DNMT1 And EZH2 Mediated Methylation Silences The MicroRNA-200b/a/429 Gene And Promotes Tumor Progression

OBJECTIVE To make clear the mechanism that DNMT1 and EZH2 in the promoter region of mi R-200b/a/429 interaction synergistic regulation of promoter methylation. To study that regulation of mi R-200b/a/429 expression and to explore the mechanism that down-regulation of mi R-200b/a/429 and the influence that epigenetics drug 5-azacytidine(5-Azacytidine) and DZNep on expression of mi R-200b/a/429 and tumor growth.METHODS MGC-803, BGC-823(three gastric cancer cells) and U87(glioblastoma cell) were selected for the research. 5-azacytidine(5-Azacytidine) or DZNep was applied to the cells, the expression of mi R-200b/a/429 was examined at the m RNA level using RT-PCR. EZH2 si RNA or DNMT1 si RNA was used to transfect cells, the expression of mi R-200b/a/429 was examined at the m RNA level using RT-PCR. 5-azacytidine, DZNep, EZH2 si RNA and DNMT1 si RNA acted MGC-803, BGC-823(three gastric cancer cells) and U87(glioblastoma cell), related protein was examined at the protein level using immunoblotting detection(Western Blot). The second part is that Co-IP technology was used to indetect EZH2 and DNMT1 in vivo interaction, CHIP analysis checked the interaction of DNMT1 and mi R-200b/a/429 promoter region.The third part is the 5- azacytidine or DZNep on nude mice animal model was used to analyze its impact on tumor growth,immunohistochemistry analysised the expression of related protein.RESULTS The histone methyltransferase inhibitor DZNep and EZH2 si RNA inhibited the expression of histone methylation related proteins, such as EZH2, EED,SUZ12, DNMT1 and H3K27me3 and reactivated of the expression of mi R-200b/a/429. DNA methyltransferase inhibitor 5-azacytidine(5-Azacytidine) and DNMT1 si RNA inhibited the expression of EZH2, SUZ12, DNMT1 and EED and up-regulated mi R-200b/a/429. Co-Ip confirmed that DNMT1 and EZH2 in vivo natural combination, both played a role in the in vivo methylation regulation.Chip analysis confirmed that DNMT1 combined with the promoter region of mi R-200b/a/429. In vivo experiments, DZNep and 5-azacytidine(5-Azacytidine)inhibited the growth of tumor, immunohistochemistry showed that DZNep and 5-azacytidine inhibited the expression of EZH2, SUZ12.CONCLUTIONS Epigenetic modification plays an important role in the regulation of mi R-200b/a/429 expression. The expression level of mi R-200b/a/429 and the absence of mi R-200b/a/429 may be related to the gene promoter hypermethylation.The down-regulation of the expression of mi R-200b/a/429 attributed the success to the interaction between DNMT1 and EZH2, in view of the complexity of epigenetic regulation, whether there is other protein or factor involved this process was not the outcome. Further research is needed.