【作者】 周旋；

【导师】 浦佩玉； 康春生；

【作者基本信息】 天津医科大学 ， 外科学， 2008， 硕士

【摘要】 脑胶质瘤是人类最常见难治性颅内恶性肿瘤,其中高度恶性的胶质母细胞瘤一年生存率仅为46%。近年发现了如EGFR基因过表达、PTEN基因表达缺失、p53基因突变等与该肿瘤发病相关的分子事件;从而认识到胶质瘤的发生是涉及细胞周期演进调控失衡、信号转导通路成员异常表达、凋亡与死亡和细胞代谢紊乱等多种基因功能发生紊乱的病理过程。联合基因治疗是胶质瘤基因治疗的发展趋势。因此,当前关于如何实现同时对多个癌基因或者抑癌基因进行调控成为肿瘤研究的核心问题。MiRNA是一类平均长度为22bp非编码小RNA。MiRNA通过和靶基因3’非翻译区种子序列完全和或不完全互补结合的形式来发挥其转录后水平基因表达的调控作用。每个miRNA具有数十种到甚至更多的蛋白表达调控靶点,而每一种蛋白表达有可能同时受到多个miRNA的调控,目前人类已经发现的四百余种miRNA可以调控三分之一基因的表达水平。MiRNA因此被认为对基因表达起着精细、广泛的调节作用,也成为了肿瘤分子生物学研究领域的新热点。本课题重点研究了胶质瘤中miRNA表达异常,应用反义miRNA寡聚核苷酸转染技术干涉异常表达的miRNA对胶质瘤恶性表型的影响以及和EGFR、PAKT-PI3K信号转导通路的关系及其机制。课题分四部分进行:方法第一部分使用晶芯^?哺乳动物miRNA微阵列芯片分析了5个胶质母细胞瘤细胞系和1个星形细胞瘤细胞系miRNA表达谱,筛选出一系列表达升高或者降低的miRNA,其中以miR-21在6个胶质瘤细胞系中表达升高最为均匀一致;并且我们使用Real-time PCR和原位杂交法对5个胶质瘤细胞系、1个正常细胞系、60例胶质瘤标本、10例髓母细胞瘤和6例正常脑组织miR-21表达进一步验证。并采取PCR方法对部分胶质瘤标本扩增miR-21基因组DNA编码区,直接测序,研究了胶质瘤中基因组DNA miRNA的扩增和突变情况。第二部分通过生物信息学手段,建立miRNA靶点选择数学模型,结合现有miRNA靶点数据库对miR-21在胶质瘤发生发展过程中下游候选靶基因进行筛选;克隆含有候选靶基因3’非翻译区全部种子序列的荧光素酶报告载体,对miR-21候选靶基因进行鉴定。第三部分在体外应用反义miRNA寡聚核苷酸转染技术以Oligofectamine介导AS-miR-21靶向miR-21转染U251人脑胶质瘤细胞系后,应用Real-time PCR和原位杂交法对miR-21表达敲低进行检测;并用免疫荧光和Western blot技术检测了miR-21敲低后,PI3K-AKT信号转导通路成员、肿瘤恶性表型相关蛋白、以及我们实验室新近验证的肿瘤生长抑制因子septin-7的表达变化;使用MTT、Annexin V法、流式细胞术及Matrigel 2D、3D生长实验等方法分析了转染前后U251细胞增殖、凋亡、细胞周期分布和侵袭能力的变化。第四部分中则通过建立裸鼠胶质瘤皮下动物模型,瘤内注射Oligofectamine和AS-miR-21混合物,动态观察肿瘤生长情况,检测反义miRNA寡聚核苷酸转染技术敲低U251裸鼠胶质瘤miR-21表达后,PI3K-AKT信号转导通路成员等蛋白表达水平的变化,并且使用TUNEL法检测细胞凋亡,对体外实验的结果进一步验证。结果MiRNA表达谱结果显示:在胶质瘤细胞系中,miR-21等8种miRNA一致表达上调超过正常脑组织的2倍;一致表达下调的miRNA包括miR-127等18种miRNA一致表达下调为正常脑组织的50%以下;Real-time PCR证实,miR-21在胶质瘤中高表达;另外,原位杂交显示在胶质瘤细胞系中miR-21高表达,在60例胶质瘤标本中miR-21表达水平和病理分级正相关,在髓母细胞瘤和正常脑组织中miR-21表达缺失。通过数学模型推测.PTEN和Septin-7是miR-21潜在作用靶点:PTEN基因mRNA得分为93.837,自由度为-7,预测种子序列起始于3’UTR第23个碱基;Septin-7基因mRNA得分为94.0019,自由度为-8,预测种子序列起始于3’UTR第515个碱基。荧光素酶报告实验显示共转染PTEN、Septin-73’UTR的荧光素酶报告载体和AS-miR-21组的荧光值显著高于单转染报告载体组。Real-time PCR和原位杂交结果表明:转染AS-miR-21后可以显著抑制miR-21表达水平。MTT结果显示转染AS-miR-21后细胞增殖率与转染无义ODN和对照组相比明显受抑（P=0.000）,通过免疫荧光和Western blot检测发现EGFR、PI3K-AKT信号转导通路的主要成员表达水平和Bcl-2等肿瘤恶性生物学表型相关蛋白均明显降低,Septin-7、TIMP-1等肿瘤生长抑制因子表达升高。应用Annexin V检测发现转染AS-miR-21组细胞凋亡率明显升高,进一步应用流式细胞术检测细胞周期分布发现细胞周期阻滞于G0/G1期,S期细胞比例降低。成功建立U251和U87胶质瘤细胞系裸鼠皮下肿瘤后,每3天测量一次肿瘤体积,并在肿瘤局部多点注射AS-miR-21和Oligofectamine混合物,发现和对照组、无义对照组相比,AS-miR-21治疗组肿瘤生长缓慢,肿瘤体积差别在观察末期（24天）最显著。应用免疫组化法检测肿瘤标本发现,miR-21表达水平被敲低后,EGFR、PAKT-PI3K等信号转导通路的主要成员（AKT-2、PAKT等）表达水平、肿瘤细胞增殖相关蛋白Ki67、肿瘤凋亡抑制相关蛋白Bcl-2、细胞周期正向演进相关蛋白CyclinD、侵袭相关蛋白MMP-9、均明显降低,Septin-7、TIMP-1等肿瘤生长抑制因子表达升高。应用TUNEL法检测原位细胞凋亡发现AS-miR-21治疗组凋亡细胞核比例明显增加。体内和体外实验的结果一致。结论1.胶质瘤中存在一系列miRNA表达异常,miR-21过表达可以被认为是人脑胶质瘤新的分子标签。2.PTEN、Septin-7基因mRNA的3’UTR是miR-21直接作用靶点。3.Oligofectamine介导转染AS-miR-21可有效敲低miR-21在U251人胶质母细胞瘤细胞系的表达,同时抑制EGFR、PI3K-AKT信号转导通路的活性,上调Septin-7表达,从而抑制了细胞的增殖和侵袭能力,出现G0/G1期阻滞,并诱导细胞凋亡。4.裸鼠皮下胶质瘤模型应用Oligofectamine介导的AS-miR-21治疗,同样可有效敲低miR-21的表达,抑制EGFR、PAKT-PI3K信号转导通路的活性,上调Septin-7表达,从而抑制肿瘤的生长,诱导细胞凋亡,与体外试验结果一致。5.miR-21可成为胶质瘤基因治疗的侯选靶点,反义miRNA寡聚核苷酸转染技术是一项有效的基因沉默技术,具有临床应用前景。更多还原

【Abstract】 Human glioma was the most frequent human brain tumor and one year survival rate of most high-grade glioma was 46%. The oncogenesis of glioma and effective therapy method was the most core theme of neurosurgery and euro-oncology. Recently, some tumor oncogenesis correlated molecular events, including EGFR over expression, PTEN and p53 gene mutant, were discovered with the primary development in molecular-biology and molecular genetics study in glioma. It had been proved that, glioma oncogenesis was a complex process which including cell cycle progression disequilibrium, abnormal expression of signaling pathway key components, cell apoptosis, death or cell metabolism disorder. With thorough study of brain tumor, the molecular-pathology difference that contributes to the occurrence of primary and secondary glioma was still obscure. Thus, to study the signaling pathway key components’ abnormal expression could better assess the essence of glioma than studying one single gene expression disorder.MiRNA was a class of～22bp small non-coding RNA which can regulate gene expression at the post-transcription level by perfect or imperfect complementarity to human the seed sequence in 3’UTR of its target gene. Each miRNA may have several or more targets and one protein expression could be regulated by several miRNA. The miRNAs that had been verified contributed to 1/3 of gene expression in nature. Therefore, because of miRNA’s fine and extensive gene expression regulation status, it had been on focus in tumor molecular biology study field.The present study emphasis on miRNA expression disorder, tumor malignant phenotype changes by targeting disorder expressed miRNA by antisense oligonucleotide, EGFR/PI3K-AKT signaling pathway interactions with miRNA and possible mechanism. The present study was clarified into 4 parts:MethodIn chapter one, miRNA microarray chip was used to analyze the miRNA expression profile in one astrocytoma and five glioblastoma cell line to scan a class of up-regulated or down-regulated miRNAs. Among these, miR-21 was the only miRNA which was over expressed across 6 glioma cell line. Real-time PCR and in-situ hybridization method was used to identify over expression status of miR-21 in 60 glioma samples, 10 medullablastoma samples and 6 glioblastoma cell lines. miR-21 genomic coding region was amplified by PCR and the PCR products were sequenced to detect the mutant status. In chapter two, miRNA target prediction model was established and combined with the classic database, we scan several candidate target mRNA. The 3’UTR of the predicted target mRNAs were cloned into a luciferase reporter and luciferase assay was conducted to identify the real target of miR-21.In chapter three, Oligofectamine reagent was used to transfect antisense miR-21 oligo nucleotide suppress U251 glioma cell line growth in vitro and the malignant phenotype was observed. Real-time PCR and in-situ hybridization method was used to prove AS-miR-21 ’s effect; immuno-fluoresence and western blot were conducted to detect PI3K-AKT signaling pathway components, malignant phenotype related proteins, and the notable identified tumor suppressor Septin-7 expression; MTT assay, Annexin V, flow-cytometry assay, 2D,3D martrigel growth assay were used to detect the U251 cell proliferation, apoptosis, cell cycle distribution and invasion changes after knocking down miR-21 expression in vitro.In chapter four, U87 and U251 nude mice xenograft tumor model was established and the mixture of AS-miR-21 and oligofectamine was injected into the xenograft tumors every three days. The tumor volume was calculated to evaluate the anti-tumor effect of AS-miR-21. Immuno-histochemistry stain and TUNEL assay were laid out to further prove the results of the present study results in vitro.ResultsIn chapter one, the miRNA expression profile indicated, 8 miRNAs, including miR-21, were 2 folds higher over expressed across the cell lines; 18 miRNAs, including miR-127, were 50% less expressed than in the normal brain control. Real-time PCR results proved that, miR-21 was over-expressed in glioma samples. In addition, in-situ hybridization method miR-21 condition in 60 glioma samples was positively correlated to the pathological classification criteria and the PI3K-AKT, EGFR signaling pathway components; and its expression was negatively correlated to PTEN, Septin-7 expression across the 60 WHO classified gliomas.In chapter two, the previous established math model shown that, PTEN and Septin-7 were the possible targets of miR-21. The score of PTEN gene mRNA was 93.837, freedom was -7, the predicted seed sequence started form the 23rd base of PTEN mRNA 3’UTR; The score of Septin-7 gene mRNA was 94.0019, freedom was -8, the predicted seed sequence started form the 515th base of Septin-7 mRNA 3’UTR. Luciferase assay replied that, luciferase activity of the pGL-3-luc and AS-miR-21 co-transfection groups were significantly higher than it in the pGL-3-luc control group.In chapter three, Real-time PCR and in-situ hybridization method shown, AS-miR-21 can suppress the miR-21 expression in U251 cell effectively. MTT curve indicated, the U251 cell survival rate is lower than that in the AS-miR-21 treated group; IF and WB results proved that EGFR, PI3K-AKT signaling pathway components proteins and Bcl-2 protein were down regulated; tumor suppressors including Septin-7, TIMP-1 were up regulated; flow-cytometry assay verified, in the AS-miR-21 treated tumor cell group, cell cycle was blocked at G0/G1 phase and the early phase was induced accordingly.In chapter four, every 3 days the mixture of oligofectamine and AS-miR-21 was injected into the U87 and U251 xenograft tumor model. By the end of observation interval, it had been proved that, the AS-miR-21 treated xenograft tumor volume was significantly inhibited than that in the control and scramble treated groups. IHC shown, EGFR, PI3K-AKT signaling pathway components proteins, tumor malignancy related protein including Ki67, CyclinD-1, MMP-9 and Bcl-2 protein were down regulated; tumor suppressors including Septin-7, TIMP-1 were up regulated. TUNEL assay indicated that the apoptosis nuclei index was higher than in the control and scramble groups. The in vivo study results were equal to the results in vitro.ConclusionA class of miRNA is abnormally expressed in human gliomas and miR-21 can be treated as a molecular signature of human glioma and glioblastoma.There are direct binding sites reside in the 3’UTR of PTEN and Septin-7 mRNA.Oligofectamine mediated AS-miR-21 transfection can effectively suppress the miR-21 expression in U251 glioblastoma cell line. ASmiR-21 can inhibit EGFR, PI3K-AKT signaling pathway activity; suppress glioma cell proliferation, invasion, and migration; block cell cycle at G0/G1 phase; induce tumor cell apoptosis.Oligofectamine mediated AS-miR-21 treatment in human gliobalstoma xenograft model can get the similar results as the in vitro study.MiR-21 can be a gene therapeutic target candidate in human glioma. Antisense oligonucleotide transfetion is an effective approach to inhibit over expressed miRNA and it has optimistic clinic trail prospects.更多还原