Liu Ru, Zhang Xiao-Huan, Zhang Kun, Li Wei, Wang Wen-Jun, Luo Di-Xian, Gao Ling
Department of Ophthalmology, the Second Xiangya Hospital Central South University, Changsha 410011, Hunan Province, China ; Department of Ophthalmology, the First People's Hospital of Chenzhou City, Chenzhou 423000, Hunan Province, China ; Institute of Translational Medicine, the First People's Hospital of Chenzhou City, Chenzhou 423000, Hunan Province, China.
Department of Ophthalmology, the Second Xiangya Hospital Central South University, Changsha 410011, Hunan Province, China.
Int J Ophthalmol. 2014 Feb 18;7(1):51-6. doi: 10.3980/j.issn.2222-3959.2014.01.09. eCollection 2014.
To investigate the effect of 5-Aza-2'-deoxycytidine (5-Aza-CdR), a DNA methyltransferase (DNMT) inhibitor, on the growth and survival of the Chinese retinoblastoma (RB) cell line HXO-RB44.
The DNA methylation status of the Ras association domain family (RASSF1A) promoter in the presence of 5-Aza-CdR at different concentrations was analyzed by methylation-specific polymerase chain reaction (MSP). RASSF1A mRNA and protein levels were measured by semiquantitative RT-PCR and immunohistochemistry staining, respectively, when cells were treated with 5.0µmol/L of 5-Aza-CdR. The effect of 5.0µmol/L 5-Aza-CdR on the proliferation and viability of HXO-RB44 cells was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry.
5-Aza-CdR efficiently induced cell cycle arrest at G0/G1 and apoptotic death in HXO-RB44 cells. MSP analysis showed that unmethylated RASSF1A DNA increased and methylated RASSF1A decreased in a dose-dependent manner in a range of 0.5-5.0µmol/L 5-Aza-CdR. Accordingly, RASSF1A expression was reactivated at both mRNA and protein levels. Incubation time of 5-Aza-CdR treatment also functioned as a factor for the demethylation status of RASSF1A promoter DNA, with a plateau on day four. 5-Aza-CdR at 5.0µmol/L completely demethylated the RASSF1A promoter in HXO-RB44 cells on day four, and as a result, RASSF1A expression increased significantly from day 4 to day 7.
5-Aza-CdR inhibits the growth of the HXO-RB44 RB cell line and induces apoptosis by demethylating the RASSF1A gene.
研究DNA甲基转移酶（DNMT）抑制剂5-氮杂-2'-脱氧胞苷（5-Aza-CdR）对人视网膜母细胞瘤（RB）细胞系HXO-RB44生长和存活的影响。
采用甲基化特异性聚合酶链反应（MSP）分析不同浓度5-Aza-CdR作用下Ras关联结构域家族（RASSF1A）启动子的DNA甲基化状态。当细胞用5.0μmol/L的5-Aza-CdR处理时，分别用半定量逆转录-聚合酶链反应（RT-PCR）和免疫组织化学染色检测RASSF1A mRNA和蛋白水平。采用3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）法和流式细胞术检测5.0μmol/L 5-Aza-CdR对HXO-RB44细胞增殖和活力的影响。
5-Aza-CdR可有效诱导HXO-RB44细胞在G0/G1期发生细胞周期阻滞并诱导凋亡。MSP分析显示，在0.5-5.0μmol/L 5-Aza-CdR范围内，未甲基化的RASSF1A DNA呈剂量依赖性增加，甲基化的RASSF1A呈剂量依赖性减少。相应地，RASSF1A在mRNA和蛋白水平均被重新激活。5-Aza-CdR处理的孵育时间也是RASSF1A启动子DNA去甲基化状态的一个影响因素，在第4天达到平台期。5.0μmol/L的5-Aza-CdR在第4天可使HXO-RB44细胞中的RASSF1A启动子完全去甲基化，结果，从第4天到第7天RASSF1A表达显著增加。
5-Aza-CdR通过使RASSF1A基因去甲基化抑制HXO-RB44 RB细胞系的生长并诱导凋亡。
