The Molecular Mechanism Of G2/M Cell Cycle Arrest In HepG2 Cells Induced By Deoxynivalenol

Deoxynivalenol(DON),also known as vomitoxin,is a type B trichothecene mycotoxin,mainly produced by Fusarium graminearum.It is a most common contaminates in cereal products such as wheat,soybean meal and corn,and it can also contaminate food products.Human and animals have a wide range of toxic effects after ingestion of food contaminated with DON.The toxicological effects of DON are shown as inhibition of DNA and protein synthesis,ribosome stress response,inflammatory response,inhibition of cell proliferation and cell cycle arrest.Studies showed that DON can induce G2/M phase cell cycle arrest in human umbilical vein endothelial cells,human colorectal cancer cells,and human intestinal epithelial cells,while its molecular mechanism is still unclear.Transcriptomic data of DON treated HepG2 cells shows that DON induces up regulation of two important transcription factors EGR1 and ATF3 which involved in cell cycle regulation.This research focused on the function and mechanism of EGR1 and ATF3 in DON induced G2/M phase cell cycle arrest,and achieved mainly results as follows:1.DON induced G2/M cell cycle arrest in HepG2 cellsHepG2 cells were treated with 2 ?g/m L and 4 ?g/m L DON for 6 h,12 h,24 h and 48 h,respectively,and using flow cytometry to detect the number of cells in different cell cycle phases.The results showed that the percentage of cells in G2/M phase was significantly increased after DON treated,and 4 ?g/m L DON was more potent than 2 ?g/m L DON,but there was no significant difference between different treatment durations of blocking effect.2.EGR1 is important for DON induced G2/M cell cycle arrestqRT-PCR and Western blot experiments demonstrated that DON induced the up regulation of EGR1 and p21 expression.The degree of DON induced p21 expression and G2/M cell cycle arrest was inhibited when EGR1 knockdown in HepG2 cells.It indicated that EGR1 is important for DON induced G2/M cell cycle arrest.3.DON induced EGR1 by up regulating the expression of ATF3?Zip2a/2bThe expression of ATF3?Zip2a/2b was up regulated when HepG2 cells were treated with 2 ?g/m L DON for 6 h,12 h,24 h and 48 h.The results showed that the overexpression of ATF3 full-length protein inhibited the degree of DON up regulated EGR1,while overexpressed ATF3?Zip2a/2b deletion isoform had an induction effect on that.It indicated that ATF3?Zip2a/2b played an important role in DON induced EGR1.4.DON induced the expression of ATF3?Zip2a/2b through H3K27 ac and H3K9acTwo histone acetylation enzyme P300 and PCAF were significantly up regulated after 2 ?g/m L DON treated HepG2 cells.P300 acetylated H3K27 site and PCAF acetylated H3K9 site.And Ch IP experiment results showed that DON enhanced the level of H3K27 ac and H3K9 ac modification in ATF3 promoter region.Respectively attenuated two modifications,the induction of ATF3?Zip2a/2b by DON was inhibited.It indicated that DON induced the expression of ATF3?Zip2a/2b by enhancing H3K27 ac and H3K9 ac modifications in its promoter region.This research cleared that EGR1 and ATF3 had an important effect on DON induced G2/M cell cycle arrest in HepG2 cells,Clarify the mechanism of DON induced the expression of ATF3?Zip2a/2b by enhancing the H3K27 ac and H3K9 ac modification of ATF3 promoter region,then up regulated the expression of EGR1 and caused G2/M cell cycle arrest in HepG2 cells.These results had an important theoretical significance for clarifying the molecular toxicology of DON.