Changes Of Histone Acetylation And Mechanism Study Of Cell Proliferation And Apoptosis By FOXO3-mediated In BEAS-2B Cell Exposed To Cigarette Smoke

Objective: We examined the differential of histone acetylation in the BEAS-2B cell exposed to cigarette smoke(CS-exposed cell).The FOXO3 was further studied as assumption of its expression regulated by histone H3K9 hypoacetylated and its role on proliferation and apoptosis in CS-exposed cell.This study is useful for understanding the mechanisms of cigarette smoke-induced lung cancer,and provide a scientific basis for prevention and treatment of lung carcinoma caused by smoking.Methods: The expression of histone deacetylase(HDAC1)and histone H3K9 acetylated protein(A-H3K9)was detected by Western Blot in CS-exposed cell,and the abnormal acetylation of histone(A-H3K9)was screened by Bioinformatics analysis.The FOXO3 gene was identified by Chip-seq,qPCR and Western Blot,to verify the relationship between histone H3K9 hypoacetylation and gene expression in CS-exposed cell,with or without TSA treatment;CCK8 assay was used to detect cell proliferation rate;flow cytometry was used to analyze cell cycle and apoptosis;caspase3/7 activity and Bax/Bcl-2 ratio were used to detect the activation state of cell apoptosis pathway;CCK8 and caspase3/7 activity were used to detect the proliferation and apoptosis in FOXO3-lentivirus transfected cells;FOXO3 and p-FOXO3 were detected by Western Blot after JNK inhibitor(JNKi)treatment.Real-time quantitative PCR and Western Blot were used to detect the effect of FOXO3 Lentivirus on the mRNA and protein expression of the target gene in the downstream of FOXO3.Results:(1)The results showed the expression of HDAC1 was up-regulated,the expression of A-H3K9 was decreased,and the histone acetylation in the genome was in a low level.The histone hypoacetylated gene mainly enriched in the PTEN dependent cell cycle arrest and apoptosis and PI3K/AKT pathways.The expression of FOXO3 in CS-exposed cells was reduced,while it is back up again after TSA treatment,suggesting that the low expression of FOXO3 gene in CS-exposed cells may be caused by H3K9 hypoacetylation.(2)The proportion of G1 phase in exposed cells was significantly lower compared to control group,while the proportion of S phase was significantly higher.The decreased in proportion of apoptosis,caspase3/7 activity and Bax/Bcl-2 expression showed that longterm CS-exposuse can induce the reduction of cell apoptosis.After transfection by FOXO3-lentivirus,cell proliferation was slowed down and the apoptotic was activated.The results suggest that FOXO3 may be involved in malignant proliferation and apoptosis inhibition in long-term CS-exposed cells.(3)In CS-exposed cell,the expression of p-JNK and FOXO3 was reduced,in line with the expression of FOXO3 and p-JNK were decreased in BEAS-2B cell treated by JNK pathway inhibitors.The mRNA and protein expression of Fasl,Bim,TRAIL and P21 genes were decreased in CS-exposed cells.While after transfected with FOXO3-lentivirus,the expression of Fasl,Bim,and P21 rised up.The results suggest that the inhibition of JNK/FOXO3/(Fasl,Bim,P21)pathway may be an important reason for the change of cell proliferation and apoptosis in CS-exposed cells.Conclusion: Long-term exposure to cigarette smoke in the bronchial epithelial cells may increase the expression of HDAC1,down-regulate the expression of FOXO3 by histone H3K9 hypoacetylation.Meantime,cigarette smoke inhibited the JNK pathway in the cell and limited transcription of FOXO3 protein,which thereby inhibited expression of the downstream target genes of FOXO3,Fas L,Bim,P21,thus promoted the bronchial epithelial cell proliferation and inhibited apoptosis,contributing to the tumorigenesis and tumour progression of lung cancer.