【作者】 谢源阳；

【导师】 袁贤瑞；

【作者基本信息】 中南大学 ， 外科学， 2009， 博士

【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（X²=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。更多还原

【Abstract】 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 gliomaObjective: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 gliomaObjective: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_proteinin＜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 cellsObjective: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.更多还原