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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重新表达作用,并进一步探讨其作用机制。应用甲基化特异性聚合酶链反应(MSP)检测PHI作用前后Molt-4细胞株p15基因甲基化状态的变化;半定量逆转录-聚合酶链反应(RT-PCR)检测Molt-4细胞经过PHI处理后DNA甲基转移酶DNMT1、DNMT3A、DNMT3B、p15基因的mRNA的表达变化;用蛋白免疫印迹(Western blotting)检测Molt-4细胞经过PHI处理后的P15蛋白的表达。结果显示,PHI作用于Molt-4细胞5d后,p15基因的甲基化程度减弱,p15基因的异常高甲基化现象被逆转,沉默的p15基因重新表达,p15mRNA、P15蛋白表达增加,并呈浓度依赖性;PHI可下调DNMT1和DNMT3B的mRNA表达(P<0.05),而对DNMT3A的mRNA表达作用不明显(P>0.05)。PHI可能通过抑制DNA甲基转移酶DNMT1和DNMT3B的活性,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发生变化,增加转录因子的进入,从而诱导p15基因重新表达。
To study the demethylation of p15 gene and the re-expression of silencing gene in Molt-4 cells of acute T lymphocytic leukemia with phenylhexyl isothiocyanate (PHI), a novel histone deacetylase inhibitor, and to further explore Its mechanism of action. The methylation status of p15 gene in Molt-4 cells before and after PHI treatment was detected by methylation-specific polymerase chain reaction (MSP). Molt-4 cells were detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) The mRNA expression of DNA methyltransferase DNMT1, DNMT3A, DNMT3B and p15 after PHI treatment was detected by Western blotting. The PHI-treated P15 protein expression in Molt-4 cells was detected by Western blotting. The results showed that the methylation of p15 gene was weakened after PHI treatment on Molt-4 cells for 5 days. The hypermethylation of p15 gene was reversed. The silencing of p15 gene was re-expressed. The expression of p15 mRNA and P15 protein was increased in a concentration-dependent manner PHI could down-regulate the mRNA expression of DNMT1 and DNMT3B (P <0.05), but had no effect on DNMT3A mRNA expression (P> 0.05). PHI may induce demethylation of p15 gene by inhibiting the activity of DNA methyltransferases DNMT1 and DNMT3B, or (or) may alter the spatial structure of chromosome by increasing the level of acetylation of histone near the p15 gene, increase the transcription Factor entry, thereby inducing p15 gene re-expression.