5-Aza-2'-deoxycytidine inhibits retinoblastoma cell by reactivating epigenetically silenced RASSF1A gene

• 作者: Di-Xian Luo Kun Zhang Ling Gao Ru Liu Wei Li Wen-Jun Wang Xiao-Huan Zhang
• 来源期刊: 国际眼科杂志：英文版
• 学科: 医学
• 关键词: 5-Aza-2’-deoxycytidine RETINOBLASTOMA METHYLATION apoptosis RAS association domain family
• 年，卷（期）: 2014,（1）
• 页码范围: 51-56
• 页数: 6页
• 分类号: R739.7

摘要

AIM: 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. ·METHODS: 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. ·RESULTS: 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 HXORB44 cells on day four,and as a result,RASSF1A expression increased significantly from day 4 to day 7.·CONCLUSION: 5-Aza-CdR inhibits the growth of the HXO-RB44 RB cell line and induces apoptosis by demethylating the RASSF1A gene.