中国地方病学杂志
中國地方病學雜誌
중국지방병학잡지
CHINESE JOURNAL OF ENDEMIOLOGY
2013年
1期
16-20
,共5页
砷中毒%基因,O(6)-甲基鸟嘌呤DNA转移酶%甲基-CpG-结合蛋白2%DNA(胞嘧啶-5-)-甲基转移酶%组蛋白脱乙酰基酶类
砷中毒%基因,O(6)-甲基鳥嘌呤DNA轉移酶%甲基-CpG-結閤蛋白2%DNA(胞嘧啶-5-)-甲基轉移酶%組蛋白脫乙酰基酶類
신중독%기인,O(6)-갑기조표령DNA전이매%갑기-CpG-결합단백2%DNA(포밀정-5-)-갑기전이매%조단백탈을선기매류
Arsenic poisoning%Genes,O(6)-methylguanine-DNA methyltransferase%Methyl-CpG-binding protein 2%DNA (Cytosine-5-)-methyltransferase%Histone deacetylases
目的 观察亚砷酸钠(NaAsO2)对人肤角质形成细胞株(HaCaT细胞)MGMT基因启动子区甲基化CpG结合蛋白-2(MeCP2)、DNA甲基转移酶1(DNMT1)及组蛋白去乙酰化酶1(HDAC1)结合情况的影响,为深化阐释砷毒作用机制提供依据.方法 分别以0.00(空白对照)、3.13、6.25、12.50、25.00 μmol/L NaAsO2重复间隔处理HaCaT细胞72 h(NaAsO2处理24h,隔天再次相同处理,重复3次),以人表皮鳞癌细胞株(A431)作为阳性对照,定量染色质免疫共沉淀技术(Q-ChIP)检测MGMT基因转录调控区ChIP1、ChIP2区域及MGMT基因编码区ChIP3区域MeCP2、DNMT1、HDAC1结合情况.结果 各组HaCaT细胞MGMT基因转录调控区ChIP1、ChIP2区域MeCP2、DNMT1、HDAC1蛋白结合水平比较,差异有统计学意义(F值分别为7.387、84.634、78.442和19.263、69.649、26.546,P均＜0.05)；其中各NaAsO2处理组ChIP1、ChIP2区域MeCP2、DNMT1、HDAC1蛋白结合水平[3.13 μmol/L NaAsO2处理组:(136.00±16.97)％、(145.00±2.83)％、(88.50±19.09)％和(106.50±37.48)％、(112.34±8.73)％、(59.71±8.49)％；6.25 μmol/L NaAsO2处理组:(130.00±42.43)％、(154.50±4.95)％、(101.00±1.27)％和(88.50±3.54)％、(134.32±2.82)％、(102.75±19.91)％;12.50 μmol/LNaAsO2处理组:(141.50±23.33)％、(161.50±7.78)％、(125.00±11.31)％和(119.50±24.75)％、(171.59±3.54)％、(167.61±10.61)％;25.00 μmol/NaAsO处理组:(134.50±43.13)％、(472.50±50.20)％、(383.50±30.41)％和(180.09±12.73)％、(348.50±27.58)％、(158.45±12.02)％]均高于空白对照组[(51.50±9.19)％、(82.00±12.73)％、(25.03±2.91)％和(37.02±4.24)％、(91.56±26.16)％、(19.09±2.90)％,P均＜0.05].各组HaCaT细胞MGMT基因编码区ChIP3区域MeCP2蛋白结合水平比较,差异无统计学意义(F=1.670,P＞0.05),而DNMT1、HDAC1蛋白结合水平比较,差异有统计学意义(F值分别为4.404、9.863,P均＜0.05),其中25.00 μmol/L NaAsO2处理组DNMT1、HDAC1蛋白结合水平[(615.85±29.63)％、(306.09±59.40)％]与空白对照组[(99.70±12.02)％、(92.45±48.79)％]比较,差异有统计学意义(P均＜0.05).结论 MeCP2可结合于砷所致高甲基化MGMT基因转录调控区,通过招募DNMT1及HDAC1使组蛋白去乙酰化,同时DNMT1可结合于MGMT基因编码区,以非甲基化DNA结合蛋白(MBD)依赖的方式招募HDAC1,通过染色质重塑方式导致MGMT基因沉默,可能是砷毒性表现的早期分子事件.
目的 觀察亞砷痠鈉(NaAsO2)對人膚角質形成細胞株(HaCaT細胞)MGMT基因啟動子區甲基化CpG結閤蛋白-2(MeCP2)、DNA甲基轉移酶1(DNMT1)及組蛋白去乙酰化酶1(HDAC1)結閤情況的影響,為深化闡釋砷毒作用機製提供依據.方法 分彆以0.00(空白對照)、3.13、6.25、12.50、25.00 μmol/L NaAsO2重複間隔處理HaCaT細胞72 h(NaAsO2處理24h,隔天再次相同處理,重複3次),以人錶皮鱗癌細胞株(A431)作為暘性對照,定量染色質免疫共沉澱技術(Q-ChIP)檢測MGMT基因轉錄調控區ChIP1、ChIP2區域及MGMT基因編碼區ChIP3區域MeCP2、DNMT1、HDAC1結閤情況.結果 各組HaCaT細胞MGMT基因轉錄調控區ChIP1、ChIP2區域MeCP2、DNMT1、HDAC1蛋白結閤水平比較,差異有統計學意義(F值分彆為7.387、84.634、78.442和19.263、69.649、26.546,P均＜0.05)；其中各NaAsO2處理組ChIP1、ChIP2區域MeCP2、DNMT1、HDAC1蛋白結閤水平[3.13 μmol/L NaAsO2處理組:(136.00±16.97)％、(145.00±2.83)％、(88.50±19.09)％和(106.50±37.48)％、(112.34±8.73)％、(59.71±8.49)％；6.25 μmol/L NaAsO2處理組:(130.00±42.43)％、(154.50±4.95)％、(101.00±1.27)％和(88.50±3.54)％、(134.32±2.82)％、(102.75±19.91)％;12.50 μmol/LNaAsO2處理組:(141.50±23.33)％、(161.50±7.78)％、(125.00±11.31)％和(119.50±24.75)％、(171.59±3.54)％、(167.61±10.61)％;25.00 μmol/NaAsO處理組:(134.50±43.13)％、(472.50±50.20)％、(383.50±30.41)％和(180.09±12.73)％、(348.50±27.58)％、(158.45±12.02)％]均高于空白對照組[(51.50±9.19)％、(82.00±12.73)％、(25.03±2.91)％和(37.02±4.24)％、(91.56±26.16)％、(19.09±2.90)％,P均＜0.05].各組HaCaT細胞MGMT基因編碼區ChIP3區域MeCP2蛋白結閤水平比較,差異無統計學意義(F=1.670,P＞0.05),而DNMT1、HDAC1蛋白結閤水平比較,差異有統計學意義(F值分彆為4.404、9.863,P均＜0.05),其中25.00 μmol/L NaAsO2處理組DNMT1、HDAC1蛋白結閤水平[(615.85±29.63)％、(306.09±59.40)％]與空白對照組[(99.70±12.02)％、(92.45±48.79)％]比較,差異有統計學意義(P均＜0.05).結論 MeCP2可結閤于砷所緻高甲基化MGMT基因轉錄調控區,通過招募DNMT1及HDAC1使組蛋白去乙酰化,同時DNMT1可結閤于MGMT基因編碼區,以非甲基化DNA結閤蛋白(MBD)依賴的方式招募HDAC1,通過染色質重塑方式導緻MGMT基因沉默,可能是砷毒性錶現的早期分子事件.
목적 관찰아신산납(NaAsO2)대인부각질형성세포주(HaCaT세포)MGMT기인계동자구갑기화CpG결합단백-2(MeCP2)、DNA갑기전이매1(DNMT1)급조단백거을선화매1(HDAC1)결합정황적영향,위심화천석신독작용궤제제공의거.방법 분별이0.00(공백대조)、3.13、6.25、12.50、25.00 μmol/L NaAsO2중복간격처리HaCaT세포72 h(NaAsO2처리24h,격천재차상동처리,중복3차),이인표피린암세포주(A431)작위양성대조,정량염색질면역공침정기술(Q-ChIP)검측MGMT기인전록조공구ChIP1、ChIP2구역급MGMT기인편마구ChIP3구역MeCP2、DNMT1、HDAC1결합정황.결과 각조HaCaT세포MGMT기인전록조공구ChIP1、ChIP2구역MeCP2、DNMT1、HDAC1단백결합수평비교,차이유통계학의의(F치분별위7.387、84.634、78.442화19.263、69.649、26.546,P균＜0.05)；기중각NaAsO2처리조ChIP1、ChIP2구역MeCP2、DNMT1、HDAC1단백결합수평[3.13 μmol/L NaAsO2처리조:(136.00±16.97)％、(145.00±2.83)％、(88.50±19.09)％화(106.50±37.48)％、(112.34±8.73)％、(59.71±8.49)％；6.25 μmol/L NaAsO2처리조:(130.00±42.43)％、(154.50±4.95)％、(101.00±1.27)％화(88.50±3.54)％、(134.32±2.82)％、(102.75±19.91)％;12.50 μmol/LNaAsO2처리조:(141.50±23.33)％、(161.50±7.78)％、(125.00±11.31)％화(119.50±24.75)％、(171.59±3.54)％、(167.61±10.61)％;25.00 μmol/NaAsO처리조:(134.50±43.13)％、(472.50±50.20)％、(383.50±30.41)％화(180.09±12.73)％、(348.50±27.58)％、(158.45±12.02)％]균고우공백대조조[(51.50±9.19)％、(82.00±12.73)％、(25.03±2.91)％화(37.02±4.24)％、(91.56±26.16)％、(19.09±2.90)％,P균＜0.05].각조HaCaT세포MGMT기인편마구ChIP3구역MeCP2단백결합수평비교,차이무통계학의의(F=1.670,P＞0.05),이DNMT1、HDAC1단백결합수평비교,차이유통계학의의(F치분별위4.404、9.863,P균＜0.05),기중25.00 μmol/L NaAsO2처리조DNMT1、HDAC1단백결합수평[(615.85±29.63)％、(306.09±59.40)％]여공백대조조[(99.70±12.02)％、(92.45±48.79)％]비교,차이유통계학의의(P균＜0.05).결론 MeCP2가결합우신소치고갑기화MGMT기인전록조공구,통과초모DNMT1급HDAC1사조단백거을선화,동시DNMT1가결합우MGMT기인편마구,이비갑기화DNA결합단백(MBD)의뢰적방식초모HDAC1,통과염색질중소방식도치MGMT기인침묵,가능시신독성표현적조기분자사건.
Objective To investigate the effect of NaAsO2 on the binding levels of methyl CpG binding protein 2(MeCP2),DNA methyltransferase 1 (DNMT1) and histone deacetylase 1(HDAC1) to the hypermethylation promoter region of MGMT gene in HaCaT cells,in order to provide a basis to deepen the interpretation of the role of arsenic poisoning mechanism.Methods HaCaT cells were treated repeatedly and interval with different concentrations of NaAsO2(3.13,6.25,12.50,25.00 μnol/L,respectively) for 72 h.Untreated HaCaT was used as blank control group and human epidermal squamous carcinoma cell line(A431 cells) as positive control group.The binding levels to the two transcription regulatory regions(ChIP1,ChIP2) and to the coding region(ChIP3) of MGMT 8ene were detected by chromatin immuno-precipitation combined with quantitative PCR.Results The differences of binding levels of MeCP2,DNMT1 and HDAC1 to ChIP1 and ChIP2 in each group were significant (F=7.387,84.634,78.442 and 19.263,69.649,26.546,all P ＜ 0.05).The binding levels of MeCP2,DNMT1 and HDAC1 to ChIP1 and ChIP2 in each NaAsO2 exposed group[3.13 μmol/L NaAsO2 exposed group:(136.00 ±16.97)％,(145.00 ± 2.83)％,(88.50 ± 19.09)％ and (106.50 ± 37.48)％,(112.34 ± 8.73)％,(59.71 ± 8.49)％;6.25 μmol/L NaAsO2 exposed group:(130.00 ± 42.43)％,(154.50 ± 4.95)％,(101.00 ± 1.27)％ and (88.50 ±3.54)％,(134.32 ± 2.82)％,(102.75 ± 19.91)％ ; 12.50 μmol/L NaAsO2 exposed group:(141.50 ± 23.33)％,(161.50 ± 7.78)％,(125.00 ± 11.31)％ and (119.50 ± 24.75)％,(171.59 ± 3.54)％,(167.61 ± 10.61)％; 25.00μmol/L NaAsO2 exposed group:(134.50 ± 43.13)％,(472.50+ 50.20)％,(383.50 ± 30.41)％ and (180.09 ±12.73)％,(348.50 ± 27.58)％,(158.45 ± 12.02)％] were higher than that in the blank control group[(51.50 ±9.19)％,(82.00 ± 12.73)％,(25.03 ± 2.91)％ and (37.02 ± 4.24)％,(91.56 ± 26.16)％,(19.09 ± 2.90)％,all P ＜ 0.05].The differences of binding levels of MeCP2 to ChIP3 in each group were not significant(F =1.670,P ＞0.05),but the differences of binding levels of DNMT1 and HDAC1 to ChIP3 were significant (F =4.404,9.863,all P ＜ 0.05),and only the binding levels in the 25.00 μmol/L NaAsO2 exposed group [(615.85 ± 29.63)％,(306.09 ± 59.40)％] were higher than that in the blank control group[(99.70 ± 12.02)％,(92.45 ± 48.79)％,all P ＜ 0.05].Conclusions MeCP2 can bind to the methylated MGMT gene transcriptional regulatory regions which are induced by arsenic and leads to histone deacetylation by the recruitment of DNMT1 and HDAC1 and,meanwhile,DNMT1 can bind to the coding region of MGMT gene to recruit HDAC1 in a methyl DNA binding protein(MBD) independence manner and media MGMT gene silencing through the chromatin remodeling way,which might be the early molecular events of arsenic poisoning.
