亚砷酸钠对HaCaT细胞MGMT基因甲基化和mRNA及蛋白表达水平的影响  

Effects of sodium arsenite on hypermethylation, transcription and expression of O6-met hylguanine-DNA methyltransferase gene in HaCaT cells

作　　者：潘雪莉[1] 张爱华[1] 

机构地区：[1]贵阳医学院卫生毒理学教研室,550004

出　　处：《中国地方病学杂志》2011年第3期273-278,共6页Chinese Jouranl of Endemiology

基　　金：基金项目：国家自然科学基金（30960337、30760225）;贵州省重大专项基金（黔科合重大专项字[2006]6016号）;贵州省科学技术基金（黔科合J字[200912188号）;贵阳医学院研究生教育创新基地建设专项经费（B200902）;贵州省卫生厅科学技术基金（gzwkj2008-1-002）

摘　　要：目的 了解不同剂量亚砷酸钠（NaAsO2）处理的人皮肤角质形成细胞（HaCaT细胞）中MGMT基因甲基化特征,以及MGMT基因甲基化对其转录和表达的调控.方法 以3.13、6.25、12.50、25.00 μmol/L NaAsO2重复间隔处理HaCaT细胞72 h.硫化测序PCR（BSP）检测MGMT基因转录起始点-329～＋93甲基化热点区域,实时荧光定量PCR（Q-PCR）及免疫印迹分析（Westem blotting）检测MGMT基因的mRNA及蛋白表达.设不染毒的HaCaT细胞为空白对照,人表皮鳞癌细胞株A431为阳性对照.结果 3.13、6.25、12.50、25.00μmol/L NaAsO2处理组,MGMT基因启动子区DNA甲基化水平分别为0.63%（1/160）、6.25%（10/160）、10.63%（17/160）和18.75%（30/160）.且甲基化的CpG位点主要位于转录起始点-249～-146区域,空白对照组未检出甲基化,组间比较差异有统计学意义（x2=76.687,P〈0.05）;3.13、6.25、12.50、25.00 μmol/L NaAsO2处理组MGMT mRNA相对表达量分别为1.518 31±0.180 54、1.425 22±0.180 39、1.014 54 ±0.096 79、0.887 72 ±0.020 00,与空白对照组（1.198 29±0.159 97）比较,差异有统计学意义（F=37.359,P〈0.05）;3.13、6.25、12.50、25.00 μmol/L NaAsO2处理组MGMT蛋白相对表达量分别为1.174 47±0.064 75、0.848 83 ±0.057 01、0.471 63±0.023 34、0.240 34±0.014 43,与空白对照组（1.066 19±0.061 24）比较,差异有统计学意义（F=20.687,P〈0.05）.结论 砷通过导致MGMT基因启动子区CpG岛高甲基化.抑制MGMT基因mRNA转录及蛋白表达,是砷致皮肤损害的重要机制之一.Objective To investigate the DNA methylation feature and DNA methylation regulation to its transcription and expression of O6-methylguanine-DNA methyltransferase gene （MGMT） in NaAsO2-treated HaCaT cells. Methods HaCaT cells were treated 72 hours at intervals and repeatedly by 3.13, 6.25,12.50, and 25.00 μmol/L NaAsO2, MGMT gene promoter region was amplified in the transcription initiation site - 329 - ＋ 93 region by bisulfate-sequencing polymerase chain reaction （BSP）, the mRNA transcription and the protein expression of MGMT was detected by real-time quantitative PCR and Western blotting. NaAsO2-untreated HaCaT cell was set as a blank control, and human epidermal squamous carcinoma cell strain A431 was set as a positive control. Results Among the groups of HaCaT cells treated with 3.13, 6.25, 12.50 and 25.00 μmol/L NaAsO2, the positive rates of the DNA methylation of promoter region in MGMT gene were 0.63%（l/160）, 6.25% （10/160）, 10.63%（ 17/160） and 18.75% （30/160）, respectively, and methylated CpG sites were mainly located in - 249--146 region relative to transcription start site. There was no DNA methylation in the blank control. There were significant differences between the blank control and the NaAsO2-treated cells （x2 = 76.687, P〈 0.05）. Average levels of MGMT mRNA were 1.518 31 ± 0.180 54, 1.425 22 ± 0.180 39, 1.014 54 ± 0.096 79 and 0.887 72 ± 0.020 00, respectively among the groups of HaCaT cells treated with 3.13, 6.25, 12.50 and 25.00 μmol/L NaAsO2, compared with the blank control cells（1.198 29 ± 0.159 97）, there were significant differences（F = 37.359, P 〈 0.05）. Average levels of MGMT protein were 1.174 47 ± 0.064 75, 0.848 83 ± 0.057 01, 0.471 63 ± 0.023 34 and 0.240 34 ± 0.014 43, respectively among the groups of HaCaT cells treated with 3.13, 6.25, 12.50 and 25.00 μmol/L NaAsO2, compared with the blank control cells （1.066 19 ± 0.061 24）, there were significant differences（F = 20.687, P 〈 0.05）. Conclusions Arsenic can cause CpC

关 键 词：砷 DNA甲基化 转录 基因 蛋白质类 O6-甲基鸟嘌呤-DNA甲基转移酶基因 

分 类 号：R1[医药卫生—公共卫生与预防医学]