镉对肝细胞中PPP2R1A启动子区甲基化及其转录水平的影响  

Effects of cadmium on promoter methylation and transcriptional level of PPP2R1A gene in hepatocytes

作　　者：张玉静[1] 陈慧峰[2] 廖昆[1] 夏斌[1] 殷花[1] 何承勇[3] 林育纯[3] 林忠宁[3] 

机构地区：[1]广东省营养膳食与健康重点实验室,中山大学公共卫生学院,广东广州510080 [2]广东省职业病防治院,广东省职业病防治重点实验室,510300 [3]厦门大学公共卫生学院,福建厦门361102

出　　处：《中国职业医学》2014年第5期496-501,共6页China Occupational Medicine

基　　金：国家自然科学基金(81172705;81072334;81130052);广东省自然科学基金重点项目(S2011020002769)

摘　　要：目的：分析镉染毒处理肝细胞中蛋白磷酸酶2A（ PP2A）-Aα支架亚基基因PPP2R1A启动子区甲基化状态及其转录水平的改变。方法采用永生化人正常肝L02细胞及肝细胞癌HepG2细胞为研究对象，对其进行以下分组和处理：①低、中和高剂量氯化镉（ CdCl2）处理组，分别予浓度为20.0、40.0和60.0 μmol/L CdCl2处理24 h；②低、中和高剂量5-氮杂-2’-脱氧胞苷（5-Aza-dC）处理组，分别予浓度为2.5、5.0和10.0 μmol/L 5-Aza-dC处理48 h；③5-Aza-dC组予浓度为5.0 μmol/L的5-Aza-dC处理48 h，CdCl2组予浓度为40.0 μmol/L 的 CdCl2处理24 h，（5-Aza-dC＋CdCl2）组予浓度为5.0 μmol/L的5-Aza-dC预处理48 h后再予浓度为40.0 μmol/L CdCl2处理24 h；④ CdCl2处理组予浓度为40.0 μmol/L CdCl2处理24 h。上述4种分组均设对照组，予等体积生理氯化钠溶液或二甲基亚砜处理。经①~③处理后的细胞采用实时荧光定量聚合酶链反应（ PCR）检测PPP2R1A、金属硫蛋白1B（MT1B）和DNA甲基转移酶3A（DNMT3A）的mRNA转录水平（以对照组水平为1.00）。经④处理后的细胞采用亚硫酸氢盐修饰后PCR扩增PPP2R1A启动子区克隆测序检测CpG岛的甲基化情况。结果 L02细胞和HepG2细胞中，不同剂量CdCl2处理组PPP2R1A mRNA转录水平随镉处理剂量增高呈剂量依赖性下降（ P〈0.05）；不同剂量5-Aza-dC处理组PPP2R1A mRNA转录水平随5-Aza-dC处理剂量增加呈剂量依赖性升高（P〈0.05）。2种细胞中，分别与对照组和5-Aza-dC处理组比较，CdCl2处理组和（5-Aza-dC＋CdCl2）处理组PPP2R1A mRNA转录水平均下降（P〈0.05），MT1B和DNMT3A的mRNA转录水平均升高（P〈0.05）；与CdCl2处理组比较，（5-Aza-dC＋CdCl2）处理组PPP2R1A mRNA转录水平均升高（P〈0.05），MT1B和DNMT3A的mRNA转录水平均下降（P〈0.05）。甲基化测序结果显示，L02细胞 CdCl2处理组 PPP2R1A 启动子区甲基化率高于对照组（6.35％ vs 2.31�Objective To analyze the effects of cadmium on the promoter methylation and transcriptional level of protein phosphatase 2A（PP2A）-Aα supported subunit gene PPP2R1A gene in hepatocytes. Methods The immortalized human fetal liver cell line L02 and the hepatocellular carcinoma cell line HepG2 were selected as the research objects：① Cells were treated with low-,medium-and high-dose（20. 0,40. 0 and 60. 0 μmol/L）cadmium chlorid（CdCl2）for 24 h. ②Cells were treated with low-,medium- and high-dose（2. 5,5. 0 and 10. 0 μmol/L）5-aza-2＆#39;-deoxycytidine（5-Aza-dC） for 48 h. ③Cells were given 5. 0 μmol/L for 48 h in 5-Aza-dC group,cells were exposed to 40. 0 μmol/L CdCl2 for 24 h in CdCl2 group and cells were exposed to 40. 0 μmol/L CdCl2 for 24 h after 48 h pretreatment of 5. 0 μmol/L 5-Aza-dC in （5-Aza-dC＋CdCl2 ）group. ④ Cells were treated with 40. 0 μmol/L CdCl2 for 24 h in CdCl2 group. The above groups were all given the controls with the same volumes of physiological sodium chloride solution or dimethyl sulfoxide. Real-time fluorescent quantitative polymerase chain reaction（ PCR）detection was used to detect the mRNA transcriptional levels of＆amp;nbsp;PPP2R1A,Metallothionein 1B（MT1B）,DNA methyltransferase 3A（DNMT3A）after treatments ①-③. After treatment④,cloning sequencing was used to detect the CpG island methylation status of PPP2R1A promoter after bisulfite sequen-cing PCR. Results In L02 and HepG2 cells,the transcriptional levels of PPP2R1A mRNA in CdCl2 group were decreased in a dose-dependent manner（P〈0. 05）. The transcriptional Levels of PPP2R1A mRNA in 5-Aza-dC group were increased in a dose-dependent manner（P〈0. 05）. In the two kinds of cells,compared with the control or 5-Aza-dC group,transcrip-tional levels of PPP2R1A mRNA in CdCl2 group and（5-Aza-dC＋CdCl2 ）group were decreased significantly respectively （P〈0. 05）,while transcriptional levels of MT1B and DNMT3A mRNA in the 2 groups were increased significantly respec-tively�

关 键 词：镉 L02细胞 HEPG2细胞 PPP2R1A 金属硫蛋白1B DNA甲基转移酶3A 启动子区甲基化 

分 类 号：R114[医药卫生—卫生毒理学] R735.7[医药卫生—公共卫生与预防医学]