亚砷酸诱导人鼻咽癌细胞Ras相关结构域家族1A基因去甲基化表达的研究

摘要

目的 研究亚砷酸诱导鼻咽癌CNE-2Z细胞株中抑癌基因Ras相关结构域家族1A基因(RAS association domain family gene 1A,RASSFIA)的表达.方法 体外培养的CNE-2Z细胞分别加入不同浓度的亚砷酸,并作用不同时间.应用台盼蓝染色法筛选出亚砷酸抑制CNE-2Z细胞生长的最佳浓度,检测IC_(50)值;流式细胞术检测细胞周期的改变;用终浓度为2 μmol/L、1 μmol/L、0.5 μmol/L的亚砷酸加入鼻咽癌CNE-2Z细胞系和不加药物的空白对照组,作用48 h后,采用甲基化特异性PCR法检测亚砷酸处理前后RASSF1A基因去甲基化的情况,采用反转录PCR检测鼻咽癌CNE-2Z细胞系中RASSFIA基因亚砷酸处理前后mRNA水平的表达情况,采用Western blot方法 检测亚砷酸处理前后RASSF1A基因蛋白质水平的表达情况.结果 亚砷酸对细胞增殖的抑制作用具有明显的时间和剂量效应.不同浓度的亚砷酸作用细胞24 h、48 h、72 h后,其IC_(50)值分别为(1.50±0.05)、(1.09±0.13)、(0.65±0.04)μmol/L,亚砷酸使细胞周期阻滞于S期和G2/M期;RASSF1A经亚砷酸作用后甲基化随着作用浓度的增高逐渐减弱,而去甲基化随着作用浓度的增高逐渐增强,且亚砷酸作用后RASSF1A在mRNA水平和蛋白质水平表达明显增强.空白对照组和不同浓度亚砷酸作用CNE-2Z细胞的处理组之间差异均有统计学意义(P值均<0.05),不同浓度亚砷酸作用CNE-2Z细胞的处理组之间差异均有统计学意义(P值均<0.05).结论 亚砷酸可诱导鼻咽癌CNE-2Z细胞株中RASSF1A表达增强,从而阻滞鼻咽癌细胞的增殖分化.%Objective To investigate the effect of arsenic trioxide (As_2O_3) on expression of anti-oncogene RAS association domain family gene 1A(RASSF1A) in nasopharyngeal carcinoma cell line CNE-2Z. Methods CNE-2Z cells were treated with various concentrations of As_2O_3 for different times. The IC_(50) values were detected by trypan blue stain assay. Cell cycle redistribution was analyzed by flow cytometry. The final concentration 2 μmol/L,1 μmol/L,0. 5 μmol/L of As_2O_3 was added to CNE-2Z cell for succedent experiments. The controls and no drugs of CNE-2Z cells were cultivated for 48 h. Methylation specific PCR was used to detect the change of methylation status of RASSF1A gene. The expression of RASSF1A gene was detected by reverse transcription PCR and Western blot at mRNA and protein level. Results The suppression of cell proliferation by As_2O_3 was time and dose-dependent. After being treated with As_2O_3,the IC_(50) values of As_2O_3 were (1.50±0.05), (1.09±0.13), (0.65±0.04) μmol/L at 24, 48, and 72 h, respectively. As_2O_3 also arrested CNE-2Z cells in G2/M phase of cell cycle. After the effect of As_2O_3, the methylation of RASSF1A gene became weaker by increasing the concentration of As_2O_3; and the expression of RASSF1A gene became stronger at mRNA and protein level. Between different concentration of As_2O_3 group and no drugs group, the differences had statistical significance (P<0.05). Along with increasing the concentration of As_2O_3, the expression of RASSF1A gene became stronger at mRNA and protein level, the methylation of RASSF1A gene became weaker and weaker. Conclusions As_2O_3 can activate the expression of RASSF1A gene to inhibit the cell cyc]e progress of uasopharyngeal carcinoma cell line.