【作者】 李天煜；

【导师】 陈利生；

【作者基本信息】 广西医科大学 ， 外科学， 2010， 博士

【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。更多还原

【Abstract】 The first part:The relationship between p33ING1b gene expression and pattern of epigenetic modificationObjectiveING1 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.MethodsFour 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.Results1.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.Conclusion1.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 expressionObjectiveThe 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.MethodsColorectal 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 Results1.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.Conclusion1.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.更多还原