Study On Screening Of Radiation Damage Markers For Underground Workers In A Mine And Its Mechanism

ObjectiveRadon is a natural radioactive gas in nature.after being inhaled into the human body;radon,a short-lived decay product,will release high-energy a particles to damage the respiratory tract and lung tissue.At the same time,some radon and its daughters will enter the blood circulation and act on the whole body.Radon and its progeny are classified as type I carcinogenic factors by the International Agency for Research on Cancer(IARC),and become the second most common cause of lung cancer after smoking.Previous studies have shown that the increased risk of lung cancer is associated with high levels of radon exposure,and the ratio of lung cancer to(ORs)increases with the increase of radon concentration.In the process of monitoring the radon concentration in the underground air of a tin mine in Yunnan Province,the research group found that the radon concentration in the underground workplace was relatively high.There has been a high incidence of lung cancer among miners in the history.It is vital to find the biomarkers of radiation damage caused by radon and explore its pathogenic mechanism.Methods and Materials1.The radon detectors were placed at the underground workplaces or offices on the ground where the miners worked or stayed for a long time,1.5m high away from the floor.The different seasonal radon concentrations in one year were detected by the chemical etching solid track method combined with the continuous measurement method.The annual exposure doses of miners exposed to radon radiation in underground and the miners surface work were estimated according to the dose-conversion parameters recommended in International Commission on Radiological Protection(ICRP)137 Report.The radon concentrations in the tap water from underground and surface office areas were measured by RAD 7 radon detector.The internal exposure dose caused by the release of radon in water was used to estimate the annual dose of internal exposure caused by radon in water.2.A cohort study of 144 miners,72 underground miners as the study group and 72 aboveground miners as the control group.A face-to-face questionnaire survey was conducted,including miners’ general conditions,health conditions,working conditions,living habits,etc.,combined with the measurement of radon concentration in the air of underground and above-ground workplaces throughout the year,and using the parameters recommended by ICRP report 137,the cumulative exposure dose that may be caused by inhalation of radon and daughters of each miner was calculated.3.Blood routine test indexes including LYM,NE,HGB,PLT,RBC and WBC,serum biochemical indexes including TP,ALB,GLO,TBIL,DBIL,IBIL,ALT and AST were detected to evaluate the blood parameters.4.The Cell cycle,the relative expression of cell cycle related regulatory proteins CDK2,CDK4,CDK6,cyclinA2,CyclinD1,CyclinEl and yH2AX in peripheral blood lymphocyte of miners were detected by flow cytometry.5.A GeneChip miRNA 4.0 Array was used to screen the plasma samples(10 samples per group)for miRNAs.The relative expression of four miRNAs was detected by real-time PCR in 144 miners,6.Five underground miners were selected as the study group and five aboveground miners as the control group.After being divided into five groups according to age,the common differentially expressed genes were analyzed by RNA-seq technique.The expression of target genes corresponding to miR-19a-3p,miR-30e-5p and miR-335-5p in the peripheral blood lymphocytes of underground miners and controls were analyzed.7.The internal relationship between potential biomarkers was analyzed by using bioinformatics and statistical methods to explore the underlying mechanism of radiation damage induced by radon.8.The data were analyzed by GraphPad Prism 8.Student t test and the nonparametric Wilcoxon test were used to assess differences in the study population characteristics and potential biomarkers between the underground and control groups.One-way analysis of variance was used to identify the differences between multiple groups.A P value<0.05 was considered statistically significant.Pearson correlation analysis was used to study the correlation between the two indexes.Results1.The average radon concentrations in the air of underground and aboveground workplaces in a year were(7.5 ± 3.1)kBq·m-3(n=39)and(0.284 ± 0,144)kBq·m-3(n=15).Based on this,the annual dose contribution ranges of radon in air were estimated to be(4.5-84.8)mSv and(1.3-12.9)mSv.2.There was no significant difference in age,BMI and smoking index between the underground group and the control group.The cumulative radon exposure in the underground group was higher than that in the control group(n underground=72,n control=72,P<0.05).The effective exposure dose ranges of radon in the underground group and the control group were(244-1845)mSv and(4-207)mSv,respectively.3.Compared with the control group,the peripheral blood LYM count and NE count in the underground group were lower,the peripheral blood hemoglobin in the underground group was higher and the differences were statistically significant(n underground=72,n cntrol=72,P<0.05).The RBC in peripheral blood of miners in the underground group was lower(n uderground=28,n contuol=60)and PLT,WBC was higher than that in the control group(n underground=72,n eontrol=72),but the differences were not statistically significant.4.The percentage of G0/Gi phase of peripheral blood lymphocyte cycle of underground miners was significantly higher than that of the control group(P<0.05);S phase was significantly lower than that of the control group(n underground=72,n control=72,P<0.05).The relative expression levels of CDK2,CDK4,CDK6,Cyclin A2,Cyclin D1 and Cyclin E1 in peripheral blood lymphocytes were higher in the underground group than those in the control group(n underground=72,n control=72,P<0.05).The relative expression levels of yH2AX in peripheral blood lymphocytes were higher in the underground group than those in the control group(n underground=28,n control=60,P<0.05).5.MicroRNA microarray screening(n underground=10,n control=10)results showed that 15 miRNAs including hsa-miR-30e-5p,hsa-miR-1246,hsa-miR-451a,hsa-miR-19a-3p,and hsa-miR-335-5p,hsa-miR-154-5p,hsa-miR-5100,hsa-miR-25-3p,hsa-miR-4701-3p,hsa-miR-338-5p,hsa-miR-505-3p,hsa-miR-4790-3p,hsa-miR-1290,hsa-miR-3976,and hsa-miR-4762-5p were downregulated in the high-radon exposure group.Eleven miRNAs including hsa-miR-488-5p,hsa-miR-1227-3p,hsa-miR-4770,hsa-miR-6724,hsa-miR-210-5p,hsa-miR-3605-3p,hsa-miR-1181,hsa-miR-6880-3p,hsa-miR-6796-3p,hsa-miR-3154,and hsa-miR-1825 were upregulated in the high-radon exposure group.Among them,5 miRNAs including hsa-miR-30e-5p,hsa-miR-1246,hsa-miR-451a,hsa-miR-19a-3p,and hsa-miR-335-5p were downregulated(fold-change>2)in the underground group.The real-time PCR detection results(n underground=72,n cntrol=72)of miR-19a,miR-30e,miR-335,and miR-451a were consistent with the screening results.6.RNA-Seq analysis showed that CCL3L3,CCL3L1,CCL4L2,LAIR2,TMED7-TICAM2,LOC11226833 7,MC1R and S100B were the common differential genes of the five groups of miners.KEGG enriched the related metabolic pathways,including cell processes,environmental information processing,human diseases,immune,endocrine and developmental systems and other organic systems.KEGG enrichment also found that the related signaling pathways including toll like receptor signaling pathway,viral protein and cytokine receptor,cytokine receptor interaction,NF-κB signaling pathway and so on.The target genes expression levels of miR-19a,miR-30e and miR-335 in the underground group were higher than those in the control group,but the differences were not statistically significant(P>0.05).7.The predicted target genes of TargetScan,miRBase and miRanda showed that CCNA2 and CCND1(encoding CyclinA2 and CyclinDl respectively)were the target genes of miR-19a.CCNE(encoding CyclinE)was the target gene of miR-30e,and CDK2(encoding CDK2)was the target gene of miR-335.Pearson correlation analysis showed that Cyclin A2 and Cyclin D1 were negatively correlated with miR-19a(P>0.05),Cyclin E1 was negatively correlated with miR-30e(P>0.05),and CDK2 was negatively correlated with miR-335(P>0.05).8.Multiple linear regression analysis showed that LYM counts,NE counts,the expression of CDK2,CDK4,CDK6,Cyclin A2,Cyclin D1 and Cyclin E1 in peripheral blood lymphocytes and the relative expression of miR-19a-3p,miR-30e-5p,miR-335-5p and miR-451a in plasma were all related to cumulative radon exposure(P<0.05).ConclusionsIn this study,gene chip technology and RNA-seq sequencing technology were used for the first time to screen underground miners’ blood samples,to find new potential biomarkers of radon-induced radiation damage,to analyze the common differentially expressed genes of miners with different cumulative radon exposure,and to explore the mechanism of radon-induced radiation damage of peripheral blood lymphocytes by bioinformatics,and the following conclusions were obtained.LYM counts and NE counts in peripheral blood may be used as indicators to evaluate radon-induced radiation damage.Proteins such as yH2AX,CDK2,CDK4,CDK6,CyclinA2,CyclinDl,CyclinE1 in peripheral blood lymphocytes and miR-19a,miR-30e,miR-335 and miR-451 a in plasma may become potential biomarkers of radon radiation damage.