多发性骨髓瘤患者p16基因甲基化及砷剂诱导去甲基化的研究

目的探讨p16基因启动子区CpG岛甲基化在多发性骨髓瘤(MM)患者发病中的作用以及三氧化二砷(As2O3)诱导p16基因的去甲基化作用。方法采用巢式甲基特异性PCR法检测MM患者以及利用As2O3作用前后人MM细胞株U266的p16基因的甲基化状态,RTPCR检测U266细胞用药前后p16基因mRNA的表达变化,生长曲线、MTT法检测As2O3对细胞的生长和增殖抑制。利用流式细胞仪检测DNA含量分析法探讨As2O3对骨髓瘤细胞系U266周期的影响。结果MM患者p16基因的甲基化比例为54.8%,U266细胞存在p16基因甲基化,p16基因不表达,As2O3作用后p16基因甲基化程度明显减弱至消失,与未处理组相比,不同剂量作用72h后p16基因表达阳性条带灰度值与βactin比值分别为0.22±0.10、0.59±0.11、0.68±0.09,阳性对照灰度比值为0.77±0.13,其差异有统计学意义(P<0.01)。结论p16基因甲基化在MM患者中较为常见,这可能为MM的诊断和治疗提供借鉴;As2O3可诱导p16基因去甲基化,使p16基因表达上调,恢复其活性,为去甲基化治疗提供新思路。

Hypermethylation of CpG island of p16 gene and arsenic trioxide induced p16 gene demethylation in multiple myeloma

OBJECTIVE: To investigate the role of hypermethylation of p16 gene in the pathogenesis of multiple myeloma (MM) and the effect of arsenic trioxide (As2O3) induced p16 gene demethylation. METHODS: Methylation status of p16 gene in MM and U266 cell line exposed to As2O3 were detected the nested-methylation specific PCR. The expression of p16 gene mRNA was determined with RT-PCR. The induced growth inhibition of U266 cell by growth curve and MTT and the DNA content of U266 cell were analyzed with flow cytometry after exposure to As2O3. RESULTS: Hypermethylation of CpG island of p16 gene was observed in 54.8% of the MM patients in our group. p16 gene fail to express in U266 cell line after methylation. As compared with beta-actin, the expression of p16 gene mRNA in U266 cell was increased to 0.22 +/- 0.10, 0.59 +/- 0.11, 0.68 +/- 0.09 after exposure to 0.5 micromol/L, 1.0 micromol/L and 2.0 micromol/L As2O3 for 72 h. CONCLUSIONS: These results indicate that methylation of p16 gene is essential important in the pathogenesis of MM and may provide a new diagnostic technique and drug target for the treatment of MM. As2O3 may activate the expression of p16 gene by demethylation.