| Background and ObjectivesBenzene is an important industrial raw material and has a very wide range of applications in daily life.However,long-term exposure to benzene can cause damage to the hematopoietic system,and long-term low-concentration benzene exposure causes a longer latency period of damage.Therefore,it is very important to explore the feasibility,high sensitivity,and high specificity of early biomarkers for the health monitoring of occupational benzene exposure populations,the screening of high-risk populations,the hazard assessment and prevention of benzene chronic poisoning cases and benzene-induced leukemia.significance.Long intergenic non-coding RNA(lincRNA)is currently one of the key research areas for studying the occurrence,development and susceptibility of diseases.Studying the specific changes of lincRNA and its regulation mechanism can not only deeply understand the pathogenesis of benzene-induced hematological toxicity,but more importantly,provide possible biomarkers for early intervention of benzene-induced hematological toxicity.Based on the results of gene sequencing and bioinformatics analysis of benzene-poisoned mice,this study screened the key molecule lincRNA-p21 in benzene-poisoned mice,and analyzed the dose-effect relationship between benzene exposure and lincRNA-p21 and the possible regulatory genes of lincRNA-p21 To study the effect of low expression of lincRNA-p21 on the cell cycle,proliferation and apoptosis of leukemia cell line K562,and the mechanism of lincRNA-p21 regulating p21-mediated cycle-related pathways through miRNA-17-5p,revealing that lincRNA-p21 is in The role and function of the regulatory network;finally,through occupational epidemiological studies,the expression of lincRNA-p21 regulatory pathway in the peripheral blood of benzene-exposed people,and its relationship with the benzene internal exposure index SPMA(S-phenyl-mercapturic acid)and oxidation The relationship between stress injury and the use of nested case-control study method to analyze the expression level of lincRNA-p21 and the predictive value of benzene-induced hematological toxicity.To clarify the possibility of lincRNA-p21 as an early biomarker of benzene-induced hematological toxicity,especially at low benzene exposure levels,which will provide biological and epidemiological theories and techniques for occupational health protection of workers exposed to benzene Support has important public health significance.Methods1.Blood toxicity of benzene-exposed mice and specific changes of lincRNA-p21 regulatory pathwayC57BL/6 mice were selected,benzene solvent was injected subcutaneously,and the dosage was set at 6,30 and 150 mg/(kg-b.w.)to construct a benzene poisoning mouse model.The automatic blood cell analyzer analyzes the peripheral blood blood cell count.The SPMA,8-OHdG and MDA kits were used to detect oxidative stress damage indicators by high-resolution mass spectrometry.Fluorescence in situ hybridization(FISH)technology was selected to detect the localization of lincRNA-p21 in mouse bone marrow cells.Combining the results of high-throughput microarrays and bioinformatics analysis to construct a lincRNA-mRNA-miRNA co-expression network,and further screen out related genes regulated by lincRNA-p21.The qRT-PCR technology was used to verify the selected target genes in the benzene poisoning mouse model samples,and the Pearson correlation analysis of the relationship between lincRNA-p21 and its downstream genes.2.The role and mechanism of lincRNA-p21 gene pathway in benzoquinone-induced cell oxidative stress damage2.1 Construct K562 cell models exposed to different concentrations(5,10,20?mol/L)of 1,4-benzoquinone,detect cell proliferation inhibition by MTT experiment and EdU experiment,and detect cell apoptosis rate and cycle distribution by flow cytometry.2.2 Construction of lincRNA-p21 low expression K562 cell line,lincRNA-p21 overexpression K562 cell line,miRNA-17-5p overexpression K562 cell line,and lincRNA-p21 and miRNA-17-5p co-transfected K562 cell line to explore lincRNA-The function and role of P21 in the pathway.First,the FISH experiment proved the location of lincRNA-p21 in the K562 cell line.The RNA binding protein immunoprecipitation(RIP)system was used for preliminary screening and the double luciferase reporter gene experiment to verify lincRNA-p21/miRNA-17-5p/p21 signal axis was used as a follow-up research target.Finally,qRT-PCR and Western blot technology were used to verify the molecular regulation mechanism of the signal axis.2.3 By constructing a K562 cell line with low expression of lincRNA-p21,using MTT and EdU to detect cell proliferation,flow cytometry to detect cell apoptosis rate and cycle distribution,combined with qRT-PCR and Western blot technology to verify that lincRNA-p21 is benzene-infected The role of cell cycle arrest in K562 cell line.3.The current health status of workers exposed to benzene in Jiangsu Province and the distribution characteristics of lincRNA-p21 gene among workers exposed to benzeneTo explore the impact of benzene on the blood system of workers,we first investigated the data of 234,437 workers in 5672 enterprises in Jiangsu Province,including 102,393 workers exposed to benzene and 132,044 non-benzene exposed workers.Through multivariate logistic regression,the risk factors related to blood routine were explored.Subsequently,four benzene-related enterprises in Yangzhou City were used as research sites to conduct occupational epidemiological surveys on benzene-exposed workers.Pearson correlation analysis was used to explore the relationship between lincRNA-p21 and its regulatory pathways and SPMA and oxidative stress damage;multiple linear regression was used Analyze the correlation between SPMA and lincRNA-p21 and the risk of blood abnormalities;analyze the mediating effect of lincRNA-p21 with the aid of the mediating effect model;finally establish a cohort model of workers exposed to benzene,adopt a nested case-control method to screen out 13 workers with abnormal blood,Randomly matched 26 controls,explored the relationship between lincRNA-p21 and other related risk factors and benzene-induced blood abnormalities,and analyzed the predictive value of lincRNA-p21 expression levels for blood abnormalities using sensitivity,specificity,and ROC curve.Result1.Blood toxicity of benzene-exposed mice and specific changes of lincRNA-p21 regulatory pathway1.1 Establishment of a mouse model of benzene poisoning and analysis of blood toxicity and oxidative damageThe number of white blood cells,neutrophils and hemoglobin in the peripheral blood of mice in the benzene exposure group was significantly lower than that in the control group(p<0.05).Benzene internal exposure index SPMA,oxidative stress damage index MDA and 8-OHdG all showed an upward trend with the increase of benzene exposure concentration,and the benzene 150mg/(kg-b.w.)group had the most significant increase(p<0.05).The results of the correlation coefficient matrix heat map show that SPMA and 8-OHdG have a positive correlation(r=0.54,p<0.05).In addition,SPMA was significantly negatively correlated with RBC(r=-0.8,p<0.05)and WBC(r=-0.68,p<0.05).1.2 The effect of benzene on the lincRNA-p21 gene pathway in mouse bone marrow cells FISH experiments proved that lincRNA-p21 is expressed in mouse bone marrow cytoplasm and nucleus,but it is mainly located in the cytoplasm.Compared with the control group,the expression level of lincRNA-p21 in the benzene exposure group increased with the increase of the exposure concentration,and there was a dose-effect relationship.The expression level of lincRNA-p21 in the benzene 150mg/(kg-bw)group mice It is 4.96 times that of control mice(p<0.05).Pearson correlation analysis found that the expression level of lincRNA-p21 has a strong positive correlation with p21(r=0.73,p<0.05),and a negative correlation with miRNA-17-p(r=-0.65,p<0.05),and p53 has no correlation(r=-0.18,p>0.05).Targetscan and miRanda online bioinformatics prediction software were used for analysis,and Cytoscape software was used to construct an endogenous competitive RNA(Competing endogenous RNA,ceRNA)co-expression network with lincRNA-p21 as the core,and successfully constructed lincRNA-p21/miRNA-17-5p/p21 signaling pathway,laying the foundation for follow-up research.2.Research on the role and mechanism of lincRNA-p21 gene pathway in benzoquinon e-induced cell oxidative stress damage2.1 The effect of benzoquinone-induced oxidative damage on the lincRNA-p21 gene pathway Benzoquinone poisoning K562 cells can cause significant proliferation inhibition,oxidative damage,apoptosis and cycle inhibition,and there is a dose-effect relationship.At the same time,the distribution of the cell cycle showed G1/S phase block.PCR results showed that as the concentration of 1,4-BQ increased,the expression levels of lincRNA-p21 and p21 showed a significant up-regulation trend,while miRNA-17-5p showed a downward trend.Pearson correlation analysis found that the expression level of lincRNA-p21 has a strong positive correlation with p21(r=0.78,p<0.01),and a negative correlation with miRNA-17-5p(r=-0.66,p<0.01),and p53 has no correlation(r=0.09,p=0.55).Compared with the control group,the expression of P21 protein in the 1,4-BQ exposure group showed a dose-dependent increase,and the trend was consistent with the results of mRNA expression.Cyclin CDK2 and Cyclin E downstream of P21 decreased significantly,showing a trend of decreasing CDK2 and Cyclin E proteins with the increase of the exposure concentration,suggesting that P21 is regulated by the polymer of Cyclin E-CDK2 in the cell cycle play a cycle-blocking role.2.2 lincRNA-p21 acts as a "molecular sponge" to adsorb miRNA-17-5p to regulate the expression of cell cycle gene p21FISH experiments proved that lincRNA-p21 was mainly located in the cytoplasm of K562 cells,and the results were consistent with those in mouse bone marrow cells.RIP combined with PCR experiments to detect the enrichment levels of lincRNA-p21 and p21,found that in the K562 cell line overexpressing miRNA-17-5p,compared with the negative control IgG antibody,the anti-AGO2 antibody significantly enriched lincRNA-p21 and p21.The results of the dual luciferase reporter gene experiment found that miRNA-17-5p can bind to the lincRNA-p21 and p21 domains and significantly reduce the fluorescence signal value of the wild-type reporter plasmid(p<0.05).The above experiments show that lincRNA-p21 can compete for adsorption of miRNA-17-5p,which in turn affects the expression level of its target gene p21.qRT-PCR and Western blot showed that overexpression of lincRNA-p21 can significantly up-regulate the level of p21(p<0.05),and knockdown of lincRNA-p21 can significantly down-regulate the level of p21(p<0.05).In order to further verify whether lincRNA-p21 can regulate the expression of p21 by adsorbing miRNA-17-5p,the K562 cell line was co-transfected with miRNA-17-5p and lincRNA-p21 overexpression plasmids.Compared with the overexpression of lincRNA-p21 plasmid group alone,The expression of lincRNA-p21 and p21 in the co-transfection group decreased by 88.25%and 63.19%respectively(p<0.01),and the expression of miRNA-17-5p increased by 2.19 times,suggesting that lincRNA-p21 may affect the expression of miRNA-17-5p To promote p21 to initiate cell cycle regulation.2.3 The regulatory role of lincRNA-p21-miRNA-17-5p-p21 signal axis in 1,4-benzoquin one-induced cytotoxicityAccording to the results of MTT and EdU experiments,it is found that 1,4-BQ interference can induce cell proliferation inhibition,which is manifested as a decrease in the relative cell proliferation rate.After transfection with si-lincRNA-p21,the relative proliferation rate of cells in the si-lincRNA-p21+1,4-benzoquinone-infected group was higher than that in the 1,4-benzoquinone-infected group,that is,lincRNA was reduced-p21 expression can promote cell growth.The cell cycle results showed that the proportion of cells in the G1 phase of the si-lincRNA-p21+1,4-benzoquinone transfection group was significantly lower than that of the 1,4-benzoquinone-treated group,and the proportion of cells in the S phase was significantly higher than that of 1,4-benzene In the quinone-exposed group,it indicates that knocking down lincRNA-p21 can restart the cell in the G1/S phase.This confirms from the opposite direction that the expression of lincRNA-p21 increases during the process of infecting K562 cells with 1,4-benzoquinone and can block it.The progression of cells in the G1/S phase.In the cell line with low expression of lincRNA-p21 in the process of benzoquinone exposure,its apoptosis rate decreased by 35.65%compared with the benzoquinone exposure group,indicating that reducing the expression level of lincRNA-p21 can effectively attenuate the process of benzoquinone exposure Increase in apoptosis.PCR and Western blot showed that the expression levels of lincRNA-p21 and the downstream gene p21 in the si-lincRNA-p21 transfection group were significantly reduced,which were 0.43 and 0.69 times higher than those in the si-NC+1,4-BQ group,respectively,while miRNA-The expression level of 17-5p increased,which was 2.60 times that of the si-NC+1,4-BQ exposure group,which was statistically different from the si-NC+1,4-BQ exposure group(p<0.05).Compared with the overexpressing cells of lincRNA-p21 low-expressing cells,the P21 protein content in lincRNA-p21 low-expressing cells increased significantly under the action of benzoquinone,but the increase was significantly smaller than the si-NC+1,4-BQ exposure group.The expression levels of cyclin CDK2 and Cyclin E in the downstream pathway of P21 increased compared with the si-NC+1,4-BQ exposure group.3.Research on the current health status of benzene-exposed workers in Jiangsu Province and the distribution characteristics of lincRNA-p21 gene in benzene-exposed workers3.1 Analysis of the health status of workers exposed to benzene in Jiangsu ProvinceThrough a survey of benzene-exposed workers in Jiangsu Province,compared with the control group,the risks of anemia,leukopenia,and neutropenia of benzene-exposed workers were increased.The OR(95%CI)was 1.10(1.05-1.16)and 1.13,respectively.(1.08-1.18),1.17(1.11-1.22).Through the analysis of benzene exposure data of benzene poisoning cases and benzene-induced leukemia cases in Jiangsu Province from 2005 to 2018,it is found that the 0-5 years working age group has the largest number of people,suggesting that benzene exposure has obvious blood system damage at the initial stage.3.2 The study of the correlation between SPMA and blood system abnormalities and oxidative stress damage in workers exposed to benzeneFour benzene-related enterprises in Yangzhou City were selected for further investigation and research,and 502 study subjects were included,of which 241,261 were in the benzene-exposed worker group and the healthy control group,respectively.A comparison of SPMA and oxidative stress damage indicators between the two groups showed that SPMA(p<0.01)and MDA(p<0.01)in the benzene exposure group were significantly higher than those in the control group;compared with the control group,the WBC and oxidative stress damage indexes of the benzene exposure group were significantly higher than those in the control group.ANC,PLT,and RBC decreased by 7.32%,13.74%,11.57%,4.57%,respectively.Among them,ANC and PLT decreased at a higher rate,followed by WBC and RBC,which were consistent with the previous survey results of benzene exposed population in Jiangsu Province.Pearson analysis showed that there was a negative correlation between SPMA and RBC,WBC,and ANC among workers in the benzene exposure group(r values were-0.17,-0.41,-0.33,and p values were all less than 0.05);SPMA and oxidative stress damage index 8-OHdG showed a positive correlation(r=0.57,p<0.01),but there was no correlation with MDA(r=0.16,p>0.05).The results of multiple linear regression analysis of blood routine indicators using the stepwise method showed that for every unit increase in SPMA level of workers exposed to benzene,WBC and ANC scores decreased by 0.031(95%CI:-0.041--0.021)and 0.018(95%Cl:-0.024--0.010)(p<0.05).3.3 Distribution characteristics of lincRNA-p21 gene in workers exposed to benzeneRT-PCR results showed that relative to the non-benzene-exposed control group,the rel ative expression of lincRNA-p21 and p21 increased by 60.59%and 48.72%,respective ly,and the differences were statistically significant(p<0.01).miRNA-17-5p showed a downward trend,and the relative expression levels decreased by 35.48%.The relative expression of p53 gene was not statistically different among the groups(p>0.05).The results are basically consistent with the trend of mouse gene expression levels.Pearson correlation analysis found that the expression level of lincRNA-p21 in workers expos ed to benzene had a strong positive correlation with p21(r=0.55,p<0.01),and a nega tive correlation with miRNA-17-5p(r=-0.40,p<0.01)).The results of multiple linear r egression analysis of blood routine indicators using the stepwise method showed that f or each increase in the expression level of lincRNA-p21 gene,the WBC and ANC sc ores decreased by 0.381(95%CI:-0.481?-0.280)and 0.252(95%CI:-0.324--0.180)(p<0.05).The results of multiple linear regression analysis of conventional indic ators using the stepwise method showed that for every unit increase in SPMA level of benzene exposed workers,the expression level of lincRNA-p21 increased by 0.017(95%CI:0.004-0.030)(p<0.05).The mediating effect model indicates that the lincRNAp21 gene expression level has a mediating effect during exposure and injury,and can predict the level of white blood cell count.The mediating effects accounted for 45.93%of the total effects,indicating that the expression level of lincRNA-p21 gene can be used as a biomarker of blood abnormalities caused by benzene exposure.3.4 A nested case-control study on the relationship between exposure indicators and lincRNA-p21 gene expression in workers exposed to benzene and abnormal blood systemThe baseline of the cohort in this study was 241 people,and 208 people entered the cohort.As of the end of the cohort follow-up on December 31,2020,the nested case-control method was adopted.Among the 208 follow-up subjects,13 cases(6.25%)had blood abnormalities and 195 cases(93.75%)were non-hematological abnormalities.From the non-blood abnormal benzene exposed workers at a ratio of 1:2,26 workers were randomly selected as the control group,and the baseline data of the two groups were compared.The expression levels of SPMA(p<0.01)and lincRNA-p21 in the blood abnormal group were significantly higher than those in the control group(p<0.01),and there was no significant difference between the two groups of p21 and miRNA-17-5p(p>0.05).Through parallel diagnostic analysis of workers exposed to blood abnormalities with benzene,it was found that the sensitivity of SPMA combined gene cluster(lincRNA-p21+p21+miRNA-17-5p)combined parallel diagnostic analysis was 92.31%.SPMA,lincRNA-p21 and combined action(SPMA+lincRNA-p21)draw ROC curve,and the results show that the area under the curve of SPMA and lincRNA-p21 are 0.769 and 0.698 respectively;the combined action can have an area under the curve of 0.784,which has the highest value in predicting blood abnormalities.Conclusion1.Benzene exposure can cause oxidative damage to mouse bone marrow cells,produce blood system toxicity,and cause abnormal expression of lincRNA-p21/miRNA-17-5p/p21.Combined with bioinformatics analysis,it is found that lincRNA-p21 and miRNA-17-5p,miRNA-17-5p and p21 have binding sites,and a ceRNA co-expression network with lincRNA-p21 as the core is established,namely lincRNA-p21/miRNA-17-5p/p21 signal network may be involved in the toxic effect of benzene poisoning mice blood system.2.Benzoquinone exposure to K562 cells leads to an increase in oxidative stress,induces an increase in the expression of lincRNA-p21,inhibits the relative cell proliferation rate,causes cell cycle G1/S block,and promotes an increase in the rate of cell apoptosis.LincRNA-p21 plays a certain regulatory role in benzoquinone-induced oxidative stress damage in K562 cells by affecting cell proliferation and cell cycle distribution.3.lincRNA-p21 acts as a "molecular sponge" for miRNA-17-5p in a base-complementary pairing manner.By competing with miRNA-17-5p's target gene p21,miRNA-17-5p reduces the amount of free miRNA-17-5p.Content,so as to realize the regulation of the target gene p21,and after lincRNA-p21 binds to miRNA-17-5p,it also serves as a target gene of miRNA-17-5p.miRNA-17-5p reduces the stability of lincRNA-p21 and promotes it degradation.4.This study found for the first time that knocking down lincRNA-p21 can partially relieve the negative regulation of benzoquinone on cell cycle arrest and proliferation inhibition of K562,promote the expression level of P21 protein,and exert the function of inhibiting the toxicity of benzoquinone.5.Occupational epidemiological studies found that the expression level of lincRNA-p21 in the peripheral blood of benzene-exposed workers was significantly higher than that of the control group.Through multiple linear regression model and mediating effect model analysis,it was suggested that the increased expression level of lincRNA-p21 might be caused by benzene.Blood system abnormalities play a certain regulatory role in the occurrence and development,and can become a potential biomarker for workers with low benzene exposure.6.A nested case-control study found that the SPMA level and target gene lincRNA-p21 of the blood abnormality group at baseline were significantly higher than those of the control group.To predict blood abnormalities by ROC curve,both SPMA and lincRNA-p21 have good predictive value,and the combined effect of predicting blood abnormalities has the highest value.This study found for the first time the ceRNA regulatory network of lincRNA-miRNA-mRNA related to benzene-induced blood system abnormalities,and combined with the verification of population data,lincRNA-p21 is a biomarker for early detection of health damage caused by benzene exposure and can be used as a low-concentration benzene exposure Workers' health monitoring indicators. |
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