【作者】 王坤；

【导师】 浦佩玉；

【作者基本信息】 天津医科大学 ， 外科学， 2011， 硕士

【摘要】 脑胶质瘤是中枢神经系统最常见的原发性肿瘤,由于恶性胶质瘤呈侵袭性生长,增殖速度快,现有的综合治疗措施如外科手术切除、术后放疗、化疗和免疫治疗难以达到根除,复发率很高,严重影响生存质量和生存期,临床预后甚差。这充分反映了目前对脑胶质瘤的病因,发病机制及其治疗手段认识的匮乏。为此,必须寻找与胶质瘤发生、发展相关的基因和信号通路,深入了解胶质瘤的分子病理机制,在此基础上开拓胶质瘤治疗的新策略和新手段。我室前期应用Atlas cDNA微阵列方法对63例不同类型脑胶质瘤的基因表达谱进行了研究,除进一步证实以往发现的胶质瘤相关基因表达异常外,还发现隔蛋白家族成员SEPT7在胶质瘤中表达显著降低,并经胶质瘤样本初步验证[。在此基础上,我们构建了SEPT7的真核表达载体,将其转染至SEPT7显著下调和缺失的胶质瘤细胞系,观察到SEPT7在体内、外均可抑制胶质瘤生长增殖和诱导凋亡,并具有抑制胶质瘤细胞侵袭的作用；故初步认定SEPT7是一个新的胶质瘤抑癌基因。此外,我们前期研究胶质瘤microRNA表达谱显示,在435个人类microRNA中多个1microRNA表达异常,其中miR-21、miR-221/222、miR-19a/b和miR-30a-5p等microRNA表达较正常组织显著上调。为了寻找可调控SEPT7表达的microRNA,我们搜索了预测microRNA靶基因网站,包括TARGETSCAN, MIRANDA等,发现有50个microRNA,包括miR-19a/b和miR-30a-5P在SEPT7 mRNA 3’UTR上有潜在的结合位点,并且和我室与南开大学数学系合作构建的预测(?)niRNA靶基因的HuMiTar数据库(Ruan J, et al., HumMiTar: A sequence-based method for prediction of human micrRNA targets. Algorithms for Molecular Biology 2008,3：16,国家自然科学基金资助项目“带microRNA调控的胶质瘤细胞信号转导加权网络”,编号A01020403)结果最为一致；故本课题重点研究了miR-19a/b和miR-30a-5p通过调控SEPT7对胶质瘤增殖、细胞周期进展、凋亡和侵袭的影响及其可能的作用机制。本课题研究分为以下三个部分：第一部分中应用Real time PCR的方法,检测了8个胶质瘤细胞系和43例不同级别人脑胶质瘤组织中miR-19a/b和miR-30a-5p的表达水平。结果表明这两个microRNA在胶质瘤中的表达较正常组织显著上调,并与肿瘤级别呈正相关。此外,我们又应用原位杂交技术对75例胶质瘤及5例正常脑组织制备的组织芯片检测两者表达,发现其与Real time PCR检测结果一致。第二部分为miR-19a/b和miR-30a-5p对SEPT7调控的研究。采用Real timePCR鉴定转染反义寡聚核苷酸抑制miR-19a/19b和miR-30a-5p表达的效果；应用荧光素酶报告试验(Luciferase Reporter Assay)检测miR-19a/19b和miR-30a-5p对SEPT7的直接调控关系；鉴于除SEPT7外,miRNA靶基因数据库预测胶质瘤中两个重要的抑癌基因PTEN和P53分别也可能是miR-19a/19b及miR-30a-5p的靶基因,因此我们也用荧光素酶报告实验检测了miR-19a/19b和(?)miR-30a-5p分别对PTEN和P53的直接调控。应用Western blot检测转染(?)miR-19a/19b和miR-30a-5p抑制物(AS-miR-19a/b和AS-miR-30a-5p)后SEPT7表达变化；RT-PCR检测转染AS-miR-19a/b.和AS-miR-30a-5p后SEPT7 mRNA表达变化。结果显示,转染AS-miR-19a/19b和AS-miR-30a-5p后肿瘤细胞miR-19a/19b和miR-30a-5p表达明显下降；SEPT7表达上调；但SEPT7 mRNA表达无明显变化,提示两者仅抑制SEPT7基因转录后的蛋白翻译。共转染AS-miR-19a或AS-miR-19b和带有在SEPT7或PTEN 3’UTR作用区的重组荧光素酶报告质粒pGL3后荧光强度增高,联合转染AS-miR-19a和AS-miR-19b时荧光强度增高更明显；共转染AS-miR-30a-5p和带有在SEPT7或P53 3’UTR作用区的重组荧光素酶报告质粒pGL3后荧光强度也增高。故可推断SEPT7和PTEN是miR-19a/b、SEPT7和P53是(?)niR-30a-5p的靶基因。在课题第三部分,‘将AS-miR-19a/b转染至人脑胶质瘤细胞系SNB19,LN229和U251中、将AS-miR-30a-5p转染至U251,LN308和U87胶质瘤细胞系。为进一步观察miR-19a/b和miR-19a/b的促癌以及SEPT7的抑癌作用,又增设联合敲低SEPT7(SEPT7 siRNA)、PTEN (PTEN siRNA)和P53组(P53 siRNA)以及单独敲低SEPT7组；分别分组如下：对照组(control),无义序列组(scramble), AS-miR-19a组,AS-miR-19b组,AS-miR-19a+AS-miR-19b组,SEPT7siRNA组,AS-miR-19a+AS-miR-19b+SEPT7 siRNA (SEPT7 si)组,AS-miR-19a+AS-miR-19b+PTEN si组(仅检测PTEN野生型的LN229细胞系)以及对照组(control),无义序列组(scramble), AS-miR-30a-5p组,SEPT7siRNA组,AS-miR-30a-5p+SEPT7 siRNA (SEPT7 si)组,AS-miR-30a-5p+P53 si组(仅检测P53野生型的U87细胞系)。采用Real time PCR检测转染后胶质瘤细胞的miR-19a/b和miR-30a-5p表达水平以鉴定抑制效果；四甲基偶氮唑蓝(MTT)实验评价细胞的增殖率；流式细胞术检测细胞周期变化；Annexin V法检测细胞早期凋亡；Transwell实验检测细胞侵袭能力变化。Real time PCR检测结果显示转染AS-miR-19a/b或AS-miR-30a-5p后,肿瘤细胞AS-miR-19a/b或miR-30a-5p表达下降,AS-miR-19a组,AS-miR-19b组及AS-miR-30a-5p组细胞增殖活性降低,细胞侵袭能力明显受到抑制,细胞周期阻滞在G0/G1期以及早期凋亡增加；AS-miR-19a+AS-miR-19b组效果更明显；转染SEPT7siRNA组则细胞增殖和细胞周期进展加速,侵袭能力增强,而转染AS-miR-19a+AS-miR-19b+ SEPT7 si组和转染AS-miR-19a+AS-miR-19b+PTEN si组；细胞增殖、周期进展、凋亡、侵袭结果无明显差异,与转染AS-miR-19a组或转染AS-miR-19b组结果也无统计学差别；而转染AS-miR-30a-5p+ SEPT7 si组或转染AS-miR-30a-5p+ P53 si组则与control组无明显差异；由此可以进一步推断SEPT7具有明显抑癌作用,与PTEN及P53不相上下；miR-19a/b及miR-30a-5p是癌微RNA (OncomiRs),可以分别通过负调控抑癌基因SEPT7和PTEN以及SEPT7和P53实现促癌作用。结论：miR-19a/b和miR-30a-5p在胶质瘤中表达上调,正常组织,低级别肿瘤与高级别肿瘤比较,其差异均具有显著性。转染AS-miR-19a/b或AS-miR-30a-5p至人胶质瘤细胞系,可抑制胶质瘤细胞的增殖活性和侵袭能力,G0/G1期出现阻滞,并促进凋亡；而这种生物学行为的改变可能通过miR-19a/b对SEPT7及PTEN的调控和miR-30a-5p对SEPT7及P53的调控实现的。由此,初步认定miR-19a/b和miR-30a-5p为癌微RNA,可成为胶质瘤基因治疗的候选靶标；并进一步认定胶质瘤中SEPT7的抑癌作用和SEPT7下调的机制之一是通过miR的调控实现的。这是首次有关(?)miR-19a/b和miR-30a-5p在人脑胶质瘤中的表达、验证其对SEPT7, PTEN和P53的调控以及对胶质瘤生物学行为的影响的研究报道,为进一步认识脑胶质瘤的分子病理机制及新一类的靶向治疗提供实验依据。更多还原

【Abstract】 Glioma is the most common primary tumor of the central nervous system. Owing to the invasive growth and rapid cell proliferation, the malignant glioma is hardly to be cured by the currently available combined therapeutic approaches, such as surgical resection, postoperative radiotherapy, chemotherapy and immunotherapy etc. Its high recurrence rate seriously affects the life quality and overall survival of the patients, and the prognosis is poor. This situation fully reflects our poor understanding of the etiology, pathogenesis and treatment strategy of gliomas. Therefore, a more comprehensive understanding of the molecular pathology of gliomas must be sought for optimizing treatment strategies and development of novel therapeutic approaches.Our previous study on the gene expression profiles of 63 glioma samples with different types and grades by Atlas cDNA microarray analysis demonstrated that the expression of SEPT7 gene, a member of septin family, was downregulated in a variety of gliomas and it was validated further in an additional group of glioma samples. On the basis of this finding, we constructed SEPT7 eukaryotic expression vector and transfected it into glioma cell lines in which SEPT7 was downregulated or deleted. It was shown that SEPT7 could inhibit the proliferation and invasion of gliomas, and induce apoptosis of tumor cells as well in vitro and in vivo. These evidences indicated that SEPT7 could be a new tumor suppressor gene. Moreover, we have previously profiled miRNA expression in glioma cell lines, and found that some human miRNAs were aberrantly expressed in gliomas, including overexpression of miR-21、miR-221/222、miR-19a/b and miR-30a-5p etc. In order to find the microRNAs in gliomas that could regulate SEPT7, we searched several microRNA target prediction database, such as TARGETSCAN and MIRANDA and 50 different miRNAs were predicted to have SEPT7 as putative target, including miR-19a/b and miR-30a-5p. The result was coincident with that found from HuMiTar algorithm for prediction of microRNA targets developed by Department of Mathematics, Nan Kai University and our lab. So the present study is to focus on exploring the modulation of SEPT7 and the regulation of malignant phenotype in gliomas by miR-19a/b and miR-30a-5p. The present study was divided into three partsIn the first part of this study, miR-19a/b and miR-30a-5p expression was examined in 8 malignant glioma cell lines and 43 freshly resected glioma samples by Real time PCR. It was found that these microRNAs were upregulated in gliomas, and their expression was positively correlated with the tumor grade. We also carried out In Situ Hybridization in tissue array to detect the microRNA expression in 75 glioma samples and 5 normal brain tissues and the result was coincident with that of Real time PCR examined.The second part of this study was focused on the validation of SEPT7 being the target of miR-19a/b and miR-30a-5p. Real time PCR was conducted to demonstrate the overexpression of miR-19a/b and miR-30a-5p in transfected glioma cells. The Luciferase reporter assay was used to identify the direct regulation of miR-19a/19b and miR-30a-5p on SEPT7. Besides SEPT7, PTEN and P53, the two tumor suppressor genes known to play important roles in gliomagenesis, also have been predicted as potential targets of miR-19a and miR-30a, respectively. So we detected the direct regulation of PTEN or P53 by miR-19a/b or miR-30a-5p simultaneously with Luciferase reporter assay. Expression of SEPT7 in glioma cells transfected with AS-miR-19a/b and AS-miR-30a-5p (Antisense oligonucleotides against miR-19a/b and miR-30a-5p) was determined by RT-PCR and Western blot analysis, It was shown that in cells transfected with AS-miR-19a/b or AS-miR-30a-5p, the expression of miR-19a/b or miR-30a-5p was significantly reduced, whereas the expression of SEPT7 was upregulated and no significanct alteration of mRNA expression of SEPT7 was observed. This finding indicates that the inhibition of SEPT7 expression is at posttranscriptional level. We also observed in the cells transfected with AS-miR-19a/b and PGL3-SEPT7 or PGL3-PTEN, the luciferase activity became stronger, and the luciferase activity was much stronger in the cells transfected with AS-miR-19a+ AS-miR-19b and PGL3-SEPT7 or PGL3-PTEN. We also found that in the cells transfected with AS-miR-30a-5p and PGL3-SEPT7 or PGL3-P53, the luciferase activity was significantly enhanced. Therefore, it can be concluded that SEPT7 and PTEN are targets of miR-19a/b and miR-30a-5p can directly target SEPT7 and P53.In the third part, we transfected AS-miR-19a/b to the human glioma cell lines SNB19, LN229, U251 and AS-miR-30a-5p to U251, LN308, U87 glioma cells. In order to observe the tumor-promoting effect of miR-19a/b (oncomiR)and miR-30a-5p (oncomiR), and tumor-suppressing effect of SEPT7, we set up cell groups in which AS-miR-19a/b combined with SEPT7 siRNA (SEPT7 si) or PTEN siRNA (PTEN si), and AS-miR-30a-5p combined with SEPT7 si or P53 siRNA (P53 si)were transfected. Thus. the glioma cells were grouped as follows:control cells, cells transfected with scramble oligonucleotides, with AS-miR-19a, with AS-miR-19b, with AS-miR-19a+ AS-miR-19b, with SEPT7 si, with AS-miR-19a+AS-miR-19b+SEPT7 si and with AS-miR-19a+AS-miR-19b+PTEN si (detected only in LN229 cells with the wild type of PTEN), and control cells, cells transfected with scramble oligonucleotides, with AS-miR-30a-5p, with SEPT7 si, with AS-miR-30a-5p+SEPT7 si, with AS-miR-30a-5p+P53 si (detected only in U87 cells with the wild type of P53). Real time PCR was conducted to detect the expression of miR-19a/b and miR-30a-5p in transfected cells. The cell proliferation rate was determined by 3-(4,5-Dime--thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cell cycle kinetics was detected by flowcytometry. The cell apoptosis was examined by Annexin V assay and invasive ability was evaluated by transwell assay. The results showed that the expression of miR-19a/b or miR-30a-5p in the cells transfected with antisense oligonucleotides was significantly downregulated. The cell proliferation activity and invasive ability were reduced, cells were arrested in G0/G1 phase, and apoptosis was induced in cell groups transfected with AS-miR-19a, AS-miR-19b or AS-miR-30a-5p as compared to those of the cells transfected with scramble oligonucleotide and control cells, and the phenotypic changes were more obvious in the AS-miR-19a+AS-miR-19b group, whereas in the SEPT7 si group that the cell proliferative rate was promoted, cell cycle accelerated and cells became more invasive. It was also shown that the effects of AS-miR-19a+AS-miR-19b+SEPT7 si group and AS-miR-19a +AS-miR-19b+PTEN si had no significant difference, and there were also no statistical significant differences among these groups and AS-miR-19a or AS-miR-19b group. Similarly, the alteration of malignant phenotype of glioma cells including cell proliferation, invasion or apoptosis showed no significant difference among AS-miR-30a-5p+SEPT7 si group, AS-miR-30a-5p+P53 si group and control group. Therefore, it can be concluded that the tumor-suppressing effect of SEPT7 is almost the same as PTEN and P53; miR-19a/b and miR-30a-5p are oncomiRNAs. Both of them can promote the growth of glioma by negatively regulating SEPT7, PTEN and SEPT7, P53, respectively.Conclusion:The results demonstrate that miR-19a/b and miR-30a-5p expression are significantly increased in the majority of the gliomas and its expression is positively correlated with the tumor grade. Transfection of AS-miR-19a/b or AS-miR-30a-5p into the glioma cells is able to inhibit the glioma cell proliferation activity, invasive ability, arrest the cells in G0/G1 pahse, and induce cell apoptosis. We also showed that miR-19a/b affected the malignant phenotype of glioma cells through the regulation of SEPT7, PTEN and miR-30a-5p by targeting SEPT7 and P53, respectively. Thus, miR-19a/b and miR-30a-5p are identified as oncomiRs which implicate that they can be candidate targets for gene therapy of gliomas. To our knowledge, this is the first comprehensive study on the expression and the potential mechanism of miR-19a/b and miR-30a-5p in gliomagenesis. These evidences provide us the molecular pathologic machnism and a new class of targeted therapeutic strategy for human gliomas.However, since one microRNA may have hundreds of targeting genes involved in a variety of cellular processes, so exploring the genes associated with gliomagenesis regulated by miR-19a/b and miR-30a-5p should be expanded. On the other hand, SEPT7 gene can be modulated by multiple miRNAs besides miR-19a/b and miR-30a-5p, and that also needs to be studied further.更多还原