结直肠癌细胞系中表观遗传修饰与p33ING1b基因转录抑制的关系
|
摘要
第一部分结直肠癌细胞系中p33ING1b基因表达与表观遗传修饰状态的关系
目的
ING1基因是1996年发现的一个肿瘤抑制基因,目前的研究发现其转录剪接体p33ING1b mRNA在的人类多种肿瘤中表达下调,但其机制并未明确。本研究旨在从p33ING1b基因启动子甲基化和组蛋白乙酰化的表观遗传修饰状态与基因表达的关系,初步探讨结直肠癌(colorectal cancer,CRC)中p33ING1b转录抑制的机制。
方法
对体外培养的结直肠癌细胞系HT29、LOVO、HCT116、COLO205,采用RT-PCR结合Real-time qPCR的方法检测其p33ING1b mRNA的表达,用巢式-甲基化特异性PCR(nested methylation specific polymerase chain reaction,nMSP)分析其p33ING1b启动子甲基化状况,用染色质免疫沉淀(chromatin immunoprecipitation,ChIP)结合Real-time qPCR方法检测其p33ING1b组蛋白乙酰化状况,用Western Blot检测其p33ING1b蛋白表达,HT29、LOVO、COLO205与HCT116比较,分析结直肠癌不同细胞系p33ING1b的表观遗传修饰状态与基因表达关系。
结果
1 HT29、LOVO、COLO205与HCT116结直肠癌细胞均有不同程度的p33ING1b mRNA表达。与HCT116比较,HT29和LOVO的p33ING1b mRNA的表达稍高(P>0.05),而COLO205的表达则较高(P<0.01),COLO205的表达比HT29和LOVO高(P<0.01)。
2结直肠癌细胞株HT29、LOVO检测为p33ING1b基因启动子半甲基化,HCT116则检测出甲基化,而COLO205则未检测出p33ING1b甲基化。
3在HT29、LOVO、COLO205与HCT116结直肠癌细胞中,p33ING1b基因启动子头部和尾部2个片段组蛋白H3乙酰化程度检测结果相似(r=0.997)。4株细胞的p33ING1b组蛋白H3乙酰化程度不同。与HCT116比较,HT29和LOVO的乙酰化水平较高(P<0.05),而COLO205的则更高(P<0.01)。COLO205的乙酰化水平较HT29和LOVO的高(P<0.01)。
4 HT29、LOVO、COLO205与HCT116结直肠癌细胞均有不同程度的p33ING1b蛋白表达。结直肠癌细胞株HCT116 p33ING1b蛋白表达较低,HT29、LOVO的则明显升高,COLO205的较高(P<0.05),而HT29、LOVO和COLO205三者无统计学差别(P>0.05)。
5在HT29、LOVO、COLO205与HCT116结直肠癌细胞株中,非甲基化细胞株COLO205的p33ING1b组蛋白H3乙酰化水平及mRNA的表达相对较高,p33ING1b蛋白表达水平亦高,而甲基化的细胞株HCT116的则较低,但在半甲基化的细胞株HT29、LOVO中p33ING1b组蛋白H3乙酰化水平及mRNA的表达中等,但其p33ING1b蛋白表达水平仍在较高水平。p33ING1b蛋白表达与p33ING1b片段1、片段2组蛋白H3乙酰化及mRNA中到高度相关(r=0.667至0.727)。
结论
1结直肠癌细胞系中有不程度的p33ING1b mRNA表达,其表达水平与蛋白表达水平正相关,提示抑癌基因p33ING1b沉默与结直肠癌的发生发展有关。
2结直肠癌细胞系中有不程度的p33ING1b基因启动子过甲基化及组蛋白乙酰化水平降低,提示抑癌基因p33ING1b启动子过甲基化、组蛋白低乙酰化参与结直肠癌的发生发展,p33ING1b启动子甲基化和组蛋白乙酰化是结直肠癌的频发事件。
3结直肠癌细胞系中不程度的p33ING1b mRNA表达水平与基因启动子甲基化负相关,与基因启动子组蛋白乙酰化水平正相关,提示结直肠癌细胞系p33ING1b mRNA表达下调与基因启动子甲基化、组蛋白乙酰化有关。
4 p33ING1b基因启动子过甲基化和组蛋白低乙酰化是导致结直肠癌细胞系p33ING1b基因转录抑制的原因之一,但可能还存在其他机制如其他表观遗传修饰或其上游转录调控机制的异常改变导致p33ING1b的转录抑制。
第二部分表观遗传学干预对结直肠癌细胞系表观遗传修饰及p33ING1b基因表达的影响
目的
研究发现抑癌基因ING1与结直肠癌关系密切,其转录剪接体p33ING1b mRNA在结直肠癌中表达下调,且存在过甲基化和去乙酰化状态,但其机制并未明确。本研究旨在通过体外表观遗传学干预,从p33ING1b启动子甲基化和组蛋白乙酰化的表观遗传状态改变与基因表达的关系,进一步探讨结直肠癌中p33ING1b转录抑制的表观遗传机制。
方法
通过曲古抑菌素A(Trichostatin A,TSA)及5-氮-2'-脱氧胞苷(5-Aza- 2'-deoxycytidine,5-Aza-2'-dc)对体外培养的结直肠癌细胞系HT29、LOVO、HCT116和COLO205行恢复乙酰化和去甲基化干预,HT29、LOVO、HCT116和COLO205实验干预TSA组(100μg/L的TSA作用细胞24h)、Aza组(1.2 mg/L的5-Aza-2'-dc作用细胞72h)、Aza+TSA组(先5-Aza-2'-dc作用细胞48h后,TSA作用细胞24h)分别与对照组(无加药干预)比较。采用RT-PCR结合Real-time qPCR的方法检测其p33ING1b mRNA表达的变化,用nMSP方法分析其p33ING1b启动子甲基化状态的改变,用ChIP方法检测其p33ING1b乙酰化状态的改变,用Western Blot检测其p33ING1b蛋白表达的变化,分析结直肠癌不同细胞系p33ING1b的表观遗传修饰状态改变与基因表达的关系,进一步探讨p33ING1b表观遗传修饰在结直肠癌中基因沉默中的机制。
结果
1单独使用TSA及5-Aza-2'-dc均轻微上调HT29、LOVO和HCT116 p33ING1b mRNA的表达水平(P<0.05),联合使用5-Aza-2'-dc及TSA可以明显上调它们p33ING1b mRNA的表达水平(P<0.01)。而单独使用TSA及5-Aza-2'-dc均轻微上调COLO205 p33ING1b mRNA的表达,但无统计学差别(P>0.05),联合使用5-Aza-2'-dc及TSA则上调COLO205 p33ING1b mRNA的表达(P<0.05)。
2用5-Aza-2'-dc及5-Aza-2'-dc+TSA干预后HT29、LOVO由p33ING1b启动子半甲基化逆转为非甲基化,HCT116由p33ING1b启动子甲基化逆转为半甲基化,干预后COLO205则仍未检测出p33ING1b甲基化。TSA干预后各结直肠癌细胞株甲基化无改变,
3表观遗传学干预对用p33ING1b片段1和片段2的组蛋白乙酰化作用相似。TSA干预后结直肠癌细胞株HT29、LOVO、HCT116和COLO205 p33ING1b染色质DNA的组蛋白H3乙酰化水平升高(P<0.05)。用5-Aza-2'-dc干预后结直肠癌细胞HT29、LOVO和HCT116 p33ING1b染色质DNA的组蛋白H3乙酰化水平轻微升高(P>0.05),而对COLO205影响不大。而用5-Aza-2'-dc+TSA干预后HT29、LOVO、HCT116和COLO205 p33ING1b染色质DNA的组蛋白H3乙酰化水平明显升高(P<0.01)。
4单独使用TSA及5-Aza-2'-dc干预后均使HT29、LOVO和HCT116 p33ING1b蛋白表达增加(P<0.01),而使COLO205 p33ING1b蛋白表达增加不显著(P<0.05),联合使用5-Aza-2'-dc及TSA可以明显增加它们p33ING1b蛋白表达(P<0.01),以HCT116作用最明显。
5对照组非甲基化细胞株COLO205 p33ING1b组蛋白H3乙酰化水平及mRNA的表达相对较高,p33ING1b蛋白表达水平亦高,用TSA、5-Aza-2'-dc及5-Aza-2'-dc+TSA干预后,其mRNA的表达、乙酰化水平及p33ING1b蛋白表达水平均有不同程度升高,以5-Aza-2'-dc+TSA干预作用明显(P<0.05)。
6对照组半甲基化细胞株HT29及LOVO p33ING1b组蛋白H3乙酰化水平、mRNA的表达及p33ING1b蛋白表达水平中等,TSA干预后其甲基化不能被逆转,5-Aza-2'-dc干预后其甲基化被逆转,其mRNA的表达、乙酰化水平及p33ING1b蛋白表达水平均有不同程度升高(P<0.05),用5-Aza-2'-dc +TSA干预后其甲基化被逆转,其乙酰化水平、mRNA的表达及p33ING1b蛋白表达水平变明显升高,差别有统计学意义(P<0.01),干预后细胞生长抑制中等。
7对照组甲基化的细胞株HCT116 p33ING1b组蛋白H3乙酰化水平及mRNA的表达相对较低,p33ING1b蛋白表达水平亦低,TSA干预后,其甲基化不能逆转,其mRNA的表达、乙酰化水平及p33ING1b蛋白表达水平轻度升高(P<0.05);5-Aza-2'-dc干预后,其甲基化被逆转,其mRNA的表达及p33ING1b蛋白表达水平同时轻度升高(P<0.05),但乙酰化水平轻微升高(P>0.05);但是经5-Aza-2'-dc干预后,再经TSA干预,其甲基化被逆转,其mRNA表达、乙酰化水平及p33ING1b蛋白表达水平明显升高(P<0.01),干预后细胞生长抑制明显,顺序反之不然。p33ING1b启动子组蛋白H3乙酰化与mRNA表达、蛋白表达中到高度正相关(r=0.564至0.713),与甲基化负相关。
结论
1结直肠癌细胞系抑癌基因p33ING1b启动子CpG岛过甲基化与p33 ING1b基因沉默相关,甲基转移酶抑制剂5-Aza-2'-dc可使抑癌基因p33 ING1b去甲基化,使p33ING1b基因表达上调。
2结直肠癌细胞系抑癌基因p33ING1b基因乙酰化与DNA甲基化负相关,组蛋白去乙酰化酶抑制剂TSA使p33ING1b基因乙酰化水平上调,部分拮抗DNA过甲基化产生的p33ING1b基因表达沉默。
3对于p33ING1b基因启动子过甲基化的结直肠癌细胞株HCT116,单用组蛋白去乙酰化酶抑制TSA作用其乙酰化水平升高,但基因表达上调不明显,但如果先用DNA甲基转移酶抑制剂5-Aza-2'-dc处理使基因获得轻度的重新表达,再用组蛋白去乙酰化酶抑制TSA处理后,则可使结肠癌细胞基因重新表达显著增强,组蛋白去乙酰化和DNA过甲基化共存于基因失活的过程之中,但DNA的高甲基化在导致p33ING1b基因沉默中可能扮演主导作用。
4在结直肠癌p33ING1b的转录抑制中,除p33ING1b基因启动子甲基化和乙酰化外,或还存在其他机制如其他表观遗传修饰机制或其上游转录调控机制的异常改变。
The first part:The relationship between p33ING1b gene expression and pattern of epigenetic modification
Objective
ING1 is a candidate tumor suppressor gene discovered in 1996. Recent research demonstrated that the expression of p33ING1b mRNA was down-regulated in many human tumors, but the mechanisms was not clear yet. This study was designed to make a initial investigate for the mechanisms of the p33ING1b transcription inhibition in colorectal cancer through analyzing the relationship between epigenetic pattern and genetic expression in the term of methylation and acetylation of p33ING1b promoter.
Methods
Four colorectal cancer cell lines(HT29, LOVO, HCT116, COLO205)were cultured in vitro and p33ING1b mRNA expression was detected by the real-time quantitative reverse transcription-polymerase chain reaction. By using nest methylation-specific PCR (nMSP),the pattern of p33ING1b promoter methylation was analyzed and the pattern of p33ING1b acetylation was detected by chromatin immunoprecipitation(ChIP) and p33ING1b protein expression was detected by Western Blot.Finally the relationship between the pattern of p33ING1b epigenetic modification and gene expression in different colorectal cancer cell lines was analyzed.
Results
1.Variable expression levels of p33ING1b mRNA could be detected in colorectal cancer cells of HT29, LOVO, COLO205 and HCT116. Compared with HCT116, the expression level of p33ING1b mRNA in HT29 and LOVO was slightly higher (P>0.05), while that in COLO205 was higher than in HT29 and LOVO (P<0.01).
2.Semi-methylation of p33ING1b promoter could be detected in colorectal cancer cell lines of HT29,LOVO,and hypermethylation in HCT116,while unmethylation was showed in COLO205.
3.The detecting results of acetylation level of two fragments of histone H3 on the front part and the rear of the promoter in the colorectal cancer cells of HT29,LOVO,COLO205 and HCT116 were similar(r=0.997).The acetylation levels in the four cell lines were different. Compared with HCT116,the acetylation levels in HT29 and LOVO were higher(P<0.05),and the level of COLO205 was the highest(P<0.01).
4.Variable protein expression levels of p33ING1b were detected in colorectal cancer cells of HT29, LOVO, COLO205 and HCT116.But the protein expression level of p33ING1b in HCT116 was lower,while that in LOVO and HT29 increased significantly, and that in COLO205 was higher (P <0.05).The protein expression levels of p33ING1b among HT29, LOVO and COLO205 had no statistical difference (P> 0.05).
5.The levels of histone H3 acetylation and mRNA expression of p33ING1b in unmethylatied cell lines COLO205 were higher relatively among HT29、LOVO、COLO205 and HCT116,also was protein expression,while that of hypermethylated cell lines HCT116 was lower.The levels of histone H3 acetylation and mRNA expression in semi-methylated cell lines HT29 and LOVO were at the medium level,but their protein expression level of p33ING1b maintained at a higher level.The p33ING1b protein expression had a moderate to high correlation with the H3 acetylation and mRNA expression.
Conclusion
1.Varible low expression levels of p33ING1b mRNA existed in human colorectal cancer cell lines,and it was positive correlation with protein expression levels,which suggested that there were intimate correlation between silencc of p33ING1b and the occurrence and development of colorectal cancer.
2. There were varible levels of hypermethylation and deacetylation of p33ING1b promoter in human colorectal cancer cell lines, which was revealed that hypermethylation and deacetylation of p33ING1b involved in the occurrence and development of colorectal cancer, and promoter methylation and acetylation of p33ING1b were frequent incidents of colorectal cancer.
3.Varible low expression levels of p33ING1b mRNA in human colorectal cancer cell lines were negatively correlated with methylation of the gene promoter,and that were positively correlated with the level of gene promoter acetylation,which suggested that the down-regulation of p33ING1b mRNA gene expression in human colorectal cancer cell lines had intimate correlation with methylation and acetylation of p33ING1b gene promoter.
4.Hypermethylation and deacetylation of p33ING1b promoter is one of the reasons that cause p33ING1b transcription inhibition of human colorectal cancer cell lines,but other mechanisms may exist,such as other epigenetic modification,or p33ING1b transcription inhibition which was caused by abnormal change of transcriptional regulatory mechanism in the upstream.
The second part:The influence of intervention on epigenetic modification of colorectal cancer cell lines and p33ING1b expression
Objective
The previous reseach showed that tumor suppressor gene ING1 was closely related to colorectal cancer.The expression of transcriptional spliceosome of p33ING1b mRNA was down-regulated in colorectal cancer,and the pattern of hypermethylation and deacetylation was detected,but its mechanism was not clear yet.This study was designed to make a further investigation for the epigenetic mechanism of the p33ING1b transcription inhibition through the external intervention and analyzed the relationship between the change of epigenetic pattern and genetic expression in p33ING1b promoter.
Methods
Colorectal cancer cell lines were cultured in vitro and divided into four groups:control group treated without any drug,and the other three experimental groups treated with TSA(TSA group),5-Aza-2'-dc (Aza group),5-Aza-2'-dc +TSA(Aza+TSA group) seperately.The expressions of p33ING1b mRNA were detected by real-time quantitative RT-PCR.The pattern of p33ING1b promoter methylation were analyzed by nMSP.Acetylation levels of p33ING1b fragment 1 and 2 were estimated by ChIP.p33ING1b protein expression was detected by Western Blot.Finally the relationship between the change of p33ING1b epigenetic modification and gene expression in different colorectal cancer cell lines was analyzed,in order to investigate a further mechanism between the p33ING1b epigenetic modification and gene silencing in colorectal cancer
Results
1.Compared with the control group respectively,group-TSA or 5-Aza-2'-dc alone could up-regulate the expression levels of p33ING1b mRNA in HT29, LOVO and HCT116 slightly(P<0.05),but combined-using of 5-Aza-2'-dc+TSA can up-regulate their p33ING1b mRNA expression levels significantly(P<0.01). Using TSA or 5-Aza-2'-dc alone could up-regulate the expression level of p33ING1b mRNA in COLO205 slightly,but there was no statistical difference (P>0.05).The combined 5-Aza-2'-dc and TSA could up-regulate the p33ING1b mRNA expression of COLO205(P<0.05).
2. 5-Aza-2'-dc and the combined 5-Aza-2'-dc and TSA resulted in de methylation which was associated with hypermethylation and Semi-methylation of p33ING1b promoter in colorectal cancer cell lines of HCT116 and HT29,LOVO.In the contrast, TSA alone did not reverse the p33ING1b promoter methylation pattern.
3.The effect of intervention to p33ING1b fragment 1 was similar with fragment 2.Using TSA alone could up-regulate acetylation levels of p33ING1b histone H3 in the colorectal cancer cell lines of HT29,LOVO,HCT116 and COLO205(P<0.05),while using 5-Aza-2'-dc alone could up-regulate acetylation levels in them slightly (P>0.05) and no effect on COLO205.The combined 5-Aza-2'-dc and TSA resulted in up-regulating acetylation levels in them significantly(P<0.01).
4.Protein expression of p33ING1b increased after intervention of using TSA or 5-Aza-2'-dc in the colorectal cancer cell lines of HT29,LOVO and HCT116(P<0.01).The combined 5-Aza-2'-dc and TSA could enhance protein expression of p33ING1b(P <0.05),and the effect on HCT116 was maximal.
5.Acetylation,mRNA and protein expression in p33ING1b were heightened by intervention to colorectal cancer cell lines.Especially in methylated cell lines of HCT116,methylation could not be reversed after using TSA alone,and the levels of acetylation,mRNA and protein expression in p33ING1b were elevated slightly (P<0.05).Then using 5-Aza-2'-dc alone,the methylation had been reversed but the levels of mRNA and protein expression in p33ING1b were also mildly elevated (P < 0.05),but acetylation level was not increased(P >0.05).While it was intervened by 5-Aza-2'-dc and then by TSA,its methylation had been reversed quickly and the levels of acetylation,mRNA and protein expression in p33ING1b raised significantly(P<0.01).The effect of intervention to cell growth inhibition was significant.
Conclusion
1.The hypermethylation of CpG islands in p33ING1b promoter in human colorectal cancer cell lines was associated with 33ING1b silencing.The methyl- transferase inhibitor,5-Aza-2'-dc could help the p33ING1b demethylated, also can up-regulate the p33ING1b expression.
2.The acetylation level of p33ING1b in human colorectal cancer cell lines had a negatively correlation with DNA methylation.The histone deacetylase inhibitor,TSA could up-regulate the level of acetylation of p33ING1b, and partly antagonize the silence of p33ING1b which was generated from p33ING1b promoter hypermethylation.
3.Regarding to cells HCT116,whose p33ING1b promoter was hypermethylation,if histone deacetyltransferase,TSA is used alone the up-regulation of gene expression would be not obvious,but if the methyltransferase inhibitor,5-Aza-2'-dc is used to make p33ING1b obtain mild re-expression firstly,then used TSA,it enable to let p33ING1b re-expression significantly in colorectal cancer cell lines.Histone deacetylation and DNA hypermethylation coexisted in the process of p33ING1b inactivation,and hypermethylation was probably responsible for silence the p33ING1b.
4.In the process of transcription inhibition of p33ING1b in colorectal cancer,except for methylation and acetylation of p33ING1b promoter,there maight have other mechanisms,such as other epigenetic modification mechanisms or abnormal changes of transcriptional regulatory mechanisms in its upstream.
目的
ING1基因是1996年发现的一个肿瘤抑制基因,目前的研究发现其转录剪接体p33ING1b mRNA在的人类多种肿瘤中表达下调,但其机制并未明确。本研究旨在从p33ING1b基因启动子甲基化和组蛋白乙酰化的表观遗传修饰状态与基因表达的关系,初步探讨结直肠癌(colorectal cancer,CRC)中p33ING1b转录抑制的机制。
方法
对体外培养的结直肠癌细胞系HT29、LOVO、HCT116、COLO205,采用RT-PCR结合Real-time qPCR的方法检测其p33ING1b mRNA的表达,用巢式-甲基化特异性PCR(nested methylation specific polymerase chain reaction,nMSP)分析其p33ING1b启动子甲基化状况,用染色质免疫沉淀(chromatin immunoprecipitation,ChIP)结合Real-time qPCR方法检测其p33ING1b组蛋白乙酰化状况,用Western Blot检测其p33ING1b蛋白表达,HT29、LOVO、COLO205与HCT116比较,分析结直肠癌不同细胞系p33ING1b的表观遗传修饰状态与基因表达关系。
结果
1 HT29、LOVO、COLO205与HCT116结直肠癌细胞均有不同程度的p33ING1b mRNA表达。与HCT116比较,HT29和LOVO的p33ING1b mRNA的表达稍高(P>0.05),而COLO205的表达则较高(P<0.01),COLO205的表达比HT29和LOVO高(P<0.01)。
2结直肠癌细胞株HT29、LOVO检测为p33ING1b基因启动子半甲基化,HCT116则检测出甲基化,而COLO205则未检测出p33ING1b甲基化。
3在HT29、LOVO、COLO205与HCT116结直肠癌细胞中,p33ING1b基因启动子头部和尾部2个片段组蛋白H3乙酰化程度检测结果相似(r=0.997)。4株细胞的p33ING1b组蛋白H3乙酰化程度不同。与HCT116比较,HT29和LOVO的乙酰化水平较高(P<0.05),而COLO205的则更高(P<0.01)。COLO205的乙酰化水平较HT29和LOVO的高(P<0.01)。
4 HT29、LOVO、COLO205与HCT116结直肠癌细胞均有不同程度的p33ING1b蛋白表达。结直肠癌细胞株HCT116 p33ING1b蛋白表达较低,HT29、LOVO的则明显升高,COLO205的较高(P<0.05),而HT29、LOVO和COLO205三者无统计学差别(P>0.05)。
5在HT29、LOVO、COLO205与HCT116结直肠癌细胞株中,非甲基化细胞株COLO205的p33ING1b组蛋白H3乙酰化水平及mRNA的表达相对较高,p33ING1b蛋白表达水平亦高,而甲基化的细胞株HCT116的则较低,但在半甲基化的细胞株HT29、LOVO中p33ING1b组蛋白H3乙酰化水平及mRNA的表达中等,但其p33ING1b蛋白表达水平仍在较高水平。p33ING1b蛋白表达与p33ING1b片段1、片段2组蛋白H3乙酰化及mRNA中到高度相关(r=0.667至0.727)。
结论
1结直肠癌细胞系中有不程度的p33ING1b mRNA表达,其表达水平与蛋白表达水平正相关,提示抑癌基因p33ING1b沉默与结直肠癌的发生发展有关。
2结直肠癌细胞系中有不程度的p33ING1b基因启动子过甲基化及组蛋白乙酰化水平降低,提示抑癌基因p33ING1b启动子过甲基化、组蛋白低乙酰化参与结直肠癌的发生发展,p33ING1b启动子甲基化和组蛋白乙酰化是结直肠癌的频发事件。
3结直肠癌细胞系中不程度的p33ING1b mRNA表达水平与基因启动子甲基化负相关,与基因启动子组蛋白乙酰化水平正相关,提示结直肠癌细胞系p33ING1b mRNA表达下调与基因启动子甲基化、组蛋白乙酰化有关。
4 p33ING1b基因启动子过甲基化和组蛋白低乙酰化是导致结直肠癌细胞系p33ING1b基因转录抑制的原因之一,但可能还存在其他机制如其他表观遗传修饰或其上游转录调控机制的异常改变导致p33ING1b的转录抑制。
第二部分表观遗传学干预对结直肠癌细胞系表观遗传修饰及p33ING1b基因表达的影响
目的
研究发现抑癌基因ING1与结直肠癌关系密切,其转录剪接体p33ING1b mRNA在结直肠癌中表达下调,且存在过甲基化和去乙酰化状态,但其机制并未明确。本研究旨在通过体外表观遗传学干预,从p33ING1b启动子甲基化和组蛋白乙酰化的表观遗传状态改变与基因表达的关系,进一步探讨结直肠癌中p33ING1b转录抑制的表观遗传机制。
方法
通过曲古抑菌素A(Trichostatin A,TSA)及5-氮-2'-脱氧胞苷(5-Aza- 2'-deoxycytidine,5-Aza-2'-dc)对体外培养的结直肠癌细胞系HT29、LOVO、HCT116和COLO205行恢复乙酰化和去甲基化干预,HT29、LOVO、HCT116和COLO205实验干预TSA组(100μg/L的TSA作用细胞24h)、Aza组(1.2 mg/L的5-Aza-2'-dc作用细胞72h)、Aza+TSA组(先5-Aza-2'-dc作用细胞48h后,TSA作用细胞24h)分别与对照组(无加药干预)比较。采用RT-PCR结合Real-time qPCR的方法检测其p33ING1b mRNA表达的变化,用nMSP方法分析其p33ING1b启动子甲基化状态的改变,用ChIP方法检测其p33ING1b乙酰化状态的改变,用Western Blot检测其p33ING1b蛋白表达的变化,分析结直肠癌不同细胞系p33ING1b的表观遗传修饰状态改变与基因表达的关系,进一步探讨p33ING1b表观遗传修饰在结直肠癌中基因沉默中的机制。
结果
1单独使用TSA及5-Aza-2'-dc均轻微上调HT29、LOVO和HCT116 p33ING1b mRNA的表达水平(P<0.05),联合使用5-Aza-2'-dc及TSA可以明显上调它们p33ING1b mRNA的表达水平(P<0.01)。而单独使用TSA及5-Aza-2'-dc均轻微上调COLO205 p33ING1b mRNA的表达,但无统计学差别(P>0.05),联合使用5-Aza-2'-dc及TSA则上调COLO205 p33ING1b mRNA的表达(P<0.05)。
2用5-Aza-2'-dc及5-Aza-2'-dc+TSA干预后HT29、LOVO由p33ING1b启动子半甲基化逆转为非甲基化,HCT116由p33ING1b启动子甲基化逆转为半甲基化,干预后COLO205则仍未检测出p33ING1b甲基化。TSA干预后各结直肠癌细胞株甲基化无改变,
3表观遗传学干预对用p33ING1b片段1和片段2的组蛋白乙酰化作用相似。TSA干预后结直肠癌细胞株HT29、LOVO、HCT116和COLO205 p33ING1b染色质DNA的组蛋白H3乙酰化水平升高(P<0.05)。用5-Aza-2'-dc干预后结直肠癌细胞HT29、LOVO和HCT116 p33ING1b染色质DNA的组蛋白H3乙酰化水平轻微升高(P>0.05),而对COLO205影响不大。而用5-Aza-2'-dc+TSA干预后HT29、LOVO、HCT116和COLO205 p33ING1b染色质DNA的组蛋白H3乙酰化水平明显升高(P<0.01)。
4单独使用TSA及5-Aza-2'-dc干预后均使HT29、LOVO和HCT116 p33ING1b蛋白表达增加(P<0.01),而使COLO205 p33ING1b蛋白表达增加不显著(P<0.05),联合使用5-Aza-2'-dc及TSA可以明显增加它们p33ING1b蛋白表达(P<0.01),以HCT116作用最明显。
5对照组非甲基化细胞株COLO205 p33ING1b组蛋白H3乙酰化水平及mRNA的表达相对较高,p33ING1b蛋白表达水平亦高,用TSA、5-Aza-2'-dc及5-Aza-2'-dc+TSA干预后,其mRNA的表达、乙酰化水平及p33ING1b蛋白表达水平均有不同程度升高,以5-Aza-2'-dc+TSA干预作用明显(P<0.05)。
6对照组半甲基化细胞株HT29及LOVO p33ING1b组蛋白H3乙酰化水平、mRNA的表达及p33ING1b蛋白表达水平中等,TSA干预后其甲基化不能被逆转,5-Aza-2'-dc干预后其甲基化被逆转,其mRNA的表达、乙酰化水平及p33ING1b蛋白表达水平均有不同程度升高(P<0.05),用5-Aza-2'-dc +TSA干预后其甲基化被逆转,其乙酰化水平、mRNA的表达及p33ING1b蛋白表达水平变明显升高,差别有统计学意义(P<0.01),干预后细胞生长抑制中等。
7对照组甲基化的细胞株HCT116 p33ING1b组蛋白H3乙酰化水平及mRNA的表达相对较低,p33ING1b蛋白表达水平亦低,TSA干预后,其甲基化不能逆转,其mRNA的表达、乙酰化水平及p33ING1b蛋白表达水平轻度升高(P<0.05);5-Aza-2'-dc干预后,其甲基化被逆转,其mRNA的表达及p33ING1b蛋白表达水平同时轻度升高(P<0.05),但乙酰化水平轻微升高(P>0.05);但是经5-Aza-2'-dc干预后,再经TSA干预,其甲基化被逆转,其mRNA表达、乙酰化水平及p33ING1b蛋白表达水平明显升高(P<0.01),干预后细胞生长抑制明显,顺序反之不然。p33ING1b启动子组蛋白H3乙酰化与mRNA表达、蛋白表达中到高度正相关(r=0.564至0.713),与甲基化负相关。
结论
1结直肠癌细胞系抑癌基因p33ING1b启动子CpG岛过甲基化与p33 ING1b基因沉默相关,甲基转移酶抑制剂5-Aza-2'-dc可使抑癌基因p33 ING1b去甲基化,使p33ING1b基因表达上调。
2结直肠癌细胞系抑癌基因p33ING1b基因乙酰化与DNA甲基化负相关,组蛋白去乙酰化酶抑制剂TSA使p33ING1b基因乙酰化水平上调,部分拮抗DNA过甲基化产生的p33ING1b基因表达沉默。
3对于p33ING1b基因启动子过甲基化的结直肠癌细胞株HCT116,单用组蛋白去乙酰化酶抑制TSA作用其乙酰化水平升高,但基因表达上调不明显,但如果先用DNA甲基转移酶抑制剂5-Aza-2'-dc处理使基因获得轻度的重新表达,再用组蛋白去乙酰化酶抑制TSA处理后,则可使结肠癌细胞基因重新表达显著增强,组蛋白去乙酰化和DNA过甲基化共存于基因失活的过程之中,但DNA的高甲基化在导致p33ING1b基因沉默中可能扮演主导作用。
4在结直肠癌p33ING1b的转录抑制中,除p33ING1b基因启动子甲基化和乙酰化外,或还存在其他机制如其他表观遗传修饰机制或其上游转录调控机制的异常改变。
The first part:The relationship between p33ING1b gene expression and pattern of epigenetic modification
Objective
ING1 is a candidate tumor suppressor gene discovered in 1996. Recent research demonstrated that the expression of p33ING1b mRNA was down-regulated in many human tumors, but the mechanisms was not clear yet. This study was designed to make a initial investigate for the mechanisms of the p33ING1b transcription inhibition in colorectal cancer through analyzing the relationship between epigenetic pattern and genetic expression in the term of methylation and acetylation of p33ING1b promoter.
Methods
Four colorectal cancer cell lines(HT29, LOVO, HCT116, COLO205)were cultured in vitro and p33ING1b mRNA expression was detected by the real-time quantitative reverse transcription-polymerase chain reaction. By using nest methylation-specific PCR (nMSP),the pattern of p33ING1b promoter methylation was analyzed and the pattern of p33ING1b acetylation was detected by chromatin immunoprecipitation(ChIP) and p33ING1b protein expression was detected by Western Blot.Finally the relationship between the pattern of p33ING1b epigenetic modification and gene expression in different colorectal cancer cell lines was analyzed.
Results
1.Variable expression levels of p33ING1b mRNA could be detected in colorectal cancer cells of HT29, LOVO, COLO205 and HCT116. Compared with HCT116, the expression level of p33ING1b mRNA in HT29 and LOVO was slightly higher (P>0.05), while that in COLO205 was higher than in HT29 and LOVO (P<0.01).
2.Semi-methylation of p33ING1b promoter could be detected in colorectal cancer cell lines of HT29,LOVO,and hypermethylation in HCT116,while unmethylation was showed in COLO205.
3.The detecting results of acetylation level of two fragments of histone H3 on the front part and the rear of the promoter in the colorectal cancer cells of HT29,LOVO,COLO205 and HCT116 were similar(r=0.997).The acetylation levels in the four cell lines were different. Compared with HCT116,the acetylation levels in HT29 and LOVO were higher(P<0.05),and the level of COLO205 was the highest(P<0.01).
4.Variable protein expression levels of p33ING1b were detected in colorectal cancer cells of HT29, LOVO, COLO205 and HCT116.But the protein expression level of p33ING1b in HCT116 was lower,while that in LOVO and HT29 increased significantly, and that in COLO205 was higher (P <0.05).The protein expression levels of p33ING1b among HT29, LOVO and COLO205 had no statistical difference (P> 0.05).
5.The levels of histone H3 acetylation and mRNA expression of p33ING1b in unmethylatied cell lines COLO205 were higher relatively among HT29、LOVO、COLO205 and HCT116,also was protein expression,while that of hypermethylated cell lines HCT116 was lower.The levels of histone H3 acetylation and mRNA expression in semi-methylated cell lines HT29 and LOVO were at the medium level,but their protein expression level of p33ING1b maintained at a higher level.The p33ING1b protein expression had a moderate to high correlation with the H3 acetylation and mRNA expression.
Conclusion
1.Varible low expression levels of p33ING1b mRNA existed in human colorectal cancer cell lines,and it was positive correlation with protein expression levels,which suggested that there were intimate correlation between silencc of p33ING1b and the occurrence and development of colorectal cancer.
2. There were varible levels of hypermethylation and deacetylation of p33ING1b promoter in human colorectal cancer cell lines, which was revealed that hypermethylation and deacetylation of p33ING1b involved in the occurrence and development of colorectal cancer, and promoter methylation and acetylation of p33ING1b were frequent incidents of colorectal cancer.
3.Varible low expression levels of p33ING1b mRNA in human colorectal cancer cell lines were negatively correlated with methylation of the gene promoter,and that were positively correlated with the level of gene promoter acetylation,which suggested that the down-regulation of p33ING1b mRNA gene expression in human colorectal cancer cell lines had intimate correlation with methylation and acetylation of p33ING1b gene promoter.
4.Hypermethylation and deacetylation of p33ING1b promoter is one of the reasons that cause p33ING1b transcription inhibition of human colorectal cancer cell lines,but other mechanisms may exist,such as other epigenetic modification,or p33ING1b transcription inhibition which was caused by abnormal change of transcriptional regulatory mechanism in the upstream.
The second part:The influence of intervention on epigenetic modification of colorectal cancer cell lines and p33ING1b expression
Objective
The previous reseach showed that tumor suppressor gene ING1 was closely related to colorectal cancer.The expression of transcriptional spliceosome of p33ING1b mRNA was down-regulated in colorectal cancer,and the pattern of hypermethylation and deacetylation was detected,but its mechanism was not clear yet.This study was designed to make a further investigation for the epigenetic mechanism of the p33ING1b transcription inhibition through the external intervention and analyzed the relationship between the change of epigenetic pattern and genetic expression in p33ING1b promoter.
Methods
Colorectal cancer cell lines were cultured in vitro and divided into four groups:control group treated without any drug,and the other three experimental groups treated with TSA(TSA group),5-Aza-2'-dc (Aza group),5-Aza-2'-dc +TSA(Aza+TSA group) seperately.The expressions of p33ING1b mRNA were detected by real-time quantitative RT-PCR.The pattern of p33ING1b promoter methylation were analyzed by nMSP.Acetylation levels of p33ING1b fragment 1 and 2 were estimated by ChIP.p33ING1b protein expression was detected by Western Blot.Finally the relationship between the change of p33ING1b epigenetic modification and gene expression in different colorectal cancer cell lines was analyzed,in order to investigate a further mechanism between the p33ING1b epigenetic modification and gene silencing in colorectal cancer
Results
1.Compared with the control group respectively,group-TSA or 5-Aza-2'-dc alone could up-regulate the expression levels of p33ING1b mRNA in HT29, LOVO and HCT116 slightly(P<0.05),but combined-using of 5-Aza-2'-dc+TSA can up-regulate their p33ING1b mRNA expression levels significantly(P<0.01). Using TSA or 5-Aza-2'-dc alone could up-regulate the expression level of p33ING1b mRNA in COLO205 slightly,but there was no statistical difference (P>0.05).The combined 5-Aza-2'-dc and TSA could up-regulate the p33ING1b mRNA expression of COLO205(P<0.05).
2. 5-Aza-2'-dc and the combined 5-Aza-2'-dc and TSA resulted in de methylation which was associated with hypermethylation and Semi-methylation of p33ING1b promoter in colorectal cancer cell lines of HCT116 and HT29,LOVO.In the contrast, TSA alone did not reverse the p33ING1b promoter methylation pattern.
3.The effect of intervention to p33ING1b fragment 1 was similar with fragment 2.Using TSA alone could up-regulate acetylation levels of p33ING1b histone H3 in the colorectal cancer cell lines of HT29,LOVO,HCT116 and COLO205(P<0.05),while using 5-Aza-2'-dc alone could up-regulate acetylation levels in them slightly (P>0.05) and no effect on COLO205.The combined 5-Aza-2'-dc and TSA resulted in up-regulating acetylation levels in them significantly(P<0.01).
4.Protein expression of p33ING1b increased after intervention of using TSA or 5-Aza-2'-dc in the colorectal cancer cell lines of HT29,LOVO and HCT116(P<0.01).The combined 5-Aza-2'-dc and TSA could enhance protein expression of p33ING1b(P <0.05),and the effect on HCT116 was maximal.
5.Acetylation,mRNA and protein expression in p33ING1b were heightened by intervention to colorectal cancer cell lines.Especially in methylated cell lines of HCT116,methylation could not be reversed after using TSA alone,and the levels of acetylation,mRNA and protein expression in p33ING1b were elevated slightly (P<0.05).Then using 5-Aza-2'-dc alone,the methylation had been reversed but the levels of mRNA and protein expression in p33ING1b were also mildly elevated (P < 0.05),but acetylation level was not increased(P >0.05).While it was intervened by 5-Aza-2'-dc and then by TSA,its methylation had been reversed quickly and the levels of acetylation,mRNA and protein expression in p33ING1b raised significantly(P<0.01).The effect of intervention to cell growth inhibition was significant.
Conclusion
1.The hypermethylation of CpG islands in p33ING1b promoter in human colorectal cancer cell lines was associated with 33ING1b silencing.The methyl- transferase inhibitor,5-Aza-2'-dc could help the p33ING1b demethylated, also can up-regulate the p33ING1b expression.
2.The acetylation level of p33ING1b in human colorectal cancer cell lines had a negatively correlation with DNA methylation.The histone deacetylase inhibitor,TSA could up-regulate the level of acetylation of p33ING1b, and partly antagonize the silence of p33ING1b which was generated from p33ING1b promoter hypermethylation.
3.Regarding to cells HCT116,whose p33ING1b promoter was hypermethylation,if histone deacetyltransferase,TSA is used alone the up-regulation of gene expression would be not obvious,but if the methyltransferase inhibitor,5-Aza-2'-dc is used to make p33ING1b obtain mild re-expression firstly,then used TSA,it enable to let p33ING1b re-expression significantly in colorectal cancer cell lines.Histone deacetylation and DNA hypermethylation coexisted in the process of p33ING1b inactivation,and hypermethylation was probably responsible for silence the p33ING1b.
4.In the process of transcription inhibition of p33ING1b in colorectal cancer,except for methylation and acetylation of p33ING1b promoter,there maight have other mechanisms,such as other epigenetic modification mechanisms or abnormal changes of transcriptional regulatory mechanisms in its upstream.
引文
1李明,顾晋.中国结直肠癌20年来发病模式的变化趋势[J].中华胃肠外科杂志,2006,7(3):214-217.
2 Jemal A,Siegel R,Ward E,et al.Cancer statistics[J].CA Cancer J Clin,2006,56:106-130.
3 Barry EL,Baron JA,Grau MV,et al.K-ras mutations in incident sporadic colorectal adenomas[J].Cancer,2006,106:1036-1040.
4 Luchtenborg M,Weijenberg MP,Wark PA,et al.Mutations in APC,CTNNB1 and K-ras genes and expression of hMLH1 in sporadic colorectal carcinomas from the Netherlands Cohort Study[J].BMC Cancer,2005,15:160.
5 Liu Y,Bodmer WF.Analysis of P53 mutations and their expression in 56 colorectal cancer cell lines[J].Proc Natl Acad Sci USA,2006,103:976-981.
6 Cho KR , Vogelstein B . Genetic alterations in the adenoma-carcinoma sequence[J].Cancer,1992,70(6 Suppl):1727-1731.
7 Fearon ER,Vogelstein B.A genetic model for colorectal tumorigenesis [J].Cell,1990,61:759-767.
8 Smith G,Carey FA,Beattie J,et al.Mutations in APC,Kirsten-ras,and p53-alternative genetic pathways to colorectal cancer[J].Proc Natl Acad Sci USA,2002,99:9433-9438.
9 Garkavtsev I,Kazarov A,Gudkov K,et al.Suppresion of the novel growthinhibtor P33/ING1 promotes neoplastic transformation[J].Nat Genet,1996,14 (4):415- 420.
10 Chen LS,Matsubara N,Yoshino T,et al.Genetic alterations of candidate tumor suppressor ING1 in human esophageal squamous cell cancer[J].Cancer Res,2001,61(11):4345-4349.
11 Chen LS,Wei JB,Zhou YC,et al.Genetic alterations and expression of inhibitorof growth 1 in human sporadic colorectal cancer[J].World J Gastroenterol,2005,11:6120-6124.
12韦建宝,陈利生,高枫.散发性结直肠癌组织中抑癌基因ING1的突变、杂合性缺失及表达[J].癌症,2005,24(2):141-144.
13 Tallen G,Kaiser I,Krabbe S,et al.No ING1 mutations in human brain tumours but reduced expression in high malignancy grades of astrocytoma[J].Int J Cancer,2004,109:476-479.
14 Takahashi M,Ozaki T,Todo S,et al.Decreased expression of the candi-date tumor suppressor gene ING1 is associated with poor prognosis in advanced neuroblastomas[J].Oncol Rep,2004,12:811-816.
15 Yu GZ,Zhu MH,Zhu Z,et al.Genetic alterations and reduced expression of tumor suppressor p33ING1b in human exocrine pancreatic carcinoma[J].World J Gastroenterol,2004,10:3597-3601.
16 Okano T,Gemma A,Hosoya Y,et al.Alterations in novel candidate tumor suppressor genes,ING1 and ING2 in human lung cancer[J].Oncol Rep,2006,15:545-549.
17 Gunduz M,Ouchida M,Fukushima K,et al.Genomic structure of the human ING1 gene and tumor-specific mutations detected in head and neck squamous cell carcinomas[J].Cancer Res,2000,60:3143-3146.
18曹云飞,陈利生,高枫.ING1启动子甲基化在散发性结直肠癌中的研究[J].中华实验外科杂志,2005,22(12):1584.
19 Walker NJ.Real-time and quantitative PCR:applications to mechanism-based toxicology[J].J Biochem Mol Toxicol,2001,15:121-127.
20 Chen WX,Chen YQ,Huang XZ,et al.Application of fluorogenic quantitative PCR in tumor research[J].J Med Mol Biol,2004,1:323-326.
21詹水凯,贾水义.荧光定量PCR数据处理方法的探讨[J].生物技术,2008,18(3):89-91.
22 Knudson AG.Hereditary cancer:two hits revisited[J].J Cancer Res Clin Oncol,1996,122:135-40.
23 Virginia AS,Jian MS,Lin L.Chromatin immunoprecipitation:a tool for studying histone acetylatlon and transcription factor binding[J].Methods,2003,31(1):67-75.
24 Wells J,Farnham PJ.Characterizing transcription factor binding sites using formaldehyde crosslinking and immunoprecipitation[J].Methods,2002,26 (1):48-56.
25 Orlando V,Strutt H,Paro R.Analysis of chromatin structure by in vivo formaldehyde cross-linking[J].Methods,1997,11(2):205-214.
26 Johnson KD,Bresnick EH.Dissecting long-range transcriptional mechanisms by chromatin immunoprecipitation[J].Methods,2002,26(1):27-36.
27 Murrell A,Rakyan VK,Beck S.From genome to epigenome[J].Hum Mol Genet,2005,14(Review Issue):R3-R10.
28 Parsons DW,Wang TL,Samuels Y,et al.Colorectal cancer:mutations in a signall- ing pathway[J].Nature,2005,436(7052):792.
29薛京伦主编.表观遗传学——原理、技术与实践[M].第1版.上海:上海科学技术出版社,2006:4.
30 Feinberg AP,Ohlsson R,Henikoff S.The epigenetic progenitor origin of human cancer[J].Nat Rev Genet,2006,7:21-23.
31 Baylin SB,Ohm JE.Epigenetic gene si1eneing in caneer-A mechanism for early oncogenic Pathway addiction?[J].Nat Rev Cenet,2006,6:107-116.
32 Jones PA,Bay1in SB.The fundamenta1 ro1e of epigenetic events in carcer [J].Nat Rev Genet,2002,3:415-428.
33孟春风,戴冬秋.组蛋白修饰与胃肠道恶性肿瘤的关系[J].中国普外基础与临床杂志,2007,14:608-611.
34朱新江,戴冬秋.表遗传学与胃肠道肿瘤[J].世界华人消化杂志,2006,14:3251-325.
35 Baylin SB,Herman JG,GraffJR,et al.Alterations in DNA methylation:a fundamental aspect of neoplasia[J].Adv Caneer Res,1998,72:141-196.
36 Murai M,Toyota M,Suzuki H,et al.Aberrant methylation and silencing of the BNIP3 gene in colorectal and gastric cancer[J].Clinical Cancer Research,2005,11:1021-1027.
37 Lind GE,Thorstensen L,Lovig T,et al.A CpG island hypermethylation profile of primary colorectal carcinomas and colon cancer cell lines[J].Molecular Cancer,2004,3:28.
38 Kim KH,Choi JS,Kim IJ,et al.Promoter hypomethylation and reactivation of MAGE-A1 and MAGE-A3 genes in colorectal cancer cell lines and cancer tissues[J].World J Gastroenterol,2006,12(35):5651-5657.
39 Esteller M.Dormant hypermethylated tumor suppressor genes:questions and answers[J].J Pathol,2005,205(2):172-180.
40 Zeremski M,Horrigan SK,Grigorian IA,et al.Localization of the candidate tumor suppressor gene ING1 to human chromosome 13q34[J].Somat Cell Mol Genet,1997,23(3):233-236.
41 Garkavtsev I,Demedtrick D,Riabowol K.Cellular localization and chromosome mapping of a novel candidate tumor suppressor gene(ING1)[J].Cytogenet Cell Genet,1997,76(3-4):176-178.
42 Cheung KJ,Li G.The tumor suppressor ING1:structure and function[J].Exp Cell Res,2001,268:1-6.
43 Shen DH,KYK C,Khoo US,et al.Epigenetic and genetic alterations of p33ING1b in ovarian cancer[J].Carcinogenesis,2005,26(4):855-863.
44 Goel A,Arnold CN,Tassone P,et al.Epigenetic inactivation of RUNX3 in microsatellite unstable sporadic colon cancers[J].Int J Cancer,2004,112(5):754-759.
45韦建宝,陈利生,高枫.2种不同ING1 mRNA剪接体在大肠癌组织中的表达研究[J].实用癌症杂志,2004,19(3):259-261.
46 Marks PA,Rifkind RA,Richon VM,et al.Histone deacetylases and cancer:causes and therapies[J].Nat Rev Cancer,2001,1(3):194-202.
47 Marks PA,Richon VM,Breslow R,et al.Histone deacetylase inhibitors as new anticancer drugs[J].Curr Opin Oncol,2001,13(6):477-483.
48 Dang WW,Steffen KK,Perry R,et al.Histone H4 lysine 16 acetylation regulates cellular lifespan[J].Nature,2009,459:802-808.
49 Fragaa MF,Esteller M.Epigenetics and aging:the targets and the marks [J].Trends Genet,2007,23(8):413-418.
50陈萦晅,房静远,陆娟,等.人结肠癌细胞中组蛋白乙酰化对细胞周期调节基因表达的影响[J].中华医学杂志,2004,84(4):312-317.
51 Kondo Y,Shen L,Issa JP.Critical role of histone methylation in tumor suppressor gene silencing in colorectal cancer[J].Mol Cell Biol,2003,23(1):206-215.
52 Bird A,Wolffe AP.Methylation-induced repression-belts braces and chromatin [J].Cell,1999,9:4512-4541.
53 Cervoni L,Egistelli L,Eufemi M,et al.DNA sequences acting as binding sites for NM23/NDPK proteins in melanoma M14 cells[J].J Cell Biochem,2006,98: 421-428.
54 Nagahama Y,Ishimaru M,Osaki M,et al.Apoptotic pathway induced by transduc- tion of RUNX3 in the human gastric carcinoma cell line MKN-1[J].Cancer Sci,2008,99:23-30.
55唐卫中,高枫,李卫.结直肠癌APC、K-ras、p53基因突变的检测[J].肿瘤,2006,26(3):282-284.
56徐艳松,唐卫中,高枫.散发性结直肠癌APC、K-ras、p53、MMR基因突变的检测[J].结直肠肛门外科,2009,15(4):229-232.
57 Jones PA,Laird PW.Cancer epigenetics comes of age[J].Nat Genet,1999,21(2):163-167.
58陈利生,李天煜,曹云飞,等.p33ING1在肛管癌中的表达及与p53和细胞凋亡的关系[J].中华胃肠外科杂志,2006,9(4):338-341.
59刘家旋,陈利生,李天煜.结直肠癌中p33ING1与VEGF蛋白表达及其临床意义[J].结直肠肛门外科,2009,15(4):252-255.
60梁君林,周永淳,陈利生.结直肠癌中抑癌基因ING1的表达及其意义[J].结直肠肛门外科,2006,12(4):270-272.
61 Dang WW,Steffen KK,Perry R,et al.Histone H4 lysine 16 acetylation regulates cellular lifespan[J].Nature,2009,459:802-808.
62 Bakin AV,Curra T.Role of DNA 5-Methylcytosine Transferase in Cell Trans- formation by fos[J].Science,1999,283(5400):387-390.
63 Murakami J,Asaumi J,Maki Y,et al.Effects of demethyhtingagent 5-Aza-2'- deoxycytidine and histone deacetylase inhibitor FR901228 on maspin gene expression in oral cancer cell lines[J].Oral Oncel,2004,40:597-603.
64 Jablonka E,Lamb MJ.The changing concept of epigenetics[J].Ann NY Acad Sci,2002,981:82-96.
65 Attwood JT,Yung RL,Richardson BC.DNA methylation and the regulation of gene transeription[J].Cell Mol Lif Sci,2002,59:241-257.
66 Finnegan EJ,Kovae KA.Plant DNA methyltransferases[J].Plant Mol Biol,2000,43:189-201.
67程苏琴,曹佳,刘晋神,等.结直肠癌中p16基因的甲基化改变与蛋白表达的关系[J].中华检验医学杂志,2004,7(5):307-310.
68 Pham AD,Sauer F.Ubiquitin-activating/conjugating activity of TAFII250,a mediator of activation of gene expression in Drosophila[J].Science,2000,289(5488):2357-2360.
69 Ng HH,Bird A.DNA methylation and chromatin modification[J].Curr Opin Genet Dev,1999,9(2):158-163.
70 Fang JY,Chen YX,Lu J,et al.Epigenetic modification regulations both expression of tumor-associated genes and cell cycle progressing in human colon cancer cell 1ines:Colo320 and SWlll6[J].Cell Res,2004,14:217-226.
71 Herman JG,Umar A,Polyak K,et al.Incidence and functional consequences of MLH1 Promoter hypermethylation in colorectal carcinoma[J].Proc Natl Acad Sci USA,1998,95:6870-6875.
72 Nguyen CT,Weisenberger DJ,Velicescu M,et al.Histone H3-Lysine 9 methylation is associated with aberrant gene silencing in cancer cells and is rapidly reversed by 5-Aza-2’-deoxycytidine[J].Cancer Research,2002,62:6456-6461.
73 Wolffe AP,Matzke MA.Epigenetics:regulation through repression[J].Science,1999,286(5439):481-486.
74 Cameron EE,Bachman KE,My?h?nen S,et al.Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer [J].Nat Genet,1999,21(1):103-107.
75 Fahrner JA,Eguchi S,Herman JG.Dependence of histone modifications and gene expression on DNA hypermethylation in cancer[J].Cancer Res,2002,62:7213- 7218.
76 Graff JR,Gabrielson E,Fujii H,et al.Methylation patterns of the E-cadherin 5'CpG island are unstable and reflect the dynamic, heterogeneous loss of E-cadherin expression during metastatic progression[J].J Biol Chem,2000,275(4):2727-2732.
77 Dobosy JR,Roberts JL,al FV.The expanding role of epigenetics in the development,diagnosis and treatment of prostate cancer and benign prostatic hyperplasia[J].J Urol,2007,177:822-831.
78 Li LC,Carroll PR,Dahiya R.Epigenetic changes in prostate cancer:implication for diagnosis and treatment[J].J Natl CancerInst,2005,97(2):103-115.
79 Digel W,Lubbert M.DNA methylation disturbances as novel therapeutic target in lung cancer: preclinical and clinical results[J].Crit Rev Oncol Hemato,2005,55(1):1-11.
1李明,顾晋.中国结直肠癌20年来发病模式的变化趋势[J].中华胃肠外科杂志,2006,7(3):214-217.
2 Cho KR,Vogelstein B.Genetic alterations in the adenoma-carcinoma sequence [J].Cancer,1992,70(6 Suppl):1727-1731.
3 Murrell A,Rakyan VK,Beck S.From genome to epigenome[J].Hum Mol Genet,2005,14(Review Issue):R3-R10.
4 Parsons DW,Wang TL,Samuels Y,et al.Colorectal cancer:mutations in a signall- ing pathway[J].Nature,2005,436(7052):792.
5 Swinburne LA,Meyer CA,Liu XS,et al.Genomic localization of RNA binding proteins reveals links between pre-mRNA processing and transcription [J].Genome Res,2006,16:912-921.
6陈浩明,薛京伦主编.医学分子遗传学[M].第1版.上海:科学出版社,2005:172-175.
7 Jablonka E,Lamb MJ.The changing concept of epigenetics[J].Ann NY Acad Sci,2002,981:82-96.
8薛京伦主编.表观遗传学——原理、技术与实践[M].第1版.上海:上海科学技术出版社,2006:4.
9 Jonas PA,Laird PW.Cancer epigenetics comes of age[J].Nature Genet,1999,21(2):163-167.
10 Esteller M.Dormant hypermethylated tumor suppressor genes:questions and answers[J].J Pathol,2005,205(2):172-180.
11 Ehrlich M . DNA methylation in cancer:too much, but also too little [J].Oncogene,2002,21(35):5400–5413.
12 Soares J,Pinto AE,Cunha CV,et al.Global DNA hypomethylation in breast carcinoma : corelation with prognostic factors and tumor progression [J].Cancer,1999,85(1):112-118.
13 Stephen BB,Joyce EO.Epigenetic gene silencing in cancer-a mechanism for early oncogenic pathway addiction?[J].Nature Reviews Cancer,2006,6:107- 116.
14 Jenuwein T,Allis CD.Translating the histone code[J].Science,2001,293(5532):1074-1080.
15 Graff JR,Gabrielson E,Fujii H,et al.Methylation patterns of the E-cadherin 5'CpG island are unstable and reflect the dynamic, heterogeneous loss of E-cadherin expression during metastatic progression[J].J Biol Chem,2000,275(4):2727-2732.
16 Marks PA,Rifkind RA,Richon VM,et al.Histone deacetylases and cancer:causes and therapies[J].Nat Rev Cancer,2001,1(3):194-202.
17 Klose RJ,Kallin EM,Zhang Y.JmjC-domain-containing proteins and histone demethylation[J].Nat Rev Genet,2006,7(9):715-727.
18 Linggi BE,Brandt SJ,Sun ZW,et al.Translating the histone code into leukemia [J].J Cell Biochem,2005,96(5):938-950.
19 Wang H,Huang ZQ,Xia L,et al.Methylation of histone H4 at arginine facilitating transcriptional activation by nuclear hormone receptor [J].Science,2001,293(5531):853-857.
20 Pham AD,Sauer F.Ubiquitin-activating/conjugating activity of TAFII250,a mediator of activation of gene expression in Drosophila[J].Science,2000,289(5488):2357-2360.
21 Nakayama J,Rice JC,Strahl BD,et al.Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly[J].Science,2001,292 (5541):110-113.
22 Dang WW,Steffen KK,Perry R,et al.Histone H4 lysine 16 acetylation regulates cellular lifespan[J].Nature,2009,459:802-808.
23 Fraga MF,Esteller M.Epigenetics and aging:the targets and the marks[J].Trends Genet,2007,23(8):413-418.
24 Cameron EE,Bachman KE,My?h?nen S,et al.Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer [J].Nat Genet,1999,21(1):103-107.
25 Selker EU.Trichostatin A causes selective loos of DNA methylation in Neuro- spora[J].Proc Natl Acad Sci USA,1998,95(16):9430-9435.
26 Kondo Y,Shen L,Issa JP.Critical role of histone methylation in tumor suppressor gene silencing in colorectal cancer[J].Mol Cell Biol,2003,23(1):206-215.
27 Bakin AV , Curra T . Role of DNA 5-Methylcytosine Transferase in Cell Transformation by fos[J].Science,1999,283(5400):387-390.
28 Ng HH,Bird A.DNA methylation and chromatin modification[J].Curr Opin Genet Dev,1999,9(2):158-163.
29 Mathers JC.Reversal of DNA hypomethylation by folic acid supple- ments: possible role in colorectal cancer prevention[J].Gut,2005,54 (5):579-581.
30 Bariol C,Suter C,Cheong K,et al.The relationship between hypomethylation and CpG island methylation in colorectal neoplasia[J].Am J Pathol,2003,162(4):1361-1371.
31 He C,Hu HQ,Braren R,et al.c-myc in the Hematopoietic Lineage is Crucial for its Angiogenic Function in the Mouse Embryo[J].Development,2008,135(14): 2467-2477.
32 Sharrard RM,Royds JA,Rogers S,et al.Patterns of methylation of the c-myc gene in human colorectal cancer progression[J].Br J Cancer,1992,65(5):667- 672.
33 Howe LR,Dannenberg AJ.A role for cyclooxygenase-2 inhibitors in the prevention and treatmention of cancer[J].Semin Oncol,2002,29(3 Suppl 11): 111-119.
34 Park GY,Joo M,Pedchenko T,et al.Regulation of macrophage cyclooxygenase-2 gene expression by modification of histon H3[J].Am J Physiol Lung cell Mol Physiol,2004,286(5):L956-L962.
35 Toyota M,Shen L,Ohe-Toyota M,et al.Aberrant methylation of the Cyclooxygen- ase 2 CpG island in colorectal tumors[J].Cancer Res,2000,60 (15):4044-4048.
36 Bargmann CI,Hung MC,Weinberg RA.The neu oncogene encodes an epidermal growth factor receptor-related protein[J].Nature,1986,319(6050):226-230.
37 Akiyama T,Sudo C,Ogawara H,et al.The product of the human C-erbB-2 gene:a 185-kilodahon glycoprotein with tyrosine kinase activity[J].Science,1986,232(4758):1644-1646.
38金中淦,袁亚,陆青.结直肠癌C-erbB-2基因启动子CpG岛甲基化状态及其与C-erbB-2蛋白表达的关系[J].检验医学,2008,23(6):594-599.
39 Paz MF,Fraga MF,Avila S,et al.A systematic profile of DNA methylation in humancancer cell lines[J].Cancer Res,2003,63(5):1114-1121.
40 Gonzalez-Zulueta M,Bender CM,Yang AS,et al.Methylation of the 5'CpG island of the p16/CDKN2 tumor suppressor gene in normal and transformed human tissues correlates with gene silencing[J].Cancer Res,1995,55(20):4531- 4535.
41 Wiencke JK,Zheng S,Lafuente A,et al.Aberrant methylation of p16INK4a in anatomic and gender-specific subtypes of sporadic colorectal cancer [J].Cancer Epidemiol,Biomarkers & Prev,1999,8(6):501-506.
42 Liang JT,Chang KJ,Chen JC,et al.Hypermethylation of the p16 gene in sporadic T3N0M0 stage colorectal cancers:association with DNA replication error and shorter survival[J].Oncology,1999,57(2):149-156.
43 Fang XM,Zheng S,Jiang CH,et al.Effects of promoter Region 5'CpG island demethylation on the biological behavior of human colorectal cancer RKO cells in vitro[J].Chin J Clin Oncol,2008,5(1):10-15.
44方晓明,孙立峰,彭佳萍,等.5-氮-2'-脱氧胞苷对RKO结肠癌细胞株pl6/CDKN2基因去甲基化的转录调节作用[J].中华医学杂志,2003,83(23):2077-2084.
45于波,李世拥,安萍.p16基因启动子甲基化与结直肠癌发生发展的关系[J].中华普通外科杂志,2003,18(1):31-33.
46程苏琴,曹佳,刘晋神,等.结直肠癌中p16基因的甲基化改变与蛋白表达的关系[J].中华检验医学杂志,2004,7(5):307-310.
47 Sato F,Harpaz N,Shibata D,et al.Hypermethylation of the p14(ARF) gene in ulcerative colitis-associated colorectal carcinogenesis[J].Cancer Res,2002,62(4):1148-1151.
48 Shen L,KondoY,Hamilton SR,et al.P14 methylation in human colon cancer is associated with microsatellite instability and wild-type p53 [J].Gastroenterology,2003,124(3):626-633.
49 Esteller M , Tortola S , Toyota M , et al . Hypermethylation-associated inactivation of p14(ARF) is independent of p16(INK4a) methylation and p53 mutational status[J].Cancer Res,2000,60(1):129-133.
50 Veigl ML,Kasturi L,Olechnowicz J,et al.Biallelic inactivation of hMLH1 by epigeneticgene silencing,a novel mechanism causing human MSI cancers [J].Proc Natl Acad Sci USA,1998,95(15):8698-8702.
51蔡国响,蔡三军.结直肠癌发病的分子机理探讨[J].临床肿瘤学杂志,2003,8(6):467-470.
52 Potocnik U,Glavac D,Golouh R,et al.Causes of microsatellite instability in colorectal tumors:implications for hereditary non-polyposis colorectal cancer screening[J].Cancer Genet Cytogenet,2001,126(2):85-96.
53 Deng G,Chen A,Hong J,et al.Methylation of CpG in a small region of the hMLH1 promoter invariably correlates with the absence of gene expression [J].Cancer Res,1999,59(9):2029-2033.
54 Arnold CN,Goel A,Boland CR.Role of hMLH1 promoter hypermethylation in drugresistance to 5-fluorouracil in colorectal cancer cell lines[J].Int J Cancer,2003,106(1):66-73.
55 Plumb JA,Strathdee G,Sludden J,et al.Reversal of drug resistance in human tumor xenografts by 2'-deoxy-5-azacytidine-induced demethylation of the hMLH1 gene promoter[J].Cancer Res,2000,60(21):6039-6044.
56 Kim SH,Bae SI,Lee HS,et al.Alteration of O(6)-methylguanine-DNA methyltransferase in colorectal neoplasms in sporadic and familial adenom- atous polyposis patients[J].Mol Carcinog,2003,37(1):32-38.
57 Nagasaka T,Sharp GB,Notohara K,et al.Hypermethylation of O(6)-me- thylguanine-DNA methyltransferase promoter may predict nonrecurrence after chemotherapy in colorectal cancer cases[J] . Clin Cancer Res , 2003 ,9(14):5306-5312.
58 Krtolica K,Krajnovic M,Usaj-Knezevic S,et al.Comethylation of p16 and MGMT genes in colorectal carcinoma:Correlation with clinclinicopath- ological features and prognostic vaIue[J].World Gastroenterol,2007,13(8): 1187-1194.
59 Garkavtsev I,Demedtrick D,Riabowol K.Cellular localization and chromosome mapping of a novel candidate tumor suppressor gene(ING1)[J].Cytogenet Cell Genet,1997,76(3-4):176-178.
60 Garkavtsev I,Riabowol K.Extension of the replicative life span of human diploid fibroblasts by inhibition of the p331NG1 candidatetumor suppressor [J].Mol Cell Biol,1997,17(4):2014-2019.
61 Oki E,Niaehara Y,Tokunaga E,et al.Reduced expression of p33(ING1) and the relationship with p53 expression in human gastric cancer[J].Cancer lett,1999,147(1-2):157-162.
62韦建宝,陈利生,高枫.散发性结直肠癌组织中抑癌基因ING1的突变、杂合性缺失及表达[J].癌症,2005,24(2):141-144.
63曹云飞,陈利生,高枫.ING1启动子甲基化在散发性结直肠癌中的研究[J].中华实验外科杂志,2005,22(12):1584.
64唐卫中,高枫,李卫,等.中国人散发性大肠癌APC基因突变的研究[J].中华实验外科杂志,2005,22(11):1357-1359.
65王夫景,杨茂鹏,于洪亮,等.抑癌基因KLF-6和APC在结直肠癌组织中的表达及临床意义[J].中华胃肠外科杂志,2006,9(5):429-432.
66汪建平,元云飞.结直肠癌DNA甲基化研究的现状[J].中华实验外科杂志,2004,21:1279-1280.
67黄美近,褚忠华.E-钙黏附素基因甲基化与结直肠癌临床病理特征的关系[J].中华实验外科杂志,2005,22(11):1355-1356.
68 Futscher BW,Oshiro MM,Wozniak RJ,et al.Role for DNA methylation in the control of cell type-specific maspin expression[J].Nat Genet,2002,31(2): 175-179.
69唐波彭,志红余,佩武,等.5-氮-2'-脱氧胞苷对RKO结肠癌细胞株maspin基因去甲基化的转录调节作用[J].中华胃肠外科杂志,2006,9(3):260-263.
70高鹏,崔铮,王静.Maspin在大肠癌中的表达及其启动子5'CpG岛甲基化状态的研究[J].广东医学,2007,28(10):1587-1589.
71 Hirai H,Maru Y,Hagiwara K,et al.A novel putative tyrosine kinase receptor encoded by the eph gene[J].Science,1987,238(4834):1717-1720.
72 Kataoka H,Igarashi H,Kanamori M,et al.Correlation of EPHA2 overexpression with high microvessel count in human primary colorectal cancer[J].Cancer Sci,2004,95(2):136-141.
73王建东,周晓军.高甲基化导致EphA7基因在结直肠癌中低表达[J].医学研究生学报,2007,20(1):6-14.
74 Toyota M,Ahuja N,Ohe-Toyota M,et al.CpG island methylator phenotype in colorectal cancer[J].Proc Natl Acad Sci USA,1999,96(15):8681-8686.
75 Toyota M,Ho C,Ahuja N,et al.Identification of differentially methylatedsequences in colorectal cancer by methylated CpG island amplification [J].Cancer Res,1999,59(10):2307-2312.
76 Van Rijnsoever M,Grieu F,Elsaleh H,et al.Characterisation of colorectal cancers showing hypermethylation at multiple CpG islands[J].Gut,2005,51(6):797-802.
77 Van Rijnsoever M,Elsaleh H,Joseph D,et al.CpG island methylator phenotype is an independent predictor of survival benefit from 5-fluorouracil in stage III colorectal cancer[J].Clin Cancer Res,2003,9(8):28988-2903.
78蔡国响,蔡三军,徐烨,等.甲基子表型阳性的散发性大肠癌的临床病理特征[J].中华消化杂志,2006,26(6):377-380.
79席亚鸣,谢庆芳,孙蓓.抑癌基因甲基化检测结直肠癌患者的研究[J].中华实验外科杂志,2003,20(3):269-270.
80陈萦亘,房静远,陆娟,等.人结肠癌细胞系中癌相关基因的表达及表型遗传修饰的影响[J].临床与实验病理学杂志,2003,19(5):533-538.
81陈萦亘,房静远,陆娟.人结肠癌细胞中组蛋白乙酰化对细胞周期调节基因表达的影响[J].中华医学杂志,2004,84(4):312-317.
82李天煜,陈利生,何纯刚,等.5-氮-2'-脱氧胞苷及TSA对人结肠癌细胞株HT29 ING1b基因表达的影响[J].广东医学,2009,30(10):1465-1467.
83 Esteller M,Garcia-Foncillas J,Andion E,et al.Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents[J].N Engl J Med,2000,343(19):1350-1354.
84 Esteller M,Hamilton SR,Burger PC,et al.Inactivation of the DNA repair gene 06-methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia[J].Cancer Res,1999,59(4):793-797.
85 Kreklau EL,Pollok KE,Bailey BJ,et al.Hematopoietic expression of O(6)-methylguanine DNA methyltransferase-P140K allows intensive treatment of human glioma xenografts with combination O(6)-benzylguanine and 1,3-bis-(2-chloroethyl)-1-nitrosourea[J].Mol Cancer Ther,2003,2(12):1321- 1329.
86 Ward IM,Minn K,Deursen v,et al.p53 Binding protein 53BP1 is required for DNA damage responses and tumor suppression in mice[J].Mol Cell Biol,2003,23(7):2556-2563.
87 Marks PA,Richon VM,Breslow R,et al.Histone deacetylase inhibitors as new anticancer drugs[J].Curr Opin Oncol,2001,13(6):477-483.
88 Shaker S,Bernstein M,Momparler IF,et al.Preclinica1 evaluation of antineoplastic activity of inhibitors of DNA methylation (5-aza-2'- deoxycytidine)and histone deacetylation (trichostatin A,depsipeptide) in combination against myeloid leukemic cells[J].Leuk Res,2003,27(5):437-444.
89 Rashid SF,Moore JS,Walker E,et al.Synergistic growth inhibition of prostate cancer cells by 1 alpha,25 Dihydroxyvitamin D(3) and its 19-nor-hexafluoride ana1ogs in combination with either sodium butyrate or trichostatin A [J].Oncogene,2001,20(15):1860-1872.
90 Esteller M,Sanchez-Cespedes M,Rosell R,et al.Detection of aberrant promoter hypermethylation of tumor suppressor genes in serum DNA from nonsmall cell lung cancer patients[J].Cancer Res,1999,59(1):67-70.
91 Toyota M,huh F,Imai K.DNA methylation and gastrointestinal malignances: functional consequences and clinical implications[J].J Gastroenterol,2000,35(10):727-734.
92 Nakayama H,Hibi K,Takase T,et al.Molecular detection of pl6 promoter methy- lation in the serum of recurrent colorectal cancer patients[J].Int J Cancer,2003,105(4):491-493.
93 Marks PA,Jiang X.Histone deacetylase inhibitors in programmed cell death and cancer therapy[J].Cell Cycle,2005,4(4):549-551.
94 Li LC,Carroll PR,Dahiya R.Epigenetic changes in prostate cancer:implicationfor diagnosis and treatment[J].J Natl CancerInst,2005,97(2):103-115.
95 Digel W,Lubbert M.DNA methylation disturbances as novel therapeutic target in lung cancer: preclinical and clinical results[J].Crit Rev Oncol Hemato,2005,55(1):1-11.
96 Espino PS,Drobic B,Dunn KL,et al.Histone modifications as aplatform for cancer therapy[J].J Cell Biochem,2005,94(6):1088-1102.
1 Strahl BD , Allis CD . The language of covalent histone modifications [J].Nature,2000,403(6765):41-45.
2 Jenuwein T,Allis CD.Translating the histone code[J].Science,2001,293 (5532):1074-1080.
3 Xu DD,Liu DP,Ji XJ,et al.In vivo DNA-protein interactions at hypersensitive site 3.5 of the human beta-globin locus control region[J].Biochem Cell Biol,2001,79(6):747-754.
4 Fu XH,Liu DP,Liang CC.Chromatin structure and transcriptional regulation of the beta-globin locus[J].Exp Cell Res,2002,278(1):1-11.
5 Spencer VA,Sun JM,Li L,et al.Chromatin immunoprecipitation:a tool for studying histone acetylation and transcription factor binding[J].Methods,2003,31(1):67-75.
6 Mayr B, Montminy M.Transcriptional regulation by thephosphorylation -dependent factor CREB[J].Nat Rev Mol Cell Biol,2001,2:599-609.
7 Brutlag D,Schlehuber C,Bonner J.Properties of formaldehyde-treated nucleo- histone[J].Biochemistry,1969,8(8):3214-3218.
8 Ilyin YV,Georgiev GP.Heterogeneity of deoxynucleoprotein particles as evidence by ultracentrifugation of cesium chloride density gradient[J].J Mol Biol,1969,41(2):299-303.
9 O'Neill LP,Turner BM.Immunoprecipitation of native chromatin: NchIP [J].Methods,2003,31(1):76-82.
10 Wells J,Farnham PJ.Characterizing transcription factor binding sites using formaldehyde crosslinking and immunoprecipitation[J].Methods,2002,26(1):48-56.
11 Orlando V,Strutt H,Paro R.Analysis of chromatin structure by in vivo formaldehyde cross-linking[J].Methods,1997,11(2):205-214.
12 Johnson KD,Bresnick EH.Dissecting long-range transcriptional mechanisms by chromatin immunoprecipitation[J].Methods,2002,26(1):27-36.
13 Kuo MH,Allis CD.In vivo cross-linking and immunoprecipitation for studying dynamic protein:DNA associations in a chromatin environment[J].Methods,1999,19(3):425-433.
14 Hecht A,Grunstein M.Mapping DNA interaction sites of chromosomal proteins using immunoprecipitation and polymerase chain reaction[J] . Methods Enzymol,1999,304:399-414.
15 Orlando V.Mapping chromosomal proteins in vivo by formaldehyde-crosslinked- chromatin immunoprecipitation[J].Trends Biochem Sci,2000,25(3):99-104.
16 Kristen M,Meisenheimer KM,Koch TH.Photocross-linking of nucleicacids to associated proteins[J].Crit Rev Biochem Mol Biol,1997,32(2):101-140.
17 Zhang L,Zhang K,Prandl R,et al.Detecting DNA-binding of proteins in vivo by UV-rosslinking and immunoprecipitation[J].Biochem Biophys Res Commun,2004,322(3):705-711.
18 Hanson RW,Reshef L.Regulation of phosphoenelpyruvate carboxykinase(GTP) gene expression[J].Ann Rev Biochem,1997,66:581-611.
19 Solomon MJ,Varshavsky A.Formaldehyde-mediated DNA-protein crosslinking:A probe for in vivo chromatin structures[J].Proc Natl Acad Sci USA,1985,82(19):6470-6474.
20 Spencer VA,Samuel SK,Davie JR.Altered Profiles in Nuclear Matrix Proteins Associated with DNA in Situ during Progression of Breast Cancer Cells [J].Cancer Res,2001,61(4):1362-1366.
21 Ren B,Robert F,Wyrick JJ,et al.Genome-wide location and function of DNAbinding proteins[J].Science,2000,290(5500):2306-2309.
22 Weinmann AS,Yan PS,Oberley MJ,et al.Isolating human transcription factor targets by coupling chromatin immunoprecipitation and CpG island microarray analysis[J].Genes Dev,2002,16(2):235-244.
23 Shannon MF,Rao S.Of chips and ChIPs[J].Science,2002,296(5568):666-669.
24 Kang SH,Vieira K,Burgert J.Combining chromatin immunoprecipitation and DNA footprinting:a novel method to analyze protein-DNA interaction in vivo[J].Nucleic Acids Res,2002,30(10):e44.
25 Niranjanakumari S,Lasda E,Brazas R,et al.Reversible cross-linking combined with immunoprecipitation to study RNA-protein interactions in vivo [J].Methods,2002,26(1):182-190.
26 Steensel BV.Mapping of genetic and epigenetic regulatory networks using microarrays[J].Nature Genetics Supplement,2005,37:s18-s24.
27 Weinmann AS.Novel ChIP-based strategies to uncover transcription factor target genes in the immune system[J].Nature Reviews Immunology,2004,4:381-386.
28 Weinmann AS,Farnham PJ.Identification of unknown target genes of human transcription factors using chromatin immunoprecipitation[J].MetIlods,2002,26(1):37-47.
29 Lei H,Juan AH,Kim MS,et al.Identification of a Hoxc8-regulated transcrip- tional network in mouse embryo fibroblast cells[J].Proc Nail Acad Sci USA,2006,103(27):10305-10309.
30 Oh SW,Mukhopadhyay A,Dixit BL,et al.Identification of direct DAF-16 targets controlling longevity,metabolism and diapause by chromatin immunoprecipit ation[J].Nat Genet,2006,38(4):251-257.
31 Hearnes JM,Mays DJ,Schavoh K,et al.Chromatin immunoprecipitation-based screen to identify functional genomic binding sites for sequence-specifictransactivators[J].Mol Cel Biol,2005,25(22):10148-10158.
32 Moss T,Dimitrov SI,Houde D.UV-laser crosslinking of proteins to DNA [J].Methods,1997,11(2):225-234.
33 Buckle M,Geiselmann J,Kolb A,et al.Protein-DNA cross-linking at the lac promoter[J].Nucleic Acids Res,1991,19(4):833-840.
34 Russmann C,Truss M,Fix A,et al.Crosslinking of progesterone receptor to DNA using tuneable nanosecond,picosecond and femtosecond UV laser pulses [J].Nucleic Acids Res,1997,25(12):2478-2484.
35 Hockensmith JW,Kubasek WL,Vorachek WR,et al.Laser cross-linking of nucleic acids to proteins:Methodology and first applications to the phage T4 DNA replication system[J].J Biol Chem,1986,261(8):3512-3518.
36 Nagaich AK,Walker DA,Wolford R,et al.Rapid periodic binding and displacement of the glucocorticoid receptor during chromatin remodeling [J].Mol Cell,2004,14(2):163-174.
37 Zhang LM,Eggers-Schumacher G,Schoffl F,et al.Analysis of heat-shock transcription factor-DNA binding in Arabidopsis suspension cultures by UV laser crosslinking[J].Plant,2001,28(2):217-223.
38 Shang Y,Hu X,DiRenzo J,et al.Cofactor dynamics and sufficiency in estrogen receptor[J].Cell,2000,103(6):843-852.
39 Shang Y,Brown M.Molecular determinants for the tissue specificity of SERMs [J].Science,2002,295(5564):2465-2468.
40 Weinmann AS,Bartley SM,Zhang T,et al.Use of chromatin immunoprecipitation to clone novel E2F target promoters[J].Mol Cell Biol,2001,21(20):6820-6832.
41 Iyer VR,Horak CE,Scafe CS,et al.Genomic binding sites of the yeast cell-cycle transcription factors SBF and MBF[J] . Nature , 2001 , 409 (6819):533-538.
42 Lieb JD,Liu X,Botstein D,et al.Promoter-specific binding of Rap1 revealedby genome-wide maps of protein-DNA association[J].Nat Genet,2001,28(4): 327-334.
43 Kapranov P,Cawley SE,Drenkow J,et al.Large-scale transcriptional activityin chromosomes 21 and 22[J].Science,2002,296(5569):916-919.
44 Zella LA,Kim S,Shevde NK,et al.Enhancers located within two introns of the vitamin D receptor gene mediate transcriptional autoregulation by 1,
25-dihydroxyvitamin D3[J].Mol Endocrinol,2006,20(6):1231-1247.
45 Banerjee N,Zhang MQ.Identifying cooperativity among transcription factors controlling the cell cycle in yeast[J].Nucleic Acids Res,2003,31(23):7024- 7031.
46 Santoro R,W?lfl S,Saluz HP.UV-Laser induced protein/DNA crosslinking reveals sequence variations of DNA elements bound by c-Jun in vivo [J].Biochem and Biophys Res Commun,1999,256(1):68-74.
47 Solano PJ,Mugat B,Martin D,et al.Genome-wide identification of in vivo drosophila engrailed-binding DNA fragments and related target genes [J].Development,2003,130(7):1243-1254.
48 Abdurashidova G,Danailov MB,Ochem A,et al.Localization of proteins bound to a replication origin of human DNA along the cell cycle[J].EMBO,2003,22:4294-4303.
49 Van Lente F,Jackson JF,Weintraub H.Identification of specific crosslinked histones after treatment of chromatin with formaldehyde[J].Cell,1975,5(1):45-50.
50 Jackson V.Studies on histone organization in the nucleosome using formalde- hyde as a reversible cross-linking agent[J].Cell,1978,15 (3):945-954.
51 Jackson V,Chalkley R.A new method for the isolation of replicative chromatin: selective deposition of histone on both new and old DNA[J].Cell,1981,23(1):121-134.
52 Jackson V,Chalkley R.Use of whole-cell fixation to visualize replicating and maturing simian virus 40: identification of new viral gene product [J].Proc Natl Acad Sci USA,1981,78(10):6081-6085.
53 Li T,Vu TH,Ulaner GA,et al.Activating and silencing histone modifications form independent allelic switch regions in the imprinted Gnas gene[J].Hum Mol Genet,2004,13(7):741-750.
54 Shan B,Xu J,Zhuo Y,et al.Induction of p53-dependent activation of the human proliferating cell nuclear antigen gene in chromatin by ionizing radiation[J].J Biol Chem,2003,278(45):44009-44017.
55 Gonzalo S,Jaco I,Fraga MF,et al.DNA methyltransferases control telomere length and telomere recombination in mammalian cells[J].Nat Cell Biol,2006,8:416-424.
56 Storre J,Schafer A,Reichert N,et al.Silencing of the meiotic genes SMC1 {beta} and STAG3 in somatic cells by E2F6[J].J Biol Chem,2005,280:41380- 41386.
57 Angelov D,VYu S,Dimitrov SI,et al.Protein-DNA crosslinking in reconstituted nucleohistone,nuclei and whole cells by piocosecond UV laser irradiation [J].Nucleic Acids Res,1988,16(10):4525-4538.
58 Lejnine S,Murnane M,Kapteyn HC,et al.Crosslinking of proteins to DNA in human nuclei using a 60 femtosecond 266nm laser[J].Nucleic Acids Res,1999,27(18):3676-3684.
59 Mutskov V,Gerber D,Angelov D,et al.Persistent interactions of core histone tails with nucleosomal DNA following acetylation and transcription factor binding[J].Mol Cell Bio,1998,18(11):6293-6304.
60 De Silanes IL,Zhan M,Lai A,et al.Identification of a target RNA motif for RNA-binding protein HuR[J].Proc Natl Acad Sci USA,2004,101 (9):2987- 2992.
61 SenGupta DJ,Zhang B,Kraemer B,et al.A three-hybrid system to detect RNA-protein interactions in vivo[J].Proc Natl Acad Sci USA,1996,93(16): 8496-8501.
62 Swinburne IA,Meyer CA,Liu XS,et al.Genomic localization of RNA binding proteins reveals links between pre-mRNA processing and transcription [J].Genome Res,2006,16:912-921.
63 Braunatein M,Rose AB,Holmes SG,et al.Transcriptional silencing in yeast is associated with reduced nucleosome acetylation[J].Genes Dev,1993,7:592-604.
64 Braunstein M,Sobel RE,Allis CD,et al.Efficient transcriptional silencing in Saccharomyces cerevisiae requires a heterochromatin histone acetylation pattern[J].Mol Cell Biol,1996,16:4349-4356.
65 Kuo MH,Zhou J,Jambeck P,et al.Histone acetyltransferase activity of yeast Gcn5p is required for the activation of target genes in vivo[J].Genes Dev,1998,12:627-639.
66 Dedon PC,Soults JA,Allis CD,et al.A simplified formaldehyde fixation and immunoprecipitation technique for studying protein-DNA interactions [J].Anal Biochem,1991,197(1):83-90.
67 Dedon PC,Soults JA,Allis CD,et al.Formaldehyde cross-linking and immunoprecipitation demonstrate developmental changes in H1 association with transcriptionally active genes[J].Mol CeLL Biol,1991,11(3):1729-1733.
68 Orlando V,Paro R.Mapping Polycomb-repressed domains in the bithorax complex using in vivo formaldehyde cross-linked chromatin[J].Cell,1993,75(6):1187- 1198.
69 Alberts AS,Geneste O,Treisman R.Activation of SRF-regulated chromosomal templates by Rho-family GTPases requires a signal that also induces H4 hyperacetylation[J].Cell,1998,92:475-487.
70 Pereira S,Grayling RA,Lurz R,et al.Archaeal nucleosomes[J].Proc Natl Acad Sci USA,1997,94(23):12633-12537.
71 Boyd KE,Wells J,Gutman J,et al.c-Myc target gene specificity is determined by a post-DNA binding mechanism[J].proc Natl Acad Sci USA,1999,95(23): 13887-13992.
72 Kondo Y,Shen L,Issa JP.Critical role of histone methylation in tumor suppressor gene silencing in colorectal cancer[J].Mol Cell Biol,2003,23(1):206-215.
73 Cameron EE,Bachman KE,My?h?nen S,et al.Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer[J].Nat Genet,1999,21(1):103-107.
74杨发达,李建明,周军,等.人大肠癌细胞PRL-3基因启动子Snail结合位点的初步研究[J].南方医科大学学报,2007,27(4):401-405.
75陈萦亘,房静远,陆娟.人结肠癌细胞中组蛋白乙酰化对细胞周期调节基因表达的影响[J].中华医学杂志,2004,84(4):312-317.
76李天煜,陈利生,何纯刚,等.5-氮-2'-脱氧胞苷及TSA对人结肠癌细胞株HT29 ING1b基因表达的影响[J].广东医学,2009,30(10):1465-1467.
77 Dang WW,Steffen KK,Perry R,et al.Histone H4 lysine 16 acetylation regulates cellular lifespan[J].Nature,2009,459:802-808.
78 Fang XM,Zheng S,Jiang CH,et al.Effects of promoter Region 5'CpG island demethylation on the biological behavior of human colorectal cancer RKO cells in vitro[J].Chin J Clin Oncol,2008,5(1):10-15.
2 Jemal A,Siegel R,Ward E,et al.Cancer statistics[J].CA Cancer J Clin,2006,56:106-130.
3 Barry EL,Baron JA,Grau MV,et al.K-ras mutations in incident sporadic colorectal adenomas[J].Cancer,2006,106:1036-1040.
4 Luchtenborg M,Weijenberg MP,Wark PA,et al.Mutations in APC,CTNNB1 and K-ras genes and expression of hMLH1 in sporadic colorectal carcinomas from the Netherlands Cohort Study[J].BMC Cancer,2005,15:160.
5 Liu Y,Bodmer WF.Analysis of P53 mutations and their expression in 56 colorectal cancer cell lines[J].Proc Natl Acad Sci USA,2006,103:976-981.
6 Cho KR , Vogelstein B . Genetic alterations in the adenoma-carcinoma sequence[J].Cancer,1992,70(6 Suppl):1727-1731.
7 Fearon ER,Vogelstein B.A genetic model for colorectal tumorigenesis [J].Cell,1990,61:759-767.
8 Smith G,Carey FA,Beattie J,et al.Mutations in APC,Kirsten-ras,and p53-alternative genetic pathways to colorectal cancer[J].Proc Natl Acad Sci USA,2002,99:9433-9438.
9 Garkavtsev I,Kazarov A,Gudkov K,et al.Suppresion of the novel growthinhibtor P33/ING1 promotes neoplastic transformation[J].Nat Genet,1996,14 (4):415- 420.
10 Chen LS,Matsubara N,Yoshino T,et al.Genetic alterations of candidate tumor suppressor ING1 in human esophageal squamous cell cancer[J].Cancer Res,2001,61(11):4345-4349.
11 Chen LS,Wei JB,Zhou YC,et al.Genetic alterations and expression of inhibitorof growth 1 in human sporadic colorectal cancer[J].World J Gastroenterol,2005,11:6120-6124.
12韦建宝,陈利生,高枫.散发性结直肠癌组织中抑癌基因ING1的突变、杂合性缺失及表达[J].癌症,2005,24(2):141-144.
13 Tallen G,Kaiser I,Krabbe S,et al.No ING1 mutations in human brain tumours but reduced expression in high malignancy grades of astrocytoma[J].Int J Cancer,2004,109:476-479.
14 Takahashi M,Ozaki T,Todo S,et al.Decreased expression of the candi-date tumor suppressor gene ING1 is associated with poor prognosis in advanced neuroblastomas[J].Oncol Rep,2004,12:811-816.
15 Yu GZ,Zhu MH,Zhu Z,et al.Genetic alterations and reduced expression of tumor suppressor p33ING1b in human exocrine pancreatic carcinoma[J].World J Gastroenterol,2004,10:3597-3601.
16 Okano T,Gemma A,Hosoya Y,et al.Alterations in novel candidate tumor suppressor genes,ING1 and ING2 in human lung cancer[J].Oncol Rep,2006,15:545-549.
17 Gunduz M,Ouchida M,Fukushima K,et al.Genomic structure of the human ING1 gene and tumor-specific mutations detected in head and neck squamous cell carcinomas[J].Cancer Res,2000,60:3143-3146.
18曹云飞,陈利生,高枫.ING1启动子甲基化在散发性结直肠癌中的研究[J].中华实验外科杂志,2005,22(12):1584.
19 Walker NJ.Real-time and quantitative PCR:applications to mechanism-based toxicology[J].J Biochem Mol Toxicol,2001,15:121-127.
20 Chen WX,Chen YQ,Huang XZ,et al.Application of fluorogenic quantitative PCR in tumor research[J].J Med Mol Biol,2004,1:323-326.
21詹水凯,贾水义.荧光定量PCR数据处理方法的探讨[J].生物技术,2008,18(3):89-91.
22 Knudson AG.Hereditary cancer:two hits revisited[J].J Cancer Res Clin Oncol,1996,122:135-40.
23 Virginia AS,Jian MS,Lin L.Chromatin immunoprecipitation:a tool for studying histone acetylatlon and transcription factor binding[J].Methods,2003,31(1):67-75.
24 Wells J,Farnham PJ.Characterizing transcription factor binding sites using formaldehyde crosslinking and immunoprecipitation[J].Methods,2002,26 (1):48-56.
25 Orlando V,Strutt H,Paro R.Analysis of chromatin structure by in vivo formaldehyde cross-linking[J].Methods,1997,11(2):205-214.
26 Johnson KD,Bresnick EH.Dissecting long-range transcriptional mechanisms by chromatin immunoprecipitation[J].Methods,2002,26(1):27-36.
27 Murrell A,Rakyan VK,Beck S.From genome to epigenome[J].Hum Mol Genet,2005,14(Review Issue):R3-R10.
28 Parsons DW,Wang TL,Samuels Y,et al.Colorectal cancer:mutations in a signall- ing pathway[J].Nature,2005,436(7052):792.
29薛京伦主编.表观遗传学——原理、技术与实践[M].第1版.上海:上海科学技术出版社,2006:4.
30 Feinberg AP,Ohlsson R,Henikoff S.The epigenetic progenitor origin of human cancer[J].Nat Rev Genet,2006,7:21-23.
31 Baylin SB,Ohm JE.Epigenetic gene si1eneing in caneer-A mechanism for early oncogenic Pathway addiction?[J].Nat Rev Cenet,2006,6:107-116.
32 Jones PA,Bay1in SB.The fundamenta1 ro1e of epigenetic events in carcer [J].Nat Rev Genet,2002,3:415-428.
33孟春风,戴冬秋.组蛋白修饰与胃肠道恶性肿瘤的关系[J].中国普外基础与临床杂志,2007,14:608-611.
34朱新江,戴冬秋.表遗传学与胃肠道肿瘤[J].世界华人消化杂志,2006,14:3251-325.
35 Baylin SB,Herman JG,GraffJR,et al.Alterations in DNA methylation:a fundamental aspect of neoplasia[J].Adv Caneer Res,1998,72:141-196.
36 Murai M,Toyota M,Suzuki H,et al.Aberrant methylation and silencing of the BNIP3 gene in colorectal and gastric cancer[J].Clinical Cancer Research,2005,11:1021-1027.
37 Lind GE,Thorstensen L,Lovig T,et al.A CpG island hypermethylation profile of primary colorectal carcinomas and colon cancer cell lines[J].Molecular Cancer,2004,3:28.
38 Kim KH,Choi JS,Kim IJ,et al.Promoter hypomethylation and reactivation of MAGE-A1 and MAGE-A3 genes in colorectal cancer cell lines and cancer tissues[J].World J Gastroenterol,2006,12(35):5651-5657.
39 Esteller M.Dormant hypermethylated tumor suppressor genes:questions and answers[J].J Pathol,2005,205(2):172-180.
40 Zeremski M,Horrigan SK,Grigorian IA,et al.Localization of the candidate tumor suppressor gene ING1 to human chromosome 13q34[J].Somat Cell Mol Genet,1997,23(3):233-236.
41 Garkavtsev I,Demedtrick D,Riabowol K.Cellular localization and chromosome mapping of a novel candidate tumor suppressor gene(ING1)[J].Cytogenet Cell Genet,1997,76(3-4):176-178.
42 Cheung KJ,Li G.The tumor suppressor ING1:structure and function[J].Exp Cell Res,2001,268:1-6.
43 Shen DH,KYK C,Khoo US,et al.Epigenetic and genetic alterations of p33ING1b in ovarian cancer[J].Carcinogenesis,2005,26(4):855-863.
44 Goel A,Arnold CN,Tassone P,et al.Epigenetic inactivation of RUNX3 in microsatellite unstable sporadic colon cancers[J].Int J Cancer,2004,112(5):754-759.
45韦建宝,陈利生,高枫.2种不同ING1 mRNA剪接体在大肠癌组织中的表达研究[J].实用癌症杂志,2004,19(3):259-261.
46 Marks PA,Rifkind RA,Richon VM,et al.Histone deacetylases and cancer:causes and therapies[J].Nat Rev Cancer,2001,1(3):194-202.
47 Marks PA,Richon VM,Breslow R,et al.Histone deacetylase inhibitors as new anticancer drugs[J].Curr Opin Oncol,2001,13(6):477-483.
48 Dang WW,Steffen KK,Perry R,et al.Histone H4 lysine 16 acetylation regulates cellular lifespan[J].Nature,2009,459:802-808.
49 Fragaa MF,Esteller M.Epigenetics and aging:the targets and the marks [J].Trends Genet,2007,23(8):413-418.
50陈萦晅,房静远,陆娟,等.人结肠癌细胞中组蛋白乙酰化对细胞周期调节基因表达的影响[J].中华医学杂志,2004,84(4):312-317.
51 Kondo Y,Shen L,Issa JP.Critical role of histone methylation in tumor suppressor gene silencing in colorectal cancer[J].Mol Cell Biol,2003,23(1):206-215.
52 Bird A,Wolffe AP.Methylation-induced repression-belts braces and chromatin [J].Cell,1999,9:4512-4541.
53 Cervoni L,Egistelli L,Eufemi M,et al.DNA sequences acting as binding sites for NM23/NDPK proteins in melanoma M14 cells[J].J Cell Biochem,2006,98: 421-428.
54 Nagahama Y,Ishimaru M,Osaki M,et al.Apoptotic pathway induced by transduc- tion of RUNX3 in the human gastric carcinoma cell line MKN-1[J].Cancer Sci,2008,99:23-30.
55唐卫中,高枫,李卫.结直肠癌APC、K-ras、p53基因突变的检测[J].肿瘤,2006,26(3):282-284.
56徐艳松,唐卫中,高枫.散发性结直肠癌APC、K-ras、p53、MMR基因突变的检测[J].结直肠肛门外科,2009,15(4):229-232.
57 Jones PA,Laird PW.Cancer epigenetics comes of age[J].Nat Genet,1999,21(2):163-167.
58陈利生,李天煜,曹云飞,等.p33ING1在肛管癌中的表达及与p53和细胞凋亡的关系[J].中华胃肠外科杂志,2006,9(4):338-341.
59刘家旋,陈利生,李天煜.结直肠癌中p33ING1与VEGF蛋白表达及其临床意义[J].结直肠肛门外科,2009,15(4):252-255.
60梁君林,周永淳,陈利生.结直肠癌中抑癌基因ING1的表达及其意义[J].结直肠肛门外科,2006,12(4):270-272.
61 Dang WW,Steffen KK,Perry R,et al.Histone H4 lysine 16 acetylation regulates cellular lifespan[J].Nature,2009,459:802-808.
62 Bakin AV,Curra T.Role of DNA 5-Methylcytosine Transferase in Cell Trans- formation by fos[J].Science,1999,283(5400):387-390.
63 Murakami J,Asaumi J,Maki Y,et al.Effects of demethyhtingagent 5-Aza-2'- deoxycytidine and histone deacetylase inhibitor FR901228 on maspin gene expression in oral cancer cell lines[J].Oral Oncel,2004,40:597-603.
64 Jablonka E,Lamb MJ.The changing concept of epigenetics[J].Ann NY Acad Sci,2002,981:82-96.
65 Attwood JT,Yung RL,Richardson BC.DNA methylation and the regulation of gene transeription[J].Cell Mol Lif Sci,2002,59:241-257.
66 Finnegan EJ,Kovae KA.Plant DNA methyltransferases[J].Plant Mol Biol,2000,43:189-201.
67程苏琴,曹佳,刘晋神,等.结直肠癌中p16基因的甲基化改变与蛋白表达的关系[J].中华检验医学杂志,2004,7(5):307-310.
68 Pham AD,Sauer F.Ubiquitin-activating/conjugating activity of TAFII250,a mediator of activation of gene expression in Drosophila[J].Science,2000,289(5488):2357-2360.
69 Ng HH,Bird A.DNA methylation and chromatin modification[J].Curr Opin Genet Dev,1999,9(2):158-163.
70 Fang JY,Chen YX,Lu J,et al.Epigenetic modification regulations both expression of tumor-associated genes and cell cycle progressing in human colon cancer cell 1ines:Colo320 and SWlll6[J].Cell Res,2004,14:217-226.
71 Herman JG,Umar A,Polyak K,et al.Incidence and functional consequences of MLH1 Promoter hypermethylation in colorectal carcinoma[J].Proc Natl Acad Sci USA,1998,95:6870-6875.
72 Nguyen CT,Weisenberger DJ,Velicescu M,et al.Histone H3-Lysine 9 methylation is associated with aberrant gene silencing in cancer cells and is rapidly reversed by 5-Aza-2’-deoxycytidine[J].Cancer Research,2002,62:6456-6461.
73 Wolffe AP,Matzke MA.Epigenetics:regulation through repression[J].Science,1999,286(5439):481-486.
74 Cameron EE,Bachman KE,My?h?nen S,et al.Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer [J].Nat Genet,1999,21(1):103-107.
75 Fahrner JA,Eguchi S,Herman JG.Dependence of histone modifications and gene expression on DNA hypermethylation in cancer[J].Cancer Res,2002,62:7213- 7218.
76 Graff JR,Gabrielson E,Fujii H,et al.Methylation patterns of the E-cadherin 5'CpG island are unstable and reflect the dynamic, heterogeneous loss of E-cadherin expression during metastatic progression[J].J Biol Chem,2000,275(4):2727-2732.
77 Dobosy JR,Roberts JL,al FV.The expanding role of epigenetics in the development,diagnosis and treatment of prostate cancer and benign prostatic hyperplasia[J].J Urol,2007,177:822-831.
78 Li LC,Carroll PR,Dahiya R.Epigenetic changes in prostate cancer:implication for diagnosis and treatment[J].J Natl CancerInst,2005,97(2):103-115.
79 Digel W,Lubbert M.DNA methylation disturbances as novel therapeutic target in lung cancer: preclinical and clinical results[J].Crit Rev Oncol Hemato,2005,55(1):1-11.
1李明,顾晋.中国结直肠癌20年来发病模式的变化趋势[J].中华胃肠外科杂志,2006,7(3):214-217.
2 Cho KR,Vogelstein B.Genetic alterations in the adenoma-carcinoma sequence [J].Cancer,1992,70(6 Suppl):1727-1731.
3 Murrell A,Rakyan VK,Beck S.From genome to epigenome[J].Hum Mol Genet,2005,14(Review Issue):R3-R10.
4 Parsons DW,Wang TL,Samuels Y,et al.Colorectal cancer:mutations in a signall- ing pathway[J].Nature,2005,436(7052):792.
5 Swinburne LA,Meyer CA,Liu XS,et al.Genomic localization of RNA binding proteins reveals links between pre-mRNA processing and transcription [J].Genome Res,2006,16:912-921.
6陈浩明,薛京伦主编.医学分子遗传学[M].第1版.上海:科学出版社,2005:172-175.
7 Jablonka E,Lamb MJ.The changing concept of epigenetics[J].Ann NY Acad Sci,2002,981:82-96.
8薛京伦主编.表观遗传学——原理、技术与实践[M].第1版.上海:上海科学技术出版社,2006:4.
9 Jonas PA,Laird PW.Cancer epigenetics comes of age[J].Nature Genet,1999,21(2):163-167.
10 Esteller M.Dormant hypermethylated tumor suppressor genes:questions and answers[J].J Pathol,2005,205(2):172-180.
11 Ehrlich M . DNA methylation in cancer:too much, but also too little [J].Oncogene,2002,21(35):5400–5413.
12 Soares J,Pinto AE,Cunha CV,et al.Global DNA hypomethylation in breast carcinoma : corelation with prognostic factors and tumor progression [J].Cancer,1999,85(1):112-118.
13 Stephen BB,Joyce EO.Epigenetic gene silencing in cancer-a mechanism for early oncogenic pathway addiction?[J].Nature Reviews Cancer,2006,6:107- 116.
14 Jenuwein T,Allis CD.Translating the histone code[J].Science,2001,293(5532):1074-1080.
15 Graff JR,Gabrielson E,Fujii H,et al.Methylation patterns of the E-cadherin 5'CpG island are unstable and reflect the dynamic, heterogeneous loss of E-cadherin expression during metastatic progression[J].J Biol Chem,2000,275(4):2727-2732.
16 Marks PA,Rifkind RA,Richon VM,et al.Histone deacetylases and cancer:causes and therapies[J].Nat Rev Cancer,2001,1(3):194-202.
17 Klose RJ,Kallin EM,Zhang Y.JmjC-domain-containing proteins and histone demethylation[J].Nat Rev Genet,2006,7(9):715-727.
18 Linggi BE,Brandt SJ,Sun ZW,et al.Translating the histone code into leukemia [J].J Cell Biochem,2005,96(5):938-950.
19 Wang H,Huang ZQ,Xia L,et al.Methylation of histone H4 at arginine facilitating transcriptional activation by nuclear hormone receptor [J].Science,2001,293(5531):853-857.
20 Pham AD,Sauer F.Ubiquitin-activating/conjugating activity of TAFII250,a mediator of activation of gene expression in Drosophila[J].Science,2000,289(5488):2357-2360.
21 Nakayama J,Rice JC,Strahl BD,et al.Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly[J].Science,2001,292 (5541):110-113.
22 Dang WW,Steffen KK,Perry R,et al.Histone H4 lysine 16 acetylation regulates cellular lifespan[J].Nature,2009,459:802-808.
23 Fraga MF,Esteller M.Epigenetics and aging:the targets and the marks[J].Trends Genet,2007,23(8):413-418.
24 Cameron EE,Bachman KE,My?h?nen S,et al.Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer [J].Nat Genet,1999,21(1):103-107.
25 Selker EU.Trichostatin A causes selective loos of DNA methylation in Neuro- spora[J].Proc Natl Acad Sci USA,1998,95(16):9430-9435.
26 Kondo Y,Shen L,Issa JP.Critical role of histone methylation in tumor suppressor gene silencing in colorectal cancer[J].Mol Cell Biol,2003,23(1):206-215.
27 Bakin AV , Curra T . Role of DNA 5-Methylcytosine Transferase in Cell Transformation by fos[J].Science,1999,283(5400):387-390.
28 Ng HH,Bird A.DNA methylation and chromatin modification[J].Curr Opin Genet Dev,1999,9(2):158-163.
29 Mathers JC.Reversal of DNA hypomethylation by folic acid supple- ments: possible role in colorectal cancer prevention[J].Gut,2005,54 (5):579-581.
30 Bariol C,Suter C,Cheong K,et al.The relationship between hypomethylation and CpG island methylation in colorectal neoplasia[J].Am J Pathol,2003,162(4):1361-1371.
31 He C,Hu HQ,Braren R,et al.c-myc in the Hematopoietic Lineage is Crucial for its Angiogenic Function in the Mouse Embryo[J].Development,2008,135(14): 2467-2477.
32 Sharrard RM,Royds JA,Rogers S,et al.Patterns of methylation of the c-myc gene in human colorectal cancer progression[J].Br J Cancer,1992,65(5):667- 672.
33 Howe LR,Dannenberg AJ.A role for cyclooxygenase-2 inhibitors in the prevention and treatmention of cancer[J].Semin Oncol,2002,29(3 Suppl 11): 111-119.
34 Park GY,Joo M,Pedchenko T,et al.Regulation of macrophage cyclooxygenase-2 gene expression by modification of histon H3[J].Am J Physiol Lung cell Mol Physiol,2004,286(5):L956-L962.
35 Toyota M,Shen L,Ohe-Toyota M,et al.Aberrant methylation of the Cyclooxygen- ase 2 CpG island in colorectal tumors[J].Cancer Res,2000,60 (15):4044-4048.
36 Bargmann CI,Hung MC,Weinberg RA.The neu oncogene encodes an epidermal growth factor receptor-related protein[J].Nature,1986,319(6050):226-230.
37 Akiyama T,Sudo C,Ogawara H,et al.The product of the human C-erbB-2 gene:a 185-kilodahon glycoprotein with tyrosine kinase activity[J].Science,1986,232(4758):1644-1646.
38金中淦,袁亚,陆青.结直肠癌C-erbB-2基因启动子CpG岛甲基化状态及其与C-erbB-2蛋白表达的关系[J].检验医学,2008,23(6):594-599.
39 Paz MF,Fraga MF,Avila S,et al.A systematic profile of DNA methylation in humancancer cell lines[J].Cancer Res,2003,63(5):1114-1121.
40 Gonzalez-Zulueta M,Bender CM,Yang AS,et al.Methylation of the 5'CpG island of the p16/CDKN2 tumor suppressor gene in normal and transformed human tissues correlates with gene silencing[J].Cancer Res,1995,55(20):4531- 4535.
41 Wiencke JK,Zheng S,Lafuente A,et al.Aberrant methylation of p16INK4a in anatomic and gender-specific subtypes of sporadic colorectal cancer [J].Cancer Epidemiol,Biomarkers & Prev,1999,8(6):501-506.
42 Liang JT,Chang KJ,Chen JC,et al.Hypermethylation of the p16 gene in sporadic T3N0M0 stage colorectal cancers:association with DNA replication error and shorter survival[J].Oncology,1999,57(2):149-156.
43 Fang XM,Zheng S,Jiang CH,et al.Effects of promoter Region 5'CpG island demethylation on the biological behavior of human colorectal cancer RKO cells in vitro[J].Chin J Clin Oncol,2008,5(1):10-15.
44方晓明,孙立峰,彭佳萍,等.5-氮-2'-脱氧胞苷对RKO结肠癌细胞株pl6/CDKN2基因去甲基化的转录调节作用[J].中华医学杂志,2003,83(23):2077-2084.
45于波,李世拥,安萍.p16基因启动子甲基化与结直肠癌发生发展的关系[J].中华普通外科杂志,2003,18(1):31-33.
46程苏琴,曹佳,刘晋神,等.结直肠癌中p16基因的甲基化改变与蛋白表达的关系[J].中华检验医学杂志,2004,7(5):307-310.
47 Sato F,Harpaz N,Shibata D,et al.Hypermethylation of the p14(ARF) gene in ulcerative colitis-associated colorectal carcinogenesis[J].Cancer Res,2002,62(4):1148-1151.
48 Shen L,KondoY,Hamilton SR,et al.P14 methylation in human colon cancer is associated with microsatellite instability and wild-type p53 [J].Gastroenterology,2003,124(3):626-633.
49 Esteller M , Tortola S , Toyota M , et al . Hypermethylation-associated inactivation of p14(ARF) is independent of p16(INK4a) methylation and p53 mutational status[J].Cancer Res,2000,60(1):129-133.
50 Veigl ML,Kasturi L,Olechnowicz J,et al.Biallelic inactivation of hMLH1 by epigeneticgene silencing,a novel mechanism causing human MSI cancers [J].Proc Natl Acad Sci USA,1998,95(15):8698-8702.
51蔡国响,蔡三军.结直肠癌发病的分子机理探讨[J].临床肿瘤学杂志,2003,8(6):467-470.
52 Potocnik U,Glavac D,Golouh R,et al.Causes of microsatellite instability in colorectal tumors:implications for hereditary non-polyposis colorectal cancer screening[J].Cancer Genet Cytogenet,2001,126(2):85-96.
53 Deng G,Chen A,Hong J,et al.Methylation of CpG in a small region of the hMLH1 promoter invariably correlates with the absence of gene expression [J].Cancer Res,1999,59(9):2029-2033.
54 Arnold CN,Goel A,Boland CR.Role of hMLH1 promoter hypermethylation in drugresistance to 5-fluorouracil in colorectal cancer cell lines[J].Int J Cancer,2003,106(1):66-73.
55 Plumb JA,Strathdee G,Sludden J,et al.Reversal of drug resistance in human tumor xenografts by 2'-deoxy-5-azacytidine-induced demethylation of the hMLH1 gene promoter[J].Cancer Res,2000,60(21):6039-6044.
56 Kim SH,Bae SI,Lee HS,et al.Alteration of O(6)-methylguanine-DNA methyltransferase in colorectal neoplasms in sporadic and familial adenom- atous polyposis patients[J].Mol Carcinog,2003,37(1):32-38.
57 Nagasaka T,Sharp GB,Notohara K,et al.Hypermethylation of O(6)-me- thylguanine-DNA methyltransferase promoter may predict nonrecurrence after chemotherapy in colorectal cancer cases[J] . Clin Cancer Res , 2003 ,9(14):5306-5312.
58 Krtolica K,Krajnovic M,Usaj-Knezevic S,et al.Comethylation of p16 and MGMT genes in colorectal carcinoma:Correlation with clinclinicopath- ological features and prognostic vaIue[J].World Gastroenterol,2007,13(8): 1187-1194.
59 Garkavtsev I,Demedtrick D,Riabowol K.Cellular localization and chromosome mapping of a novel candidate tumor suppressor gene(ING1)[J].Cytogenet Cell Genet,1997,76(3-4):176-178.
60 Garkavtsev I,Riabowol K.Extension of the replicative life span of human diploid fibroblasts by inhibition of the p331NG1 candidatetumor suppressor [J].Mol Cell Biol,1997,17(4):2014-2019.
61 Oki E,Niaehara Y,Tokunaga E,et al.Reduced expression of p33(ING1) and the relationship with p53 expression in human gastric cancer[J].Cancer lett,1999,147(1-2):157-162.
62韦建宝,陈利生,高枫.散发性结直肠癌组织中抑癌基因ING1的突变、杂合性缺失及表达[J].癌症,2005,24(2):141-144.
63曹云飞,陈利生,高枫.ING1启动子甲基化在散发性结直肠癌中的研究[J].中华实验外科杂志,2005,22(12):1584.
64唐卫中,高枫,李卫,等.中国人散发性大肠癌APC基因突变的研究[J].中华实验外科杂志,2005,22(11):1357-1359.
65王夫景,杨茂鹏,于洪亮,等.抑癌基因KLF-6和APC在结直肠癌组织中的表达及临床意义[J].中华胃肠外科杂志,2006,9(5):429-432.
66汪建平,元云飞.结直肠癌DNA甲基化研究的现状[J].中华实验外科杂志,2004,21:1279-1280.
67黄美近,褚忠华.E-钙黏附素基因甲基化与结直肠癌临床病理特征的关系[J].中华实验外科杂志,2005,22(11):1355-1356.
68 Futscher BW,Oshiro MM,Wozniak RJ,et al.Role for DNA methylation in the control of cell type-specific maspin expression[J].Nat Genet,2002,31(2): 175-179.
69唐波彭,志红余,佩武,等.5-氮-2'-脱氧胞苷对RKO结肠癌细胞株maspin基因去甲基化的转录调节作用[J].中华胃肠外科杂志,2006,9(3):260-263.
70高鹏,崔铮,王静.Maspin在大肠癌中的表达及其启动子5'CpG岛甲基化状态的研究[J].广东医学,2007,28(10):1587-1589.
71 Hirai H,Maru Y,Hagiwara K,et al.A novel putative tyrosine kinase receptor encoded by the eph gene[J].Science,1987,238(4834):1717-1720.
72 Kataoka H,Igarashi H,Kanamori M,et al.Correlation of EPHA2 overexpression with high microvessel count in human primary colorectal cancer[J].Cancer Sci,2004,95(2):136-141.
73王建东,周晓军.高甲基化导致EphA7基因在结直肠癌中低表达[J].医学研究生学报,2007,20(1):6-14.
74 Toyota M,Ahuja N,Ohe-Toyota M,et al.CpG island methylator phenotype in colorectal cancer[J].Proc Natl Acad Sci USA,1999,96(15):8681-8686.
75 Toyota M,Ho C,Ahuja N,et al.Identification of differentially methylatedsequences in colorectal cancer by methylated CpG island amplification [J].Cancer Res,1999,59(10):2307-2312.
76 Van Rijnsoever M,Grieu F,Elsaleh H,et al.Characterisation of colorectal cancers showing hypermethylation at multiple CpG islands[J].Gut,2005,51(6):797-802.
77 Van Rijnsoever M,Elsaleh H,Joseph D,et al.CpG island methylator phenotype is an independent predictor of survival benefit from 5-fluorouracil in stage III colorectal cancer[J].Clin Cancer Res,2003,9(8):28988-2903.
78蔡国响,蔡三军,徐烨,等.甲基子表型阳性的散发性大肠癌的临床病理特征[J].中华消化杂志,2006,26(6):377-380.
79席亚鸣,谢庆芳,孙蓓.抑癌基因甲基化检测结直肠癌患者的研究[J].中华实验外科杂志,2003,20(3):269-270.
80陈萦亘,房静远,陆娟,等.人结肠癌细胞系中癌相关基因的表达及表型遗传修饰的影响[J].临床与实验病理学杂志,2003,19(5):533-538.
81陈萦亘,房静远,陆娟.人结肠癌细胞中组蛋白乙酰化对细胞周期调节基因表达的影响[J].中华医学杂志,2004,84(4):312-317.
82李天煜,陈利生,何纯刚,等.5-氮-2'-脱氧胞苷及TSA对人结肠癌细胞株HT29 ING1b基因表达的影响[J].广东医学,2009,30(10):1465-1467.
83 Esteller M,Garcia-Foncillas J,Andion E,et al.Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents[J].N Engl J Med,2000,343(19):1350-1354.
84 Esteller M,Hamilton SR,Burger PC,et al.Inactivation of the DNA repair gene 06-methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia[J].Cancer Res,1999,59(4):793-797.
85 Kreklau EL,Pollok KE,Bailey BJ,et al.Hematopoietic expression of O(6)-methylguanine DNA methyltransferase-P140K allows intensive treatment of human glioma xenografts with combination O(6)-benzylguanine and 1,3-bis-(2-chloroethyl)-1-nitrosourea[J].Mol Cancer Ther,2003,2(12):1321- 1329.
86 Ward IM,Minn K,Deursen v,et al.p53 Binding protein 53BP1 is required for DNA damage responses and tumor suppression in mice[J].Mol Cell Biol,2003,23(7):2556-2563.
87 Marks PA,Richon VM,Breslow R,et al.Histone deacetylase inhibitors as new anticancer drugs[J].Curr Opin Oncol,2001,13(6):477-483.
88 Shaker S,Bernstein M,Momparler IF,et al.Preclinica1 evaluation of antineoplastic activity of inhibitors of DNA methylation (5-aza-2'- deoxycytidine)and histone deacetylation (trichostatin A,depsipeptide) in combination against myeloid leukemic cells[J].Leuk Res,2003,27(5):437-444.
89 Rashid SF,Moore JS,Walker E,et al.Synergistic growth inhibition of prostate cancer cells by 1 alpha,25 Dihydroxyvitamin D(3) and its 19-nor-hexafluoride ana1ogs in combination with either sodium butyrate or trichostatin A [J].Oncogene,2001,20(15):1860-1872.
90 Esteller M,Sanchez-Cespedes M,Rosell R,et al.Detection of aberrant promoter hypermethylation of tumor suppressor genes in serum DNA from nonsmall cell lung cancer patients[J].Cancer Res,1999,59(1):67-70.
91 Toyota M,huh F,Imai K.DNA methylation and gastrointestinal malignances: functional consequences and clinical implications[J].J Gastroenterol,2000,35(10):727-734.
92 Nakayama H,Hibi K,Takase T,et al.Molecular detection of pl6 promoter methy- lation in the serum of recurrent colorectal cancer patients[J].Int J Cancer,2003,105(4):491-493.
93 Marks PA,Jiang X.Histone deacetylase inhibitors in programmed cell death and cancer therapy[J].Cell Cycle,2005,4(4):549-551.
94 Li LC,Carroll PR,Dahiya R.Epigenetic changes in prostate cancer:implicationfor diagnosis and treatment[J].J Natl CancerInst,2005,97(2):103-115.
95 Digel W,Lubbert M.DNA methylation disturbances as novel therapeutic target in lung cancer: preclinical and clinical results[J].Crit Rev Oncol Hemato,2005,55(1):1-11.
96 Espino PS,Drobic B,Dunn KL,et al.Histone modifications as aplatform for cancer therapy[J].J Cell Biochem,2005,94(6):1088-1102.
1 Strahl BD , Allis CD . The language of covalent histone modifications [J].Nature,2000,403(6765):41-45.
2 Jenuwein T,Allis CD.Translating the histone code[J].Science,2001,293 (5532):1074-1080.
3 Xu DD,Liu DP,Ji XJ,et al.In vivo DNA-protein interactions at hypersensitive site 3.5 of the human beta-globin locus control region[J].Biochem Cell Biol,2001,79(6):747-754.
4 Fu XH,Liu DP,Liang CC.Chromatin structure and transcriptional regulation of the beta-globin locus[J].Exp Cell Res,2002,278(1):1-11.
5 Spencer VA,Sun JM,Li L,et al.Chromatin immunoprecipitation:a tool for studying histone acetylation and transcription factor binding[J].Methods,2003,31(1):67-75.
6 Mayr B, Montminy M.Transcriptional regulation by thephosphorylation -dependent factor CREB[J].Nat Rev Mol Cell Biol,2001,2:599-609.
7 Brutlag D,Schlehuber C,Bonner J.Properties of formaldehyde-treated nucleo- histone[J].Biochemistry,1969,8(8):3214-3218.
8 Ilyin YV,Georgiev GP.Heterogeneity of deoxynucleoprotein particles as evidence by ultracentrifugation of cesium chloride density gradient[J].J Mol Biol,1969,41(2):299-303.
9 O'Neill LP,Turner BM.Immunoprecipitation of native chromatin: NchIP [J].Methods,2003,31(1):76-82.
10 Wells J,Farnham PJ.Characterizing transcription factor binding sites using formaldehyde crosslinking and immunoprecipitation[J].Methods,2002,26(1):48-56.
11 Orlando V,Strutt H,Paro R.Analysis of chromatin structure by in vivo formaldehyde cross-linking[J].Methods,1997,11(2):205-214.
12 Johnson KD,Bresnick EH.Dissecting long-range transcriptional mechanisms by chromatin immunoprecipitation[J].Methods,2002,26(1):27-36.
13 Kuo MH,Allis CD.In vivo cross-linking and immunoprecipitation for studying dynamic protein:DNA associations in a chromatin environment[J].Methods,1999,19(3):425-433.
14 Hecht A,Grunstein M.Mapping DNA interaction sites of chromosomal proteins using immunoprecipitation and polymerase chain reaction[J] . Methods Enzymol,1999,304:399-414.
15 Orlando V.Mapping chromosomal proteins in vivo by formaldehyde-crosslinked- chromatin immunoprecipitation[J].Trends Biochem Sci,2000,25(3):99-104.
16 Kristen M,Meisenheimer KM,Koch TH.Photocross-linking of nucleicacids to associated proteins[J].Crit Rev Biochem Mol Biol,1997,32(2):101-140.
17 Zhang L,Zhang K,Prandl R,et al.Detecting DNA-binding of proteins in vivo by UV-rosslinking and immunoprecipitation[J].Biochem Biophys Res Commun,2004,322(3):705-711.
18 Hanson RW,Reshef L.Regulation of phosphoenelpyruvate carboxykinase(GTP) gene expression[J].Ann Rev Biochem,1997,66:581-611.
19 Solomon MJ,Varshavsky A.Formaldehyde-mediated DNA-protein crosslinking:A probe for in vivo chromatin structures[J].Proc Natl Acad Sci USA,1985,82(19):6470-6474.
20 Spencer VA,Samuel SK,Davie JR.Altered Profiles in Nuclear Matrix Proteins Associated with DNA in Situ during Progression of Breast Cancer Cells [J].Cancer Res,2001,61(4):1362-1366.
21 Ren B,Robert F,Wyrick JJ,et al.Genome-wide location and function of DNAbinding proteins[J].Science,2000,290(5500):2306-2309.
22 Weinmann AS,Yan PS,Oberley MJ,et al.Isolating human transcription factor targets by coupling chromatin immunoprecipitation and CpG island microarray analysis[J].Genes Dev,2002,16(2):235-244.
23 Shannon MF,Rao S.Of chips and ChIPs[J].Science,2002,296(5568):666-669.
24 Kang SH,Vieira K,Burgert J.Combining chromatin immunoprecipitation and DNA footprinting:a novel method to analyze protein-DNA interaction in vivo[J].Nucleic Acids Res,2002,30(10):e44.
25 Niranjanakumari S,Lasda E,Brazas R,et al.Reversible cross-linking combined with immunoprecipitation to study RNA-protein interactions in vivo [J].Methods,2002,26(1):182-190.
26 Steensel BV.Mapping of genetic and epigenetic regulatory networks using microarrays[J].Nature Genetics Supplement,2005,37:s18-s24.
27 Weinmann AS.Novel ChIP-based strategies to uncover transcription factor target genes in the immune system[J].Nature Reviews Immunology,2004,4:381-386.
28 Weinmann AS,Farnham PJ.Identification of unknown target genes of human transcription factors using chromatin immunoprecipitation[J].MetIlods,2002,26(1):37-47.
29 Lei H,Juan AH,Kim MS,et al.Identification of a Hoxc8-regulated transcrip- tional network in mouse embryo fibroblast cells[J].Proc Nail Acad Sci USA,2006,103(27):10305-10309.
30 Oh SW,Mukhopadhyay A,Dixit BL,et al.Identification of direct DAF-16 targets controlling longevity,metabolism and diapause by chromatin immunoprecipit ation[J].Nat Genet,2006,38(4):251-257.
31 Hearnes JM,Mays DJ,Schavoh K,et al.Chromatin immunoprecipitation-based screen to identify functional genomic binding sites for sequence-specifictransactivators[J].Mol Cel Biol,2005,25(22):10148-10158.
32 Moss T,Dimitrov SI,Houde D.UV-laser crosslinking of proteins to DNA [J].Methods,1997,11(2):225-234.
33 Buckle M,Geiselmann J,Kolb A,et al.Protein-DNA cross-linking at the lac promoter[J].Nucleic Acids Res,1991,19(4):833-840.
34 Russmann C,Truss M,Fix A,et al.Crosslinking of progesterone receptor to DNA using tuneable nanosecond,picosecond and femtosecond UV laser pulses [J].Nucleic Acids Res,1997,25(12):2478-2484.
35 Hockensmith JW,Kubasek WL,Vorachek WR,et al.Laser cross-linking of nucleic acids to proteins:Methodology and first applications to the phage T4 DNA replication system[J].J Biol Chem,1986,261(8):3512-3518.
36 Nagaich AK,Walker DA,Wolford R,et al.Rapid periodic binding and displacement of the glucocorticoid receptor during chromatin remodeling [J].Mol Cell,2004,14(2):163-174.
37 Zhang LM,Eggers-Schumacher G,Schoffl F,et al.Analysis of heat-shock transcription factor-DNA binding in Arabidopsis suspension cultures by UV laser crosslinking[J].Plant,2001,28(2):217-223.
38 Shang Y,Hu X,DiRenzo J,et al.Cofactor dynamics and sufficiency in estrogen receptor[J].Cell,2000,103(6):843-852.
39 Shang Y,Brown M.Molecular determinants for the tissue specificity of SERMs [J].Science,2002,295(5564):2465-2468.
40 Weinmann AS,Bartley SM,Zhang T,et al.Use of chromatin immunoprecipitation to clone novel E2F target promoters[J].Mol Cell Biol,2001,21(20):6820-6832.
41 Iyer VR,Horak CE,Scafe CS,et al.Genomic binding sites of the yeast cell-cycle transcription factors SBF and MBF[J] . Nature , 2001 , 409 (6819):533-538.
42 Lieb JD,Liu X,Botstein D,et al.Promoter-specific binding of Rap1 revealedby genome-wide maps of protein-DNA association[J].Nat Genet,2001,28(4): 327-334.
43 Kapranov P,Cawley SE,Drenkow J,et al.Large-scale transcriptional activityin chromosomes 21 and 22[J].Science,2002,296(5569):916-919.
44 Zella LA,Kim S,Shevde NK,et al.Enhancers located within two introns of the vitamin D receptor gene mediate transcriptional autoregulation by 1,
25-dihydroxyvitamin D3[J].Mol Endocrinol,2006,20(6):1231-1247.
45 Banerjee N,Zhang MQ.Identifying cooperativity among transcription factors controlling the cell cycle in yeast[J].Nucleic Acids Res,2003,31(23):7024- 7031.
46 Santoro R,W?lfl S,Saluz HP.UV-Laser induced protein/DNA crosslinking reveals sequence variations of DNA elements bound by c-Jun in vivo [J].Biochem and Biophys Res Commun,1999,256(1):68-74.
47 Solano PJ,Mugat B,Martin D,et al.Genome-wide identification of in vivo drosophila engrailed-binding DNA fragments and related target genes [J].Development,2003,130(7):1243-1254.
48 Abdurashidova G,Danailov MB,Ochem A,et al.Localization of proteins bound to a replication origin of human DNA along the cell cycle[J].EMBO,2003,22:4294-4303.
49 Van Lente F,Jackson JF,Weintraub H.Identification of specific crosslinked histones after treatment of chromatin with formaldehyde[J].Cell,1975,5(1):45-50.
50 Jackson V.Studies on histone organization in the nucleosome using formalde- hyde as a reversible cross-linking agent[J].Cell,1978,15 (3):945-954.
51 Jackson V,Chalkley R.A new method for the isolation of replicative chromatin: selective deposition of histone on both new and old DNA[J].Cell,1981,23(1):121-134.
52 Jackson V,Chalkley R.Use of whole-cell fixation to visualize replicating and maturing simian virus 40: identification of new viral gene product [J].Proc Natl Acad Sci USA,1981,78(10):6081-6085.
53 Li T,Vu TH,Ulaner GA,et al.Activating and silencing histone modifications form independent allelic switch regions in the imprinted Gnas gene[J].Hum Mol Genet,2004,13(7):741-750.
54 Shan B,Xu J,Zhuo Y,et al.Induction of p53-dependent activation of the human proliferating cell nuclear antigen gene in chromatin by ionizing radiation[J].J Biol Chem,2003,278(45):44009-44017.
55 Gonzalo S,Jaco I,Fraga MF,et al.DNA methyltransferases control telomere length and telomere recombination in mammalian cells[J].Nat Cell Biol,2006,8:416-424.
56 Storre J,Schafer A,Reichert N,et al.Silencing of the meiotic genes SMC1 {beta} and STAG3 in somatic cells by E2F6[J].J Biol Chem,2005,280:41380- 41386.
57 Angelov D,VYu S,Dimitrov SI,et al.Protein-DNA crosslinking in reconstituted nucleohistone,nuclei and whole cells by piocosecond UV laser irradiation [J].Nucleic Acids Res,1988,16(10):4525-4538.
58 Lejnine S,Murnane M,Kapteyn HC,et al.Crosslinking of proteins to DNA in human nuclei using a 60 femtosecond 266nm laser[J].Nucleic Acids Res,1999,27(18):3676-3684.
59 Mutskov V,Gerber D,Angelov D,et al.Persistent interactions of core histone tails with nucleosomal DNA following acetylation and transcription factor binding[J].Mol Cell Bio,1998,18(11):6293-6304.
60 De Silanes IL,Zhan M,Lai A,et al.Identification of a target RNA motif for RNA-binding protein HuR[J].Proc Natl Acad Sci USA,2004,101 (9):2987- 2992.
61 SenGupta DJ,Zhang B,Kraemer B,et al.A three-hybrid system to detect RNA-protein interactions in vivo[J].Proc Natl Acad Sci USA,1996,93(16): 8496-8501.
62 Swinburne IA,Meyer CA,Liu XS,et al.Genomic localization of RNA binding proteins reveals links between pre-mRNA processing and transcription [J].Genome Res,2006,16:912-921.
63 Braunatein M,Rose AB,Holmes SG,et al.Transcriptional silencing in yeast is associated with reduced nucleosome acetylation[J].Genes Dev,1993,7:592-604.
64 Braunstein M,Sobel RE,Allis CD,et al.Efficient transcriptional silencing in Saccharomyces cerevisiae requires a heterochromatin histone acetylation pattern[J].Mol Cell Biol,1996,16:4349-4356.
65 Kuo MH,Zhou J,Jambeck P,et al.Histone acetyltransferase activity of yeast Gcn5p is required for the activation of target genes in vivo[J].Genes Dev,1998,12:627-639.
66 Dedon PC,Soults JA,Allis CD,et al.A simplified formaldehyde fixation and immunoprecipitation technique for studying protein-DNA interactions [J].Anal Biochem,1991,197(1):83-90.
67 Dedon PC,Soults JA,Allis CD,et al.Formaldehyde cross-linking and immunoprecipitation demonstrate developmental changes in H1 association with transcriptionally active genes[J].Mol CeLL Biol,1991,11(3):1729-1733.
68 Orlando V,Paro R.Mapping Polycomb-repressed domains in the bithorax complex using in vivo formaldehyde cross-linked chromatin[J].Cell,1993,75(6):1187- 1198.
69 Alberts AS,Geneste O,Treisman R.Activation of SRF-regulated chromosomal templates by Rho-family GTPases requires a signal that also induces H4 hyperacetylation[J].Cell,1998,92:475-487.
70 Pereira S,Grayling RA,Lurz R,et al.Archaeal nucleosomes[J].Proc Natl Acad Sci USA,1997,94(23):12633-12537.
71 Boyd KE,Wells J,Gutman J,et al.c-Myc target gene specificity is determined by a post-DNA binding mechanism[J].proc Natl Acad Sci USA,1999,95(23): 13887-13992.
72 Kondo Y,Shen L,Issa JP.Critical role of histone methylation in tumor suppressor gene silencing in colorectal cancer[J].Mol Cell Biol,2003,23(1):206-215.
73 Cameron EE,Bachman KE,My?h?nen S,et al.Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer[J].Nat Genet,1999,21(1):103-107.
74杨发达,李建明,周军,等.人大肠癌细胞PRL-3基因启动子Snail结合位点的初步研究[J].南方医科大学学报,2007,27(4):401-405.
75陈萦亘,房静远,陆娟.人结肠癌细胞中组蛋白乙酰化对细胞周期调节基因表达的影响[J].中华医学杂志,2004,84(4):312-317.
76李天煜,陈利生,何纯刚,等.5-氮-2'-脱氧胞苷及TSA对人结肠癌细胞株HT29 ING1b基因表达的影响[J].广东医学,2009,30(10):1465-1467.
77 Dang WW,Steffen KK,Perry R,et al.Histone H4 lysine 16 acetylation regulates cellular lifespan[J].Nature,2009,459:802-808.
78 Fang XM,Zheng S,Jiang CH,et al.Effects of promoter Region 5'CpG island demethylation on the biological behavior of human colorectal cancer RKO cells in vitro[J].Chin J Clin Oncol,2008,5(1):10-15.