear regression model,we performed a linear regression analysis of the relationship between Ionizing radiation dose and the content changes of 11 miRNA.The results showed that there was a linear relationship between the content of 3 specific miRNA(miR-200 b,miR-150 and miR-548d)and the dose of ionizing radiation.Three unary regression models and one multivariate regression model were constructed to estimate the dose of ionizing radiation.The determination coefficients R2 of the unary regression models constructed by miR-200 b,miR-150 and miR-548 d were 0.733,0.789 and 0.755,respectively,all greater than 0.5,indicating that the models had good degree of fitting.The R2 of the multiple regression model constructed by3 specific miRNA was significantly increased,reaching 0.866,indicating that the goodness of fit of the multiple regression model was further improved compared with the unitary regression model.7.Through the verification of ROC curve,we can see that the three unary regression models all have good sensitivity and specificity in the prediction of ionizing radiation dose.The sensitivity and specificity of the multiple regression model were significantly higher than that of the unary regression models.This suggests that miR-200 b,miR-150 and miR-548 d have the potential to become ionizing radiation markers.ConclusionAfter a radiation accident,the first problem we have to face is how to assess the radiation dose accurately.The ideal detection method should be sensitive and specific,relatively easy and simple,and could be widely applied to people early after ionizing radiation.Our study found that miR-200 b,miR-150 and miR-548 d can be used as new ionizing radiation markers,and the radiation dose prediction model constructed by the three miRNA can comparatively accurately evaluate the radiation dose.This may contribute to the construction of nuclear radiation emergency management system of our country.Innovations1.When nuclear radiation and nuclear contamination occur,human beings are usually exposed to ionizing radiation all over their bodies,therefore,patients who receive total body irradiation(TBI)are more representative of the victims of nuclear radiation.However,in the previous studies on miRNA as a biological marker of ionizing radiation dose,the research objects were animals(mainly mice)and human bodies receiving local radiotherapy.The research field of miRNA as a biological marker of ionizing radiation dose is still in the blank stage,with people receiving total body radiotherapy as the research object.Our study may make up for this blank field to some extent.2.The detection range of chromosomal aberration detection is 0.1 ? 5Gy;the accuracy is reduced,when the dose of ionizing radiation is over 5Gy.The dose points of total body radiotherapy in our study were 5Gy and 10 Gy.The ROC curve verified that the multiple linear regression model had a good prediction of the ionizing radiation dose of 5 ? 10 Gy.It can be seen that,when the dose of ionizing radiation is among 5 ? 10 Gy,our study may make up for the inaccuracy of chromosome aberration detection to a certain extent.3.Among the existing detection methods of radiation dose,the simple and rapid methods(cell detection,molecular chemical detection,etc.)have poor specificity and sensitivity,and the dose assessment is not accurate;the methods that can accurately assess radiation dose(chromosomal aberration detection,etc.)are complicated,time-consuming,limited and difficult to be widely used.In contrast,the real-time fluorescence quantitative PCR method is fast,accurate,simple and high-throughput,and can be widely used in the detection of large populations.