Study Of MicroRna Regulating Toxic Pathways Of Benzo[a]pyrene Induced Lung Injury

Objective:Polycyclic aromatic hydrocarbons(PAHs)are a class of carcinogenic organic pollutants.Epidemiological studies have proved that PAHs can cause a variety of lung damages,including inflammation,decreased lung function,fibrosis,lung cancer,etc.,but there are still gaps in the mechanism.It has been found that micro RNAs(mi RNAs)are not only involved in a variety of biological processes,but also sensitive to environmental responses as epigenetic factors,so mi RNAs play an important role in the occurrence and development of lung injury caused by PAHs.With the development of systems toxicology,pathway-based methods are becoming more and more popular.This study adopts a new "Bottom-up" strategy which was based on previously established key toxicity pathways,combined omics technology and verification through in vitro experiments,and validation in patients from publications with related diseases,for the purpose of identification between mi RNAs that played a disease drivers role on illness(as “drivers”)and the ones that acted as subordinate symptoms of exposure(as “symptoms”).Supplemented by a benchmark dose analysis for pathway perturbations,this study provides new ideas for other similar studies and new clues about the role of mi RNAs in risk assessment.Methods:Five key toxic pathways in aryl hydrocarbon receptor activation were used as the basis.(1)In this study,we used RNA and small RNA sequencing data of 16HBECYP1A1 cells exposed to multiple doses of benzo[a]pyrene(Ba P)to investigate mi RNA regulation affecting pathway perturbations.Specifically,Target Scan,TCGA,RNA Hybrid and other databases were used to screen highly expressed mi RNAs and predict target genes.(2)The binding and regulation of mi RNA and m RNAs were verified by cytological experiments,including transfection,q RT-PCR and FREMSA.Based on the mi RNA: m RNA expression-regulation relationship,eight regulatory patterns were integrated to screen out the “drivers” mi RNAs.(3)BMDexpress software was used for dose analysis,and the Point of Departure(Po D)was calculated.The regulation effect of exogenous mi RNA on pathway perturbation was confirmed.(4)According to the published data,the expression of relevant mi RNAs in the tissues of lung disease patients was verified.Results:Previous studies constructed the five most relevant molecular response toxicity pathways for Ba P to lung injury,including AHR pathway,IL-6 pathway,P53 pathway,NRF2 pathway,and fibrosis pathway.(1)In this study,TCGA,Target Scan,and RNA hybrid databases were used for screening,and a total of 69 pairs of candidate mi RNA: m RNA relationships were obtained.Then,based on transfection mimics and q RT-PCR experiments,the regulatory relationship of 26 pairs of mi RNAs: m RNA(including 7 mi RNAs and 23 m RNAs)was determined.And by integrating the expression differences and regulatory relationships of mi RNAs/target genes,all experimental phenomena were summarized into eight mi RNAs:m RNA expression-regulation mode.(2)These eight patterns specifically include the positive/negative regulation of mi RNA on target genes,and the up/down regulation of mi RNA is accompanied by the up/down regulation of target genes.Four patterns of mi RNAs that acted as disease drivers were identified,5 “drivers” mi RNAs,including mi R-1343-3p,mi R-3173-5p,mi R-629-3p,mi R-219a-1-3p and mi R-9-5p.(3)According to regulation patterns,co-transfection of three “drivers” mi RNAs(mi R-1343-3p,mi R-3173-5p,mi R-629-3p)mimics to research the pathways perturbation.After co-transfection,fibrosis pathways BMD was increased and Po D was inrceased.Thus,three “drivers” mi RNAs actually act as strong regulation in fibrosis pathways.(4)We confirmed one of the five “drivers” mi RNAs identified in published population studies of lung disease.Conlusion:Based on the five key toxic pathways of BAP-induced lung injury,this study summarized eight mi RNAs: The molecular regulation mechanism of m RNA,and five strongly regulated mi RNAs(mi R-1343-3p,mi R-629-3p,mi R-219a-1-3p,mi R-3173-5p and mi R-9-5p)as disease drivers,The expression trend of the five mi RNAs in the cells was consistent with that in the lung related diseases.The co-transfection of mi R-1343-3p,mi R-629-3p and mi R-3173-5p mimics plays a key regulatory role in the overall disturbance of Fibrosis pathway caused by Ba P exposure.