Epigenetic Changes Of BEAS-2B Cells In Neoplastic Transformation 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 methylation status and mRNA expression of tumor suppressor gene and repeats LINE1promoter in the process of malignant transformation, and the expression of DNA methltransferase.Methods:The immortalized human bronchial epithelial cells (BEAS-2B) in exponential growth phase were planted onto Transwell membrane with5×104per well. After adherence, cells were directly exposed to cigarette smoke with oxygen concentration of21%and temperature at37℃. Three transwell membrane were exposed to cigarette smoke simultaneously. The optimal time and concentration for inducing the malignant transformation of BEAS-2B cells were determined by CCK-8assay. After the exposure, cells were cultured until to30passages. The exposed cells were trypsinized into dishes for further growth to40passages. Biological characteristics between the control group and1generations,10generations,20generations,30generations and their40generations cells were compared for cell cycle, apoptotic rate and ROS levels. The extend of malignant transformation was detected by soft agar colony formation and nude mice experiment. The expression of DNMT1, DNMT3a, DNMT3b were detected by Q-PCR. The methylation status of the RASSFlA、p16. RARβ、hMLHl、FHIT and LINE-1. Genes were observed by methylation-specific PCR, based on modified genome DNA by bisulphate. Then the mRNA level of RASSF1A, p16and FHIT were detected by Q-PCR.Results:(1) CCK-8data showed that with the increase of smoking concentration and time, cell proliferation inhibition rate was rising, the final concentration and time exposured to cigarette smoke was20%and10minutes, under these conditions, the rate of cell proliferation was about30%.(2) Detection of basic indicators:The percentage of G1phase in each exposure group were decreased (P<0.05), and the percentage of S phase have risen (P<0.05). After the first exposure the apoptotic rate was significantly increased (P<0.05), but became significantly lower in cells after generation. Compared with the control group cells, ROS levels of each exposure group had a upward trend and does-dependent effect.(3) Changes of malignant transformation ability:BEAS-2B cells had capacity of colony formation rate about10%, after exposure for30passages capacity of colony formation rate was up to50%(P<0.05). Sm30-40cells were proved to totally malignant transformed by nude mice experiment. Pathology analysis revealed that tumor cells with different size were distributed diffusely, however, none tumor cells were find in control group.(4) Gene expression and methylation status:Cells exposed to cigarette smoke after passage appeared DNMT1expression levels decline (P<0.05), the DNMT3a gene expression was lower in the control group than the exposure group (P<0.05). Methylation specific PCR was used to analyze the DNA methylation of promoter region of RASSFIA、p16、RARβ、hMLH1、FHIT and LINE-lgene in the control group and exposure groups. The RASSF1A and FHIT methylation were found in the exposure groups but not in control groups. LINE-1was highly expressed in the control group, and the expression levels of the exposure groups were much lower.Conclution:1. A model of malignant transformation of human cells in vitro induced by cigarette smoke was established.2. Cell proliferation was uncontrolled after exposure, showed that the cells had a malignant characteristics and tumorigenicity in nude mice.3. Hypermethylation of the promoter of RASSF1A and FHIT, hypomethylation of LINE-1genes, meanwhile their expression have changed in the exposure groups. This may be one mechanism of lung cancer caused by cigarette smoke and provide a theoretical basis for screening cigarette smoke-induced lung cancer early molecular markers.