Research On DNA Methylation Silencing Genes Reltated To Malignant Transfo-rmation Of Human Bronchial Epithelial Cells Induced By Glycidyl Methacrylate

Glycidyl methacrylate (GMA) is the main component of acrylic plastic, which contains double carbon bonds and expoxy clusters, and could participate in ionic and radical reaction with high activity. It is widely used in resin, coating, adhesive and plastic industries. Previous studies showed that, GMA was low toxic, had reproductive and developmental toxicity, and could induce SHE cell, normal human fetal lung fibroblasts,16HBE and other kinds of cells malignant transformation, and had potentially carcinogenic, and the results of a number of mutagenicity test in vitro were positive,. In the process of16HBE cells malignant transformation induced by GMA, there was also accompanied by changes in DNA methylation genes. DNA methylation was always found in the promoter of tumor suppressor genes, and affected gene expression directly or indirectly. This DNA modification didn’t change the sequence of gene, but it could turn off the activity of certain genes, leading to cell invasion, abnormal proliferation and tumor occurrence. Gene silence induced by DNA methylation was prevalent in the process of tumor occurence and progression. In contrast to genetic changes associated with tumor, DNA methylation was reversible.In this study, by screening DNA methylation silencing genes in the process of16HBE cells malignant transformation induced by GMA at different time points and analysis of the changes of DNA methylation silencing gene status, we aimed to further explore the DNA methylation mechanism associated with16HBE cells malignant transformation induced by GMA.16HBE cells were exposed to8μg/ml GMA (DMSO as solvent control),72h, interval24h, three times. After the end of exposure, countit as the first generation of transformed cells, continuously culture cells to30th generations. Biological characteristics of malignant transformation were identified by the tests of ConA and colony forming frequency on soft agar. GMA infected cells without and with the treat of methylation transferase inhibitor (5mol/L,5-Aza-cdr) as well as synchronization solvent control (DMSO) cells were collected at the time point of10th generation,20th generation,30th generation respectively. Adopt"NimbleGen HG18 CpG Promoter Microarray Methlation" produced by NimbleGen company to screen DNA methylation silencing genes in the different transformation stage and analyze its KEEG pathway; analyze status changes of DNA methylation silencing gene, use qPCR to detect OPCML gene expression levels at different points; apply quantitative DNA methylation detection kit (MethylFlashTM Methylated DNA Quantification Kit (Colorimetric)) to detect the level of genome-wide DNA methylation at different time points.1Analysis of DNA methylation silencing genes at different stages of malignant transformation of16HBE cells induced by GMAIn the process of malignant transformation of16HBE cells induced by GMA, there were821,75,44DNA methylation silencing genes at time point of10th generation,20th generation,30th generation respectively, no DNA methylation silencing genes were found at all transformed stages.There were26,1,3statistically significant pathways involved in DNA methylation silencing genes at the time point of10th generation,20th generation,30th generation respectively.2Analysis the status change of DNA methylation silencing gene in the process of16HBE cells malignant transformation induced by GMAIn the process of malignant transformation of16HBE cells induced by GMA,801genes were silent in10th generation while not in20th generation or30th generation;66genes were silent in20th generation while not in10th generation or30th generation;30genes were silent in30th generation while not in10th generation or20th generation;8genes were silent in10th generation and20th generation while not in30th generation;14genes were silent in30th generation and10th generation while not in20th generation;1gene was silent in20th generation and30th generation while not in10th generation.DNA methylation silencing genes in10th generation while not in20th generation or30th generation were involved in25statistically significant pathways; DNA methylation silencing genes in20th generation while not in10th generation or30th generation were involved in1statistically significant pathway; DNA methylation silencing genes in30th generation while not in10th generation or20th generation were involved in3statistically significant pathways.OPCML gene was occurred to DNA methylation at all different points. Compared with DMSO group, OPCML gene expression levels were down in20th generation and30th generation. OPCML gene was also occurred to DNA methylation in GMA group treated with methylation transferase inhibitor in the10th generation and20th generation, and compared to GMA group not treated with methylation transferase inhibitor, OPCML gene expression levels went upward.3Analysis of genome-wide DNA methylation level in the process of16HBE cells malignant transformation induced by GMAThe genome-wide methylation level in GMA group not treated with methylation transferase inhibitor was lower than that in the same generation of DMSO group in10th generation,20th generation and30th generation; the genome-wide DNA methylation level in GMA group treated with methylation transferase inhibitor was higher than that in untreated GMA group in10th generation and20th generation.In summary, in the process of16HBE cells malignant transformation induced by GMA, DNA methylation silencing genes were changed at different points, and the gene espression level was impacted by DNA methylation, and DNA methylation can be eliminated by demethylation agent in the gene hypermethylation promoter region, making silencing genes especially tumor suppressor genes express high level, thereby reactivating;The low level of the genome-wide methylation was occurred in untreated group cells at different points, and it had occurred in10th generation malignant transformation cell.