摘要
本研究旨在探讨三氧化二砷(As2O3)逆转恶性淋巴瘤CA46细胞株p16基因甲基化状态和调节转录作用及其可能的机制。以高甲基化的恶性淋巴瘤细胞株CA46作为研究基因甲基化与表达关系的实验对象。采用SRB法检测As2O3对人恶性淋巴瘤CA46细胞系增殖、活力的影响;nMSP法检测药物作用后p16甲基化状态变化;RT-PCR法检测p16、甲基转移酶DNMT1、DNMT3A、DNMT3B mRNA的表达;利用流式细胞术DNA倍体分析法探讨As2O3对恶性淋巴瘤细胞周期的影响。结果表明:①As2O3作用CA46细胞株72小时后p16基因甲基化程度明显减弱,p16基因异常甲基化的现象被逆转;②未处理组细胞p16基因呈微弱表达,As2O3作用72小时后p16基因表达增强,0.5μmol/L组、1.0μmol/L组和2.0μmol/L组p16基因表达阳性条带与β-actin灰度的比值分别为(0.33±0.10)、(0.57±0.11)、(0.67±0.09),阳性对照灰度比值为(0.73±0.13),差异有统计学意义(p<0.01);③与未处理组相比,As2O3作用72小时后甲基转移酶DNMT3A、DNMT3B的表达下降并呈浓度依赖性,而DNMT1表达不受影响;④与对照组相比,3组不同浓度As2O3均能明显抑制肿瘤细胞生长,G0/G1期细胞增加。结论:As2O3可能通过抑制甲基转移酶DNMT3A、DNMT3B或(和)直接逆转p16基因甲基化状态,使p16基因表达上调,并恢复其活性,从而实现其细胞周期调控功能,将细胞阻滞于G0/G1期,抑制肿瘤细胞的增长。
This study was aimed to investigate the reversing effect of arsenic trioxide （As2O3 ） on methylation status and the regulatory effect on transcription of malignant lymphoma cell line CA46 pl6 gene as well as their possibe mechanisms. The hypermethylated malignant lymphoma cell fine CA46 was used as a subject of experiment for studying relation of gene methylation with expression. The effect of As2O3 on the proliferation and viability of CA46 was detected by SRB method, the change of pl6 methylation status after exposure to As2O3 was determined by nMSP, the expressions of p16, DNMT1, DNMT3A, DNMT3B mRNA were assayed by RT-PCR,the influence of As203 on CA46 cell cycle was analyzed by flow cytometry using analytical method for DNA ploidy. The results showed that the methylation level of p16 gene was obviously reduced after treatment with As203 for 72 hours and the hypermethylation of p16 gene was successfully reversed; the expression of p16 gene in untreated （control） group was low while it was enhanced in treated groups; the gray scale ratios of pl6 gene to β-actin in groups treated withAs2O3 of concentration 0. 5, 1.0 and 2.0 μmol/L were 0.33 ＋0.10, 0.57 ＋0.11 and 0.67 ＋0.09 respectively, exhibiting a significant difference in comparison with 0.73 ＋0.13 of positive control （p 〈0.01 ） ; as compared with the untreated group, the expression of DNM＇IBA and DNMT3B in treated groups was obviously down-regulated in a concentration-dependent manner, while expression of DNMT1 was nearly unchanged; as compared with control, all the 3 different concentrations of As2O3 could inhibit the proliferation of CA46 cells and increase the cell number in G0/G1 phase. It is concluded that the As2O3 may up-regulate the expression of pl6 gene, recover the activity of pl6 gene, thereby promote the regulatory function on cell cycle resulring in arrest of cells in G0/G1 phase and inhibit growth of tumor cells through depressing the expression of DNMT3A and DNMT3B and/or directly reversing the methylation status of
出处
《中国实验血液学杂志》
CAS
CSCD
2010年第2期403-409,共7页
Journal of Experimental Hematology
基金
福建省百千万人才工程基金资助项目
编号303052801
福建省自然科学基金资助项目
编号CO540014
福建医科大学教授基金资助项目
编号JS06080
