Differential Effects Of DNA Methylation In Malignant Transformation Of BEAS-2B Cells Induced By Radon And Cigarette Smoking

Objective:To investigate the mechanisms of lung cancer caused by radon and cigarette smoking, BEAS-2B cells were induced to malignant transformation by radon and cigarette smoking in vitro to establish a cell model of lung cancer and gene expression of DNMTs and DNA methylation status of all gene levels were detected. Using Illumina450 k methylation chip screened abnormal methylation specific genes to provide theoretical basis for the prevention and treatment of lung cancer.Methods:The immortalized human bronchial epithelial cells(BEAS-2B) in exponential growth phase were planted onto Transwell membrane with 1.5×105 per well. After adherence, cells were directly exposed to radon and cigarette smoking with oxygen concentration of 21% and temperature at 37℃.Three transwell membrane were exposed to radon a simultaneously.The optimal time and concentration for inducing the malignant transformation of BEAS-2B cells were determined by CCK-8 assay. After the exposure, cells were cultured until to 15 passages. The exposed cells were trypsinized into dishes for further growth to 20 passages. Biological characteristics between the control group and different passages cells were compared for cell cycle, apoptosis by using flow cytometry and the extend of malignant transformation was detected by soft agar colony formation. The m RNA expression of DNMT1, DNMT3 A, DNMT3 B were detected by Q-PCR. The methylation status of gene levels were detected by enzyme standard instrument. Using Illumina450 k methylation chip screened abnormal methylation specific genes. Then the m RNA level of SPDEF, CDK5RAP1, PTPRM and ABCG1 were detected by Q-PCR.Results:(1)The establishment of cell model. The survival rates of BEAS-2B cells were related to concentration and time of exposure. Appropriate exposure of radon was 20000Bq/m3 and 30 mins and of cigarette smoking was 20% and 10 min. A series of sequential steps emerged among cells which exposed to radon and cigarette smoking for 15 to 20 passages, showed similar characteristics of cancer cells.The percentage of G1 phase percentage in the radon group cells was significantly lower and the percentage of S phase was significantly higher.(2)The apoptosis were significantly lower in cells after generation. The capacity of colony formation rate was significantly lower in combined group compared to control group and was up to 24.00%±0.31% in soft ager after exposure for 15 passages and generation for 20 passages(P < 0.05).(2)Changes of methylation status.The DNA methylation status of all gene levels were higher in the control group than the exposure group, The m RNA level of DNMT1 gene was higher in the control group than the exposure group, The DNMT3 A and DNMT3 B gene expression was lower in the control group than the exposure group.(3)Using Illumina450 k methylation chip screened abnormal methylation specific genes. CDK5RAP1 genes were much lower in the combined group, SPDEF genes were highly expressed in the single group, ABCG1 genes were highly expressed in the radon group, PTPRM genes were much lower in the cigarette smoking group.Conclution:1. A model of malignant transformation of human cells in vitro induced by radon and cigarette smoking was established 2. Methylation status may have changed due to DNMTs genes expression alteration during the malignant transformation. 3. Abnormal methylation genes may play different role during the malignant transformation of BEAS-2B cells induced by radon and cigarette smoking in vitro.