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
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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。P<0.05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(P<0.01),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(P<0.01)。
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,(P<0.01)；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,(P<0.01)。
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。
Background
The occurrence and development of malignant tumors are complex process that have multiple genes and a variety of signaling pathways participate in, and a variety of related developmental genes play an extremely important role in this process. As one of the important factors of tumor development, the expression and regulation of microRNAs is becoming more and more attention and research subject. MicroRNAs are a class of small molecules RNA with length18-22nt and non-coding, which have gene regulatory functions. MiRNAs disorders are often associated with the development and progression of cancer. MiRNAs target mRNAs by completely or not completely complementary binding, causing degradation of target mRNAs, or inhibit translation, which exerts its biological effects.
Glioblastoma (Glioblastoma, WHO IV grade), easily called glioblastoma multiforme tumor (Glioblastoma multiforme, GBM), are the most common and most malignant primary tumors in the central nervous system tumors. The treatment of glioblastoma, is troubled by the problem of neurosurgery, the main treatment is to maximize the clinical surgical resection and postoperative supplemented by means of radiotherapy and chemotherapy. Although some aspects of treatment have made new progress in recent years, but because of its high degree of malignancy, easy to relapse after surgery, the prognosis remains poor. Therefore, to explore new glioblastoma pathogenesis and find new and more effective treatments is still a major research focus at present.
Microvascular proliferation is a unique pathological features of GBM, however, the function of the proliferative of microvascular endothelial cells is poorly understood in the currentAlmost all studies and chemotherapy drugs are targeted to GBM cells, and act on the anti-angiogenic effects of chemotherapy drugs are not expected to occur.Because GBM microvessel only to a very small part in the tumor, previous studies that by extracting the entire tumor total RNA or genomic DNA to find GBM microvascular specific gene is clearly inappropriate.This study will extract non-tumor brain tissue microvascular and GBM microvessels, compared the differences in miRNA expression profiling in GBM microvascular and brain microvascular with micro-chip technology, apply of modern bioinformatics tools to analyze and integrate of miRNA expression profiling,and study certain factors that can enhance or inhibit the proliferation of GBM microvessels on miRNA, mRNA and protein levels, in order to find a more effective inhibition of GBM vascular proliferation potential targets.
Objective
1. Extracting and purificating of microvascular from GBM and normal brain tissue to detect differences miRNAs between them;
2. Screened significant differences miRNA, to study its effects on vascular endothelial cells biological behavior;
3. To study the function and mechanism of the filter out significant differences miRNA and provide experimental evidence for clear whether its can be used as a new target for the treatment of GBM.
Methods
1. Applying dextran sedimentation density gradient centrifugation, extract and purify of GBM microvascular and non-tumor normal brain tissue capillaries. Look microvascular as the specimen, and extract the total RNA from it.
2. By miRNA microarray technology, screening differences expression microRNAs in GBM microvascular and brain tissue microvascular, using quantitative real-time PCR to further validate the gene chip result, verify its expression in the microvasculature organization and human umbilical vein endothelial cells (HUV-EC-C).
3. By bioinformatics tools(TagetScan5.1, MicroCosm Targets Version5) prediction the target genes of differences miRNA, and perform KEGG pathway analysis for the specific target genes. Select OK the significant differences microRNA and their target genes that related to the regulation in the biological behavior of endothelial cells.
4. Construct the lentiviral vectors with over express the desired gene and its negative control vector; perform packaging and viral titer in the293T cells;
5. Using scratch test, MTT assay and flow cytometry, to observe the effect of target gene expression in vitro migration, proliferation and cell cycle on vascular endothelial cells.
6. Cloned the predicted target genes3'-UTR into the XbaI site GV272and construct luciferase reporter gene, with the miRNA lentivirus and negative control lentivirus co-transfected293T cells, by analyzing the expression of luciferase to preliminary screening target genes of these highly expressed miRNA in endothelial cells. Using Western Blot to further confirmed the regulation of miRNAs to their target gene in cells and tissues.
7. All results are expressed as mean plus or minus standard deviation. Comparisons between groups were performed with the t test. The relationship between miR-7-5p and RAF1expression was explored by Pearson's method. P<0.05was considered to be statistically significant. Statistical analyses were performed with SPSS software (version16.0).
Results
1. Applying dextran sedimentation density gradient centrifugation the microvascular organization was successfully extracted and purified from GBM and normal brain tissue, using trypan blue stain them, and then observed them under the microscope, we can find the normal brain tissue microvascular complete and clear each branch obvious, and however the GBM capillaries present broken, lumps and branch clutter.
2. By microarray analysis showed that miR-7-5p significantly reduced in GBM microvessels relative to normal brain tissue microvascular (P<0.01), and reduce2.61 times relative to the normal non-tumor brain tissue microvascular. In all five cases of GBM microvascular and brain microvascular, the results of qPCR verify and the gene chip analyses have exactly the same.
3. Bioinformatics prediction results show, the3'-UTR of RAF1that participate in the biological behavior of endothelial cells and miR-7-5p5'end "seed sequence" have better matching sites. Determination of luciferase activity shows that the RAF1is a direct target of the miR-7-5p downstream (P<0.01).
4. MTT assay, cell cycle experiments showed that miR-7-5p induced endothelial cell cycle arrest in the G1phase, and inhibited the proliferation of endothelial cell in vitro. Scratch test showed that miR-7-5p has no effect on vascular endothelial cells in migration in vitro.
5. Western blot analysis showed that the infection of LV-miR-7-5p leaded to a decrease of RAF1protein expression in HUV-EC-C cells. Actin was used as internal (P<0.01); In the GBM and normal brain tissue capillaries, the expression of miR-7-5p and RAF1shows negatively correlated. Pearson's correlation analysis with statistical significance. Correlation coefficient was-0.786.(P<0.01).
Conclusions
Dextran sedimentation density gradient centrifugation methods can be successfully extracted microvascular from GBM normal brain tissue, by trypan blue staining, and microscopy found a significant difference between GBM and normal brain microvascular in the form, this is in line with the objective law. The study began with the GBM and normal brain tissue microvascular. Therefore, there have a good reference value for the pinpoint valuable microRNAs and their targets gene that play a vital role for the occurrence of GBM microvascular, and may provide a more meaningful experimental basis for the treatment of GBM. In the current study of the gene chips, we revealed the miR-7-5p significantly downregulated in GBM microvascular, and verified the result by qPCR in the GBM microvascular tissue and HUV-EC-C cell lines. Moreover, we successfully packaged the lentivirus of over express miR-7-5p (LV-miR-7-5p) and its negative control lentivirus (LV-miR-NC), and performed the MTT assay, cell cycle test and scratch test in vitro. Experimental results showed that the overexpression of miR-7-5p can significantly inhibit the proliferation of the HUV-EC-C cells by induce the cell cycle arrest in G1phase, but for the migration of vascular endothelial cells had no effect. These results strongly supported the miR-7-5p may be an inhibitor of GBM angiogenesis.
To explore the mechanism of miR-7-5p inhibition GBM microvascular endothelial cell growth, we have identified the potential target genes of miR-7-5p.Bioinformatics analysis shows that the RAF1may the be potential target of miR-7-5p.Moreover, RAF1plays an important role in the development and occur for many types of cancer. Therefore, we apply the luciferase activity assay to identify the role of miR-7-5p in expression of the RAF1.The luciferase activity assay shows that miR-7-5p can directly targeting the3'-UTR of RAF1. Western blot results showed that the overexpression of miR-7-5p can significantly reduced the expression of RAF1in HUV-EC-C cells. We have carried out western blot experiments in five-groups of GBM microvascular and normal brain tissue microvascular, the result shows that the RAF1upregulated in GBM tissue capillaries, however downregulated in the brain microvessels, and there have a negative correlation appeared between the miR-7-5p and the RAF1.These data confirm the miR-7-5p can downregulate the expression of RAF1, which also shows RAF1may be a contributing factor in angiogenesis of GBM. This study suggests that miR-7-5p may play a role of tumor suppressor genes in the formation and development of microvessels in GBM through the regulation of RAF1which downstream target genes, and play an important effect in the regulation of proliferation of endothelial cells.
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。P<0.05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(P<0.01),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(P<0.01)。
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,(P<0.01)；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,(P<0.01)。
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。
Background
The occurrence and development of malignant tumors are complex process that have multiple genes and a variety of signaling pathways participate in, and a variety of related developmental genes play an extremely important role in this process. As one of the important factors of tumor development, the expression and regulation of microRNAs is becoming more and more attention and research subject. MicroRNAs are a class of small molecules RNA with length18-22nt and non-coding, which have gene regulatory functions. MiRNAs disorders are often associated with the development and progression of cancer. MiRNAs target mRNAs by completely or not completely complementary binding, causing degradation of target mRNAs, or inhibit translation, which exerts its biological effects.
Glioblastoma (Glioblastoma, WHO IV grade), easily called glioblastoma multiforme tumor (Glioblastoma multiforme, GBM), are the most common and most malignant primary tumors in the central nervous system tumors. The treatment of glioblastoma, is troubled by the problem of neurosurgery, the main treatment is to maximize the clinical surgical resection and postoperative supplemented by means of radiotherapy and chemotherapy. Although some aspects of treatment have made new progress in recent years, but because of its high degree of malignancy, easy to relapse after surgery, the prognosis remains poor. Therefore, to explore new glioblastoma pathogenesis and find new and more effective treatments is still a major research focus at present.
Microvascular proliferation is a unique pathological features of GBM, however, the function of the proliferative of microvascular endothelial cells is poorly understood in the currentAlmost all studies and chemotherapy drugs are targeted to GBM cells, and act on the anti-angiogenic effects of chemotherapy drugs are not expected to occur.Because GBM microvessel only to a very small part in the tumor, previous studies that by extracting the entire tumor total RNA or genomic DNA to find GBM microvascular specific gene is clearly inappropriate.This study will extract non-tumor brain tissue microvascular and GBM microvessels, compared the differences in miRNA expression profiling in GBM microvascular and brain microvascular with micro-chip technology, apply of modern bioinformatics tools to analyze and integrate of miRNA expression profiling,and study certain factors that can enhance or inhibit the proliferation of GBM microvessels on miRNA, mRNA and protein levels, in order to find a more effective inhibition of GBM vascular proliferation potential targets.
Objective
1. Extracting and purificating of microvascular from GBM and normal brain tissue to detect differences miRNAs between them;
2. Screened significant differences miRNA, to study its effects on vascular endothelial cells biological behavior;
3. To study the function and mechanism of the filter out significant differences miRNA and provide experimental evidence for clear whether its can be used as a new target for the treatment of GBM.
Methods
1. Applying dextran sedimentation density gradient centrifugation, extract and purify of GBM microvascular and non-tumor normal brain tissue capillaries. Look microvascular as the specimen, and extract the total RNA from it.
2. By miRNA microarray technology, screening differences expression microRNAs in GBM microvascular and brain tissue microvascular, using quantitative real-time PCR to further validate the gene chip result, verify its expression in the microvasculature organization and human umbilical vein endothelial cells (HUV-EC-C).
3. By bioinformatics tools(TagetScan5.1, MicroCosm Targets Version5) prediction the target genes of differences miRNA, and perform KEGG pathway analysis for the specific target genes. Select OK the significant differences microRNA and their target genes that related to the regulation in the biological behavior of endothelial cells.
4. Construct the lentiviral vectors with over express the desired gene and its negative control vector; perform packaging and viral titer in the293T cells;
5. Using scratch test, MTT assay and flow cytometry, to observe the effect of target gene expression in vitro migration, proliferation and cell cycle on vascular endothelial cells.
6. Cloned the predicted target genes3'-UTR into the XbaI site GV272and construct luciferase reporter gene, with the miRNA lentivirus and negative control lentivirus co-transfected293T cells, by analyzing the expression of luciferase to preliminary screening target genes of these highly expressed miRNA in endothelial cells. Using Western Blot to further confirmed the regulation of miRNAs to their target gene in cells and tissues.
7. All results are expressed as mean plus or minus standard deviation. Comparisons between groups were performed with the t test. The relationship between miR-7-5p and RAF1expression was explored by Pearson's method. P<0.05was considered to be statistically significant. Statistical analyses were performed with SPSS software (version16.0).
Results
1. Applying dextran sedimentation density gradient centrifugation the microvascular organization was successfully extracted and purified from GBM and normal brain tissue, using trypan blue stain them, and then observed them under the microscope, we can find the normal brain tissue microvascular complete and clear each branch obvious, and however the GBM capillaries present broken, lumps and branch clutter.
2. By microarray analysis showed that miR-7-5p significantly reduced in GBM microvessels relative to normal brain tissue microvascular (P<0.01), and reduce2.61 times relative to the normal non-tumor brain tissue microvascular. In all five cases of GBM microvascular and brain microvascular, the results of qPCR verify and the gene chip analyses have exactly the same.
3. Bioinformatics prediction results show, the3'-UTR of RAF1that participate in the biological behavior of endothelial cells and miR-7-5p5'end "seed sequence" have better matching sites. Determination of luciferase activity shows that the RAF1is a direct target of the miR-7-5p downstream (P<0.01).
4. MTT assay, cell cycle experiments showed that miR-7-5p induced endothelial cell cycle arrest in the G1phase, and inhibited the proliferation of endothelial cell in vitro. Scratch test showed that miR-7-5p has no effect on vascular endothelial cells in migration in vitro.
5. Western blot analysis showed that the infection of LV-miR-7-5p leaded to a decrease of RAF1protein expression in HUV-EC-C cells. Actin was used as internal (P<0.01); In the GBM and normal brain tissue capillaries, the expression of miR-7-5p and RAF1shows negatively correlated. Pearson's correlation analysis with statistical significance. Correlation coefficient was-0.786.(P<0.01).
Conclusions
Dextran sedimentation density gradient centrifugation methods can be successfully extracted microvascular from GBM normal brain tissue, by trypan blue staining, and microscopy found a significant difference between GBM and normal brain microvascular in the form, this is in line with the objective law. The study began with the GBM and normal brain tissue microvascular. Therefore, there have a good reference value for the pinpoint valuable microRNAs and their targets gene that play a vital role for the occurrence of GBM microvascular, and may provide a more meaningful experimental basis for the treatment of GBM. In the current study of the gene chips, we revealed the miR-7-5p significantly downregulated in GBM microvascular, and verified the result by qPCR in the GBM microvascular tissue and HUV-EC-C cell lines. Moreover, we successfully packaged the lentivirus of over express miR-7-5p (LV-miR-7-5p) and its negative control lentivirus (LV-miR-NC), and performed the MTT assay, cell cycle test and scratch test in vitro. Experimental results showed that the overexpression of miR-7-5p can significantly inhibit the proliferation of the HUV-EC-C cells by induce the cell cycle arrest in G1phase, but for the migration of vascular endothelial cells had no effect. These results strongly supported the miR-7-5p may be an inhibitor of GBM angiogenesis.
To explore the mechanism of miR-7-5p inhibition GBM microvascular endothelial cell growth, we have identified the potential target genes of miR-7-5p.Bioinformatics analysis shows that the RAF1may the be potential target of miR-7-5p.Moreover, RAF1plays an important role in the development and occur for many types of cancer. Therefore, we apply the luciferase activity assay to identify the role of miR-7-5p in expression of the RAF1.The luciferase activity assay shows that miR-7-5p can directly targeting the3'-UTR of RAF1. Western blot results showed that the overexpression of miR-7-5p can significantly reduced the expression of RAF1in HUV-EC-C cells. We have carried out western blot experiments in five-groups of GBM microvascular and normal brain tissue microvascular, the result shows that the RAF1upregulated in GBM tissue capillaries, however downregulated in the brain microvessels, and there have a negative correlation appeared between the miR-7-5p and the RAF1.These data confirm the miR-7-5p can downregulate the expression of RAF1, which also shows RAF1may be a contributing factor in angiogenesis of GBM. This study suggests that miR-7-5p may play a role of tumor suppressor genes in the formation and development of microvessels in GBM through the regulation of RAF1which downstream target genes, and play an important effect in the regulation of proliferation of endothelial cells.
引文
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