Investigation Of The Molecular Mechanism Underlying The Carcinogenicity Of Musk Xylene Using Cell Models And Computer Prediction Methods

Musk xylene(MX)is a kind of widely used synthetic musk.Due to its slow degradation rates,low polarity,lipophilic property,and bio-accumulative ability,MX can enter the human body and accumulate in the body through skin absorption and air inhalation.Thus MX has become a new type of environmental contaminant in recent years.Existing researches have confirmed that MX is related to the occurrence of various cancers and the migration of tumor cells,but the molecular mechanism of MX involved in cancer is still unclear.In this study,the normal human hepatic cell line L02 was used to investigate the long-term carcinogenicity of MX.At first,the effect of MX on cell morphology was observed,and constructed a model of MX-induced malignant transformation of L02 cells;Then,the gene expression profiles of normal L02 cells and malignantly transformed L02 cells were detected by using gene chip technology.And further analysis through gene ontology(GO)analysis and KEGG pathway analysis were performed to explore the target genes and signaling pathways that MX affected.In addition,molecular dynamics(MD)simulation was also used to predict and screen the potential receptors of MX.Finally,the expression level of the downstream genes of the target genes were detected through the RT-PCR(Real time Polymerase Chain Reaction)technology.(1)After MX exposure for 24 h,some irregular fusiform,protuberances and multinucleated cells were observed,and the proportion of abnormal cells increased with cell passage.Indefinite cell proliferation,ability of anchorage-independent proliferation,migration and invision were then observed in subsequent experiments,which suggested the positive effects of MX on cell malignant transformation in vitro.(2)Gene chip results shown that the gene expression pattern of MXtransformed L02 cells is significant different from that of the control group,with a total of 1831 up-regulated genes and 1670 down-regulated genes.Further GO analysis and KEGG pathway analysis results indicated that differentially expressed genes are involved in the regulation of many biological functions,including MAPK(mitogen-activated protein kinase)pathway and many biological processes that are related to cancer occurrence and development.(3)Molecular dynamics simulation results shown that MX can bind to EGFR,FGFR1 and TGFβR three receptors which involved in the MAPK pathway.In the binding pattern,the binding site of MX with the three proteins were similar,and the bonding mainly consisted of π-H bond and hydrogen bond.From the perspective of average binding free energy,the binding strength was in order:EGFR>TGFβR>FGFR1.(4)RT-PCR results showed that compared with the control group,the expression levels of Grb2,Sos1,Sos2,downstream genes of EGFR and FGFR1,in the MX malignant transformation group were up-regulated.In this paper,the molecular mechanism and target genes of MX carcinogenesis were systematically analyzed.This is essential to establish a quality control policy for musk xylene levels in the environment,and analyze and evaluate the effects of xylene musk on human health.At the same time,it is also of great significance to explore the environmental causes of human cancer,prevent and treat cancer and to design anti-tumor drugs.