【作者】 南阳；

【导师】 钟跃；

【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,具有侵袭性生长,肿瘤细胞增殖快,易复发的特点。脑胶质瘤本质上是一种多基因异常疾病,涉及癌基因的过表达和抑癌基因的突变、缺失等多环节、多通路的异常。传统治疗方法(包括手术、化疗和放疗)治疗效果差,又可能产生中枢神经系统的毒、副作用。由此,从根本上纠正与脑胶质瘤发生、发展相关的基因异常的基因治疗已成为医学研究领域的热点。MiRNA是一类长约22nt的非编码RNA,能够与靶基因的3’端非转录区域(3’untranslated region,3’UTR)特异性结合而导致靶mRNA降解或抑制其翻译,从而对基因进行转录后调控。在人类细胞中约30%的蛋白编码基因受miRNA的调控,具有高度的保守性、时空和组织特异性。同一miRNA可以调控多个mRNA基因,不同miRNA分子也可以协同调控同—mRNA基因。因此,miRNA与其靶标基因之间组成了一个复杂的调控网络,控制着细胞及个体的重要生命活动。MiR-451位于染色体17q11.2,与原癌基因人类表皮生长因子受体2的17q11.2-21区域临近。近年的研究发现,异常表达的miR-451在乳腺癌、卵巢癌、胃癌和结肠直肠癌和胶质瘤中均有报道。Gal等研究表明miR-451在恶性胶质瘤干细胞中低表达,miR-451联合抗肿瘤药甲磺酸伊马替尼能明显抑制恶性胶质瘤干细胞的生长和增殖。有趣的是,Godlewski等研究表明胶质瘤细胞在葡萄糖充足时miR-451表达水平上升,促进肿瘤细胞增殖,而在葡萄糖不足的情况下miR-451表达水平调低从而使LKB1/AMPK水平上升,继而减慢了细胞增殖而增强了肿瘤细胞的迁移能力,使肿瘤细胞适应于代谢环境所带来的压力。另外,在乳腺癌多药耐药细胞系MCF-7/DOX中,过表达miR-451通过直接调控耐药基因MDR1的表达,增强细胞对阿霉素的敏感性。但也有研究报道称卵巢癌多药耐药的癌细胞系A2780DX5和KB-V1中抑制miR-451或miR-27a的表达可导致MDR1的mRNA和蛋白水平的下降,从而增加细胞系对药物(长春碱、阿霉素)的敏感程度。在胃癌和结肠直肠癌中,miR-451通过直接靶标癌基因巨噬细胞迁移抑制因子(macrophage migration inhibitory factor, MIF)发挥抑癌基因的作用。MiR-451高表达时能降低细胞增殖能力,增加细胞放疗的敏感性,而低表达时与患者较差的预后密切相关。本课题组在预实验研究中通过miRNA微阵列芯片杂交发现：6个胶质瘤细胞系中miR-451表达下调为正常脑组织的50%以下,并结合Gal等研究初步认定miR-451基因与胶质瘤发生相关。本研究利用miR-451拟似物(miR-451 mimics)作用于恶性胶质瘤细胞系,观察其对肿瘤生长的抑制作用,并探讨可能存在的机制,以期为胶质瘤的综合治疗提供新的靶标。本课题研究分为以下二个部分：第一部分中应用寡核苷酸miR-451 mimics转染人脑胶质瘤细胞系A172、LN229和U251,并通过Real-time PCR检测miR-451的表达,然后通过MTT法分析肿瘤细胞增殖活性,流式细胞仪分析细胞周期,Transwell法检测胶质瘤细胞迁移和侵袭能力,Annexin V-FITC检测细胞凋亡,Western blot检测相关蛋白的表达。结果显示,miR-451在人脑胶质瘤细胞系A172、LN229和U251中低表达。上调脑胶质瘤细胞中miR-451的表达后,自培养第2天起,细胞增殖速率呈下降趋势,且低于对照组和scramble组(均P<0.05)。与对照组和scramble组相比,miR-451 mimics组细胞周期阻滞在G0/G1期,G2/M期减少(均P<0.05)；细胞迁移和侵袭能力降低(均P<0.05)；凋亡率明显升高(均P<0.05)。同时,与对照组和scramble组相比,miR-451 mimics组Akt1、CyclinD1、MMP-2、MMP-9和Bcl-2蛋白表达显著降低(均P<0.05),而p27蛋白显著表达升高(P<0.05)。第二部分建立LN229裸鼠皮下荷瘤模型,将模型分为对照组、scramble组和miR-451 mimics组。每组6只,共18只,并在肿瘤局部多点注射治疗,每3天测量一次肿瘤体积,动态测量肿瘤生长。免疫组化法检测肿瘤标本的相关蛋白表达情况分析可能的抑癌机制。结果表明,miR-451 mimics组对裸鼠移植瘤人脑胶质瘤的抑制作用较对照组及scramble组均明显增强(均P<0.01)；与对照组和scramble组相比,miR-451 mimics组Akt1、CyclinD1、MMP-2、MMP-9和Bcl-2蛋白表达显著降低(均P<0.05),而p27蛋白显著表达升高(P<0.05)。结论：体、内外研究中与对照组和scramble组相比,miR-451 mimics组作用于人脑胶质瘤细胞系后,肿瘤细胞的增值、侵袭和迁移能力受到明显的抑制,而细胞凋亡能力增强。其分子机制可能为：miR-451可能通过抑制PI3K/Akt信号通路,下调了肿瘤细胞增值、侵袭相关蛋白以及凋亡抑制蛋白的表达,从而发挥了抑制脑胶质瘤细胞生长的作用。更多还原

【Abstract】 Glioma is the most common primary malignant tumor in the central nervous system. Owing to the feature of its invasive growth, fast cell proliferation and high recurrence rate. The molecular development of glioma is a complex process which involved the activation of proto-oncogenes and inactivation of tumor suppressor genes and the abnormal expression of mutiple cytokines in the cellular message transmission pathway. Glioma is hard to be removed completely by the surgical resection. The postoperative radiotherapy, chemotherapy and immunotherapy can improve the part of life quality and overall survival of the patients. Therefore, the gene therapy for correcting the aberration of genetic events in glioma may be the best method, which has been the hot spots in the study of glioma.MicroRNAs (miRNAs) are small, non-coding RNAs about 22 nucleotides in length that negatively regulate gene expression at the post-transcriptional and/or translational level by binding loosely complimentary sequences in the 3’-untranslated regions (UTRs) of target mRNAs. MicroRNAs are predicted to regulate the expression of approximately one-third of all human genes. Some miRNA can target hundreds of genes, and some genes may be targeted by multiple miRNAs, suggesting that miRNAs play important roles in coordinating many cellular processes. As such, miRNA-mediated gene regulation is now considered an important role in biologic processes of human cells.MiR-451 is located on chromosome 17q11.2, a region known to be amplified in certain types of cancers, and is in close proximity to HER2 (17q12). In recent years, studies have found that abnormal expression of miR-451 in breast cancer, ovarian cancer, gastric cancer, colorectal cancer and glioma has been reported. Gal reported that transfection of glioblastoma (GBM) cells with the mature miR-451 could inhibited neurosphere formation, and inhibited GBM cell growth. Furthermore, transfection of miR-451 combined with Imatinib mesylate treatment had a cooperative effect in dispersal of GBM neurospheres, however, the regulatory mechanism of miR-451 is unclear. Another report showed that miR-451, down-regulated in migrating GBM cells, could regulate LKB1/AMPK signaling by directly regulating CAB39 expression in GBM cells. Noticeably, it showed that miR-451 could reduce GBM cell migration but pomote its proliferation, which were partly contrary to Gal H’s results. In breast cancer, Olga showed that miR-451 could regulated the·expression of multidrug resistance 1 gene. Transfection of the MCF-7/DOX-resistant cells with miR-451 resulted in the increased sensitivity of cells to DOX. In ovarian cancer, expressions of miR-27a and miR-451 were up-regulated in multidrug resistant (MDR) cancer cell lines A2780DX5 and KB-V1, as compared with their parental lines A2780 and KB-3-1. Treatment of A2780DX5 cells with the antagomirs of miR-27a or miR-451 decreased the expression of P-glycoprotein and MDR1 mRNA. In contrast, the mimics of miR-27a and miR-451 increased MDR1 expression in the parental cells A2780. The sensitivity to and intracellular accumulation of cytotoxic drugs that are transported by P-glycoprotein were enhanced by the treatment with the antagomirs of miR-27a or miR-451. In gastrointestinal cancer Cells, down-regulation of miR-451 was associated with worse prognosis. MiR-451 was decreased expression in gastric and colorectal cancer versus nontumoral tissues. Overexpression of miR-451 in gastric and colorectal cancer cells reduced cell proliferation and increased sensitivity to radiotherapy. Microarray and bioinformatic analysis identified the novel oncogene macrophage migration inhibitory factor (MIF) as a potential target of miR-451. In fact, overexpression of miR-451 down-regulated mRNA and protein levels of MIF and decreased expression of reporter genes with MIF target sequences.In our previous studies, we profiled miRNA expression in five GBM cell lines, one astrocytoma cell line, and normal brain tissue. Our data revealed that the miRNA most significantly decreased in GBM compared to normal brain tissue in these studies was miR-451. In the present study, we used 2’-O-methyl-oligonucleotides to overexpress miR-451 in the human GBM cell lines, then we observed whether there were jointly or collaborative enhancement to suppress the proliferation, invasion and apoptosis of tumor, and then explored the possible mechanisms with a view to provide a new direction for the comprehensive treatment of glioma.The present study was divided into two parts.In the first part of this study, we used 2’-O-methyl-oligonucleotides to overexpress miR-451 in A172, LN229 and U251 GBM cell lines, then miR-451 mRNA was quantified by real time PCR. MTT method was used to evaluate cell proliferation rate. Flow cytometry and the invasion and migration ability was detected by Transwell analysis and Scarification test. Annexin V-FITC were used for cell cycle apoptosis analysis respectively. Western blot was used to evaluate the expression of proteins. The result showed that miR-451 was upregulation following in miR-451 mimics transfection of A172, LN229 and U251 cells. The cell multiplication rate in miR-451 mimics group presented decreasing trend since the second day in culture (P<0.05), as compared with control and scramble group. The number of cells inhibited at G0/G1 phase in miR-451 mimics group was more than any other group (P<0.05). Transwell and Scarification test indicated that the invasion and migration ability of the cells in miR-451 mimics group were decreased obviously (P<0.05). The apoptosis rate in miR-451 mimics group was higher than that in the other two groups (P0.05). The protein expression of AKT1, Cyclin D1, MMP2, MMP9 and bcl-2 were lower in miR-451 mimics group, while p27 was significantly higher than those in the other two groups (P<0.05).In the second part, in vivo study was carried out to further investigate the miR-451 and its concrete mechanism in glioma. Eighteen athymic mice were randomly divided into 3 groups (control, scramble and miR-451 mimics group), and were treated respectively. Athymic mice xenogeneic transplant model was established by inoculation (sc) with LN229 glioma cells. Body mass (BM) and diameter of tumor mass were measured. Furthermore, The protein expressions of AKT1, CyclinD1, p27, MMP2, MMP9 and Bcl-2 in tumor tissues were analyzed with immunohistochemistry. The results show that the tumor-inhibiting rate of miR-451 mimics was significantly higher than the control (P<0.05) and scramble (P<0.05). The protein expression of Akt1, Cyclin D1, MMP2, MMP9 and bcl-2 were lower in miR-451 mimics group, while p27 was significantly higher than those in the other two groups (P<0.05).Conclusion:The suppressive effect of miR-451 mimics group is better than control and scramble group in vitro and vivo, which indicate that miR-451 may function as tumor suppressor in human gliomas. Its molecular mechanism may be as followed:the tumor suppressor activity of miR-451 may be regulated by the PI3K/AKT pathway to inhibit cell proliferation and invasion and induce cell apoptosis in GBM.更多还原