Study Of MiRNA And γ-H2AX As Biomarkers Of Radiation Dose

Objective:With the development of the times,nuclear technology is gradually used in all aspects of life,such as nuclear power supply and heating,medical treatment,X-rays detection,food sterilization and disinfection.The continuous development of nuclear technology applications increases the possibility of nuclear exposure and other emergencies,so it is necessary to screen biomarkers that can meet the needs of fast and high throughput.In addition,occupational population is exposed to low-dose radiation environment for a long time,so it is particularly important to carry out dose detection for such population.At present,rapid and high-throughput dose prediction methods are still being explored,and long-term low dose detection methods have not been fully established.Therefore,in this study,molecular markers of miRNA andγ-H2AX in peripheral blood of mice irradiated by ⁶⁰Coγ-rays were screened and verified,providing experimental evidence for the establishment of miRNA as biomarkers of radiation dose andγ-H2AX as biomarkers of long-term low dose radiation dose.Methods:This topic is studied from two aspects.（1）Study on miRNA as potential biomarkers of radiation dose:High-throughput sequencing technology was used to identify miRNA in peripheral blood leukocytes of BALB/c mice at 4 h after 0 Gy,1 Gy,2 Gy,4 Gy and 6Gy ⁶⁰Coγ-rays irradiation,and potential biomarkers of radiation dose were screened based on miRNA multiples.The basic biological functions of target genes were analyzed by KEGG pathway analysis,hierarchical clustering analysis and GO analysis.Finally,quantitative real-time PCR was used to verify the miRNA in mouse peripheral blood and human peripheral blood in vitro,providing experimental basis for the establishment of miRNA as biomarkers of radiation dose.（2）Study onγ-H2AX as a biomarker of long-term low-dose radiation dose:γ-H2AX content in peripheral blood lymphocytes of mice exposed to long-term low-dose radiation of 0 Gy×10,0.01 Gy×10,0.02 Gy×10 and0.05 Gy×10 at the first month,the second month and the third month after exposure was analyzed by immunofluorescence technique,and dose-effect and time-effect analysis were conducted.In order to establishγ-H2AX as a biomarker of long-term low dose radiation dose.Results:（1）Screening of dose-responsive miRNAsTo|log Fold change|>1 and P<0.05 for filters with different dose groups（1,2,4and 6 Gy）and control group（0 Gy）of high-throughput sequencing results were analyzed,and the 1,2,4 and 6 Gy dose group screening to the radiation-sensitive miRNAs with 20,42,63 and 40.After that,the radiation-sensitive miRNAs of each dose group were collected by using R language to screen the dose-responsive miRNAs,which included miR-150-5p,miR-150-3p,miR-6916-5p,miR-122-5p and miR-212-3p.Further KEGG and GO analysis of dose-responsive miRNA target genes showed that they tended to be enriched in radiation damage related pathways such as AMPK signal pathway and PI3K-Akt signal pathway.（2）Verification of dose response to miRNAsFirstly,this study was verified in peripheral blood of mice.Quantitative real-time PCR results showed that mmu-miR-150-5p and mmu-miR-122-5p had a significant dose-effect relationship in peripheral blood leukocytes of mice.This study was further verified in human peripheral blood.In this study,5 male volunteers with no recent history of exposure were recruited and the miR-150-5p and miR-122-5p screened at the animal level were verified by quantitative real-time PCR.The results showed that hsa-miR-150-5p and hsa-miR-122-5p in human peripheral blood lymphocytes also had significant dose-effect relationship,and the results of dose-effect fitting curve showed that the R²of hsa-miR-122-5p was 0.8802.In addition to the reported biomarker miR-150-5p,miR-122-5p also has the potential to become a biomarker of radiation dose.（3）Validation ofγ-H2AX as a biomarker marker of long-term low-dose radiationγ-H2AX induced by long-term low-dose radiation could be observed in 0.01 Gy×10,0.02 Gy×10 and 0.05 Gy×10 groups at all time points.γ-H2AX showed good dose-effect relationship at the second month and the third month after irradiation.In addition,the number of focal points ofγ-H2AX increased with the increase of time after exposure,suggesting thatγ-H2AX may be a potential biomarker of long-term low-dose radiation damage.Conclusion:MiR-122-5p expression levels have a dose-effect relationship,which is a potential biomarker of radiation dose.γ-H2AX expression levels have dose-effect and time-effect relationships,which is potential biomarkers of long-term low-dose radiation dose.