抑瘤基因RUNX3在胶质瘤中的表达及启动子甲基化的研究
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摘要
脑胶质细胞瘤是颅内常见的原发肿瘤,预后不良。尽管采取了手术、放疗和化疗等综合治疗措施,仍然难以取得满意的疗效。RUNX3基因是人类Runt结构域转录因子家族成员,在神经轴突形成、细胞增殖和凋亡等方面发挥重要的作用。最近的研究表明RUNX3基因在多种肿瘤中表达下调,是一候选抑瘤基因,其表达的调控多与基因启动子甲基化状态有关。但目前关于RUNX3基因在胶质瘤中的表达特点还不清楚,其在胶质瘤中的表达调控机制有待进一步研究。本研究运用免疫组化检测RUNX3蛋白在胶质瘤和正常脑组织中的表达,分析其表达与各临床资料的关系;随后运用MSP检测胶质瘤中RUNX3基因启动子甲基化的状态,初步探讨其在胶质瘤中表达下调的分子机制;最后运用特异性去甲基化药物5-氮胞苷干预胶质瘤细胞株,试图通过逆转甲基化状态恢复RUNX3基因的表达,并检测胶质瘤细胞的增殖和侵袭能力,观察5-氮胞苷对胶质瘤细胞株的影响。
第一章RUNX3蛋白在胶质瘤中的表达及临床意义
目的:研究RUNX3蛋白在正常脑组织和胶质瘤中表达,探索RUNX3基因在胶质瘤中的作用,以及RUNX3蛋白在胶质瘤中异常表达与临床病理资料之间的关系。
方法:运用免疫组化的方法检验了40例脑胶质瘤和8例正常脑组织石蜡标本中RUNX3蛋白的表达情况,并比较RUNX3蛋白在各临床病理特征分组间的差异。
结果:正常脑组织中RUNX3蛋白均为阳性或强阳性表达,而在17/4.0(42.5%)胶质瘤中为阴性或弱阳性表达,其表达水平在胶质瘤中较正常脑组织中明显下降(P=0.024)。RUNX3蛋白在胶质瘤中的表达随着肿瘤恶性程度的增加表达进行性降低(r=-0.478,P=0.002),低级别(Ⅰ-Ⅱ级)胶质瘤中IRS为7.70±3.20,高级别(Ⅲ-Ⅳ级)胶质瘤中IRS为4.75±2.88,RUNX3蛋白在低级别和高级别胶质瘤中表达强度有明显差异(X~2=8.697,P=0.020)。但RUNX3蛋白的表达与胶质瘤的病理类型、患者年龄、性别、肿瘤部位等因素无显著相关性。
结论:RUNX3蛋白在胶质瘤组织中存在明显的表达缺失或下调,并且其表达水平随着肿瘤级别的升高而降低。RUNX3基因可能作为一抑瘤基因参与了胶质瘤的发生和发展。
第二章胶质瘤中RUNX3基因的转录表达及其启动子甲基化状态的研究
目的:检测RUNX3基因启动子甲基化情况,探索影响RUNX3基因在胶质瘤和正常脑组织中表达差异的分子机制。
方法:分别运用RT-PCR和Western blot方法检测10例正常脑组织和35例胶质瘤新鲜标本中RUNX3 mRNA和蛋白的表达水平,分析其与临床病理资料之间的关系,运用特异性甲基化PCR(MSP)检测同批标本的RUNX3基因启动子甲基化情况,分析RUNX3基因表达水平与其启动子甲基化之间的关系。
结果:35例胶质瘤中RUNX3 mRNA和蛋白的光密度比值分别为0.303±0.23 1和0.401±0.217。10例正常脑组织RUNX3 mRNA和蛋白的光密度比值分别为0.767±0.047和0.753±0.042。RUNX3 mRNA和蛋白的表达在正常脑组织和胶质瘤中有显著区别(P＜0.01)。RUNX3 mRNA和蛋白在低级别胶质瘤(Ⅰ-Ⅱ级)中表达为0.428±0.241和0.553±0.196,高级别胶质瘤(Ⅲ-Ⅳ级)中分别为0.208±0.175和0.287±0.154,表明RUNX3 mRNA和蛋白的表达水平随着胶质瘤恶性程度的增加而降低(r_(mRNA)=-0.598,P_(mRNA)＜0.01;r_(protein)=-0.703,P_(protein)＜0.01),并且RUNX3 mRNA和蛋白的表达水平呈正相关(r=0.965,P＜0.01)。MSP显示35例胶质瘤标本中有42.8%(15/35)RUNX3基因启动子发生甲基化,10例正常脑组织中未检测到RUNX3启动子的甲基化,RUNX3启动子甲基化在正常脑组织和胶质瘤中比较有显著的统计学差异(P＜0.01),并且RUNX3启动子甲基化与肿瘤的恶性程度相关,在低级别胶质瘤中只有20%(3/15)RUNX3启动子甲基化,而在高级别胶质瘤中甲基化率为60%(12/20)。15例RUNX3基因甲基化的胶质瘤中mRNA和蛋白分别为0.1 13±0.098和0.213±0.105,20例未甲基化胶质瘤中mRNA和蛋白分别为0.445±0.197和0.612±0.169,RUNX3启动子高甲基化与mRNA及蛋白表达呈显著负相关(P＜0.01)。
结论:RUNX3基因启动子在胶质瘤中异常高甲基化。RUNX3基因启动子高甲基化是RUNX3基因在胶质瘤中表达下调的重要原因,并且RUNX3基因启动子高甲基化在高级别胶质瘤中更为常见。
第三部分5-氮胞苷诱导胶质瘤细胞株RUNX3表达和对细胞凋亡、侵袭的影响
目的:探讨特异性去甲基化药物5-氮胞苷对胶质瘤母细胞株U-251增殖、凋亡和侵袭能力的影响,以及作用前后RUNX3基因表达和启动子甲基化的情况。
方法:常规方法培养U-251细胞。取对数生长期的细胞作为研究对象。运用MTT检测不同浓度5-氮胞苷处理U-251细胞株12小时、24小时、48小时和72小时后对细胞增殖的影响。PI染色经流式细胞仪检测分别用5μmol/L,10μmol/L,20μmol/L和50μmol/L5-氮胞苷处理U-251细胞株后的凋亡情况。运用Transwell检测U-251细胞经过5-氮胞苷处理后的侵袭能力。运用RT-PCR,Western blot和MSP检测药物干预前后RUNX3基因表达和甲基化的情况。
结果:U-251细胞株经过不同浓度5-氮胞苷处理后,随着药物浓度的上升和作用时间的延长,药物抑制细胞增殖的作用逐渐增强,即具有量效和时效依赖性。经流式细胞仪检测分析结果显示:经过不同浓度5-氮胞苷处理后,细胞凋亡率由对照组的1.32±0.21%上升至50μmol/L时的39.21±1.32%。各干预浓度下的凋亡率同对照组相比均有明显区别(P＜0.01)。Transwell侵袭小室结果显示随着去甲基化药物作用浓度的上升,U-251细胞侵袭细胞数由阴性对照组的51.3±2.6下降至20μmol/L干预组的29.2±2.3,抑制率达到了43.13%。RT-PCR,Western blot和MSP显示5-氮胞苷作用前,U-251细胞RUNX3启动子是甲基化的,RUNX3mRNA和蛋白表达缺失,在药物干预后,可以检测到RUNX3基因mRNA和蛋白的表达,RUNX3基因启动子甲基化状态被部分逆转,即甲基化与非甲基化并存。
结论:5-氮胞苷在体外能有效抑制U-251细胞的增殖、侵袭,并促进其凋亡。5-氮胞苷通过去甲基化作用恢复U-251细胞中RUNX3基因的表达。
Brain glioma is a common primary intracranial tumor,gradeⅡ-Ⅳare regarded as malignant and prognosis remains dismal.Although surgical treatment combined with radiotherapy and/or chemotherapy were taken, the curative effect is still not satisfied.
RUNX3 gene is a member of runt domain-containing family of transcription factors and plays an important role in axon guidance,cell proliferation and induction of apoptosis.Previous studies indicate that RUNX3 expression is down-regulated in many human tumors and may be a putativ tumor suppressor gene.But RUNX3 expression and its characteristics in glioma are still unknown,its mechanism of transcriptional expression needs further investigation.We first use IHC to detect RUNX3 protein expression in normal brain and glioma and analyze its relationship with clinical data.Then we use MSP to detect RUNX3 promotor methylation status in glioma specimens and reveal the regulatory mechanism of RUNX3 gene.Finally,5-azaC was used to reverse methylation status in U-251 glioma cell line and invasive ability and apoptosis of tumor cell were also investigated.RUNX3 maybe a new therapeutic target gene.
Part 1 The expression and clinical significance of RUNX3 protein in glioma
Objective:To investigate the expression of RUNX3 protein and its potential clinical significance in human brain and glioma.
Methods:RUNX3 protein expression was determined in 8 normal brains and 40 gliomas paraffin embedded specimens by immunohistochemical method and the differences of RUNX3 protein expression among clinical pathological groups were also investigated.
Results:In all normal brain tissues,the RUNX3 protein expressions were either positive or strong positive,but in 42.5%(17/40)of glioma specimens were weak positive or negative,the expression of RUNX3 protein was significantly down-regulated in glioma(P=0.024).Compared among different pathological groups,RUNX3 expression was higher in low-grade glioma(IRS=7.70±3.20)than in high-grade glioma (IRS=4.75±2.88),which is statistically significant different.The increase of RUNX3 correlated negatively with tumor grade(γ=-0.478,P=0.002). No relationship was observed between the expression of RUNX3 protein and sex,age,pathological types,tumor location.
Conclusion:The expression of RUNX3 protein was down-regulated in gliomas when compared with normal brain tissues and its increase correlated negatively with tumor grade.RUNX3 may be functioned as a tumor suppressor gene in the occurrence and development of glioma.
Part 2 The experimental study of transcriptional expression and promoter methylation status of RUNX3 gene in glioma
Objective:To detect the promoter methylation status of RUNX3 gene and investigate the molecular mechanism of RUNX3 gene down-regulation in glioma.
Methods:Total RNA and protein were extracted from 35 human gliomas and 10 normal brain tissue specimens.RT-PCR,Western blot and MSP were used to detect the RUNX3 mRNA expression,RUNX3 protein expression and promoter methylation status.In the same time,the correlations between clinic pathological parameters and the expression and methylation status of RUNX3 gene were analyzed.
Results:Relative expression of RUNX3 mRNA and protein in 35 cases of glioma specimens were 0.303±0.231 and 0.401±0.271 respectively,but in 10 normal brain tissues were 0.767±0.047 and 0.753±0.042 respectively,the difference of expression was significant between normal brain and glioma(P＜0.01).Relative expression of RUNX3 mRNA and protein in low-grade glioma were 0.428±0.241 and 0.553±0.196 respectively,in high-grade glioma were 0.208±0.175 and 0.287±0.154 respectively.The decreased expression of RUNX3 mRNA and protein correlated positively with tumor grade(r_(mRNA)=-0.598, P_(mRNA)＜0.01;r_(protein)=-0.703,P_(protein)in＜0.01)and the RUNX3 mRNA and protein were positively associated.The prevalence of RUNX3 promoter methylation in glioma tissue was 42.8%(15/35),while in normal brain tissue no methylaiton was detected,the difference between glioma and normal brain was statistically signiflcant(P＜0.01).RUNX3 promoter methylation status was also statistically different between low-grade(20%) and high-grade(60%)glioma tissues(P=0.037).Expression of RUNX3 mRNA and protein in 15 cases of RUNX3 promoter methylation glioma were 0.113±0.098 and 0.213±0.105 respectively,in 20 cases of non-methylation glioma were 0.445±0.197and 0.612±0.169 respectively. RUNX3 promoter methylation was statistically associated with the down-regulation of RUNX3 mRNA and protein.
Conclusion:Aberrant RUNX3 gene promoter methylaiton was found in glioma and epigenetic silencing of RUNX3 gene expression by promoter hypermethylation could play an important role in glioma. RUNX3 promoter hypermethylation was more common in high-grade glioma.
Part 3 5-azacytidine induced RUNX3 re-expression and impacts on apoptosis and invasive ability of tumor cells
Objective:To explore the transcription expression and promoter methylation status of RUNX3 gene in U-251 cell line in vitro exposed to the specific demethylation agent--5-azacytidine.Tumor cellular growth suppression and invasive potential in vitro were also observed.
Methods:MTT colorimetric assay was performed to detect the inhibition effect on U-251 cellular growth after treated with 5-azacytindine after 12hrs,24hrs,48hrs and 72hrs respectively.Apoptosis was analyzed by flow cytometry through PI stain.Transwell chamber in vitro invasive assay was used to examine the effects of inhibiting tumor cell invasive abilities.RT-PCR,Western blot and MSP were used to detect RUNX3 mRNA,protein and promoter methylation status before and after treatment with 5-azacytidine.
Results:Inhibitory effects of 5-azacytidine on proliferation of U-251 cells observed by MTT colorimetric survival assay,was that treatment with 5-azacytidine inhibited growth of U-251 cells in a time and dose-dependent manner.5-azacytidine-induced apoptosis in a dose-dependent manner was determined by flow cytometry.Compared with control group,the apoptosis ratio of U-251 cell raise from 1.32±0.21%to 39.21±1.32%,which is treated with 50μmol/L 5-azacytidine,significant difference was noticed when compared with control group(P＜0.01).The result of Transwell assay exhibited that cell number through the artificial basal membrane became less compared with the control group,from 51.3±2.6 to 29.2±2.3.By using RT-PCR,Western blot and MSP techniques,we found out that RUNX3 gene promoter was methylated,RUNX3 mRNA and protein expression were absent in U-251 cell line before 5-azacytidine treatment,but after treated with 5-azacytidine,RUNX3 mRNA and protein were re-expressed and RUNX3 promoter methylation status were partially reversed.
Conclusion:5-azacytidine can inhibit proliferation and invasive ability and increase apoptosis of U-251 tumor cell line.5-azacytidine can reactivate RUNX3 expression by demethylation RUNX3 promoter methylation status.
第一章RUNX3蛋白在胶质瘤中的表达及临床意义
目的:研究RUNX3蛋白在正常脑组织和胶质瘤中表达,探索RUNX3基因在胶质瘤中的作用,以及RUNX3蛋白在胶质瘤中异常表达与临床病理资料之间的关系。
方法:运用免疫组化的方法检验了40例脑胶质瘤和8例正常脑组织石蜡标本中RUNX3蛋白的表达情况,并比较RUNX3蛋白在各临床病理特征分组间的差异。
结果:正常脑组织中RUNX3蛋白均为阳性或强阳性表达,而在17/4.0(42.5%)胶质瘤中为阴性或弱阳性表达,其表达水平在胶质瘤中较正常脑组织中明显下降(P=0.024)。RUNX3蛋白在胶质瘤中的表达随着肿瘤恶性程度的增加表达进行性降低(r=-0.478,P=0.002),低级别(Ⅰ-Ⅱ级)胶质瘤中IRS为7.70±3.20,高级别(Ⅲ-Ⅳ级)胶质瘤中IRS为4.75±2.88,RUNX3蛋白在低级别和高级别胶质瘤中表达强度有明显差异(X~2=8.697,P=0.020)。但RUNX3蛋白的表达与胶质瘤的病理类型、患者年龄、性别、肿瘤部位等因素无显著相关性。
结论:RUNX3蛋白在胶质瘤组织中存在明显的表达缺失或下调,并且其表达水平随着肿瘤级别的升高而降低。RUNX3基因可能作为一抑瘤基因参与了胶质瘤的发生和发展。
第二章胶质瘤中RUNX3基因的转录表达及其启动子甲基化状态的研究
目的:检测RUNX3基因启动子甲基化情况,探索影响RUNX3基因在胶质瘤和正常脑组织中表达差异的分子机制。
方法:分别运用RT-PCR和Western blot方法检测10例正常脑组织和35例胶质瘤新鲜标本中RUNX3 mRNA和蛋白的表达水平,分析其与临床病理资料之间的关系,运用特异性甲基化PCR(MSP)检测同批标本的RUNX3基因启动子甲基化情况,分析RUNX3基因表达水平与其启动子甲基化之间的关系。
结果:35例胶质瘤中RUNX3 mRNA和蛋白的光密度比值分别为0.303±0.23 1和0.401±0.217。10例正常脑组织RUNX3 mRNA和蛋白的光密度比值分别为0.767±0.047和0.753±0.042。RUNX3 mRNA和蛋白的表达在正常脑组织和胶质瘤中有显著区别(P＜0.01)。RUNX3 mRNA和蛋白在低级别胶质瘤(Ⅰ-Ⅱ级)中表达为0.428±0.241和0.553±0.196,高级别胶质瘤(Ⅲ-Ⅳ级)中分别为0.208±0.175和0.287±0.154,表明RUNX3 mRNA和蛋白的表达水平随着胶质瘤恶性程度的增加而降低(r_(mRNA)=-0.598,P_(mRNA)＜0.01;r_(protein)=-0.703,P_(protein)＜0.01),并且RUNX3 mRNA和蛋白的表达水平呈正相关(r=0.965,P＜0.01)。MSP显示35例胶质瘤标本中有42.8%(15/35)RUNX3基因启动子发生甲基化,10例正常脑组织中未检测到RUNX3启动子的甲基化,RUNX3启动子甲基化在正常脑组织和胶质瘤中比较有显著的统计学差异(P＜0.01),并且RUNX3启动子甲基化与肿瘤的恶性程度相关,在低级别胶质瘤中只有20%(3/15)RUNX3启动子甲基化,而在高级别胶质瘤中甲基化率为60%(12/20)。15例RUNX3基因甲基化的胶质瘤中mRNA和蛋白分别为0.1 13±0.098和0.213±0.105,20例未甲基化胶质瘤中mRNA和蛋白分别为0.445±0.197和0.612±0.169,RUNX3启动子高甲基化与mRNA及蛋白表达呈显著负相关(P＜0.01)。
结论:RUNX3基因启动子在胶质瘤中异常高甲基化。RUNX3基因启动子高甲基化是RUNX3基因在胶质瘤中表达下调的重要原因,并且RUNX3基因启动子高甲基化在高级别胶质瘤中更为常见。
第三部分5-氮胞苷诱导胶质瘤细胞株RUNX3表达和对细胞凋亡、侵袭的影响
目的:探讨特异性去甲基化药物5-氮胞苷对胶质瘤母细胞株U-251增殖、凋亡和侵袭能力的影响,以及作用前后RUNX3基因表达和启动子甲基化的情况。
方法:常规方法培养U-251细胞。取对数生长期的细胞作为研究对象。运用MTT检测不同浓度5-氮胞苷处理U-251细胞株12小时、24小时、48小时和72小时后对细胞增殖的影响。PI染色经流式细胞仪检测分别用5μmol/L,10μmol/L,20μmol/L和50μmol/L5-氮胞苷处理U-251细胞株后的凋亡情况。运用Transwell检测U-251细胞经过5-氮胞苷处理后的侵袭能力。运用RT-PCR,Western blot和MSP检测药物干预前后RUNX3基因表达和甲基化的情况。
结果:U-251细胞株经过不同浓度5-氮胞苷处理后,随着药物浓度的上升和作用时间的延长,药物抑制细胞增殖的作用逐渐增强,即具有量效和时效依赖性。经流式细胞仪检测分析结果显示:经过不同浓度5-氮胞苷处理后,细胞凋亡率由对照组的1.32±0.21%上升至50μmol/L时的39.21±1.32%。各干预浓度下的凋亡率同对照组相比均有明显区别(P＜0.01)。Transwell侵袭小室结果显示随着去甲基化药物作用浓度的上升,U-251细胞侵袭细胞数由阴性对照组的51.3±2.6下降至20μmol/L干预组的29.2±2.3,抑制率达到了43.13%。RT-PCR,Western blot和MSP显示5-氮胞苷作用前,U-251细胞RUNX3启动子是甲基化的,RUNX3mRNA和蛋白表达缺失,在药物干预后,可以检测到RUNX3基因mRNA和蛋白的表达,RUNX3基因启动子甲基化状态被部分逆转,即甲基化与非甲基化并存。
结论:5-氮胞苷在体外能有效抑制U-251细胞的增殖、侵袭,并促进其凋亡。5-氮胞苷通过去甲基化作用恢复U-251细胞中RUNX3基因的表达。
Brain glioma is a common primary intracranial tumor,gradeⅡ-Ⅳare regarded as malignant and prognosis remains dismal.Although surgical treatment combined with radiotherapy and/or chemotherapy were taken, the curative effect is still not satisfied.
RUNX3 gene is a member of runt domain-containing family of transcription factors and plays an important role in axon guidance,cell proliferation and induction of apoptosis.Previous studies indicate that RUNX3 expression is down-regulated in many human tumors and may be a putativ tumor suppressor gene.But RUNX3 expression and its characteristics in glioma are still unknown,its mechanism of transcriptional expression needs further investigation.We first use IHC to detect RUNX3 protein expression in normal brain and glioma and analyze its relationship with clinical data.Then we use MSP to detect RUNX3 promotor methylation status in glioma specimens and reveal the regulatory mechanism of RUNX3 gene.Finally,5-azaC was used to reverse methylation status in U-251 glioma cell line and invasive ability and apoptosis of tumor cell were also investigated.RUNX3 maybe a new therapeutic target gene.
Part 1 The expression and clinical significance of RUNX3 protein in glioma
Objective:To investigate the expression of RUNX3 protein and its potential clinical significance in human brain and glioma.
Methods:RUNX3 protein expression was determined in 8 normal brains and 40 gliomas paraffin embedded specimens by immunohistochemical method and the differences of RUNX3 protein expression among clinical pathological groups were also investigated.
Results:In all normal brain tissues,the RUNX3 protein expressions were either positive or strong positive,but in 42.5%(17/40)of glioma specimens were weak positive or negative,the expression of RUNX3 protein was significantly down-regulated in glioma(P=0.024).Compared among different pathological groups,RUNX3 expression was higher in low-grade glioma(IRS=7.70±3.20)than in high-grade glioma (IRS=4.75±2.88),which is statistically significant different.The increase of RUNX3 correlated negatively with tumor grade(γ=-0.478,P=0.002). No relationship was observed between the expression of RUNX3 protein and sex,age,pathological types,tumor location.
Conclusion:The expression of RUNX3 protein was down-regulated in gliomas when compared with normal brain tissues and its increase correlated negatively with tumor grade.RUNX3 may be functioned as a tumor suppressor gene in the occurrence and development of glioma.
Part 2 The experimental study of transcriptional expression and promoter methylation status of RUNX3 gene in glioma
Objective:To detect the promoter methylation status of RUNX3 gene and investigate the molecular mechanism of RUNX3 gene down-regulation in glioma.
Methods:Total RNA and protein were extracted from 35 human gliomas and 10 normal brain tissue specimens.RT-PCR,Western blot and MSP were used to detect the RUNX3 mRNA expression,RUNX3 protein expression and promoter methylation status.In the same time,the correlations between clinic pathological parameters and the expression and methylation status of RUNX3 gene were analyzed.
Results:Relative expression of RUNX3 mRNA and protein in 35 cases of glioma specimens were 0.303±0.231 and 0.401±0.271 respectively,but in 10 normal brain tissues were 0.767±0.047 and 0.753±0.042 respectively,the difference of expression was significant between normal brain and glioma(P＜0.01).Relative expression of RUNX3 mRNA and protein in low-grade glioma were 0.428±0.241 and 0.553±0.196 respectively,in high-grade glioma were 0.208±0.175 and 0.287±0.154 respectively.The decreased expression of RUNX3 mRNA and protein correlated positively with tumor grade(r_(mRNA)=-0.598, P_(mRNA)＜0.01;r_(protein)=-0.703,P_(protein)in＜0.01)and the RUNX3 mRNA and protein were positively associated.The prevalence of RUNX3 promoter methylation in glioma tissue was 42.8%(15/35),while in normal brain tissue no methylaiton was detected,the difference between glioma and normal brain was statistically signiflcant(P＜0.01).RUNX3 promoter methylation status was also statistically different between low-grade(20%) and high-grade(60%)glioma tissues(P=0.037).Expression of RUNX3 mRNA and protein in 15 cases of RUNX3 promoter methylation glioma were 0.113±0.098 and 0.213±0.105 respectively,in 20 cases of non-methylation glioma were 0.445±0.197and 0.612±0.169 respectively. RUNX3 promoter methylation was statistically associated with the down-regulation of RUNX3 mRNA and protein.
Conclusion:Aberrant RUNX3 gene promoter methylaiton was found in glioma and epigenetic silencing of RUNX3 gene expression by promoter hypermethylation could play an important role in glioma. RUNX3 promoter hypermethylation was more common in high-grade glioma.
Part 3 5-azacytidine induced RUNX3 re-expression and impacts on apoptosis and invasive ability of tumor cells
Objective:To explore the transcription expression and promoter methylation status of RUNX3 gene in U-251 cell line in vitro exposed to the specific demethylation agent--5-azacytidine.Tumor cellular growth suppression and invasive potential in vitro were also observed.
Methods:MTT colorimetric assay was performed to detect the inhibition effect on U-251 cellular growth after treated with 5-azacytindine after 12hrs,24hrs,48hrs and 72hrs respectively.Apoptosis was analyzed by flow cytometry through PI stain.Transwell chamber in vitro invasive assay was used to examine the effects of inhibiting tumor cell invasive abilities.RT-PCR,Western blot and MSP were used to detect RUNX3 mRNA,protein and promoter methylation status before and after treatment with 5-azacytidine.
Results:Inhibitory effects of 5-azacytidine on proliferation of U-251 cells observed by MTT colorimetric survival assay,was that treatment with 5-azacytidine inhibited growth of U-251 cells in a time and dose-dependent manner.5-azacytidine-induced apoptosis in a dose-dependent manner was determined by flow cytometry.Compared with control group,the apoptosis ratio of U-251 cell raise from 1.32±0.21%to 39.21±1.32%,which is treated with 50μmol/L 5-azacytidine,significant difference was noticed when compared with control group(P＜0.01).The result of Transwell assay exhibited that cell number through the artificial basal membrane became less compared with the control group,from 51.3±2.6 to 29.2±2.3.By using RT-PCR,Western blot and MSP techniques,we found out that RUNX3 gene promoter was methylated,RUNX3 mRNA and protein expression were absent in U-251 cell line before 5-azacytidine treatment,but after treated with 5-azacytidine,RUNX3 mRNA and protein were re-expressed and RUNX3 promoter methylation status were partially reversed.
Conclusion:5-azacytidine can inhibit proliferation and invasive ability and increase apoptosis of U-251 tumor cell line.5-azacytidine can reactivate RUNX3 expression by demethylation RUNX3 promoter methylation status.
引文
[1]周良辅。现代神经外科学,第一版。上海:复旦大学出版社,2001,376.
[2]Asher A,Burger PC,Croteau D,et al.A Primer of brain tumors:a patient's reference.USA,ABTA,2004:8-10.
[3]Koul D.PTEN signaling pathways in glioblastoma.Cancer Biol Ther,2008,7(9):1321-5.
[4]Furnari FB,Fenton T,Bachoo RM,et al.Malignant astrocytic glioma:genetics,biology,and paths to treatment.Genes Dev,2007,21(21):2683-710.
[5]Brandes AA,Tosoni A,Franceschi E,et al.Glioblastoma in adults.Crit Rev Oncol Hematol.2008,67(2):139-52.
[6]Herman JG,Baylin SB.Gene sileneing in cancer in associate with promoter hyermethylation.NewEngl J Med,2003,349:2042-2054.
[7]Jones PA,Takai D.The role of DNA methylation in mammalion epigenetics.Science,2001,293(5532):1068-1070.
[8]Feinberg AP.The epigenetic of cancer etiology.Semi Cancer Biol,2004,14:427-432.
[9]Bangsow C,Rubins N,Glusman G,et al.The RUNX3 gene-sequence,structure and regulated expression.Gene,2001,279(2):221-32.
[10]Kato N,Tamura G,Fukase M,et al.Hypermethylation of the RUNX3 gene promoter in testicular yolk sac tumor of infants.Am J Pathol,2003,163(2):387-391.
[11]Miyazono K,Maeda S,Imamura T.Coordinate regulation of cell growth and differentiation by TGF-beta super family and Runx proteins.Oncogene,2004,23(24):4232-4237.
[12]Wei D,Gong W,Oh SC,et al.Loss of RUNX3 expression significantly affects the clinical outcome of gastric cancer patients and its restoration causes drastic suppression of tumor growth and metastasis.Cancer Res,2005,65(11):4809-4816.
[13]Kim TY,Lee HJ,Hwang KS,et al.Methylation of RUNX3 in various types of human cancers and premalignant stages of gastric carcinoma.Lab Invest,2004,84(4):479-484.
[14]Imamura Y,Hibi K,Koike M,et al.RUNX3 promoter region is specifically methylated in poorly-differentiated colorectal cancer.Anticancer Res,2005,25(4): 2627-2630.
[15] Black PM. Brain tumor, part l.NewEngl J Med, 1991,324:1471-1476.
[16]van Wijnen AJ, et al. Nomenclature for Runt-related (RUNX) proteins.Oncogene,2004,23,4209-4210.
[17] Li QL, Ito K, Sakakura C, et al. Causal relationship between the loss of RUNX3 expression and gastric cancer. Cell, 2002,109:113-124.
[18]Naoyuki Homma, Gen Tamura, Teichiro Honda. Spreading of methylation within RUNX3 CpG island in gastric cancer. Cancer Sci. 2006,97(1):51-6.
[19] Kim WJ, Kim EJ, Jeong P, et al. RUNX3 inactivation by point mutations and aberrant DNA methylation in bladder tumors. Cancer Res. 2005, 65(20):9347-54.
[20]Kleihues P, Cavenee WK, et al. World Health Organization classification of tumors:Pathology and genetics of tumors of the nervous system. Lyon: IARC Press.2000.
[21]Remmele W, Stegner HE. Recommendation for uniform definition of an immunoreactive score (IRS) for immunohistochemical estrogen receptor detection(ER-CIA) in breast cancer tissue. Pathology, 1987,8:138-140.
[22] Berardi MJ, Sun C, Zehr M, et al. The Ig fold of the core binding factor alpha Runt domain is a member of a family of structurally and functionally related Ig-fold DNA-binding domains. Structure 1999,7:1247-56.
[23] Blyth K, Cameron ER, Neil JC.The RUNX genes: gain or loss of function in cancer. Nature Reviews Cancer 2005,5(5): 376-387.
[24] Kramer I, Sigrist M, de Nooij JC, et al. A role for Runx transcription factor signaling in dorsal root ganglion sensory neuron diversification. Neuron,2006,49(3):379-93.
[25] Inoue K, Ito K, Osato M, Lee B, et al. The transcription factor Runx3 represses the neurotrophin receptor TrkB during lineage commitment of dorsal root ganglion neurons. J Biol Chem,2007,282(33):24175-84.
[26] Grueter B, Petter M, Egawa T, Laule-Kilian K, et al. Runx3 regulates integrin alpha E/CD103 and CD4 expression during development of CD4-/CD8+ T cells. J Immunol, 2005 ,175(3):1694-705.
[27]Hasegawa K, Yazumi S, Wada M, et al. Restoration of RUNX3 enhances transforming growth factor-beta-dependent p21 expression in a biliary tract cancer cell line. Cancer Sci. 2007,98(6):838-43.
[28] Chi XZ, Yang JO, Lee KY, et al. I RUNX3 suppresses gastric epithelial cell growth by inducing p21(WAF1/Cip1)expression in cooperation with transforming growth factor {beta}-activated SMAD.Mol Cell Biol.2005,25(18):8097-107.
[29]Sakakura C,Hasegawa K,Miyagawa K,et al.Possible Involvement of RUNX3Silencing in the Peritoneal Metastases of Gastric Cancers.Clin Cancer Res 2005,11(18):6479-88.
[30]Hesson L,Bieche I,Krex D et al.Frequent epigenetic inactivation of RASSF1A and BLU genes located within the critical 3p21.3 region in gliomas.Oncogene.2004,23(13):2408-19.
[31]Annegret Kunitz,Marietta Wolter,et al.DNA hypermethylation and aberrant expression of the EMP3 Gene at 19q13.3 in human gliomas.Brain Pathol,2007,17(4):363-370.
[32]Song BC,Chung YH,Kim JA,et al.Transforming growth factor-beta,as a useful serologic marker of small hepatocel1u1ar carcinoma.Cancer,2002,94(1):175-180.
[33]Hiramatsu T,Osaki M,Ito Y,et al.Expression of RUNX3 Protein in human Esophageal Mucosa and Squamous Cell.Carcinoma.Pathobiology,2005,72(6):316-324.
[34]Yanagawa N,Tamura G,Oizumi H,et al.Promoter hypermethylation of tumor suppressor and tumor-related genes in non-small cell lung cancers.Cancer Sci,2003,94(7):589-592.
[35]倪志.5-Aza-CdR对人结肠癌Lovo细胞增殖调亡及抑瘤基因RUNX3表达的影响.世界华人消化杂志,2006,14(2):184-155.
[36]Mori T,Nomoto S,Koshikawa K,et al.Decreased expression and frequent allelic inactivation of the RUNX3 gene at 1p36 in human hepatocellular carcinoma.Liver Int,2005,25(2):380-388.
[37]Seoane M,Iglesias P,Gonzalez T,et al.Retinoblastoma loss modulates DNA damage response favoring tumor progression.PLoS ONE.2008;3(11):e3632
[38]Kirito K,Sakoe K,Shinoda D,et al.A novel RUNX1 mutation in familial platelet disorder with propensity to develop myeloid malignancies.Haematologica,2008,93(1):155-6.
[39]Tanne Y,Tanimoto K,Tanaka N,et al.Expression and activity of Runx2 mediated by hyaluronan during chondrocyte differentiation.Arch Oral Bioi,2008,53(5):478-87.
[40]Woolf E,Xiao C,Fainaru O,Lotem J,et al.Runx3 and Runx1 are required for CD8 T cell development during thymopoiesis.Proc Natl Acad Sci USA.2003,100(13):7731-6.
[41]Kang S,Kim JW,Kang GH,Park NH,Song YS,Kang SB,Lee HP.Polymorphism in folate-and methionine-metabolizing enzyme and aberrant CpG island hypermethylation in uterine cervical cancer.Gynecol Oncol.2005,96(1):173-80.
[42]肖文华,刘为纹.肝细胞癌RUNX3基因甲基化与杂合缺失的分析及意义。中华肝脏病杂志,2004,12(4)227-23.
[43]Takahashi T,Shivapurkar N,Riquelme E,et al.Aberrant promoter hypermethylation of multiple genes in gallbladder carcinoma and chronic cholecystitis.Clin Cancer Res,2004,10:6126-33.
[44]Sakakura C,Hagiwara A,Miyagawa K,et al.Frequent downregulation of the runt domain transcription factors RUNX1,RUNX3 and their cofactor CBFβ in gastric cancer.Int J Cancer,2005,113(2):221-8
[45]Kurkjian C,Kummar S,Murgo AJ.DNA methylation:its role in cancer development and therapy.Curr Probl Cancer,2008,32(5):187-235.
[46]Jones PA,Baylin SB.The fundamental role of epigenetic events in cancer.Nat Rev Genet,2002,3(6):415-428.
[47]Ehrlich M.DNA methylation in cancer:too much,but also too little.Oncogene,2002,21(35):5400-5413.
[48]Esteller M.DNA methylation and cancer therapy:new developments and expectations.Curr Opin Oneol,2005,17(1):55-60.
[49]Das PM,Singal R.DNA methylation and cancer.J Clin Oncol,2004,22(22):4632-4642.
[50]Davis CD,Uthus EO.DNA methylation,cancer susceptibility,and nutrient interactions.Exp Biol Med,2004,229(10):985-995.
[51]卢圣栋主编.现代分子生物学实验技术.北京:高等教育出版社,1993.465-497.
[52]Eads CA,Laird PW.Combined bisulfite restriction analysis(COBRA).Methods Mol Biol.2002;200:71-85.
[53]Perry AS,Liyanage H,Lawler M,et al.Discovery of DNA hypermethylation using a DHPLC screening strategy.Epigenetics.2007,2(1):43-9.
[54]邓大军,邓国仁等。变性高效液相色谱法检测CpG岛胞嘧啶甲基化。中华医学杂志,2001,81:158-161。
[55]Wu Z,Luo J,Ge Q,et al.Microarray-based Ms-SNuPE:near-quantitative analysis for a high-throughput DNA methylation.Biosens Bioelectron,2008,23(9):1333-9.
[56]Herman JG,Graff JR,Myohanen S,et al.Methylation-specific PCR:a novel PCR assay for methylaiton status of CpG island.Proc Natl Acad Sci,1996,93(18):9821-9826.
[57]von Deimling A,Fimmers R,Schmidt MC,Bender B,Fassbender F,Nagel Jet al.Comprehensive allelotype and genetic anaysis of 466 human nervous system tumors.J Neuropathol Exp Neurol,2000,59(6):544-558.
[58]Schmidt MC,Antweiler S,Urban N,et al.Impact of genotype and morphology on the prognosis of glioblastoma.J Neuro-pathol Exp Neurol,2002,61(4):321-328.
[59]Costello JF,Fruhwald MC,Smiraglia DJ,et al.Aberrant CpG-island methylation has non-random and tumor-type-specific patterns.Nat Genet,2000,24(2):132-138.
[60]Wada M,Yazumi S,Takaishi S,et al.Frequent loss of RUNX3 gene expression in human bile duct and pancreatic cancer cell lines.Oncogene,2004,23(13):2401-2407.
[61]Waki T,Tamura G,Sato M,et al.Age-related methylation of tumor suppressor and tumor-related genes:an analysis of autopsy samples.Oncogene.2003,22(26):4128-33.
[62]Weaver KD,Grossman SA,Heman JG.Methylated tumor specific DNA as a plasm biomarker in patients with glioma.Cancer Investigation,2006,24(1):35-40.
[63]Nomoto S,Kinoshita T,Mori T,et al.Adverse prognosis of epigenetic inactivation in RUNX3 gene at 1p36 in human pancreatic cancer.Br J Cancer.2008,98(10):1690-5.
[64]Peng Z,Tang H,Wang X,et al.Inhibition of the growth and metastasis of human colon cancer by restoration of RUNX3 expression in cancer cells..Int J Oncol.2008,33(5):979-84.
[65]Park WS,Cho YG,Kim C J,et al.Hypermethylation of the RUNX3 gene in hepatocellular carcinoma.Exp Mol Med,2005,37(4):276-281.
[66]Baylin SB,Herman JG,Graff JR,et al.Alterations in DNA methylation:a fundamental aspect of neoplasia.Adv Cancer Res,1998,72:141-196.
[67]杜红玲,戚豫,石永进,等.脱乙酞化酶抑制剂与去甲基化制剂联合诱导UZ“细胞凋亡和p16基因重新表达。癌症,2002,21(10):1057-1061.
[68] Jones PA. Altering gene repression with 5-azacytidine. Cell, 1985, 40(3): 485-486.
[69] Jones PA, Taylor SM. Hemimethylated duplex DNAs prepared from 5-azacytidine treated cells. Nucleic Acids Res, 1981, 9(12):2933-47.
[70] Mueller W, Nutt CL, Ehrich M, et al. Downregulation of RUNX3 and TES by hypermethylation in glioblastoma. Oncogen,2007,26,583-593.
[71] Ku JL, Kang SB, Shin YK, et al. Promoter hypermethylation down regulates RUNX3 gene expression in colorectal cancer cell lines. Oncogene, 2004, 23(40):6736-6742.
[72]Qing-Lin Li, Hye-Ryeon Kim, Wun-Jae Kim et al. Transcriptional silencing of the RUNX3 gene by CpG hypermethylation is associated with lung cancer.Biochem Biophys Res Commun. 2004,314(1):223-8.
[73] Mosmann T. Rapid colorimetric assay for cellular growth and survival .'application to proliferation and cytotoxicity assays. J Immunol Methods,1983,65:55-61.
[74] Albini A, Iwamoto Y, Kleinman HK, et al. A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res, 1987,47:3239-3245.
[75] Yasuko Yamamura, Lee WL, Inoue KI, et al. RUNX3 cooperates with FoxO3a to induce apoptosis in gastric cancer cells. J Biol Chem.2006,24;281(8):5267-76.
[76]Lau QC, Raja E, Salto-Tellez M. RUNX3 Is frequently inactivated by dual mechanisms of protein mislocalization and promoter hypermethylation in breast cancer. Cancer Res. 2006,66(13):6512-20.
[77] Peng Z, Wei D, Wang L,et al.RUNX3 inhibits the expression of vascular endothelial growth factor and reduces the angiogenesis, growth, and metastasis of human gastric cancer. Clin Cancer Res. 2006,12(21):6386-94.
[78] Bender CM, Pao MM, Jones PA. Inhibition of DNA methylation by 5-aza-2'-deoxycytidine suppresses the growth of human tumor cell lines. Cancer Res.1998, 58(1):95-101.
[79] Christman JK. 5-Azacytidine and 5-aza-2'-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy.Oncogene, 2002, 21(35): 5483-5495
[80]Hennessy BT, Garcia-Manero G, Kantarjian HM, et al. DNA methylation in hematological malignancies: the role of decitabine. Expert Opin Investig Drugs,2003, 12(12):1985-1993.
[81]Wijermans P, et al. Low-dose 5-aza-2'-deoxycytidine, a DNA hypomethylating agent , for the treatment of high risks myelodysplastic syndrome : a multicenter pahse 2 study in elderly patients. J Clin Oncol,2000,18(5) :956-962.
[82]Sorm F, Piskala A, Cihak A, et al. 5-azacytidine, a new, highly effective cancerostatic. Experientia,1964,20(4) :202-203.
[83]Yoshida M, Kijima M, Akita M, et al. Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A. J Biol Chem, 1990,265(28) :17174-17179.
[84]Pina IC, Gautschi JT, Wang GY, et al. Psammaplins from the sponge Pseudoceratina purpurea rinhibition of both histone deacetylase and DNA methyltransferase. J Org Chem, 2003,68(10) :3866-73.
[85] Berletch JB, Liu C, Love WK, Epigenetic and genetic mechanisms contribute to telomerase inhibition by EGCG J Cell Biochem. 2008, 103(2):509-19.
[86]Coffrnan JA, Dickey-Sims C, Haug JS, et al. Evaluation of developmental phenotypes produced by morpholino antisense targeting of a sea urchin Runx gene.BMC Biol, 2004,7,2 :6.
[1]Wolffe AP,Matzke MA.Epigenetics:regulation through repression.Science,1999,286(5439):481-486.
[2] Lewin B. GeneⅧ. Perarson PrencHall press. 2004.
[3] Jaenisch R, Bird A. Epigenetic regulation of gene expression: how the genomeintegrates intrinsic and environmental signals. Nat Genet,2003,33:245-254.
[4] Couzin J. Small RNAs make big splash.Science,2002,29(56):2296-2297.
[5] Giacinti L, Vici P, Lopez M. Epigenome: A new target in cancer therapy. Clin Ter,2008,159(5):347-60.
[6] Baylin SB, Esteller M, Rountree MR, et al. Aberrant patterns of DNA methylation, chromatin formation and gene expression in cancer. Hum Mol Genet,2001,10(7):687-692.
[7] Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer. Nat Rev Genet, 2002, 3(6):415-428.
[8] Cross SH, Bird AP. CpG islands and genes. Curr Opin Genet Dev, 1995, 5(3):309-314.
[9] Jones PA, Laird PW. Cancer epigenetics comes of age. Nat Genet, 1999,21(2):163-167.
[10] Ehrlich M. DNA methylation in cancer: too much, but also too little. Oncogene,2002,21(35):5400-5413.
[11] Eads CA, Laird PW. Combined bisulfite restriction analysis (COBRA). Methods Mol Biol,2002,200:71-85.
[12] Wong IH. Qualitative and quantitative polymerase chain reaction-based methods for DNA methylation analyses. Methods Mol Biol,2006,336:33-43.
[13] Campan M, Weisenberger DJ, Trinh B, et al. Methylight. Methods Mol Biol.2009,507:325-37.
[14] Perry AS, Liyanage H, Lawler M, et al. Discovery of DNA hypermethylation using a DHPLC screening strategy. Epigenetics,2007,2(1):43-9.
[15] Wu Z, Luo J, Ge Q, et al. Microarray-based Ms-SNuPE: near-quantitative analysis for a high-throughput DNA methylation. Biosens Bioelectron,2008,23(9):1333-9.
[16] Costello JF, Hong C, Plass C, et al. Restriction landmark genomic scanning: analysis of CpG islands in genomes by 2D gel electrophoresis. Methods Mol Biol.2009,507:131-48.
[17] Huang TH, Perry MR, Laux DE. Methylation profiling of CpG islands in human breast cancer cells. HumMol Genet, 1999,8(3):459.
[18] Gitan RS, Shi H, Chen CM, et al. Methylation-specific oligonucleotide microarray: a new potential for high-throughput methylation analysis. Genome Res, 2002, 12(1):158.
[19] Shi H, Wei SH, Leu YW, et al. Triple analysis of the cancer epigenome: an integrated microarray system for assessing gene expression, DNA methylation,and histone acetylation. Cancer Res, 2003, 63(9):2164.
[20] Donninger H, Vos MD, Clark GJ. The RASSF1A tumor suppressor. J Cell Sci,2007,120(18):3163-72
[21] Hesson L, Bieche I, Krex D et al. Frequent epigenetic inactivation of RASSF1A and BLU genes located within the critical 3p21.3 region in gliomas. Oncogene,2004,23(13):2408-19.
[22] Horiguchi K, Tomizawa Y, Tosaka M, et al. Epigenetic inactivation of RASSF1A candidate tumor suppressor gene at 3p21.3 in brain tumors. Oncogene,2003,22(49):7862-5.
[23] Taylor V, Suter U. Epithelial membrane protein-2 and epithelial membrane protein-3: two novel members of the peripheral myelin protein 22 gene family.Gene, 1996,175(1-2):115-20.
[24] Kunitz A, Wolter M, van den Boom J, et al. DNA hypermethylation and aberrant expression of the EMP3 Gene at 19ql 3.3 in human gliomas. Brain Pathol,2007,17(4):363-370.
[25] Alaminos M, Davalos V, Ropero S, et al. EMP3 a myelin-related gene located in the critical 19p13.3 region, is epigenetically silenced and exhibits features of a candidate tumor suppressor in glioma and neuroblastoma. Cancer Res,2005,65(7):2565-71.
[26] Kamb A, Gruis NA, Weaver-Feldhaus J, et al. A cell cycle regulator potentially involved in genesis of many tumor types. Science, 1994, 264(5): 436-440.
[27] Izumoto S, Arita N, Ohnishi T, et al. Homozygous deletions of p16INK4A/MTS1 and p15INK4B/MTS2 genes in glioma cells and primary glioma tissues. Cancer Lett. 1995, 97(2):241-7.
[28] Costello JF, Berger MS, Huang HS, et al. Silencing of pl6/CDKN2 expression in human gliomas by methylation and chromatin condensation. Cancer Res, 1996,56(10):2405-10.
[29] Nakamura M, Watanabe T, Yonekawa Y, et al. Promoter methylaiton of the DNA repair gene MGMT in astrocytomas is frequently associated with G:C-A:T mutation of the TP53 tumor suppressor gene. Carcinogenesis, 2001,22(10):1715-1719.
[30] Silber JR, Bobola MS, Ghatan S, et al. O6-methylguanine-DNA methyltransferase activity in adult gliomas: relation to patient and tumor characteristics. Cancer Res, 1998,58,(5):1068-1073.
[31] WE Farrell, DJ Simpson, SJ Frost, et al. Methylation mechanisms in pituitary tumorigenesis. Endocr Relat Cancer, 1999,6(4): 437-447.
[32] Simpson DJ, Bicknell JE, McNicol AM, et al. Hypermethylation of the P16/CDKN2/MTST gene and loss of protein expression is associated with nonfunctional pituitary adenomas but not somatotrophinomas. Genes Chromosomes Cancer. 1999,24(4):221-224.
[33] Bello MJ, De Campos JM, Isla A, et al. Promoter CpG methylation of multiple genes in pituitary adenomas: Frequent involvement of Caspase-8. Oncol Rep,2006,15(2): 443-448.
[34] Yoshino A, Katayama Y, Ogino A, et al. Promoter hypermethylation profile of cell cycle regulator genes in pituitary adenomas. J Neurooncol,2007,83(2):153-162.
[35] Zhao J, Dahle D, Zhou Y, et al. Hypermethylation of the promoter region is associated with the loss of MEG3 gene expression in human pituitary tumors. J Clin Endocrinol Metab, 2005,90(4):2179-86.
[36] Liu Y, Pang JC, Dong S, et al. Aberrant CpG island hypermethylation profile is associated with atypical and anaplastic meningiomas. Hum Pathol,2005,36(4):416-425.
[37] Tse JY, Ng HK, Lo KW, et al. Analysis of cell cycle regulators: p16INK4A, pRb,and CDK4 in low-grade and high-grade meningiomas. Hum Pathol.1998,29(11):1200-7.
[38]Nakane Y, Natsume A, et al. Malignant transformation-related genes in meningiomas: allelic loss on Ip36 and methylation status of p73 and RASSF1A.J Neurosurg,2007,107(2):398-404.
[39] de Robles P, Mclntyre J, Kalra S, et al. Methylation status of MGMT gene promoter in meningiomas. Cancer Genet Cytogenet,2008,187(1):25-7.
[40] Inda MM, Castresana JS. RASSF1A promoter is highly methylated in primitive neuroectodermal tumors of the central nervous system. Neuropathology,2007,27(4):341-6.
[41] Gonzalez-Gomez P, Bello MJ, Inda MM. Deletion and aberrant CpG island methylation of Caspase 8 gene in medulloblastoma. Oncol Rep.2004,12(3):663-6.
[42] Lassaletta L, Patron M, Gonzalez T, et al.RASSF1A methylation and cyclin D1 expression in vestibular schwannomas. Acta Neuropathol,2007, 114(4):431-3.
[43] Losi-Guembarovski R, Kuasne H, Guembarovski AL, et al. DNA methylation patterns of the CDH1, RARB, and SFN genes in choroid plexus tumors. Cancer Genet Cytogenet. 2007, 179(2): 140-5.
[44] Yu L, Liu C, Vandeusen J, et al. Global assessment of promoter methylation in a mouse model of cancer identifies ID4 as a putative tumor suppressor gene in human leukemia. Nat Genet, 2005, 37(3):265-274.
[45] Tsou JA, Hagen JA, Carpenter CL, et al. DNA methylation analysis: a powerful new tool for lung cancer diagnosis. Oncogene, 2002, 21(35): 5450-5461.
[46] Sidransky D. Emerging molecular markers of cancer. Nat Rev Cancer, 2002,2(3):210-219.
[47] Honorio S, Agathanggelou A, Schuermann M, et al. Detection of RASSF1A aberrant promoter hypermethylation in sputum from chronic smokers and ductal carcinoma in situ from breast cancer patients. Oncogene, 2003, 22(1):147-150.
[48] Wakabayashi T, Natsume A, Hatano H, et al. p16 promoter methylation in the serum as a basis for the molecular diagnosis of gliomas. Neurosurgery.2009;64(3):455-61;
[49] Weaver KD, Grossman SA, Heman JG. Methylated tumor specific DNA as a plasma biomarker in patients with glioma. Cancer Investigation, 2006,24(1):35-40.
[50] Tao L, Yang S, Xie M, et al. Hypomethylation and over-expression of c-jun and c-myc protoonco genes and increased DNA methyltransferase activity in dichloroacetic and trichloroacetic acid-promoted mouse liver tumors. Cancer Lett,2000,158(2):185-193.
[51] Cadieux B, Ching TT, VandenBerg SR, et al. Genome-wide hypomethylation in human glioblastomas associated with specific copy number alteration, methylenetetrahydrofolate reductase allele status, and increased proliferation.Cancer Res,2006, 66(17):8469-76.
[52] Bredel M. Anticancer drug resistance in primary human brain tumors. Brain Res,2001,35, 161-204.
[53] Hegi ME, Diserens AC, Gorlia T et al. MGMT gene silencing and benefit from temozolomide in glioblastoma. N Eng J Med,2005, 352(10), 997-1003.
[54] Blanc JL, Wager M, Guilhot J, et al. Correlation of clinical features and methylation status of MGMT gene promoter in glioblastoma. J Neuro-Oncology,2004, 68(3):275-283.
[55] Patel R, Shervington L, Lea R, et al. Epigenetic silencing of telomerase and a non-alkylating agent as a novel therapeutic approach for glioma. Brain Res,2008,1188:173-81.
[56] Couillard J, Demers M, Lavoie G, et al. The role of DNA hypomethylation in the control of stromelysin gene expression. Biochem Biophys Res Commun, 2006,342(4):1233-9.
[57] Soffietti R, Leoncini B, Ruda R. New developments in the treatment of malignant gliomas.. Curr Opin Oncol. Expert Rev Neurother, 2Q07,7(10):1313-26.
[58] Siegelin MD, Habel A, Gaiser T. Epigalocatechin-3-gallate (EGCG) down regulates PEA15 and thereby augments TRAIL-mediated apoptosis in malignant glioma. Neurosci Lett, 2008,448(1):161-5.
[59] Pi(?)a IC, Gautschi JT, Wang GY, et al. Psammaplins from the sponge Pseudoceratina purpurea: inhibition of both histonedeacetylase and DNA methyltransferase. J Org Chem,2004, 68(10): 3866-3873.
[60] Saikawa Y, Kubota T, Maeda S, et al. Inhibitor of DNA methyltransferase by antisense oligodeoxynucleotide modifies cell characteristics in gastric cancer cell lines. Oncol Rep, 2004,12(3):527-531.
[61] Robert MF, Morin S, Beaulieu N, et al. DNMT1 is required to maintain CpG methylation and aberrant gene silencing in human cancer cells. Nat Genet,2003,33(1):61-65.
[62] Lindsey JC, Lusher ME, Anderton JA,et al. Identification of tumor-specific epigenetic events in medulloblastoma development by hypermethylation profiling.Carcinogenesis,2004,25(5):661-8.
[1]Bangsow C,Rubins N,Glusman G,et al.The RUNX3 gene-sequence,structure and regulated expression.Gene,2001,279(2):221-32.
[2]Kato N,Tamura G;Fukase M,et al.Hypermethylation of the RUNX3 gene promoter in testicular yolk sac tumor of infants.Am J Pathol,2003,163(2):387-391.
[3]Miyazono K,Maeda S,Imamura T.Coordinate regulation of cell growth and differentiation by TGF-beta super family and Runx proteins.Oncogene,2004,23(24):4232-4237.
[4]Li QL,Ito K,Sakakura C,et al.Causal relationship between the loss of RUNX3expression and gastric cancer.Cell,2002,109(1):113-24.
[5]Wei D,Gong W,Oh SC,et al.Loss of RUNX3 expression significantly affects the clinical outcome of gastric cancer patients and its restoration causes drastic suppression of tumor growth and metastasis.Cancer Res,2005,65(11):4809-4816.
[6]Drissi H,Pouliot A,Koolloos C,et al.1,25-(OH)2-vitamin D3 suppresses the bone-related Runx2/Cbfa1 gene promoter.Exp Cell Res.2002,274(2):323-33.
[7] Spender LC, Whiteman HJ, Karstegl CE, Farrell PJ. Transcriptional cross-regulation of RUNX1 by RUNX3 in human B cells. Oncogene,2005,24(11):1873-81.
[8] Massague J. The transforming growth factor-beta family. Annu. Rev. Cell Biol.1990,6,597-641.
[9] Hogan BL. Bone morphogenetic proteins: multifunctional regulators of vertebrate development. Genes Dev,1996,10(13):1580-1594.
[10] Reddi AH. Bone and cartilage differentiation. Curr Opin Genet Dev, 1994,4(5) :737-744.
[11] Massague J. TGF-beta signal transduction. Annu Rev Biochem,1998,67,753-791.
[12] Zhang Y, Derynck R. Regulation of Smad signaling by protein associations and signalling crosstalk. Trends Cell Biol, 1999,9(7) :274-279.
[13] Hanai J, Chen LF, Kanno T, et al. Interaction and functional cooperation of PEBP2/CBF with Smads. Synergistic induction of the immunoglobulin germline Calpha promoter. J Biol. Chem,1999,274(44):31577-31582.
[14] Ito Y. Oncogenic potential of the RUNX gene family: 'overview'.Oncogene,2004,23(24) :4198-4208.
[15] Miyazono K, Maeda S, Imamura T. Coordinate regulation of cell growth and differentiation by TGF-beta superfamily and Runx proteins. Oncogene, 2004,23(24) :4232-4237.
[16] Chi XZ, Yang JO, Lee KY, et al. RUNX3 suppresses gastric epithelial cell growth by inducing p21(WAFl /Cipl) expression in cooperation with transforming growth factor beta-activated SMAD. MolCellBio, 2005, 25(18): 8097-8107.
[17] Coffman JA, Dickey-Sims C, Haug JS, et al. Evaluation of developmental phenotypes produced by morpholino antisense targeting of a sea urchin Runx gene.BMC Biol, 2004,7,2 :6.
[18] Spender LC, Cornish GH, Sullivan A, et al. Expression of transcription factor AML-2 (RUNX3, CBF(alpha)-3) is induced by Epstein-Barr virus EBNA-2 and correlates with the B-cell activation phenotype. J Virol, 2002,76(10):4919-27.
[19] Levanon D, et al. Spatial and temporal expression pattern of Runx3 (Aml2) and Runx1 (Aml1) indicates non-redundant functions during mouse embryogenesis.Mechanisms of Develop, 2001, 109(2): 413-417.
[20] Kramer I, Sigrist M, de Nooij JC, et al. A role for Runx transcription factor signaling in dorsal root ganglion sensory neuron diversification. Neuron,2006,49(3):379-93.
[21] Nakamura S, Senzaki K, Yoshikawa M, et al. Dynamic regulation of the expression of neurotrophin receptors by Runx3. Development,2008,135(9):1703-11.
[22] Inoue K, Ito K, Osato M, et al. The transcription factor Runx3 represses the neurotrophin receptor TrkB during lineage commitment of dorsal root ganglion neurons. J Biol Chem, 2007,282(33):24175-84
[23] Taniuchi I, Osato M, Egawa T, et al. Differential requirements for Runx proteins in CD4 repression and epigenetic silencing during T lymphocyte development. Cell, 2002,111(5):621-33.
[24] Woolf E, Xiao C, Fainaru O, et al. Runx3 and Runxl are required for CD8 T cell development during thymopoiesis. Proc Natl Acad Sci USA,2003,24;100(13):7731-6.
[25] Grueter B, Petter M, Egawa T, et al. Runx3 regulates integrin alpha E/CD103 and CD4 expression during development of CD4-/CD8+ T cells. J Immunol, 2005,175(3):1694-705.
[26] Telfer JC, Hedblom EE, Anderson MK, et al. Localization of the domains in runx transcription factors required for the repression of CD4 in thymocytes. J Immunol.2004,172(7):4359-70.
[27] Meng-Jiao Shi, Janet Stavnezer. CBF 3 (AML2) Is Induced by TGF-β1 to Bind and Activate the Mouse Germline Ig Promoter. The Journal of Immunology, 1998,161:6751-6760.
[28] Park SR, Lee EK, Kim BC, et al. p300 cooperates with Smad3/4 and Runx3 in TGFbetal-induced IgA isotype expression. Eur J Immunol,2003;33(12):3386-92.
[29] Dominguez-Soto A, Relloso M, Vega MA, et al. RUNX3 regulates the activity of the CDlla and CD49d integrin gene promoters. Immunobiology,2005,210(2-4):133-9.
[30] Puig-Kroger A, Sanchez-Eisner T, Ruiz N, et al. RUNX/AML and C/EBP factors regulate CDlla integrin expression in myeloid cells through overlapping regulatory elements. Blood,2003,102(9):3252-61.
[31] Li QL, Ito K, Sakakura C, et al. Causal relationship between the loss of RUNX3 expression and gastric cancer. Cell, 2002, 109(1): 113-24.
[32] Guo WH, Weng LQ, Ito K, et al. Inhibition of growth of mouse gastric cancer cells by RUNX3,a novel tumor suppressor.Oncogene,2002,21(54):8351-8355.
[33]Wei D,Gong W,Oh SC,et al.Loss of RUNx3 expression significantly affects the clinical outcome of gastric cancer patients and its restoration causes drastic suppression of tumor growth and metastasis.Cancer Res,2005,65(11):4809-4816.
[34]Nomoto S,Kinoshita T,Mori T,et al.Adverse prognosis of epigenetic inactivation in RUNX3 gene at 1p36 in human pancreatic cancer.Br J Cancer.2008,98(10):1690-5
[35]Xiao WH,Liu WW.Hemizygous deletion and hypermethylation of RUNX3 gene in hepatocellular carcinoma.World J Gastroenterol,2004,10(3):376-380.
[36]Peng Z,Tang H,Wang X,et al.Inhibition of the growth and metastasis of human colon cancer by restoration of RUNX3 expression in cancer cells.Int J Oncol,2008;33(5):979-84.
[37]Oshimo Y,Oue N,Mitani Y,et al.Frequent loss of RUNX3 expression by promoter hypermethylation in gastric carcinoma.Pathobiology,2004,71(3):137-143.
[38]Kim TY,Lee HJ,Hwang KS,et al.Methylation of RUNX3 in various types of human cancers and premalignant stages of gastric carcinoma.Lab Invest,2004,84(4):479-484.
[39]Sato K,Tomizawa Y,Iijima H,et al.Epigenetic inactivation of the RUNX3 gene in lung cancer.Oncol Rep,2006,15(1):129-35.
[40]Sakaura C,Hasegawa K,Miyagawa K,et al.Possible involvement of RUNX3silencing in the peritoneal metastases of gastric cancers.Clin Cancer Res,2005,11(18):6479-88.
[41]肖文华,刘为纹.肝细胞癌RUNX3基因甲基化与杂合缺失的分析及其意义.中华肝脏病杂志,2004,12(4):227-230.
[42]Li J,Kleeff J,Guweidhi A,et al.RUNX3 expression in primary metastatic pancatic cancer.J Clin Pathol,2004,57(3):294-299.
[43]Yanagawa N,Tamura G,Oizumi H,et al.Promoter hypermethylation of RASSF1A and RUNX3 genes as an independent prognostic prediction marker in surgically resected non-small cell lung cancers.Lung Cancer,2007,58(1):131-8.
[44]Ku JL,Kang SB,Shin YK,et al.Promoter hypermethylation downregulates RUNX3 gene expression in colorectal cancer cell lines.Oncogene,2004;23:6736-6742.
[45] Hiramatsu T, Osaki M, Ito Y, et al. Expression of RUNX3 protein in human esophageal mucosa and squamous cell. Carcinoma. Pathobiology,2005,72(6):316-324.
[46] Long C, Yin B, Lu Q, et al. Promoter hypermethylation of the RUNX3 gene in esophageal squamous cell carcinoma. Cancer Invest. 2007;25(8):685-90.
[47] Tonomoto Y, Tachibana M, Dhar DK,et al. Differential expression of RUNX genes in human esophageal squamous cell carcinoma: downregulation of RUNX3 worsens patient prognosis. Oncology. 2007;73(5-6):346-56.
[48] J Li, J Kleeff, A Guweidhi, I Esposito, PO Berberat, et al. RUNX3 expression in primary and metastatic pancreatic cancer. J Clin Pathol. 2004, 57(3):294-9.
[49] Kirito K, Sakoe K, Shinoda D, et al. A novel RUNX1 mutation in familial platelet disorder with propensity to develop myeloid malignancies, Haematologica,2008 ,93(1):155-6.
[50] Tanne Y, Tanimoto K, Tanaka N, et al. Expression and activity of Runx2 mediated by hyaluronan during chondrocyte differentiation. Arch Oral Biol,2008,53(5):478-87.
[51] Nevadunsky NS, Barbieri JS, Kwong J, et al.RUNX3 protein is overexpressed in human epithelial ovarian cancer. Gynecol Oncol, 2009,112(2):325-30.
[52] Jiang Y, Tong D, Lou G, et al. Expression of RUNX3 gene, methylation status and clinicopathological significance in breast cancer and breast cancer cell lines. Pathobiology,2008,75(4):244-51.
[53] Peng Z, Wei D, Wang L, et al. RUNX3 inhibits the expression of vascular endothelial growth factor and reduces the angiogenesis, growth, and metastasis of human gastric cancer. Clin Cancer Res. 2006,12(21):6386-94.
[2]Asher A,Burger PC,Croteau D,et al.A Primer of brain tumors:a patient's reference.USA,ABTA,2004:8-10.
[3]Koul D.PTEN signaling pathways in glioblastoma.Cancer Biol Ther,2008,7(9):1321-5.
[4]Furnari FB,Fenton T,Bachoo RM,et al.Malignant astrocytic glioma:genetics,biology,and paths to treatment.Genes Dev,2007,21(21):2683-710.
[5]Brandes AA,Tosoni A,Franceschi E,et al.Glioblastoma in adults.Crit Rev Oncol Hematol.2008,67(2):139-52.
[6]Herman JG,Baylin SB.Gene sileneing in cancer in associate with promoter hyermethylation.NewEngl J Med,2003,349:2042-2054.
[7]Jones PA,Takai D.The role of DNA methylation in mammalion epigenetics.Science,2001,293(5532):1068-1070.
[8]Feinberg AP.The epigenetic of cancer etiology.Semi Cancer Biol,2004,14:427-432.
[9]Bangsow C,Rubins N,Glusman G,et al.The RUNX3 gene-sequence,structure and regulated expression.Gene,2001,279(2):221-32.
[10]Kato N,Tamura G,Fukase M,et al.Hypermethylation of the RUNX3 gene promoter in testicular yolk sac tumor of infants.Am J Pathol,2003,163(2):387-391.
[11]Miyazono K,Maeda S,Imamura T.Coordinate regulation of cell growth and differentiation by TGF-beta super family and Runx proteins.Oncogene,2004,23(24):4232-4237.
[12]Wei D,Gong W,Oh SC,et al.Loss of RUNX3 expression significantly affects the clinical outcome of gastric cancer patients and its restoration causes drastic suppression of tumor growth and metastasis.Cancer Res,2005,65(11):4809-4816.
[13]Kim TY,Lee HJ,Hwang KS,et al.Methylation of RUNX3 in various types of human cancers and premalignant stages of gastric carcinoma.Lab Invest,2004,84(4):479-484.
[14]Imamura Y,Hibi K,Koike M,et al.RUNX3 promoter region is specifically methylated in poorly-differentiated colorectal cancer.Anticancer Res,2005,25(4): 2627-2630.
[15] Black PM. Brain tumor, part l.NewEngl J Med, 1991,324:1471-1476.
[16]van Wijnen AJ, et al. Nomenclature for Runt-related (RUNX) proteins.Oncogene,2004,23,4209-4210.
[17] Li QL, Ito K, Sakakura C, et al. Causal relationship between the loss of RUNX3 expression and gastric cancer. Cell, 2002,109:113-124.
[18]Naoyuki Homma, Gen Tamura, Teichiro Honda. Spreading of methylation within RUNX3 CpG island in gastric cancer. Cancer Sci. 2006,97(1):51-6.
[19] Kim WJ, Kim EJ, Jeong P, et al. RUNX3 inactivation by point mutations and aberrant DNA methylation in bladder tumors. Cancer Res. 2005, 65(20):9347-54.
[20]Kleihues P, Cavenee WK, et al. World Health Organization classification of tumors:Pathology and genetics of tumors of the nervous system. Lyon: IARC Press.2000.
[21]Remmele W, Stegner HE. Recommendation for uniform definition of an immunoreactive score (IRS) for immunohistochemical estrogen receptor detection(ER-CIA) in breast cancer tissue. Pathology, 1987,8:138-140.
[22] Berardi MJ, Sun C, Zehr M, et al. The Ig fold of the core binding factor alpha Runt domain is a member of a family of structurally and functionally related Ig-fold DNA-binding domains. Structure 1999,7:1247-56.
[23] Blyth K, Cameron ER, Neil JC.The RUNX genes: gain or loss of function in cancer. Nature Reviews Cancer 2005,5(5): 376-387.
[24] Kramer I, Sigrist M, de Nooij JC, et al. A role for Runx transcription factor signaling in dorsal root ganglion sensory neuron diversification. Neuron,2006,49(3):379-93.
[25] Inoue K, Ito K, Osato M, Lee B, et al. The transcription factor Runx3 represses the neurotrophin receptor TrkB during lineage commitment of dorsal root ganglion neurons. J Biol Chem,2007,282(33):24175-84.
[26] Grueter B, Petter M, Egawa T, Laule-Kilian K, et al. Runx3 regulates integrin alpha E/CD103 and CD4 expression during development of CD4-/CD8+ T cells. J Immunol, 2005 ,175(3):1694-705.
[27]Hasegawa K, Yazumi S, Wada M, et al. Restoration of RUNX3 enhances transforming growth factor-beta-dependent p21 expression in a biliary tract cancer cell line. Cancer Sci. 2007,98(6):838-43.
[28] Chi XZ, Yang JO, Lee KY, et al. I RUNX3 suppresses gastric epithelial cell growth by inducing p21(WAF1/Cip1)expression in cooperation with transforming growth factor {beta}-activated SMAD.Mol Cell Biol.2005,25(18):8097-107.
[29]Sakakura C,Hasegawa K,Miyagawa K,et al.Possible Involvement of RUNX3Silencing in the Peritoneal Metastases of Gastric Cancers.Clin Cancer Res 2005,11(18):6479-88.
[30]Hesson L,Bieche I,Krex D et al.Frequent epigenetic inactivation of RASSF1A and BLU genes located within the critical 3p21.3 region in gliomas.Oncogene.2004,23(13):2408-19.
[31]Annegret Kunitz,Marietta Wolter,et al.DNA hypermethylation and aberrant expression of the EMP3 Gene at 19q13.3 in human gliomas.Brain Pathol,2007,17(4):363-370.
[32]Song BC,Chung YH,Kim JA,et al.Transforming growth factor-beta,as a useful serologic marker of small hepatocel1u1ar carcinoma.Cancer,2002,94(1):175-180.
[33]Hiramatsu T,Osaki M,Ito Y,et al.Expression of RUNX3 Protein in human Esophageal Mucosa and Squamous Cell.Carcinoma.Pathobiology,2005,72(6):316-324.
[34]Yanagawa N,Tamura G,Oizumi H,et al.Promoter hypermethylation of tumor suppressor and tumor-related genes in non-small cell lung cancers.Cancer Sci,2003,94(7):589-592.
[35]倪志.5-Aza-CdR对人结肠癌Lovo细胞增殖调亡及抑瘤基因RUNX3表达的影响.世界华人消化杂志,2006,14(2):184-155.
[36]Mori T,Nomoto S,Koshikawa K,et al.Decreased expression and frequent allelic inactivation of the RUNX3 gene at 1p36 in human hepatocellular carcinoma.Liver Int,2005,25(2):380-388.
[37]Seoane M,Iglesias P,Gonzalez T,et al.Retinoblastoma loss modulates DNA damage response favoring tumor progression.PLoS ONE.2008;3(11):e3632
[38]Kirito K,Sakoe K,Shinoda D,et al.A novel RUNX1 mutation in familial platelet disorder with propensity to develop myeloid malignancies.Haematologica,2008,93(1):155-6.
[39]Tanne Y,Tanimoto K,Tanaka N,et al.Expression and activity of Runx2 mediated by hyaluronan during chondrocyte differentiation.Arch Oral Bioi,2008,53(5):478-87.
[40]Woolf E,Xiao C,Fainaru O,Lotem J,et al.Runx3 and Runx1 are required for CD8 T cell development during thymopoiesis.Proc Natl Acad Sci USA.2003,100(13):7731-6.
[41]Kang S,Kim JW,Kang GH,Park NH,Song YS,Kang SB,Lee HP.Polymorphism in folate-and methionine-metabolizing enzyme and aberrant CpG island hypermethylation in uterine cervical cancer.Gynecol Oncol.2005,96(1):173-80.
[42]肖文华,刘为纹.肝细胞癌RUNX3基因甲基化与杂合缺失的分析及意义。中华肝脏病杂志,2004,12(4)227-23.
[43]Takahashi T,Shivapurkar N,Riquelme E,et al.Aberrant promoter hypermethylation of multiple genes in gallbladder carcinoma and chronic cholecystitis.Clin Cancer Res,2004,10:6126-33.
[44]Sakakura C,Hagiwara A,Miyagawa K,et al.Frequent downregulation of the runt domain transcription factors RUNX1,RUNX3 and their cofactor CBFβ in gastric cancer.Int J Cancer,2005,113(2):221-8
[45]Kurkjian C,Kummar S,Murgo AJ.DNA methylation:its role in cancer development and therapy.Curr Probl Cancer,2008,32(5):187-235.
[46]Jones PA,Baylin SB.The fundamental role of epigenetic events in cancer.Nat Rev Genet,2002,3(6):415-428.
[47]Ehrlich M.DNA methylation in cancer:too much,but also too little.Oncogene,2002,21(35):5400-5413.
[48]Esteller M.DNA methylation and cancer therapy:new developments and expectations.Curr Opin Oneol,2005,17(1):55-60.
[49]Das PM,Singal R.DNA methylation and cancer.J Clin Oncol,2004,22(22):4632-4642.
[50]Davis CD,Uthus EO.DNA methylation,cancer susceptibility,and nutrient interactions.Exp Biol Med,2004,229(10):985-995.
[51]卢圣栋主编.现代分子生物学实验技术.北京:高等教育出版社,1993.465-497.
[52]Eads CA,Laird PW.Combined bisulfite restriction analysis(COBRA).Methods Mol Biol.2002;200:71-85.
[53]Perry AS,Liyanage H,Lawler M,et al.Discovery of DNA hypermethylation using a DHPLC screening strategy.Epigenetics.2007,2(1):43-9.
[54]邓大军,邓国仁等。变性高效液相色谱法检测CpG岛胞嘧啶甲基化。中华医学杂志,2001,81:158-161。
[55]Wu Z,Luo J,Ge Q,et al.Microarray-based Ms-SNuPE:near-quantitative analysis for a high-throughput DNA methylation.Biosens Bioelectron,2008,23(9):1333-9.
[56]Herman JG,Graff JR,Myohanen S,et al.Methylation-specific PCR:a novel PCR assay for methylaiton status of CpG island.Proc Natl Acad Sci,1996,93(18):9821-9826.
[57]von Deimling A,Fimmers R,Schmidt MC,Bender B,Fassbender F,Nagel Jet al.Comprehensive allelotype and genetic anaysis of 466 human nervous system tumors.J Neuropathol Exp Neurol,2000,59(6):544-558.
[58]Schmidt MC,Antweiler S,Urban N,et al.Impact of genotype and morphology on the prognosis of glioblastoma.J Neuro-pathol Exp Neurol,2002,61(4):321-328.
[59]Costello JF,Fruhwald MC,Smiraglia DJ,et al.Aberrant CpG-island methylation has non-random and tumor-type-specific patterns.Nat Genet,2000,24(2):132-138.
[60]Wada M,Yazumi S,Takaishi S,et al.Frequent loss of RUNX3 gene expression in human bile duct and pancreatic cancer cell lines.Oncogene,2004,23(13):2401-2407.
[61]Waki T,Tamura G,Sato M,et al.Age-related methylation of tumor suppressor and tumor-related genes:an analysis of autopsy samples.Oncogene.2003,22(26):4128-33.
[62]Weaver KD,Grossman SA,Heman JG.Methylated tumor specific DNA as a plasm biomarker in patients with glioma.Cancer Investigation,2006,24(1):35-40.
[63]Nomoto S,Kinoshita T,Mori T,et al.Adverse prognosis of epigenetic inactivation in RUNX3 gene at 1p36 in human pancreatic cancer.Br J Cancer.2008,98(10):1690-5.
[64]Peng Z,Tang H,Wang X,et al.Inhibition of the growth and metastasis of human colon cancer by restoration of RUNX3 expression in cancer cells..Int J Oncol.2008,33(5):979-84.
[65]Park WS,Cho YG,Kim C J,et al.Hypermethylation of the RUNX3 gene in hepatocellular carcinoma.Exp Mol Med,2005,37(4):276-281.
[66]Baylin SB,Herman JG,Graff JR,et al.Alterations in DNA methylation:a fundamental aspect of neoplasia.Adv Cancer Res,1998,72:141-196.
[67]杜红玲,戚豫,石永进,等.脱乙酞化酶抑制剂与去甲基化制剂联合诱导UZ“细胞凋亡和p16基因重新表达。癌症,2002,21(10):1057-1061.
[68] Jones PA. Altering gene repression with 5-azacytidine. Cell, 1985, 40(3): 485-486.
[69] Jones PA, Taylor SM. Hemimethylated duplex DNAs prepared from 5-azacytidine treated cells. Nucleic Acids Res, 1981, 9(12):2933-47.
[70] Mueller W, Nutt CL, Ehrich M, et al. Downregulation of RUNX3 and TES by hypermethylation in glioblastoma. Oncogen,2007,26,583-593.
[71] Ku JL, Kang SB, Shin YK, et al. Promoter hypermethylation down regulates RUNX3 gene expression in colorectal cancer cell lines. Oncogene, 2004, 23(40):6736-6742.
[72]Qing-Lin Li, Hye-Ryeon Kim, Wun-Jae Kim et al. Transcriptional silencing of the RUNX3 gene by CpG hypermethylation is associated with lung cancer.Biochem Biophys Res Commun. 2004,314(1):223-8.
[73] Mosmann T. Rapid colorimetric assay for cellular growth and survival .'application to proliferation and cytotoxicity assays. J Immunol Methods,1983,65:55-61.
[74] Albini A, Iwamoto Y, Kleinman HK, et al. A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res, 1987,47:3239-3245.
[75] Yasuko Yamamura, Lee WL, Inoue KI, et al. RUNX3 cooperates with FoxO3a to induce apoptosis in gastric cancer cells. J Biol Chem.2006,24;281(8):5267-76.
[76]Lau QC, Raja E, Salto-Tellez M. RUNX3 Is frequently inactivated by dual mechanisms of protein mislocalization and promoter hypermethylation in breast cancer. Cancer Res. 2006,66(13):6512-20.
[77] Peng Z, Wei D, Wang L,et al.RUNX3 inhibits the expression of vascular endothelial growth factor and reduces the angiogenesis, growth, and metastasis of human gastric cancer. Clin Cancer Res. 2006,12(21):6386-94.
[78] Bender CM, Pao MM, Jones PA. Inhibition of DNA methylation by 5-aza-2'-deoxycytidine suppresses the growth of human tumor cell lines. Cancer Res.1998, 58(1):95-101.
[79] Christman JK. 5-Azacytidine and 5-aza-2'-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy.Oncogene, 2002, 21(35): 5483-5495
[80]Hennessy BT, Garcia-Manero G, Kantarjian HM, et al. DNA methylation in hematological malignancies: the role of decitabine. Expert Opin Investig Drugs,2003, 12(12):1985-1993.
[81]Wijermans P, et al. Low-dose 5-aza-2'-deoxycytidine, a DNA hypomethylating agent , for the treatment of high risks myelodysplastic syndrome : a multicenter pahse 2 study in elderly patients. J Clin Oncol,2000,18(5) :956-962.
[82]Sorm F, Piskala A, Cihak A, et al. 5-azacytidine, a new, highly effective cancerostatic. Experientia,1964,20(4) :202-203.
[83]Yoshida M, Kijima M, Akita M, et al. Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A. J Biol Chem, 1990,265(28) :17174-17179.
[84]Pina IC, Gautschi JT, Wang GY, et al. Psammaplins from the sponge Pseudoceratina purpurea rinhibition of both histone deacetylase and DNA methyltransferase. J Org Chem, 2003,68(10) :3866-73.
[85] Berletch JB, Liu C, Love WK, Epigenetic and genetic mechanisms contribute to telomerase inhibition by EGCG J Cell Biochem. 2008, 103(2):509-19.
[86]Coffrnan JA, Dickey-Sims C, Haug JS, et al. Evaluation of developmental phenotypes produced by morpholino antisense targeting of a sea urchin Runx gene.BMC Biol, 2004,7,2 :6.
[1]Wolffe AP,Matzke MA.Epigenetics:regulation through repression.Science,1999,286(5439):481-486.
[2] Lewin B. GeneⅧ. Perarson PrencHall press. 2004.
[3] Jaenisch R, Bird A. Epigenetic regulation of gene expression: how the genomeintegrates intrinsic and environmental signals. Nat Genet,2003,33:245-254.
[4] Couzin J. Small RNAs make big splash.Science,2002,29(56):2296-2297.
[5] Giacinti L, Vici P, Lopez M. Epigenome: A new target in cancer therapy. Clin Ter,2008,159(5):347-60.
[6] Baylin SB, Esteller M, Rountree MR, et al. Aberrant patterns of DNA methylation, chromatin formation and gene expression in cancer. Hum Mol Genet,2001,10(7):687-692.
[7] Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer. Nat Rev Genet, 2002, 3(6):415-428.
[8] Cross SH, Bird AP. CpG islands and genes. Curr Opin Genet Dev, 1995, 5(3):309-314.
[9] Jones PA, Laird PW. Cancer epigenetics comes of age. Nat Genet, 1999,21(2):163-167.
[10] Ehrlich M. DNA methylation in cancer: too much, but also too little. Oncogene,2002,21(35):5400-5413.
[11] Eads CA, Laird PW. Combined bisulfite restriction analysis (COBRA). Methods Mol Biol,2002,200:71-85.
[12] Wong IH. Qualitative and quantitative polymerase chain reaction-based methods for DNA methylation analyses. Methods Mol Biol,2006,336:33-43.
[13] Campan M, Weisenberger DJ, Trinh B, et al. Methylight. Methods Mol Biol.2009,507:325-37.
[14] Perry AS, Liyanage H, Lawler M, et al. Discovery of DNA hypermethylation using a DHPLC screening strategy. Epigenetics,2007,2(1):43-9.
[15] Wu Z, Luo J, Ge Q, et al. Microarray-based Ms-SNuPE: near-quantitative analysis for a high-throughput DNA methylation. Biosens Bioelectron,2008,23(9):1333-9.
[16] Costello JF, Hong C, Plass C, et al. Restriction landmark genomic scanning: analysis of CpG islands in genomes by 2D gel electrophoresis. Methods Mol Biol.2009,507:131-48.
[17] Huang TH, Perry MR, Laux DE. Methylation profiling of CpG islands in human breast cancer cells. HumMol Genet, 1999,8(3):459.
[18] Gitan RS, Shi H, Chen CM, et al. Methylation-specific oligonucleotide microarray: a new potential for high-throughput methylation analysis. Genome Res, 2002, 12(1):158.
[19] Shi H, Wei SH, Leu YW, et al. Triple analysis of the cancer epigenome: an integrated microarray system for assessing gene expression, DNA methylation,and histone acetylation. Cancer Res, 2003, 63(9):2164.
[20] Donninger H, Vos MD, Clark GJ. The RASSF1A tumor suppressor. J Cell Sci,2007,120(18):3163-72
[21] Hesson L, Bieche I, Krex D et al. Frequent epigenetic inactivation of RASSF1A and BLU genes located within the critical 3p21.3 region in gliomas. Oncogene,2004,23(13):2408-19.
[22] Horiguchi K, Tomizawa Y, Tosaka M, et al. Epigenetic inactivation of RASSF1A candidate tumor suppressor gene at 3p21.3 in brain tumors. Oncogene,2003,22(49):7862-5.
[23] Taylor V, Suter U. Epithelial membrane protein-2 and epithelial membrane protein-3: two novel members of the peripheral myelin protein 22 gene family.Gene, 1996,175(1-2):115-20.
[24] Kunitz A, Wolter M, van den Boom J, et al. DNA hypermethylation and aberrant expression of the EMP3 Gene at 19ql 3.3 in human gliomas. Brain Pathol,2007,17(4):363-370.
[25] Alaminos M, Davalos V, Ropero S, et al. EMP3 a myelin-related gene located in the critical 19p13.3 region, is epigenetically silenced and exhibits features of a candidate tumor suppressor in glioma and neuroblastoma. Cancer Res,2005,65(7):2565-71.
[26] Kamb A, Gruis NA, Weaver-Feldhaus J, et al. A cell cycle regulator potentially involved in genesis of many tumor types. Science, 1994, 264(5): 436-440.
[27] Izumoto S, Arita N, Ohnishi T, et al. Homozygous deletions of p16INK4A/MTS1 and p15INK4B/MTS2 genes in glioma cells and primary glioma tissues. Cancer Lett. 1995, 97(2):241-7.
[28] Costello JF, Berger MS, Huang HS, et al. Silencing of pl6/CDKN2 expression in human gliomas by methylation and chromatin condensation. Cancer Res, 1996,56(10):2405-10.
[29] Nakamura M, Watanabe T, Yonekawa Y, et al. Promoter methylaiton of the DNA repair gene MGMT in astrocytomas is frequently associated with G:C-A:T mutation of the TP53 tumor suppressor gene. Carcinogenesis, 2001,22(10):1715-1719.
[30] Silber JR, Bobola MS, Ghatan S, et al. O6-methylguanine-DNA methyltransferase activity in adult gliomas: relation to patient and tumor characteristics. Cancer Res, 1998,58,(5):1068-1073.
[31] WE Farrell, DJ Simpson, SJ Frost, et al. Methylation mechanisms in pituitary tumorigenesis. Endocr Relat Cancer, 1999,6(4): 437-447.
[32] Simpson DJ, Bicknell JE, McNicol AM, et al. Hypermethylation of the P16/CDKN2/MTST gene and loss of protein expression is associated with nonfunctional pituitary adenomas but not somatotrophinomas. Genes Chromosomes Cancer. 1999,24(4):221-224.
[33] Bello MJ, De Campos JM, Isla A, et al. Promoter CpG methylation of multiple genes in pituitary adenomas: Frequent involvement of Caspase-8. Oncol Rep,2006,15(2): 443-448.
[34] Yoshino A, Katayama Y, Ogino A, et al. Promoter hypermethylation profile of cell cycle regulator genes in pituitary adenomas. J Neurooncol,2007,83(2):153-162.
[35] Zhao J, Dahle D, Zhou Y, et al. Hypermethylation of the promoter region is associated with the loss of MEG3 gene expression in human pituitary tumors. J Clin Endocrinol Metab, 2005,90(4):2179-86.
[36] Liu Y, Pang JC, Dong S, et al. Aberrant CpG island hypermethylation profile is associated with atypical and anaplastic meningiomas. Hum Pathol,2005,36(4):416-425.
[37] Tse JY, Ng HK, Lo KW, et al. Analysis of cell cycle regulators: p16INK4A, pRb,and CDK4 in low-grade and high-grade meningiomas. Hum Pathol.1998,29(11):1200-7.
[38]Nakane Y, Natsume A, et al. Malignant transformation-related genes in meningiomas: allelic loss on Ip36 and methylation status of p73 and RASSF1A.J Neurosurg,2007,107(2):398-404.
[39] de Robles P, Mclntyre J, Kalra S, et al. Methylation status of MGMT gene promoter in meningiomas. Cancer Genet Cytogenet,2008,187(1):25-7.
[40] Inda MM, Castresana JS. RASSF1A promoter is highly methylated in primitive neuroectodermal tumors of the central nervous system. Neuropathology,2007,27(4):341-6.
[41] Gonzalez-Gomez P, Bello MJ, Inda MM. Deletion and aberrant CpG island methylation of Caspase 8 gene in medulloblastoma. Oncol Rep.2004,12(3):663-6.
[42] Lassaletta L, Patron M, Gonzalez T, et al.RASSF1A methylation and cyclin D1 expression in vestibular schwannomas. Acta Neuropathol,2007, 114(4):431-3.
[43] Losi-Guembarovski R, Kuasne H, Guembarovski AL, et al. DNA methylation patterns of the CDH1, RARB, and SFN genes in choroid plexus tumors. Cancer Genet Cytogenet. 2007, 179(2): 140-5.
[44] Yu L, Liu C, Vandeusen J, et al. Global assessment of promoter methylation in a mouse model of cancer identifies ID4 as a putative tumor suppressor gene in human leukemia. Nat Genet, 2005, 37(3):265-274.
[45] Tsou JA, Hagen JA, Carpenter CL, et al. DNA methylation analysis: a powerful new tool for lung cancer diagnosis. Oncogene, 2002, 21(35): 5450-5461.
[46] Sidransky D. Emerging molecular markers of cancer. Nat Rev Cancer, 2002,2(3):210-219.
[47] Honorio S, Agathanggelou A, Schuermann M, et al. Detection of RASSF1A aberrant promoter hypermethylation in sputum from chronic smokers and ductal carcinoma in situ from breast cancer patients. Oncogene, 2003, 22(1):147-150.
[48] Wakabayashi T, Natsume A, Hatano H, et al. p16 promoter methylation in the serum as a basis for the molecular diagnosis of gliomas. Neurosurgery.2009;64(3):455-61;
[49] Weaver KD, Grossman SA, Heman JG. Methylated tumor specific DNA as a plasma biomarker in patients with glioma. Cancer Investigation, 2006,24(1):35-40.
[50] Tao L, Yang S, Xie M, et al. Hypomethylation and over-expression of c-jun and c-myc protoonco genes and increased DNA methyltransferase activity in dichloroacetic and trichloroacetic acid-promoted mouse liver tumors. Cancer Lett,2000,158(2):185-193.
[51] Cadieux B, Ching TT, VandenBerg SR, et al. Genome-wide hypomethylation in human glioblastomas associated with specific copy number alteration, methylenetetrahydrofolate reductase allele status, and increased proliferation.Cancer Res,2006, 66(17):8469-76.
[52] Bredel M. Anticancer drug resistance in primary human brain tumors. Brain Res,2001,35, 161-204.
[53] Hegi ME, Diserens AC, Gorlia T et al. MGMT gene silencing and benefit from temozolomide in glioblastoma. N Eng J Med,2005, 352(10), 997-1003.
[54] Blanc JL, Wager M, Guilhot J, et al. Correlation of clinical features and methylation status of MGMT gene promoter in glioblastoma. J Neuro-Oncology,2004, 68(3):275-283.
[55] Patel R, Shervington L, Lea R, et al. Epigenetic silencing of telomerase and a non-alkylating agent as a novel therapeutic approach for glioma. Brain Res,2008,1188:173-81.
[56] Couillard J, Demers M, Lavoie G, et al. The role of DNA hypomethylation in the control of stromelysin gene expression. Biochem Biophys Res Commun, 2006,342(4):1233-9.
[57] Soffietti R, Leoncini B, Ruda R. New developments in the treatment of malignant gliomas.. Curr Opin Oncol. Expert Rev Neurother, 2Q07,7(10):1313-26.
[58] Siegelin MD, Habel A, Gaiser T. Epigalocatechin-3-gallate (EGCG) down regulates PEA15 and thereby augments TRAIL-mediated apoptosis in malignant glioma. Neurosci Lett, 2008,448(1):161-5.
[59] Pi(?)a IC, Gautschi JT, Wang GY, et al. Psammaplins from the sponge Pseudoceratina purpurea: inhibition of both histonedeacetylase and DNA methyltransferase. J Org Chem,2004, 68(10): 3866-3873.
[60] Saikawa Y, Kubota T, Maeda S, et al. Inhibitor of DNA methyltransferase by antisense oligodeoxynucleotide modifies cell characteristics in gastric cancer cell lines. Oncol Rep, 2004,12(3):527-531.
[61] Robert MF, Morin S, Beaulieu N, et al. DNMT1 is required to maintain CpG methylation and aberrant gene silencing in human cancer cells. Nat Genet,2003,33(1):61-65.
[62] Lindsey JC, Lusher ME, Anderton JA,et al. Identification of tumor-specific epigenetic events in medulloblastoma development by hypermethylation profiling.Carcinogenesis,2004,25(5):661-8.
[1]Bangsow C,Rubins N,Glusman G,et al.The RUNX3 gene-sequence,structure and regulated expression.Gene,2001,279(2):221-32.
[2]Kato N,Tamura G;Fukase M,et al.Hypermethylation of the RUNX3 gene promoter in testicular yolk sac tumor of infants.Am J Pathol,2003,163(2):387-391.
[3]Miyazono K,Maeda S,Imamura T.Coordinate regulation of cell growth and differentiation by TGF-beta super family and Runx proteins.Oncogene,2004,23(24):4232-4237.
[4]Li QL,Ito K,Sakakura C,et al.Causal relationship between the loss of RUNX3expression and gastric cancer.Cell,2002,109(1):113-24.
[5]Wei D,Gong W,Oh SC,et al.Loss of RUNX3 expression significantly affects the clinical outcome of gastric cancer patients and its restoration causes drastic suppression of tumor growth and metastasis.Cancer Res,2005,65(11):4809-4816.
[6]Drissi H,Pouliot A,Koolloos C,et al.1,25-(OH)2-vitamin D3 suppresses the bone-related Runx2/Cbfa1 gene promoter.Exp Cell Res.2002,274(2):323-33.
[7] Spender LC, Whiteman HJ, Karstegl CE, Farrell PJ. Transcriptional cross-regulation of RUNX1 by RUNX3 in human B cells. Oncogene,2005,24(11):1873-81.
[8] Massague J. The transforming growth factor-beta family. Annu. Rev. Cell Biol.1990,6,597-641.
[9] Hogan BL. Bone morphogenetic proteins: multifunctional regulators of vertebrate development. Genes Dev,1996,10(13):1580-1594.
[10] Reddi AH. Bone and cartilage differentiation. Curr Opin Genet Dev, 1994,4(5) :737-744.
[11] Massague J. TGF-beta signal transduction. Annu Rev Biochem,1998,67,753-791.
[12] Zhang Y, Derynck R. Regulation of Smad signaling by protein associations and signalling crosstalk. Trends Cell Biol, 1999,9(7) :274-279.
[13] Hanai J, Chen LF, Kanno T, et al. Interaction and functional cooperation of PEBP2/CBF with Smads. Synergistic induction of the immunoglobulin germline Calpha promoter. J Biol. Chem,1999,274(44):31577-31582.
[14] Ito Y. Oncogenic potential of the RUNX gene family: 'overview'.Oncogene,2004,23(24) :4198-4208.
[15] Miyazono K, Maeda S, Imamura T. Coordinate regulation of cell growth and differentiation by TGF-beta superfamily and Runx proteins. Oncogene, 2004,23(24) :4232-4237.
[16] Chi XZ, Yang JO, Lee KY, et al. RUNX3 suppresses gastric epithelial cell growth by inducing p21(WAFl /Cipl) expression in cooperation with transforming growth factor beta-activated SMAD. MolCellBio, 2005, 25(18): 8097-8107.
[17] Coffman JA, Dickey-Sims C, Haug JS, et al. Evaluation of developmental phenotypes produced by morpholino antisense targeting of a sea urchin Runx gene.BMC Biol, 2004,7,2 :6.
[18] Spender LC, Cornish GH, Sullivan A, et al. Expression of transcription factor AML-2 (RUNX3, CBF(alpha)-3) is induced by Epstein-Barr virus EBNA-2 and correlates with the B-cell activation phenotype. J Virol, 2002,76(10):4919-27.
[19] Levanon D, et al. Spatial and temporal expression pattern of Runx3 (Aml2) and Runx1 (Aml1) indicates non-redundant functions during mouse embryogenesis.Mechanisms of Develop, 2001, 109(2): 413-417.
[20] Kramer I, Sigrist M, de Nooij JC, et al. A role for Runx transcription factor signaling in dorsal root ganglion sensory neuron diversification. Neuron,2006,49(3):379-93.
[21] Nakamura S, Senzaki K, Yoshikawa M, et al. Dynamic regulation of the expression of neurotrophin receptors by Runx3. Development,2008,135(9):1703-11.
[22] Inoue K, Ito K, Osato M, et al. The transcription factor Runx3 represses the neurotrophin receptor TrkB during lineage commitment of dorsal root ganglion neurons. J Biol Chem, 2007,282(33):24175-84
[23] Taniuchi I, Osato M, Egawa T, et al. Differential requirements for Runx proteins in CD4 repression and epigenetic silencing during T lymphocyte development. Cell, 2002,111(5):621-33.
[24] Woolf E, Xiao C, Fainaru O, et al. Runx3 and Runxl are required for CD8 T cell development during thymopoiesis. Proc Natl Acad Sci USA,2003,24;100(13):7731-6.
[25] Grueter B, Petter M, Egawa T, et al. Runx3 regulates integrin alpha E/CD103 and CD4 expression during development of CD4-/CD8+ T cells. J Immunol, 2005,175(3):1694-705.
[26] Telfer JC, Hedblom EE, Anderson MK, et al. Localization of the domains in runx transcription factors required for the repression of CD4 in thymocytes. J Immunol.2004,172(7):4359-70.
[27] Meng-Jiao Shi, Janet Stavnezer. CBF 3 (AML2) Is Induced by TGF-β1 to Bind and Activate the Mouse Germline Ig Promoter. The Journal of Immunology, 1998,161:6751-6760.
[28] Park SR, Lee EK, Kim BC, et al. p300 cooperates with Smad3/4 and Runx3 in TGFbetal-induced IgA isotype expression. Eur J Immunol,2003;33(12):3386-92.
[29] Dominguez-Soto A, Relloso M, Vega MA, et al. RUNX3 regulates the activity of the CDlla and CD49d integrin gene promoters. Immunobiology,2005,210(2-4):133-9.
[30] Puig-Kroger A, Sanchez-Eisner T, Ruiz N, et al. RUNX/AML and C/EBP factors regulate CDlla integrin expression in myeloid cells through overlapping regulatory elements. Blood,2003,102(9):3252-61.
[31] Li QL, Ito K, Sakakura C, et al. Causal relationship between the loss of RUNX3 expression and gastric cancer. Cell, 2002, 109(1): 113-24.
[32] Guo WH, Weng LQ, Ito K, et al. Inhibition of growth of mouse gastric cancer cells by RUNX3,a novel tumor suppressor.Oncogene,2002,21(54):8351-8355.
[33]Wei D,Gong W,Oh SC,et al.Loss of RUNx3 expression significantly affects the clinical outcome of gastric cancer patients and its restoration causes drastic suppression of tumor growth and metastasis.Cancer Res,2005,65(11):4809-4816.
[34]Nomoto S,Kinoshita T,Mori T,et al.Adverse prognosis of epigenetic inactivation in RUNX3 gene at 1p36 in human pancreatic cancer.Br J Cancer.2008,98(10):1690-5
[35]Xiao WH,Liu WW.Hemizygous deletion and hypermethylation of RUNX3 gene in hepatocellular carcinoma.World J Gastroenterol,2004,10(3):376-380.
[36]Peng Z,Tang H,Wang X,et al.Inhibition of the growth and metastasis of human colon cancer by restoration of RUNX3 expression in cancer cells.Int J Oncol,2008;33(5):979-84.
[37]Oshimo Y,Oue N,Mitani Y,et al.Frequent loss of RUNX3 expression by promoter hypermethylation in gastric carcinoma.Pathobiology,2004,71(3):137-143.
[38]Kim TY,Lee HJ,Hwang KS,et al.Methylation of RUNX3 in various types of human cancers and premalignant stages of gastric carcinoma.Lab Invest,2004,84(4):479-484.
[39]Sato K,Tomizawa Y,Iijima H,et al.Epigenetic inactivation of the RUNX3 gene in lung cancer.Oncol Rep,2006,15(1):129-35.
[40]Sakaura C,Hasegawa K,Miyagawa K,et al.Possible involvement of RUNX3silencing in the peritoneal metastases of gastric cancers.Clin Cancer Res,2005,11(18):6479-88.
[41]肖文华,刘为纹.肝细胞癌RUNX3基因甲基化与杂合缺失的分析及其意义.中华肝脏病杂志,2004,12(4):227-230.
[42]Li J,Kleeff J,Guweidhi A,et al.RUNX3 expression in primary metastatic pancatic cancer.J Clin Pathol,2004,57(3):294-299.
[43]Yanagawa N,Tamura G,Oizumi H,et al.Promoter hypermethylation of RASSF1A and RUNX3 genes as an independent prognostic prediction marker in surgically resected non-small cell lung cancers.Lung Cancer,2007,58(1):131-8.
[44]Ku JL,Kang SB,Shin YK,et al.Promoter hypermethylation downregulates RUNX3 gene expression in colorectal cancer cell lines.Oncogene,2004;23:6736-6742.
[45] Hiramatsu T, Osaki M, Ito Y, et al. Expression of RUNX3 protein in human esophageal mucosa and squamous cell. Carcinoma. Pathobiology,2005,72(6):316-324.
[46] Long C, Yin B, Lu Q, et al. Promoter hypermethylation of the RUNX3 gene in esophageal squamous cell carcinoma. Cancer Invest. 2007;25(8):685-90.
[47] Tonomoto Y, Tachibana M, Dhar DK,et al. Differential expression of RUNX genes in human esophageal squamous cell carcinoma: downregulation of RUNX3 worsens patient prognosis. Oncology. 2007;73(5-6):346-56.
[48] J Li, J Kleeff, A Guweidhi, I Esposito, PO Berberat, et al. RUNX3 expression in primary and metastatic pancreatic cancer. J Clin Pathol. 2004, 57(3):294-9.
[49] Kirito K, Sakoe K, Shinoda D, et al. A novel RUNX1 mutation in familial platelet disorder with propensity to develop myeloid malignancies, Haematologica,2008 ,93(1):155-6.
[50] Tanne Y, Tanimoto K, Tanaka N, et al. Expression and activity of Runx2 mediated by hyaluronan during chondrocyte differentiation. Arch Oral Biol,2008,53(5):478-87.
[51] Nevadunsky NS, Barbieri JS, Kwong J, et al.RUNX3 protein is overexpressed in human epithelial ovarian cancer. Gynecol Oncol, 2009,112(2):325-30.
[52] Jiang Y, Tong D, Lou G, et al. Expression of RUNX3 gene, methylation status and clinicopathological significance in breast cancer and breast cancer cell lines. Pathobiology,2008,75(4):244-51.
[53] Peng Z, Wei D, Wang L, et al. RUNX3 inhibits the expression of vascular endothelial growth factor and reduces the angiogenesis, growth, and metastasis of human gastric cancer. Clin Cancer Res. 2006,12(21):6386-94.