表没食子儿茶素没食子酸酯对Reh细胞增殖及RUNX3基因甲基化状态的影响

中国临床药理学与治疗学 ›› 2012, Vol. 17 ›› Issue (6): 609-615.

表没食子儿茶素没食子酸酯对Reh细胞增殖及RUNX3基因甲基化状态的影响

吴明彩¹, 韦中玲², 蒋明³, 方基勇¹, 毕富勇¹

¹皖南医学院生物化学教研室,芜湖 241002,安徽;
²皖南医学院弋矶山医院血液内科,芜湖 241001,安徽;
³二炮芜湖鸠江干休所卫生所,芜湖 241000,安徽

收稿日期:2012-04-25 修回日期:2012-05-05 出版日期:2012-06-26 发布日期:2012-06-25
通讯作者: 毕富勇,男,教授,硕士生导师,研究方向:肿瘤生化。Tel: 18900538928 E-mail: bifuyong@yahoo.com.cn
作者简介:吴明彩,女,硕士,讲师,研究方向:肿瘤生化。Tel: 13855333606 E-mail: williyia@wnmc.edu.cn
基金资助:
安徽高校省级自然科学研究项目(KJ2010B249)

Effects of Epigallocatechin-3-galate on growth inhibition of Reh cell line and methylation of RUNX3 gene

WU Ming-cai¹, WEI Zhong-ling², JIANG Ming³, FANG Ji-yong¹, BI Fu-yong¹

¹Department of Biochemistry, Wannan Medical College, Wuhu 241001, Anhui, China;
²Department of Hematology, Yijishan Hospital, Wannan Medical College, Wuhu 241001, Anhui, China;
³Cadres Sanatorium, Second Artillery of PLA, Wuhu 241000, Anhui, China

Received:2012-04-25 Revised:2012-05-05 Online:2012-06-26 Published:2012-06-25

摘要/Abstract

摘要： 目的: 观察表没食子儿茶素没食子酸酯(EGCG)对白血病细胞系Reh细胞增殖抑制作用及对RUNX3基因启动子区甲基化状态及表达的影响,探讨Reh细胞RUNX3基因的失活机制及EGCG对RUNX3基因表达的调控。方法: 体外培养Reh细胞,用5、10、15和 20 μg/mL EGCG与Reh细胞孵育,采用MTT法检测EGCG对Reh细胞增殖活性的影响; 采用流式细胞术检测EGCG对Reh细胞凋亡的影响;采用甲基化特异性PCR(MSP)检测RUNX3基因启动子区域的甲基化情况;采用RT-PCR、Western blot法分别检测RUNX3 mRNA、蛋白的表达。结果: EGCG对Reh细胞生长有明显的抑制作用,并有时间及剂量依赖性; EGCG可促进Reh细胞发生凋亡,随着EGCG浓度的增加,细胞凋亡率逐渐增加;MSP法结果显示EGCG处理后RUNX3基因启动子高甲基化的区域发生去甲基化,EGCG能够诱导Reh细胞RUNX3基因mRNA和蛋白的重新表达,具剂量依赖性。结论: EGCG可明显抑制Reh细胞的增殖, 并诱导Reh细胞中异常甲基化的RUNX3基因去甲基化,使RUNX3基因恢复表达, 这可能是其抗肿瘤的重要机制。

关键词: 表没食子儿茶素没食子酸酯, RUNX3, 甲基化

Abstract: AIM: To investigate the effects of epigallocatechin-3-gallate on the proliferation and the methylation status of Reh cells,and to explore the mechanism of the silencing of tumor suppressor gene RUNX3 and the regulating effect of EGCG on RUNX3 gene expression. METHODS: The cultured Reh cells were incubated with EGCG for different concentrations of 5, 10, 15 and 20 μg/mL, MTT assay was used to analyse the inhibition of cell proliferation by EGCG, cell apoptosis index was examined by flow cytometry, methylation-specific PCR (MSP) analysis was evaluated for the promoter region methylation status of the RUNX3 gene in Reh cell line. The mRNA and protein level of RUNX3 was detected by RT-PCR and western blot. RESULTS: EGCG of different concentrations could significantly inhibit proliferation and increase the apoptosis index of Reh cells in dose-and-time dependent manners. After treated with EGCG, the promoter region methylation status of the RUNX3 gene representing partial demethylation could be detected in Reh cells.RT-PCR and western blot also showed the mRNA level of RUNX3 was increased in dose-and-time dependent manner. CONCLUSION: EGCG could depress the proliferation rate of Reh. The possible mechanism might be its reversing the hypermethylation of RUNX3 gene and activiting the expression of RUNX3 gene.

Key words: Epigallocatechin-3-gallate, RUNX3, Methylation

中图分类号:

R965.2

吴明彩, 韦中玲, 蒋明, 方基勇, 毕富勇. 表没食子儿茶素没食子酸酯对Reh细胞增殖及RUNX3基因甲基化状态的影响[J]. 中国临床药理学与治疗学, 2012, 17(6): 609-615.

WU Ming-cai, WEI Zhong-ling, JIANG Ming, FANG Ji-yong, BI Fu-yong. Effects of Epigallocatechin-3-galate on growth inhibition of Reh cell line and methylation of RUNX3 gene[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2012, 17(6): 609-615.

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