| Objective: Coking operations such as coal preparation,coking,benzene refining and other production tasks are prone to produce coke oven emissions,dust,carbon monoxide and other occupational risk factors.The emission characteristics are extremely complex with high level of environmental risks,so it is necessary to accurately assess the environmental exposure levels of different pollutants.However,environmental monitoring or biological testing are still the common methods for the exposure assessment of occupational pollutants in China,which would waste manpower and time,and are limited for the extrapolation of research results due to the heterogenity of different workplaces and individuals.In addition,coking workers exposed to a large number of environmental pollutants in production activities tend to be susceptible to various diseases.Therefore,regular monitoring of the exposure levels of occupational pollutants in coking industry and the health status of the occupational population as well as qualitative and quantitative analysis are urgent for health hazard control of coking workers.Although a series of researches about the role of coking pollutants in the occurrence and development of human organ system diseases have been conducted,the exact pathogenesis caused by coking pollutants still needs to be further clarified.In recent years,as a common epigenetic modification,the role and function of RNA methylation are increasingly being explored.The RNA m6 A modification,as the most abundant RNA methylation modification in eukaryotes,has become a new research hotspot.In addition,miRNA,as a class of small non-coding single stranded RNA with a length of 19-25 nucleotides,specifically binds to the 3 'untranslated region of the target gene m RNA at the post transcriptional level,and participates in cell proliferation,differentiation,apoptosis and other biological processes by regulating the translation process,which plays an important role in the occurrence and development of tumors.However,whether m6 A modification and miRNAs are involved in the malignant transformation of human bronchial epithelial cells induced by coke oven emissions and the specific mechanism are not clear.Therefore,this study aims to construct time and occupation/job-specific job exposure matrice according to the types of pollutants.In addition,this study evaluates the risks of different exposure levels of pollutants on respiratory system injury for coking workers.What's more,the role,function and pathway of m6 A related enzymes,miRNAs and their targeted regulatory genes in the malignant transformation of human bronchial epithelial cells induced by coke oven emissions are explored,so as to further evaluate the health damage effect of coke oven emissions and improve the health protection for coking workers.Methods: In this study,the job exposure matrice of major occupational pollutants in coking industry were constructed to evaluate the exposure level of each pollutant,so as to explore the effects of different pollutant exposure on the lung injury of coking workers.Different experiments in vitro were conducted to explore the potential biological changes and the effects of m6 A related enzymes,miRNA as well as the targeted regulatory genes in the malignant transformation of human bronchial epithelial cells induced by coke oven emissions to clarify the role and mechanism of coke oven emissions in the induction of malignant transformation of human bronchial epithelial cells.In the first part of this research,through expert consultation and literature review,the main occupational risk factors in coking industry were determined,and the occupational exposure data of corresponding pollutants were collected.And then these collected data was randomly divided into training and validation set based on the ratio of 4:1.Linear mixed effect model was used to construct the job exposure matrice for different pollutants with the training set.Finally,the correlation analysis and Bland-Altman diagram were conducted between the predicted concentration of pollutants in the established job exposure matrice and the measured concentration in the validation set as well as the internal exposure level of polycyclic aromatic hydrocarbons in urine for coking workers to evaluate the stability and reliability of these job exposure matrice.In the second part,1810 coking workers from a coking plant in northeast China were enrolled as the studied population.Their basic demographic information,living habits,past medical history and occupational history were investigated.Based on the above job exposure matrice,individual cumulative exposure levels of the pollutants were calculated.In addition,the lung function status were detected,which were then taken as dependent variable while age,body mass index,smoking history,drinking history,initial working age,cumulative working years as well as the cumulative exposure levels of pollutants were enrolled as independent variables to construct logistic regression models in order to identify occupational risk factors for lung dysfunction of coking workers.In the third part of this study,the malignant transformation of human bronchial epithelial cells was induced by the organic extract of coke oven emissions at the concentration of 20 mg/ml.And then CCK-8 test and cell cycle analysis were conducted to detect the changes of cell proliferation and cell cycle.In addition,7-AAD and Annexin V-APC were applied to detect the changes of cell apoptosis.Then,the expression levels of METTL3,METTL14 and miR-92a-3p in malignant transformed cells were detected by qRT-PCR and Western Blot.Meanwhile,the downstream target gene of miR-92a-3p was predicted by Starbase and Linkedomics databases.The double luciferase reporter gene test,qRT-PCR and Western Blot were used to validate the targeted regulatory relationship between miR-92a-3p and the target gene.And then the expression levels of targeted gene were detected in malignant transformed cells.In addition,we transfected the overexpressed METTL14 plasmid into human bronchial epithelial cells to investigate the regulatory effect of METTL14 on miR-92a-3p and its downstream target genes by qRT-PCR and Western Blot.SAS 9.4 was used to construct the job exposure matrice,and Stata 14.0 software was used to calculate the cumulative exposure levels of different pollutants.In addition,the Bland-Altman diagram was drawn by medcalc 18.2.1 while SPSS 22.0 was used for Spearman rank correlation analysis and the construction of logistic regression models.Student's t test was performed to compare the quantitative data of two groups with continuous normal distributions;while analysis of variance was conducted to compare the quantitative data of three and more groups.Dunnett method was applied for pairwise comparison between experimental groups and the control group and chi square test was performd for the analysis of categorical variables.All tests were bilateral,with a statistical significance of P < 0.05.Results: 1.We enrolled coke oven emissions,dust,carbon monoxide,heat and noise as the main occupational pollutants in the coking industry under the guidence of the experts,and finally collected 6021 time weighted average concentration data and 2356 short-term exposure level data,which included 27 occupation categories and 35 job categories,covering the years from 1977 to 2018.By fitting the linear mixed effect model,the corresponding job exposure matrice were constructed for the five pollutants according to the exposure types.Results of Spearman rank correlation test and Bland-Altman diagram indicated a generally good consistency between the measured and predicted values except for the job exposure matrix for noise short-term exposure levels.What's more,correlation analysis for the internal exposures showed that except for9-hydroxyphenanthrene,the concentrations of 1-hydroxypyridine and1-hydroxynaphthalene in urine had good correlations with the predicted values of job exposure matrix of coke oven emissions with a statistical significance.2.Through logistic regression model,this study found that with the increase of age,the risk of pulmonary ventilation dysfunction for coke oven workers also increased,the odds ratio(OR)with its 95% confidence interval(95% CI)was 1.092(1.057-1.129).Compared with non-smokers,smokers were more likely to have pulmonary ventilation dysfunction,the odds ratio was 1.359(1.079-1.710).However,working years might be a protective factor for the occurrence of pulmonary dysfunction(odds ratio: 0.716(0.586-0.874)).In addition,with the increase of cumulative exposure to COE and noise,the risk of pulmonary ventilation disorders also significantly increased.The cumulative exposure levels of COE and noise increased by 1 unit,the risk of pulmonary ventilation disorder increased to 1.295 and 1.002 times,respectively.Compared with the individuals with cumulative exposure levels below 200?,the risk of pulmonary ventilation dysfunction in individuals with cumulative exposure levels of 200-500?,500-800? and above 800? was 3.717 times,3.839 times and 5.015 times respectively.In addition,compared with non-smokers,smoking workers were more likely to develop obstructive pulmonary ventilation disorder with the odds ratio of 1.342(1.045-1.722).The OR for the cumulative exposure to coke oven emissions and noise were 1.513(1.259-1.818)and1.003(1.000-1.006),respectively.Compared with workers with cumulative heat exposure less than 200?,the ORs and 95% CIs were 4.950(2.935-8.348),5.728(2.539-12.923)and 8.081(2.459-26.552)for workers exposed to 200-500?,500-800?and 800? and above,respectively.However,with the increase of accumulated working years,the risk of obstructive pulmonary ventilation disorder decreased(OR = 0.677,P <0.001).When the model was fitted with restrictive pulmonary ventilation dysfunction as dependent variable,the risk effects of cumulative working years,cumulative exposure of coke oven emissions,noise were not statistically significant.However,smoking status and cumulative heat exposure with 200-500? still increased the risk of restrictive pulmonary ventilation dysfunction with OR of 1.401(1.070-1.835)and 1.771(1.160-2.703),respectively.In addition,the age was also a risk facor for restrictive pulmonary ventilation dysfunction with OR of 1.050(P < 0.001).3.Human bronchial epithelial cells were exposed to the organic extract of coke oven emissions to establish malignant transformation model.The results of functional experiments in vitro showed that compared with negative control group,the cell cycle process and proliferation ability of malignant transformation group were significantly promoted,while the apoptosis process had no significant change.The expression levels of METTL3,METTL14 and miR-92a-3p were detected by qRT-PCR and Western Blot,which showed that the expression of METTL14 and miR-92a-3p were significantly up-regulated in malignant transformation group,but no change of METTL3 expression was observed.Next,we predicted the downstream target gene of miR-92a-3p using Targetscan and Starbase databases,and found that RGS3 may be a potential targeted gene of miR-92a-3p.Through the analysis of Linkedmics online website,we found that there was a significant negative correlation between the expression of miR-92a-3p and RGS3.Then,results of double luciferase reporter gene experiment,qRT-PCR and Western Blot confirmed that miR-92a-3p could directly bind to the 3'-UTR region of RGS3 and inhibit the expression of RGS3.Compared with the negative control group,the expression level of RGS3 in malignant transformation group was significantly decreased as well.In addition,we found the expression levels of miR-92a-3p and its target gene were affected after transfection of METTL14 overexpressed plasmid into cells.The expression of miR-92a-3p was significantly up-regulated,while the expression of RGS3 was inhibited.Conclusions: 1.The job exposure matrice of five major occupational pollutants(coke oven emissions,dust,carbon monoxide,heat and noise)in coking industry were constructed.2.The cumulative exposure of coke oven emissions,noise and heat can significantly increase the risk of lung dysfunction in coking workers.3.Coke oven emissions can promote cell circle and cell proliferation to maintain the malignant status of human bronchial epithelial cells and play an important role through regulating the METTL14/miR-92a-3p/RGS3 pathway. |
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