| Background and ObjectivesBenzene is an important environmental pollutant,mainly from indoor decoration coatings,organic solvents,automobile exhaust and tobacco combustion.Chronic benzene poisoning is caused by long-term low-level exposure,which can cause severe blood system damage,such as whole blood cell reduction.After benzene enters the body through different pathways,it undergoes a series of metabolism and finally generates the final carcinogen 1,4-benzoquinone in bone marrow.Studies have found that reactive oxygen species generated in the process of benzene metabolism can cause direct toxic effects,which reacts with biological macromolecules such as DNA,RNA and protein on the biofilm to cause oxidative damage,causing changes in body genetics and epigenetics,and further causing dysfunction of the blood system.Microribonucleic acid,known as micro RNA in English,is a highly conserved endogenous non-coding RNA that regulates gene expression by binding to the complementary sequence of the3’-UTR of the target gene m RNA.The current evidence shows that a large part of the human genome is regulated by mi RNA,including cell proliferation,apoptosis and cancer.The let-7 family is the first identified mi RNAs in mammals,and is also recognized as a tumor suppressor gene,which has an important impact on the proliferation and differentiation of stem cells.Our previous study found that benzene exposure can inhibit the expression of let-7 in mouse bone marrow cells.Bioinformatics prediction showed that let-7e-5p can bind to apoptosis gene Caspase-3 and cell cycle-related gene p21,let-7 may be involved in benzene-induced hematopoietic inhibition.In this study,C57BL/6 wild-type mice were used to establish a benzene poisoning mouse model,and the effects of benzene exposure on the toxicity and oxidative damage of mouse blood system were studied.The specific expressions of let-7e-5p,p21 and Caspase-3 in the oxidative damage of mouse hematopoietic cells induced by benzene exposure were investigated.Furthermore,let-7e-5p overexpression cell line was constructed by using human chronic myeloid leukemia K562 cell line,and its regulation mechanism on 1,4-BQ-induced cytotoxicity was explored to verify whether let-7e-5p overexpression regulated 1,4-BQ-induced cytotoxicity by targeting p21 and Caspase-3.Finally,through the comparison of blood routine,internal exposure dose and oxidative damage index between benzene exposure group and control group,combined with the difference of let-7e-5p,p21 and Caspase-3 RNA expression in peripheral blood of the two groups,the correlation between benzene exposure level,let-7e-5p and its pathway gene level,oxidative damage and hematological indexes was analyzed,so as to provide scientific basis for the feasibility of abnormal expression of let-7e-5p and its pathway gene as a biological marker of benzene-induced hematopoietic toxicity.Methods and Results1.Hemotoxicity of benzene-exposed mice and specific changes of let-7e-5p regulatory pathwayC57BL/6 mice were divided into four groups with six mice in each group.The 28 day benzene exposure experiment was conducted by subcutaneous injection.The exposure concentrations were 0,6,30 and 150mg/(kg-b.w.).The weight of mice was weighed during exposure.After the exposure experiment,the limbs and main organs of mice were stripped.The organ coefficients of mice in each group were calculated to analyze the effects of benzene on oxidative damage-related indexes,let-7e-5p regulatory pathway gene expression levels and blood routine.The results showed that the weight gain of mice in 150mg/(kg-b.w.)benzene exposure group was significantly slowed down.The chest-to-body ratio and kidney-to-body ratio of mice decreased,while the liver-to-body ratio and spleen-to-body ratio increased.Peripheral white blood cells,red blood cells,neutrophils and lymphocytes were significantly decreased.The content of phenylmercapto uric acid in urine increased with the increase of exposure concentration.In the detection of oxidative damage index,it was found that the level of 8-hydroxyguanosine in the urine of mice in the benzene exposure group was increased.At the same time,the expression level of let-7e-5p gene in bone marrow cells of benzene-exposed mice decreased,and the expression levels of Caspase-3 and p21 genes and proteins increased.The above results showed that benzene exposure could increase oxidative stress level and inhibit hematopoietic function in mice,and at the same time,abnormal expression of let-7e-5p,Caspase-3 and p21 genes occurred.2.The role and mechanism of let-7e-5p in the cytotoxicity of benzene metabolite benzoquinoneK562 cells were treated with 0,5,10 and 20μmol/L 1,4-BQ.The relative proliferation rate,oxidative stress level,apoptosis and cell cycle were detected by MTT,Ed U and flow cytometry.In addition,RT-PCR and Western Blot were used to detect the expression of target genes and related pathway proteins.The results showed that the relative proliferation rate of K562 cells exposed to 20μmol/L 1,4-BQ decreased significantly at 24 h and 48 h,respectively,which was consistent with the results of EDU experiment.ROS level increased with the increase of exposure.There was a dose-response relationship between apoptosis rate and 1,4-BQ concentration.Under the action of1,4-BQ,the cell cycle results showed that most cells were arrested in the G1/S phase.Moreover,RT-PCR and Western Blot showed that with the increase of exposure concentration,let-7e-5p expression decreased,and p21 and Caspase-3 m RNA and protein expression increased,which were manifested as promoting cell apoptosis and inhibiting cell proliferation.All results were statistically different at the concentration of 20μmol/ L of 1,4-BQ.K562 cells were transfected with mimics to obtain let-7e-5p overexpression cell lines and NC control cell lines.The transfection efficiency was verified by RT-PCR and Western Blot.Two cells were treated with 20μmol/L 1,4-BQ,and the relative proliferation rate was also detected by MTT assay.Flow cytometry was used to detect apoptosis and cell cycle distribution.RT-PCR was used to detect the expression of target genes and Western blotting was used to detect the expression of genes and proteins.PCR results showed that the relative expression level of let-7e-5p in let-7e-5p overexpression cells was significantly higher than that in NC cells,indicating that the construction of let-7e-5p overexpression cell lines was successful.Next,20μmol/L 1,4-BQ was selected to treat the two cells.The apoptosis rate of let-7e-5p overexpression cells was significantly lower than that of NC cells,and the cell cycle arrest at G1/S phase was restored.Western Blot results showed that the expression levels of p21 and Caspase-3 proteins in let-7e-5p overexpression cell group were significantly decreased in both groups after exposure to 20μmol/L 1,4-BQ.Combined with bioinformatics software,RIP experiment and double luciferase experiment,let-7e-5p can simultaneously target and regulate p21 and Caspase-3 at the molecular level to play biological functions.In conclusion,1,4-BQ can inhibit cell proliferation and promote cell apoptosis.Moreover,let-7e-5p can regulate the cytotoxicity caused by 1,4-BQ by targeting p21 and Caspase-3 dual pathways.3.Study on the relationship between let-7e-5p and regulatory genes and occupational benzene exposure and hematological abnormalitiesWe first evaluated the occupational health risks under low benzene exposure,and selected a paint factory in Jiangsu Province to understand the benzene occupational exposure of each post through questionnaires and field hygiene monitoring.The occupational health risk assessment of benzene exposed population in the factory was carried out by using the occupational hazard classification standard and EPA model in China’s workplace.The results showed that the exposure concentration of each post did not exceed the occupational exposure limit in China,and the health risk of occupational hazard classification in China’s workplace was relatively harmless.However,EPA model assessment results show that there are high carcinogenicity and non-carcinogenicity.Therefore,it is of great significance to carry out risk assessment under low benzene exposure,health monitoring and early injury markers for docking benzene population.In order to explore the relationship between let-7e-5p and pathway genes and occupational benzene exposure and abnormal blood system.A occupational epidemiological survey was conducted among 159 benzene-exposed workers in benzene-related enterprises in Jiangsu Province.At the same time,1:1 matching was conducted according to gender and age.The healthy population without benzene-exposed was taken as the control group,and the basic data,peripheral blood and urine of the population were collected.RNA was extracted from peripheral blood leukocytes to analyze the expression of let-7e-5p,p21 and Caspase-3.The contents of S-PMA and 8-OHd G were detected in the collected urine samples.Compared with the control group,the white blood cells,platelets,neutrophils and red blood cells in the benzene exposure group were decreased.The serum malondialdehyde content was increased in the benzene exposure group,and the contents of S-PMA and 8-OHd G in urine were significantly higher than those in the control group.PCR results showed that the expression of let-7e-5p in peripheral white blood cells of benzene-exposed population was decreased,and the m RNA expression of p21 and Caspase-3 was increased,which was statistically different from that of the control group.The above results suggest that the abnormal expression of let-7e-5p,p21 and Caspase-3 has a certain correlation with benzene-induced hematopoietic injury,which can be used as a potential biomarker for benzene-induced hematopoietic injury.ConclusionIn conclusion,benzene exposure can cause changes in oxidative damage related indicators,blood routine and gene expression levels in mice,which are characterized by increased oxidative stress level,abnormal expression of let-7e-5p,Caspase-3 and p21 genes and abnormal peripheral blood.At the same time,overexpression of let-7e-5p can reduce the proliferation inhibition and apoptosis promotion of cells by 1,4-BQ,and its mechanism is mainly through the apoptosis pathway regulated by let-7e-5p targeting Caspase-3 and the cell cycle pathway regulated by p21.The expression of let-7e-5p and its related pathway genes Caspase-3 and p21 in white blood cells of benzene exposed workers was abnormal,and they were correlated with benzene exposure dose,oxidative damage index and hematological indexes.It indicated that let-7e-5p might be a potential biomarker for oxidative damage and hematopoietic toxicity of benzene exposure. |
