| Glycidyl methacrylate (GMA), as a comonomer, has good characteristics of anti-UV, water resistance and heat resistance, it is widely used in resin, coating, adhesive and plastic industries. Many researches have been reported that GMA has mutagenicity, it could induce malignant transformation in several types of mammalian or human cells. All these results have demonstrated its potential carcinogenicity.In vitro cell transformation test is an effective way to study the carcinogenic mechanisms of various physical and chemical factors.16HBE, an SV40 large T antigen-immortalized human airway epithelial cell line, retains the phenotype of the differentiated lineage of the parent. This cell line has contact inhibition and rarely produce tumors when injected into nude mice, which provides us an ideal model for detecting potential carcinogenicity of chemicals and for studying on the mechanisms of malignant transformation.Epigenetic, which refers to heritable differences of genes without changing their DNA sequences, is critical in maintaining the stability and integrity of the expression profiles of different cell types by modifying DNA methylation and histone methylation. Epigenetic events have a close relationship with the development and progression of tumors. Many researches showed that the disorder of DNA methylation level and pattern exisited in tumor occurrence and development.In vitro malignant transformation model of 16HBE induced by GMA, treated three times at low concentration, had been established. The Agilent whole genome oligo microarray was used to detect the change of the expression of methylation-related genes during malignant transforming stages of 16HBE cells induced by GMA, RT-PCR was used to reexaminate the differentially expressed genes, then DNA methylation patterns of tumor-related genes promoter were determined by methylation-specific PCR (MSP) assay, in order to discuss the epigenetic mechanism of its potential carcinogenicity.1. The expressing alteration of DNA methyltransferase enzymes (DNMTs) and methyl-CpG binding proteins genes in malignant transformation of 16HBE cells induced by GMA.1.1 Screen the differentially expressed genes of DNMTs and methyl-CpG binding proteins by gene chip during malignant transforming stages of 16HBE cells induced by GMA.The result of gene chip showed that the expressions of DNMT1, MBD1 and MBD3 were up-regulated compared with control group in the transformed 10th-generation (protophase) cells (Ratio>2), no significant differences were observed in the transformed 30th-generation (anaphase) cells.1.2 The expressing alteration of DNMT1 and MBD1 during malignant transforming stages of 16HBE cells induced by GMA.The expressions of DNMT1 and MBD1 mRNA were detected with RT-PCR in the malignant transformation protophase cells. The result showed that the levels of DNMT1 and MBD1 mRNA were up-regulated by 80.60%,79.10%. Methylation may be play an important role during malignant transformation of 16HBE induced by GMA, DNMT1 and MBD1 play an important role in the inactivation-mechanism of methylation-related genes in malignant transformation of 16HBE induced by GMA.2. The expressing alteration and DNA methylation of Tumor-related genes during malignant transforming stages of 16HBE cells induced by GMA.2.1 Screen the differentially expression of Tumor-related genes during malignant transforming stages of 16HBE cells induced by GMA.The result of gene chip showed that the expressions of CACNA2D3, LRRC4 genes were down-regulated compared with control group in malignant transformation protophase and anaphase cells. The expression of CTGF gene was observed up-regulated expression in malignant transformation protophase cells while down-regulated expression in malignant transformation anaphase cells. The expressions of H19,THBS1 genes were down-regulated in malignant transformation anaphase cells.The mechanisms of expressing alteration of these genes need further study.2.2 Analysis DNA methylation of tumor-related genes during malignant transforming stages of 16HBE cells induced by GMA.According to the differentially expression of genes and literature, we selected eight genes (P16, APC, LRRC4, H19, hMSH2, MGMT, CTGF, THBS1), DNA methylation patterns of these genes were detected by MSP during malignant transforming stages of 16HBE cells induced by GMA (early stage, protophase, anaphase). The results showed that there was abnormally methylation in CpG island of P16 gene promoter during malignant transforming stages of 16HBE cells induced by GMA, P16 gene can be considered as an early sensitive index of malignant transformation of 16HBE cells induced by GMA. hMSH2, CTGF, THBS1 genes promoter had not occurred methylation, suggesting that they were not sensitive genes during malignant transformation of 16HBE cells. APC, MGMT genes were observed to occur methylaion in solvent control and GMA group, so they were not specific genes during malignant transformation of 16HBE cells. LRRC4, H19 genes need further study.In summary, the changes of the expression of methylation-related genes during malignant transforming stages of 16HBE cells induced by GMA were analyzed, and the differentially expressed genes were reexaminated, then DNA methylation patterns of tumor-related genes promoter were determined by MSP.All results showed that methylation may be play an important role during malignant transformation of 16HBE induced by GMA, DNMT1 and MBD1 play an important role in the inactivation-mechanism of methylation-related genes in malignant transformation of 16HBE induced by GMA, and DNA methylation of Tumor-related genes can be considered as an early sensitive index of malignant transformation of 16HBE cells induced by GMA. It provides new cues on carcinogenicity risk evaluation of GMA, epidemiologic monitoring and mechanism study on chemical carcinogenesis. |
