PHI诱导急性T淋巴细胞性白血病Molt-4细胞p15基因去甲基化的实验研究
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摘要
【目的】研究新型组蛋白去乙酰化酶抑制剂异硫氰酸苯己酯(PHI)对急性T淋巴细胞性白血病Molt-4细胞p15基因的去甲基化作用及诱导沉默基因从新(de novo)表达作用,并进一步探讨其去甲基化作用的机制,为PHI作为一种新型抗肿瘤药物提供理论依据。
【方法】采用甲基化特异性聚合酶链反应(MSP)法检测PHI作用前后Molt-4细胞株甲基化状态的变化情况,并对PCR产物进行基因测序鉴定,以经典的DNA甲基化抑制剂5-杂氮胞苷(5-Aza)和经典的组蛋白去乙酰化酶抑制剂古曲抑菌素(TSA)为对照;半定量逆转录-聚合酶链反应(RT-PCR)检测Molt-4细胞经过不同浓度PHI处理后DNA甲基转移酶1(DNMT1)、3A(DNMT3A)、3B(DNMT3B)、p15基因的mRNA的表达变化;用蛋白免疫印迹法(Western Blotting)检测Molt-4细胞经过不同浓度PHI处理后的P15蛋白的表达变化等。【结果】(1)甲基化特异性聚合酶链反应检测提示Molt-4细胞p15基
因发生高甲基化而失活。不同浓度PHI作用于Molt-4细胞5天后, PHI各组p15基因的甲基化程度减弱,p15基因的异常甲基化现象被逆转,沉默的p15基因从新表达,并呈明显的浓度依赖性。
(2)不同浓度PHI处理5天后,与空白对照组相比,各组的DNMT1和DNMT3B的mRNA表达下降(p<0.05);DNMT3A的mRNA表达则无明显变化(p>0.05)。
(3)PHI处理Molt-4细胞5天后,与空白对照组相比,各组的P15蛋白表达增加,且随着药物浓度的增加,其表达增强。
【结论】(1)新型组蛋白去乙酰化酶抑制剂PHI有DNA去甲基化的作用,能诱导沉默的p15基因从新表达。
(2)PHI可能是通过降低DNA甲基转移酶DNMT1和DNMT3B的活性从而诱导p15基因产生去甲基化,或者(和)是通过改变p15基因附近组蛋白的乙酰化水平,导致染色体空间结构的变化,诱导p15基因产生去甲基化。
Objects To investigate the effect of Phenylhexyle Isothiocyanate (PHI) on demethylation and activation transcription of p15 in acute leukemia cell line Molt-4, which has been proved a novel HDACi recently,and to further study its potential mechanism of demethylation.
Methods DNA sequencing and Modified Methylation Specific PCR (MSP) were used to screen p15-M and p15-U mRNA after Molt-4 cells had been treated with PHI, 5-Aza and TSA. DNA methyltransferase 1(DNMT1), 3A (DNMT3A), 3B (DNMT3B) and p15 mRNA were measured by RT-PCR. P15 protein was detected by Western Blotting.
Results The gene p15 in Molt-4 was hypermethylated and gene silence. Hypermethylation of gene p15 was apparently attenuated and p15 gene was de novo activation after 5days exposure to PHI in a concentration dependent manner. DNMT1 and DNMT3B were inhibited by exposure to PHI for 5 days (P<0.05). Alteration of DNMT3A was not significant at those concentrations.
Conclusions We have demonstrated that PHI could active demethylation and activation transcription. Hypermethylation of gene p15 was reversed and activation transcription could be de novo by PHI in Molt-4. It may result from down-regulating DNA methyltransferases, DNMT1 and DNMT3B, or up-regulating the histone acetylation that allow chromatin to unfold and the accessibility of regulators in the p15 promoter for transcription activation.
【方法】采用甲基化特异性聚合酶链反应(MSP)法检测PHI作用前后Molt-4细胞株甲基化状态的变化情况,并对PCR产物进行基因测序鉴定,以经典的DNA甲基化抑制剂5-杂氮胞苷(5-Aza)和经典的组蛋白去乙酰化酶抑制剂古曲抑菌素(TSA)为对照;半定量逆转录-聚合酶链反应(RT-PCR)检测Molt-4细胞经过不同浓度PHI处理后DNA甲基转移酶1(DNMT1)、3A(DNMT3A)、3B(DNMT3B)、p15基因的mRNA的表达变化;用蛋白免疫印迹法(Western Blotting)检测Molt-4细胞经过不同浓度PHI处理后的P15蛋白的表达变化等。【结果】(1)甲基化特异性聚合酶链反应检测提示Molt-4细胞p15基
因发生高甲基化而失活。不同浓度PHI作用于Molt-4细胞5天后, PHI各组p15基因的甲基化程度减弱,p15基因的异常甲基化现象被逆转,沉默的p15基因从新表达,并呈明显的浓度依赖性。
(2)不同浓度PHI处理5天后,与空白对照组相比,各组的DNMT1和DNMT3B的mRNA表达下降(p<0.05);DNMT3A的mRNA表达则无明显变化(p>0.05)。
(3)PHI处理Molt-4细胞5天后,与空白对照组相比,各组的P15蛋白表达增加,且随着药物浓度的增加,其表达增强。
【结论】(1)新型组蛋白去乙酰化酶抑制剂PHI有DNA去甲基化的作用,能诱导沉默的p15基因从新表达。
(2)PHI可能是通过降低DNA甲基转移酶DNMT1和DNMT3B的活性从而诱导p15基因产生去甲基化,或者(和)是通过改变p15基因附近组蛋白的乙酰化水平,导致染色体空间结构的变化,诱导p15基因产生去甲基化。
Objects To investigate the effect of Phenylhexyle Isothiocyanate (PHI) on demethylation and activation transcription of p15 in acute leukemia cell line Molt-4, which has been proved a novel HDACi recently,and to further study its potential mechanism of demethylation.
Methods DNA sequencing and Modified Methylation Specific PCR (MSP) were used to screen p15-M and p15-U mRNA after Molt-4 cells had been treated with PHI, 5-Aza and TSA. DNA methyltransferase 1(DNMT1), 3A (DNMT3A), 3B (DNMT3B) and p15 mRNA were measured by RT-PCR. P15 protein was detected by Western Blotting.
Results The gene p15 in Molt-4 was hypermethylated and gene silence. Hypermethylation of gene p15 was apparently attenuated and p15 gene was de novo activation after 5days exposure to PHI in a concentration dependent manner. DNMT1 and DNMT3B were inhibited by exposure to PHI for 5 days (P<0.05). Alteration of DNMT3A was not significant at those concentrations.
Conclusions We have demonstrated that PHI could active demethylation and activation transcription. Hypermethylation of gene p15 was reversed and activation transcription could be de novo by PHI in Molt-4. It may result from down-regulating DNA methyltransferases, DNMT1 and DNMT3B, or up-regulating the histone acetylation that allow chromatin to unfold and the accessibility of regulators in the p15 promoter for transcription activation.
引文
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