Screening And Functional Analysis Of MiRNAs Related To DNA Damage Response Induced By Hexavalent Chromium

ObjectiveThe aim of this study is to screen and validate differential expressed miRNAs related to hexavalent chromium exposure in population investigation as well as in vitro study,which also include functional analysis.This study will identify sensitive biomarkers for workers occupationally exposed to hexavalent chromium,providing a basis for the mechanism of toxicity of hexavalent chromium.MethodsIn vitro experiments,we used 5 μM K2Cr2O7 to treat human lymphoblast B cell line for 24 hours.After washing,the cells were resuspended in fresh medium,incubating for another 7 days.Cell cycle,cell apoptosis and DNA damage were detected in cells from control group,cells treated with K2Cr2O7 for 24 hours,and cells after detoxication for 3 days and 7 days by Muse flow cytometry.Total RNA were extracted from these cells samples for miRNA sequencing.We chose 26 differentially expressed miRNAs for validation by RT-qPCR.In the study of population,we selected 66 electroplating workers as exposure group,who exposed to hexavalent chromium,and the 66 age and gender matched cases of control group was chosen from the people with no hexavalent chromium exposure,and they were not exposed to harmful chemicals and ionizing radiation in recent three months.After collection of anticoagulation peripheral blood,ICP-MS was used to detect blood chromium concentrations.We selected 3 cases of control and 3 cases of exposure for miRNAs sequencing after extracting total RNA form whole blood.Based on the results of in vitro experiments and population investigation,we selected 10 significantly differential expressed miRNAs for validation in a larger number of population.The relationship between expression levels of miRNAs and blood concentrations of hexavalent chromium,exposure time were analyzed.Finally,according to the results of the validation,we selected 3 miRNAs for functional research.Firstly,we treated K2Cr2O7 as we mentioned above.Then we adopted corresponding miRNAs mimic or inhibitor respectively,and analyzed the influence of miRNAs on cell cycle and cell apoptosis after hexavalent chromium treatment.ResultsExposure to hexavalent chromium can induce G0/G1 arrest and the arrest will reduce with the time of detoxification.Twenty-four hours and 3 days of detoxification,the early apoptosis rate,the late apoptosis rate and the total apoptosis rate of the cells were higher than those of the control group(P<0.05).The early apoptosis rate was the highest on the 3 day after detoxification,and the late apoptosis rate and total apoptosis rate reached highest at 24 hours after detoxification.DNA damage reached the highest at 24 hours after hexavalent chromium treatment.After detoxification,the DNA damage decreased gradually,and there was no difference between the exposure group and the control group at 7 days after detoxification.In the results of cell screening,48 miRNAs were differential expressed both in the 24 hours after treatment and 7 days after detoxification,and the number of differentially expressed miRNA in 3 days after detoxification was 145.In the cell verification experiment,there were 9 miRNAs differential expressed in 3 days after detoxification between the control group and the exposed group(P<0.05),which were miR-101-3p,miR-142-3p,miR-146a-3p,miR-148a-3p,miR-181a-2-3p,miR-21-5p,miR-221-3p,miR-27a-5p,miR-424-3p.Among them,the experimental verification results of miR-146a-3p,miR-148a-3p,miR-21-5p,miR-221-3p and miR-424-3p are consistent with the trend of screening results.The blood chromium concentration of the exposed group and the control group was 13.78 + 16.45(ppb),3.37 + 3.78(ppb)respectively,and the exposure group was significantly higher than that of the control group.Among the results of population screening,there were differences in the expression of 45 miRNAs between control group and exposure group(P<0.05),of which 12 miRNAs were up-regulation,and 33 miRNAs were down-regulation.In the population verification,there were 6 differential expressed miRNAs between the exposure group and the control group,which were miR-142-3p,miR-19a-3p,miR-19b-3p,miR-21-5p,miR-424-3p and miR-941.Among them,the results of miR-424-3p in population verification were consistent with the results in cell verification.The results of miR-941 in population verification were consistent with the trend of population screening.Spearnman correlation analysis showed that the expression of miR-142-3p,miR-148a-3p,miR-19a-3p,miR-19b-3p had a negative correlation with the concentration of chromium(ppb)(P<0.05),and the expression of miR-21-5p,miR-590-3p,miR-941 was positively correlated with the concentration of chromium(P<0.05).There is a positive correlation between miR-19a-3p and working years.Transfecting mimic of miR-146a-3p,miR-148a-3p and inhibitor of miR-221-3p in the cells of hexavalent chromium detoxication,three were no difference of cell cycle and cell apoptosis comparing to negative control 24 hours and 3 days later.ConclusionHexavalent chromium treatment could significantly increase DNA damage of HMy2·CIR cells.DNA damage response including Cell cycle and cell apoptosis changed dynamically during repair process.The expression levels of 5 miRNAs such as miR-146a-3p were significantly abnormal on the 3 day after detoxification,and the expression returned to normal at 7 days after detoxification through screening and validation.Functional studies showed that miR-146a-3p,miR-148a-3p and miR-221-3p had no significant effect on DDR induced by hexavalent chromium.miR-142-3p,miR-19a-3p,miR-19b-3p and miR-941 were significantly correlated with the concentration of chromium,and miR-19a-3p was significantly related to working years in the study of population.