Effects Of Histone Acetylation In Malignant Transformation Of BEAS-2B Cells Induced By Cigarette Smoke

Objective:To investigate the mechanisms of lung cancer caused by cigarette smoke and provide a theoretical basis for screening cigarette smoke-induced lung cancer early molecular markers, immortalized human bronchial epithelial cells(BEAS-2B cells) were induced to malignant transformation by cigarette smoke in vitro to establish a cell model of lung cancer.We observed the different sites of histone H3K9 acetylation and the expression of some tumor suppressor genes.Methods:BEAS-2B cells were cultured in 37 ℃, 5% CO2 incubator with the LHC-8 serum-free medium, then planting them onto the Transwell membrane of 1.5×105 per well when they are in exponential growth phase. 24 hours later cells were directly exposed to cigarette smoke with oxygen concentration of 21% and at 37℃.Three transwells were exposed to cigarette smoke simultaneously. The optimal time and concentration on inducing the malignant transformation of BEAS-2B cells were determined by CCK-8 assay. After the exposure, cells were cultured until to 30 passages. The exposed cells were trypsinized into dishes for further growth of generation to 40 passages. The control group is treated the samecondition without cigarette smoke. Biological characteristics between the control group and different passages cells were compared for cell morphology, cell cycle,apoptotic rate and ROS levels by using flow cytometry and the extend of malignant transformation was detected by soft agar colony formation. Screening the different sites of histone H3K9 acetylation between the control group and the cells after exposure for 30 passages and generation for 40 passages(Sm30-40) by using ChIP-seq. The optimal time and concentration of trichostatin A were determined by CCK-8 assay. The expression ofMGMT、P21WAF1、FHIT、RARβ、PTEN were detected by Q-PCR before and after the control group and different passages cells were treated with TSA.Results:(1)CCK-8 data showed that with the increase of smoking concentration and time, cell proliferation inhibition rate was increased. Appropriate exposure conditions of cigarette smoke were was 20% and exposure time was 10 minutes, Under these conditions, the rate of cell proliferation inhibition was about 30%.(2) Detection of general index: A series of sequential steps emerged among cells which exposed to cigarette smoke for 10 passages and generation for 40 passages, including altered cell morphology, heteromorphism and lost of contact inhibition. With increasing of passages, morphological changes and cell growth were similar to cancer cells. The percentage of G1 phase in exposure group cells was significantly lower and the percentage of S phase was significantly higher compared to control group. The apoptotic rates were significantly lower in cells after generation.The ROS levels of each exposure group had an upward trend and does-dependent effect.(3)Changes of malignant transformation ability: BEAS-2B cells had capacity of colony formation and the rate was about 1.7%, after exposure for 30 passages and generation for40 passages the capacity of colony formation rate was up to over 30%( P<0.05).(4)High-throughput detection of histone H3K9 acetylation: Compared with the control group,there were 155 genes which histone H3K9 acetylation levels were increased and 892 genes which histone H3K9 acetylation levels were decreased in the cells exposure to cigarette smoke.(5) Some changes in relative gene expression: The mRNA levels of MGMT, P21WAF1, FHIT, RARβ,PTEN were lower in the exposure group than the control group. After treated the cells with TSA of 75nmol/L for 24 hours, no significant change was found in mRNA levels of the five genes in the control group. while in the exposure group, there was no change in the expression of FHIT, RARβ and the expression levels of MGMT,P21WAF1, PTEN were increased after treated with TSA.Conclusion:1. A model of malignant transformation of human bronchial epithelial cells in vitro induced by cigarette smoke was established.2. Histone H3K9 acetylation was related to the expression alteration of partial genes during the malignant transformation.This may provide a theoretical basis for screening theearly molecular markers of cigarette smoke-induced lung cancer.