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目的探讨5-氮-2′-脱氧胞嘧啶(5-Aza-CdR)诱导骨髓瘤细胞系U266 p16基因DNA 5′CpG岛去甲基化作用及对U266细胞增生的影响。方法采用巢式甲基特异性PCR法(n-MSP)、DNA序列分析、RT-PCR、细胞生长曲线、流式细胞仪DNA含量分析法检测5-Aza-CdR对U266细胞p16基因去甲基化作用及其对U266细胞的生长、增生及细胞周期的影响。结果(1)5-Aza-CdR能够逆转U266细胞p16基因异常甲基化;(2)5-Aza-CdR能激活p16基因沉默的再转录;(3)5-Aza-CdR能下调U266细胞甲基转移酶DNMT1、DNMT3A、DNMT3B的表达并呈浓度依赖性;(4)5-Aza-CdR作用的U266细胞被阻滞于G_0～G_1期。结论5-Aza-CdR可能通过抑制甲基转移酶直接对p16基因去甲基化,逆转U266细胞DNA异常甲基化,并有效地激活因高甲基化所致p16基因沉默的再转录。
Objective To investigate the demethylation of 5’CpG island DNA of U266 p16 gene in 5-Aza-CdR-induced myeloma cell line U626 and its effect on proliferation of U266 cells. Methods N-Methyl-specific PCR (n-MSP), DNA sequence analysis, RT-PCR, cell growth curve and flow cytometry were used to detect the demethylation of p16 gene in U266 cells Effect and its effect on the growth, proliferation and cell cycle of U266 cells. Results: (1) 5-Aza-CdR can reverse the abnormal methylation of p16 gene in U266 cells; (2) 5-Aza-CdR can activate the transcription of p16 gene silencing; (3) The expression of DNMT1, DNMT3A and DNMT3B was in a concentration-dependent manner. (4) U266 cells treated with 5-Aza-CdR were arrested in G_0 ~ G_1 phase. Conclusion 5-Aza-CdR may directly demethylate p16 gene by inhibiting methyltransferase, reversing DNA abnormal methylation in U266 cells and effectively activating transcriptional silencing of p16 gene due to hypermethylation.