恶性血液病患者mdr1基因启动子区甲基化状态及其与表达的关系

Methylation Status of Multidrug Resistance (mdr1) Gene and Its Correlation with Expression of mdr1 Gene in Patients with Hematologic Malignancies

为了探讨多药耐药 (mdr1)基因启动子区的甲基化状态及其与表达的关系 ,采用甲基化敏感的HpaⅡ酶切结合竞争性定量PCR检测 5 4例急性白血病 (AL)和 9例多发性骨髓瘤 (MM)患者mdr1基因启动子区位于转录起始点上游 - 110和 - 5 0bp处两个CCGG位点 (I区和Ⅱ区 )的甲基化率 ;半定量RT PCR检测mdr1基因的表达水平。结果显示I区、Ⅱ区及总甲基化率均与表达呈负相关 ,I区比Ⅱ区相关密切 (r分别为 - 0 .6 4和 - 0 .4 0 )。低甲基化组(n=36 )mdr1高表达率显著增高 (P <0 .0 0 1)。与化疗敏感组 (n =8)相比 ,耐药组 (n=16 )I区 (P =0 0 5 )、Ⅱ区 (P <0 .0 5 )和总甲基化率 (P <0 .0 5 )均减低。与未化疗组 (n =36 )相比 ,化疗后组 (n =14 )I区和总甲基化率均减低 (P <0 .0 5 ) ,Ⅱ区甲基化率也减低 ,但无统计学意义 (P >0 .0 5 )。化疗后组随甲基化率减低 ,mdr1表达升高 ,但尚无统计学意义 (P =0 .0 6 )。结论 :AL和MM患者骨髓mdr1基因表达水平与基因转录起始点上游 - 110和- 5 0处两个CCGG位点的甲基化程度呈负相关 ,且与 - 110处关系更密切。化疗后及耐药病人mdr1基因甲基化水平相对降低。化疗后mdr1基因表达增高与其甲基化水平降低有关。

To investigate the correlation between methylation and expression of multidrug resistance (mdr1) gene,restriction endonuclease HpaⅡ combined with competitive PCR technique was used to quantitatively detect the methylation status of two CCGG sites located at -110 and -50 bp(region I and Ⅱ) up to the transcription start site in mdr1 promoter in 54 AL and 9 MM patients. Semi-quantitative RT-PCR was used to detect the expression level of mdr1 gene. The results showed that inverse correlation between methylation rate of either region or total methylation rate and expression of mdr1 gene was observed. The correlation in the region I ( r =-0.64) was closer than that in the region Ⅱ ( r =-0.4). High expression rate of mdr1 ascended significantly in low methylation group ( n =36) ( P <0.001). In comparison with chemotherapy sensitive group ( n =8), the methylation rate in refractory AL patients ( n =16) was lower ( P =0.05) in the region I, P <0.05 in the region Ⅱ and total regions. Comparing with the untreated patients ( n =36) , the methylation rate in the region I and total methylation rate were lower in the patients with chemotherapy (n=14) ( P <0.05). The methylation rate in the region Ⅱ was also decreased after chemotherapy, however, no statistical significance was shown ( P >0.05). Increased mdr1 expression level accompanying with decreased methylation rate after chemotherapy was found, although no significant difference was shown ( P =0.06). It is concluded that the expression level of mdr1 gene was associated with the methylation status of CCGG in -110 and -50 bp upstream to the transcription start site, especially the-110 site. In both the patients treated with chemotherapy and the refractory patients, the methylation level of mdr1 gene decreased relatively. The rising expression of mdr1 gene after chemotherapy was associated with the decrease of methylation level.