MicroRNA-7-5p在胶质母细胞瘤微血管内皮细胞的表达及其功能的研究

发布时间：2018-02-24 05:28

本文关键词： 微血管内皮细胞增殖 MiR-7-5p 胶质母细胞瘤 RAF1 慢病毒包装　出处：《山东大学》2014年博士论文　论文类型：学位论文

【摘要】：背景恶性肿瘤的发生与发展是一个极为复杂的过程,多种基因、多种信号通路参与其中,多种相关的发育基因在此过程中发挥着异常重要的作用。MicroRNAs的表达与调控作用,作为肿瘤发生发展的重要因素之一,被日益受到重视和越来越多的研究。MicroRNAs (miRNAs)是一类长度为18-22nt、具有基因调控功能的小分子非编码RNA。微RNAs (microRNAs, miRNAs)的失调往往与癌症的发生和发展相关。miRNAs靶向作用于其靶基因mRNAs,并与其3'-UTR完全或者不完全互补结合,引起靶mRNAs的降解,或者抑制其翻译,从而发挥其生物学作用。胶质母细胞瘤(Glioblastoma, WHO IV级),易称为多形性胶质母细胞瘤(Glioblastoma multiforme, GBM),无论是从发病率还是恶性程度,都是中枢神经系统肿瘤中最高的原发性肿瘤。多年来,胶质母细胞瘤的治疗,一直是神经外科学领域的一大难题,目前主要的临床治疗方案仍然是手术中最大限度的切除及辅以术后放疗、化疗等手段。虽然近几年来对GBM的有些治疗取得了新的进展,但由于其高度恶性,高术后复发率,预后仍然较差。因此,目前探索新的GBM的发病机制及寻找其新的更为有效的治疗方法仍然是一大研究热点。微血管增殖是GBM特有的病理特征,但是,目前对于增殖的GBM微血管内皮细胞的功能知之甚少。几乎所有的研究与化疗药物均是靶向作用于GBM细胞,而作用于抗肿瘤血管生成的化疗药物未出现预期的效果。GBM中微血管只占到肿瘤很小的一部分,以往研究通过提取整个肿瘤的总RNA或基因组DNA来寻找GBM微血管特异性差异基因显然是不适当的。本研究将提取非肿瘤脑组织微血管和GBM微血管,用微芯片技术分析对比GBM微血管和脑组织微血管的miRNA表达谱的差异性,应用现代生物学信息工具分析整合miRNA表达谱,在miRNA、mRNA及蛋白水平上研究能够加强或抑制GBM微血管增殖的某些因子,以期发现更有效的潜在抑制GBM微血管增殖的新靶点。目的 1.提取纯化GBM及正常脑组织的微血管,检测二者的差异miRNAs; 2.筛选出差异显著的miRNA,研究其对血管内皮细胞生物学行为的影响； 3.对筛选出的差异显著的miRNA进行功能及其机制的研究,为明确其是否可作为GBM治疗的新靶点提供实验依据。方法 1.应用葡聚糖沉淀梯度密度离心法,提取纯化GBM微血管和非肿瘤正常脑组织微血管。以此为基点提取总RNA。 2.用miRNA基因芯片技术,筛查在GBM微血管和脑组织微血管中的差异表达的microRNAs,应用实时定量PCR进一步对其进行验证,验证其在微血管组织及在人脐静脉血管内皮细胞系(HUV-EC-C)中的表达情况。 3.运用生物信息学工具TagetScan5.1、MicroCosm Targets Version5预测差异miRNA的靶基因,并对其特异性靶基因进行KEGG pathway分析。选择确定差异显著的参与调控血管内皮细胞生物学行为的microRNA及其靶基因。 4.构建过表达目的基因的慢病毒载体和阴性对照慢病毒载体；应用293T细胞进行病毒包装和滴度测定；慢病毒载体感染HUV-EC-C细胞；应用qPCR检测慢病毒载体目的基因的表达。 5.采用划痕实验、MTT实验、流式细胞术,观察目的基因的表达对血管内皮细胞体外迁移能力、增殖能力及其细胞周期的影响。 6.将预测的靶基因的野生型或突变型3'-UTR克隆入GV272的XbaI位点构建萤光素酶报告基因,与过表达miRNA慢病毒或对照组慢病毒质粒共转染HUV-EC-C细胞,通过分析萤光素酶的表达以初步判定miRNA的靶基因。应用Western Blot在细胞及组织中检测,进一步确认miRNA对其靶基因的调控关系。 7.所有结果用均数加减标准差来表示。t检验进行组间比较。MiR-7-5p和RAF1表达之间的关系通过皮尔森相关性统计方法来探究。P0.05为差异有统计学意义。所有统计学分析用SPSS16.0软件进行。结果 1.应用葡聚糖沉淀梯度密度离心法从GBM和正常脑组织中成功纯化提取出微血管组织,并应用台盼兰染色,镜下可见正常脑组织微血管每条清晰完整,分支明显；GBM微血管破碎,呈团状,分支杂乱。 2.通过基因芯片分析显示,在GBM微血管中miR-7-5p相对于正常脑组织微血管呈现大幅下调(P0.01),相对于正常非肿瘤脑组织微血管减少2.616倍。在各5例GBM微血管和脑组织微血管中,应用qPCR验证符合基因芯片分析结果。之后,我们又在人脐静脉内皮细胞系(HUV-EC-C)中验证miR-7-5p的表达情况,经qPCR检测,HUV-EC-C细胞中miR-7-5p明显低表达。 3.生物信息学预测结果表明,参与血管内皮细胞生物学行为的RAF1的3'-UTR与miR-7-5p的5’端“种子序列”有很好的匹配位点。荧光素酶活性测定结果显示RAF1是miR-7-5p下游的直接靶点(P0.01)。 4.MTT实验、细胞周期实验显示,在体外miR-7-5p诱导血管内皮细胞周期停滞于G1期,从而抑制血管内皮细胞增值。划痕实验表明在体外miR-7-5p对血管内皮细胞的迁移能力无影响。 5. Western blot分析显示,在HUV-EC-C细胞感染过表达miR-7-5p病毒能降低RAF1的蛋白水平,Actin作为内参,(P0.01)；在GBM和正常脑组织微血管中,miR-7-5p和RAF1的表达呈负相关关系。Pearson's相关分析具有统计学‘意义。相关性系数为-0.786,(P0.01)。结论葡聚糖沉淀梯度密度离心法能够成功提取GBM微血管与正常脑组织微血管,台盼兰染色,镜下发现二者在形态上有明显的差别,这符合客观规律。本研究从纯化的GBM微血管和正常脑组织的微血管为基点开始。因此,对于明确在GBM微血管的发生发展中起至关重要的有意义的microRNAs及其它们的作用靶点,具有更好的参考价值,对GBM的治疗可能提供更有意义的实验依据。在目前基因芯片研究中,我们揭示了miR-7-5p在GBM微血管中明显下调,并应用qPCR在GBM微血管组织及HUVEC-C细胞株中进行了验证。此外,我们成功地包装了过表达miR-7-5p及其对照组慢病毒,并进行了体外MTT实验、细胞周期实验及划痕实验。实验结果表明niR-7-5p的过度表达能够通过诱导HUV-EC-C细胞在G1期的细胞周期停滞而显著抑制其增殖,而对血管内皮细胞在体外的迁移能力无影响。这些结果有力地支持了niR-7-5p可能是GBM微血管增值的一种抑制物。为了探究引起由miR-7-5p介导的GBM微血管内皮细胞生长受抑制的机制,我们接下来开始鉴别miR-7-5p潜在的靶基因。生物信息学分析显示,RAF1可能是]miR-7-5p潜在的下游靶点。而且,RAF1在多种癌症的发生发展中起着重要作用。因此,我们应用荧光素酶活性测定法来鉴定miR-7-5p在RAF1表达上的作用。荧光素酶活性测定数据显示miR-7-5p能够直接靶向作用于RAF1的3'-UTR。蛋白免疫印迹结果显示,在HUV-EC-C细胞中miR-7-5p的过度表达明显下调了RAF1的表达。我们又对5组GBM微血管和正常脑组织微血管进行了western blot实验,结果显示RAF1在GBM微血管组织中上调,在脑组织微血管中下调,并且和miR-7-5p的表达呈负相关关系。这些数据证实了miR-7-5p能够下调RAF1的表达,这也表明了RAF1在GBM微血管的形成中可能是一个促进因素。本研究提示,miR-7-5p通过对下游靶基因RAF1的调控,在GBM微血管的形成和发展中可能扮演抑癌基因的角色,并在调控血管内皮细胞的增殖等恶性生物学特性中发挥着重要作用。
[Abstract]:Background MicroRNAs ( microRNAs ) are a kind of small molecule non - coding RNAs with a length of 18 - 22 nt and have gene regulation function . MicroRNAs ( microRNAs ) are a kind of small molecule non - coding RNAs with a length of 18 - 22nt and have gene regulation function . MicroRNAs ( microRNAs ) are a kind of small molecule non - coding RNAs with length of 18 - 22nt . Glioblastoma ( WHO grade IV ) , known as Glioblastoma multiforme , is one of the most important primary tumors in the central nervous system tumor . Microchip technique is used to study the difference of miRNA expression profiles of microvessels and brain tissues of non - tumor tissues . Purpose ( 1 ) extracting and purifying the microvessels of the purified and normal brain tissues , and detecting the difference miRNA of the two ; 2 . screening the miRNA with obvious difference to study the effect of the miRNA on the biological behavior of vascular endothelial cells ; 3 . The study of the function and mechanism of miRNA with significant difference in screening can provide an experimental basis for determining whether it can be used as a new target for the treatment . method 1 . Using dextran precipitation gradient density centrifugation method , the microvessels of the microvessels and non - tumor normal brain tissues were purified and purified . The total RNA was extracted by using the method as the base point . 2 . Using miRNA gene chip technique to screen microRNAs expressed differentially expressed in microvessels and brain tissue , real - time quantitative PCR was used to further verify the expression of microRNAs in microvascular tissue and human umbilical vein endothelial cell line ( HUV - EC - C ) . 3 . Using the bioinformatic tool TagetScan5.1 , MicroCosm Targets Version 5 , the target gene of the difference miRNA was predicted , and its specific target gene was analyzed . The microRNAs and their target genes involved in regulating the biological behavior of vascular endothelial cells were selected . 4 . The lentivirus vector and the negative control slow virus vector expressing the target gene were constructed ; the virus packaging and the titer determination were carried out by the expression target gene ; the lentivirus vector was infected with HUV - EC - C cells ; and the expression of the target gene of the lentivirus vector was detected by qPCR . 5 . MTT assay and flow cytometry were used to observe the effect of gene expression on migration , proliferation and cell cycle of vascular endothelial cells in vitro . 6 . The luciferase reporter gene was constructed by cloning the wild - type or mutant 3 ' - terminal of the predicted target gene into the Xba I site of GV272 . HUV - EC - C cells were co - transfected with a slow virus plasmid expressing the miRNA lentivirus or the control group . The expression of luciferase was analyzed to determine the target gene of miRNA . Western Blot was used to detect the target gene in the cells and tissues , and the regulatory relationship between miRNA and its target gene was further confirmed . 7 . All results were expressed by mean addition and subtraction standard deviation . The relationship between MiR - 7 - 5p and RAF1 expression was investigated by Pearson correlation statistical method . All statistical analyses were performed by SPSS 16.0 software . Results 1 . Microvascular tissue was successfully purified by dextran precipitation gradient density centrifugation and trypan blue staining was used in normal brain tissue . 2 . The expression of miR - 7 - 5p in human umbilical vein endothelial cell line ( HUV - EC - C ) was significantly downregulated ( P0.01 ) , and the expression of miR - 7 - 5p in human umbilical vein endothelial cell line ( HUV - EC - C ) was verified by qPCR , and the expression of miR - 7 - 5p in HUV - EC - C cells was significantly lower than that in normal non - tumor tissue . 3 . The results of bioinformatics predict that the 3 ' - untranslated region of RAF1 participating in the biological behavior of vascular endothelial cells has a good matching site with the 5 ' - terminal " seed sequence " of miR - 7 - 5p . The luciferase activity assay results show that RAF1 is a direct target downstream of miR - 7 - 5p ( P0.01 ) . 4 . MTT assay and cell cycle experiment show that miR - 7 - 5p induces vascular endothelial cell cycle arrest in G1 phase in vitro , thereby inhibiting the value of vascular endothelial cells . The scratch test shows that miR - 7 - 5p has no effect on the migration ability of vascular endothelial cells in vitro . 5 . Western blot analysis showed that the expression of miR - 7 - 5p in HUV - EC - C cells decreased RAF1 protein level and Actin as internal reference , ( P0.01 ) . Conclusion The results show that the overexpression of niR - 7 - 5p can significantly inhibit the proliferation of vascular endothelial cells and the migration ability of vascular endothelial cells in vitro . These results strongly support niR - 7 - 5p , which may be a kind of inhibitor of the value - added of the microvascular endothelial cells . In order to investigate the mechanism of miR - 7 - 5p mediated inhibition of expression of miR - 7 - 5p , we began to identify the potential target genes of miR - 7 - 5p .

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.41

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