外泌体内的miR-221通过靶向调控DNM3促进神经胶质瘤恶性进展和化疗抵抗

发布时间：2018-05-11 01:28

本文选题：胶质瘤 + 外泌体　；参考：《河北医科大学》2017年博士论文

【摘要】：脑胶质瘤在中枢神经系统中高发,且预后很差。即使给予术后放疗、化疗或联合治疗,脑胶质瘤患者的复发率仍然很高,治疗效果始终不能令人满意。随着基因水平研究的不断深入,有关胶质瘤的基因分子机制得到深入的探索。O6-甲基鸟嘌呤-DNA甲基转移酶(O6-methylguanine-DNA methyhransferase,MGMT)启动子甲基化、1p/19q杂合性缺失、异柠檬酸脱氢酶1(Isocitrate dehydrogenase 1,IDH1)基因突变、ATRX表达缺失等均已经成为胶质瘤病理学诊断的重要分子生物学标记,也丰富了人们对于肿瘤基因变异的认识。Stephen Paget于1889年提出肿瘤生长的“种子-土壤”学说,奠定了肿瘤微环境的概念。外泌体是肿瘤微环境中的重要成分。它通过释放自身携带的细胞因子参与了肿瘤的发生、增殖、侵袭和化疗抵抗等生物学过程。而肿瘤细胞来源的外泌体(Tumor cells derived exosome,Texo)由于它的结构特征和运输功能已经成为近年来肿瘤领域的研究热点。2013年,美国、德国3位科学家凭借他们所发现的细胞囊泡运输的调节机制,荣获2013年诺贝尔生理学或医学奖。外泌体(exosome)作为人体内一类重要运输囊泡,也开始受到越来越多的关注。科学家们已发现,外泌体会参与到肿瘤细胞的生长、增殖、侵袭、凋亡、血管生成、炎症反应、免疫应答等重要的生物过程,细胞会通过分泌外泌体,将一些信号分子分泌到较远的组织或细胞中,以起到调控作用。外泌体作为药物传递系统(Drug delivery system,DDS)为高效药物投递提供天然的内源性纳米级载体,其靶向作用的潜力也逐渐被发现。miRNA是一类内源性的具有调控功能的非编码RNA,可以与靶mRNA的3'非翻译区(Untranslated Regions,UTR)结合从而在转录后水平调节靶基因的表达。有研究显示,miR-221在直肠癌、胰腺癌、乳腺癌和膀胱癌中是一种促癌基因。利用高通量筛选测序平台筛选脑胶质瘤组织miRNA差异表达情况,筛选出了胶质瘤组织内miR-221高表达,特别是胶质母细胞瘤组织。考虑到胶质瘤细胞外泌体内miR-221的表达情况以及调节机制尚未进行过研究,故在本实验中我们选取胶质瘤细胞来源的外泌体miR-221作为研究对象,进行体内外细胞和外泌体miR-221表达的检测,并通过CCK-8实验,流式细胞技术以及Transwell实验探讨其对于胶质瘤的作用。然后进一步寻找miRNA的靶基因。DNM3(Dynamin 3)是发动蛋白超家族(包括DNM1、DNM2、DNM3)中的一员,参与许多膜转运功能,例如胞质分裂、吞噬作用、转运囊泡出芽和细胞流动等。最近的研究表明,DNM3的高表达可抑制细胞增殖及诱导凋亡。基于对目前已有文献的充分分析,我们推测miRNA-DNM3途径有可能成为抑制神经胶质瘤生长及放化疗增敏的治疗靶点。由于DNM3出现在Targetscan的预测结果中,我们进一步研究其与miR-221的关系。实验中我们确定了miR-221的靶基因DNM3以及miR-221的转录调控因子RELA,并通过双荧光素酶报告基因实验得以证实,并分析它们表达量的相关性,最后通过病毒转染实验证实miR-221对胶质瘤的作用是通过靶向调控DNM3引起的,并受上游RELA的转录调控。第一部分miR-221在人脑胶质瘤组织和GBM源性外泌体内的表达及其与胶质瘤WHO病理分级的关系目的:研究miR-221在人脑胶质瘤组织和GBM源性外泌体内的表达,并分析miR-221的表达与胶质瘤WHO病理分级的关系。方法:1 48例胶质瘤患者及11例非肿瘤患者组织标本均于2015年采集自河北医科大学第二医院神经外科。包括23名女性,36名男性,年龄从30至68岁。标本采集后迅速放至液氮中冷冻,然后-80℃低温保存。诊断结果均由2位病理医师根据WHO分级指导得出。2抽取上述患者的静脉血,室温静置后获得血清或者采集后立即离心获得血浆上清液,提取外泌体。3 SHG-44、U87MG、HEB和U251的培养,及细胞外泌体的提取。4定量反转录聚合酶链反应(qRT-PCR)方法测定神经胶质瘤组织或外泌体内miR-221含量。5应用t检验分析神经胶质瘤中miR-221含量与胶质瘤级别或不同细胞系之间的关系。结果:1 miRNA-221在人脑胶质瘤组织中表达上调:与对照组织比较,胶质瘤组织中miRNA-221均呈高表达(P0.01),其中高度恶性胶质瘤组织中miRNA-221表达量均明显高于低度恶性者(P0.01)。在WHO III和WHO IV胶质瘤中表达量较WHO II或非肿瘤组织明显增高(P0.01)。miR-221的表达与胶质瘤的病理分级(WHO分级)明显相关。2比较在无细胞的血清和血浆外泌体内miR-221的差异表达,可见胶质瘤患者与非肿瘤患者miR-221表达量具有显著性差异(P0.01)。3血浆中的检测结果与组织检测结果相似:胶质瘤患者(n=48)与非肿瘤患者(n=11)相比,血浆外泌体miR-221表达量具有显著性差异(P0.01)。胶质瘤患者血浆外泌体miR-221表达量随胶质瘤病理级别升高表达量显著升高。4 miR-221在四种胶质瘤细胞系中均高表达,且miR-221在U87MG细胞来源的外泌体中表达最高,U251、SHG-44、HEB细胞外泌体内表达依次降低。结论:1 miR-221在人脑胶质瘤组织中高表达,在血浆来源或胶质瘤细胞系来源的外泌体内也存在高表达,且其表达量随着胶质瘤WHO病理分级呈递增趋势。高级别(III和IV级)胶质瘤miR-221的表达量明显高于低级别(II级)胶质瘤,差别具有统计学意义。2 miR-221的表达量与胶质瘤WHO病理分级呈显著的正相关,预示外泌体miR-221可能用于胶质瘤相关的临床检测。第二部分U87MG细胞源性的外泌体miR-221对SHG-44细胞生物学活性的影响目的:研究U87MG细胞源性外泌体miR-221对SHG-44细胞的增殖、侵袭、化疗抵抗等方面的影响。方法:1培养U87MG和SHG-44细胞系,提取U87MG细胞系外泌体。2向SHG-44细胞系转染anti-miR-221(miR-221 ASO)以降低细胞内miR-221的表达,转染anti-miR-NC(Ctrl ASO)作对照。实验组SHG-44细胞培养基中同时加入外泌体。3进行CCK-8实验研究降低miR-221表达后胶质瘤细胞增殖能力的变化。4进行流式细胞实验研究降低miR-221表达对胶质瘤细胞凋亡能力和替莫唑胺耐药的变化。5进行Transwell实验和划痕实验研究降低miR-221表达后胶质瘤细胞侵袭能力的变化。结果:1 qRT-PCR结果显示,转染anti-miR-221后,SHG-44细胞内miR-221的表达量显著降低(P0.01)。2 CCK-8实验结果显示,anti-miR-NC+Exo组的增殖活力最高,anti-miR-221组的增殖活力最低,即敲低miR-221的表达后,胶质瘤细胞SHG-44的增殖能力显著降低(P0.01)。3流式细胞实验结果显示,敲低miR-221表达后,SHG-44细胞凋亡能力无明显改变,但是对替莫唑胺的敏感性显著增高(P0.01)。4 Transwell实验和划痕实验结果显示,敲低miR-221表达后,SHG-44细胞侵袭能力显著降低(P0.01)。结论:敲低miR-221表达,可以使SHG-44细胞增殖及侵袭能力显著降低,凋亡能力虽无改变,但是对替莫唑胺的敏感性增高。miR-221可以影响胶质瘤细胞的增殖、侵袭及对替莫唑胺的化疗耐药。第三部分miR-221对下游靶基因DNM3的调控以及RELA对miR-221转录能力的影响目的:寻找miR-221的下游作用靶点以及miR-221的转录调控因子,深入了解miR-221对胶质瘤生物学影响的作用机制。方法:1使用Targetscan数据库预测miR-221的下游作用靶点,并搜集相关文献筛选有意义结果。2构建miR-221过表达慢病毒质粒,建立miR-221过表达稳定SHG-44细胞系,进行稳定细胞系的筛选。3双荧光素酶报告基因检测用于验证miR-221与DNM3的作用关系。4应用qRT-PCR及蛋白质印迹(Western blot)方法检测miR-221与DNM3在SHG-44细胞中的表达量,并分析表达量的相关性。5通过CCK-8实验、流式细胞术、Transwell迁移和侵袭实验等方法,研究上调DNM3对胶质瘤SHG-44细胞增殖、侵袭和凋亡等生物学行为的影响。6通过生物信息学方法,预测miR-221启动子区域的转录因子结合位点;利用荧光素酶报告实验,在SHG-44细胞中验证此转录因子与miR-221启动子的结合情况;通过上调或下调这一转录因子,采用实时定量PCR方法检测miR-221的表达变化。结果:1根据Targetscan的预测结果,并结合相关文献资料,发现DNM3上存在两个与miR-221的作用靶点。2成功构建了miR-221过表达慢病毒载体和稳转胶质瘤细胞系,miR-221的表达水平显著升高(P0.01),是对照组的45.5倍。3双荧光素酶报告检测结果表明,相对于miR-NC,miR-221可以显著抑制荧光素酶的活性(P0.01),而在突变靶位点后此现象消失(P0.01)。4 qRT-PCR检测胶质瘤组织中的DNM3表达量:DNM3表达量随胶质瘤病理级别升高表达量反而降低。Spearman秩相关分析表明,胶质瘤细胞DNM3与miR-221的表达量呈负相关(Spearman r=-0.908,P0.01)。5转染miR-221过表达载体后,在高表达miR-221的细胞中,DNM3低表达;CCK-8实验提示,增加DNM3的表达后,细胞增殖能力显著降低。流式细胞技术检测发现,加入替莫唑胺后,DNM3促进了胶质瘤细胞的早期凋亡(P0.01)。Transwell实验和划痕实验结果表明,转染DNM3能够抑制胶质瘤细胞的迁移和侵袭能力。6通过生物信息学方法,预测到miR-221编码基因的启动子区域存在RELA的结合位点;双荧光素酶报告基因检测证实RELA能够与miR-221基因的启动子区域相结合;实时定量PCR结果表明,转染RELA后,能诱导miR-221的过表达。结论:DNM3的mRNA中存在miR-221在胶质瘤中的靶向作用位点,转录因子RELA调节miR-221基因的转录。第四部分miR-221促进SHG-44人脑胶质瘤细胞系生长的体内研究目的:动物体内证实miR-221对胶质瘤生物学活性影响。方法:1稳定转染miR-221的SHG-44胶质瘤细胞系,进行稳定细胞系的筛选,对照组转染miR-NC。2应用反转录定量聚合酶链反应(qRT-PCR)方法检测miR-221在SHG-44细胞中的表达量。3裸小鼠皮下人脑胶质瘤模型的建立。4肿瘤生长情况观察:每隔4天用游标卡尺测量一次肿瘤的长径(a)及宽径(b),计算肿瘤体积。结果:1成功构建miR-221过表达的SHG-44胶质瘤细胞系,qRT-PCR检测稳定细胞系内miR-221的相对表达量为47±1.45,明显高于Vector转染细胞系(P0.01)。2 miR-221过表达脑胶质瘤模型证实肿瘤生长速度明显增快。带瘤生存20天后取下肿瘤组织,发现miR-221过表达组肿瘤重量为对照组肿瘤重量的2倍余(P0.05)。结论:miR-221过表达促进胶质瘤细胞体内生长,miR-221是一种促癌因子。
[Abstract]:The brain glioma is high in the central nervous system and has poor prognosis. The recurrence rate of the patients with glioma is still high and the therapeutic effect is still not satisfactory even after the postoperative radiotherapy, chemotherapy or combined therapy. The molecular mechanism of glioma has been deeply explored with the molecular mechanism of the gene level..O6- methyl guanine is deeply explored. Methotrexate -DNA methyltransferase (O6-methylguanine-DNA methyhransferase, MGMT) promoter methylation, 1p/19q heterozygosity deletion, ISO citrate dehydrogenase 1 (Isocitrate dehydrogenase 1, IDH1) gene mutation and ATRX expression loss have become important molecular biomarkers for pathological diagnosis of glioma, and also enrich people's tumor base. .Stephen Paget proposed the "seed soil" theory of tumor growth in 1889, which established the concept of tumor microenvironment. Exocrine is an important component of tumor microenvironment. It participates in the biological processes of tumor occurrence, proliferation, invasion and chemotherapeutic resistance by releasing its own cytokine, and tumor cells. Tumor cells derived exosome (Texo), because of its structural characteristics and transport function, has become a research hotspot in the field of cancer in recent years.2013. In the United States, 3 scientists in Germany were awarded the 2013 Nobel prize for physiology or medicine for their discovery of cellular vesicle transport. The exocrine (exosome) was awarded. As an important type of transport vesicles in the human body, more and more attention has been paid. Scientists have found that exocrine experience is involved in the biological processes such as growth, proliferation, invasion, apoptosis, angiogenesis, inflammatory response, immune response and other important biological processes, and the cells secrete some signal molecules into more distant tissues. Drug delivery system (DDS) provides natural endogenous nanoscale carriers for high efficiency drug delivery. The potential of its targeting is also gradually found to be a kind of endogenous non coded RNA with regulatory function, which can be used with the 3'non translation region of the target mRNA (Untrans). Lated Regions, UTR) combined to regulate the expression of target genes at post transcriptional levels. Studies have shown that miR-221 is a oncogene in rectal cancer, pancreatic cancer, breast and bladder cancer. High throughput screening sequencing platform is used to screen the differential expression of miRNA in brain glioma tissue, and the high expression of miR-221 in glioma tissues is screened, especially in glioma tissues. It is a glioblastoma tissue. Considering the expression of miR-221 in the extracellular secretory of glioma cells and the regulation mechanism has not been studied, in this experiment, we selected the external secretory miR-221 derived from glioma cells as the research object to detect the expression of miR-221 in the cells and exosecrete in vitro and in vivo, and through the CCK-8 experiment, flow formula. Cell technology and Transwell experiments explore its role in glioma. Then further search for the target gene.DNM3 (Dynamin 3) of miRNA is a member of the superfamily of the promoter protein (including DNM1, DNM2, DNM3), and participates in many membrane transport functions, such as cytokinesis, phagocytosis, transport vesicle buds and cell flow. Recent studies have shown that The high expression of DNM3 inhibits cell proliferation and induces apoptosis. Based on a full analysis of current literature, we speculate that the miRNA-DNM3 pathway may be a therapeutic target for inhibiting the growth of glioma and chemosensitization. As DNM3 appears in the prediction of Targetscan, we further study the relationship with miR-221. We identified the target gene DNM3 of miR-221 and the transcriptional regulator RELA of miR-221, and confirmed by the double luciferase reporter gene experiment, and analyzed the correlation of their expression. Finally, the effect of miR-221 on the glioma was confirmed by the targeting regulation of DNM3 by the virus transfection experiment, and was regulated by the upstream RELA. Control. Part 1 expression of miR-221 in human glioma tissue and GBM derived exocrine and its relationship with pathological grade of glioma WHO: To study the expression of miR-221 in human glioma tissue and GBM derived exocrine, and to analyze the relationship between the expression of miR-221 and the pathological grade of glioma WHO. Methods: 148 patients with glioma and 11 cases of glioma. The tissue specimens of non tumor patients were collected from the Department of Neurosurgery at the second hospital of Hebei Medical University in 2015, including 23 women, 36 men, aged from 30 to 68. The specimens were quickly frozen in liquid nitrogen after collection, and then stored at -80 C. The diagnosis results were guided by 2 pathologists according to the guidance of.2 to extract the above patients. Blood serum, after room temperature statics, obtained serum or immediately after collection, centrifugation to obtain plasma supernatant, extract exocrine.3 SHG-44, U87MG, HEB and U251, and extract.4 quantitative reverse transcriptional polymerase chain reaction (qRT-PCR) method for the determination of glioma tissue or exocrine miR-221 content.5 application of t test and analysis of T analysis of neuroglia The relationship between the miR-221 content of the tumor and the grade of glioma or different cell lines. Results: 1 miRNA-221 was up-regulated in the human glioma tissue. Compared with the control tissue, the expression of miRNA-221 in the glioma tissues was highly expressed (P0.01), and the expression of miRNA-221 in the high malignant glioma tissues was significantly higher than that of the low-grade malignant (P0.01). In WHO The expression of III and WHO IV glioma was significantly higher than that of WHO II or non tumor tissue (P0.01), and the expression of.MiR-221 was significantly correlated with the pathological grade of glioma (WHO grading). The difference of.2 in the expression of miR-221 in the non cellular and plasma exocrine bodies showed significant difference between the miR-221 expression of glioma and non tumor patients (P0). .01) the detection results in.3 plasma were similar to that of tissue detection. Compared with non tumor patients (n=11), the miR-221 expression of plasma exocrine miR-221 was significantly different (P0.01). The expression of miR-221 expression in plasma exocrine miR-221 in glioma patients increased with the pathological grade of glioma significantly increased.4 miR-221 in four gliomas The expression of miR-221 in the Exocyst of U87MG cells was the highest, and the expression of U251, SHG-44 and HEB cells decreased in turn. Conclusion: 1 miR-221 is highly expressed in human glioma tissue, and high expression in the external secretory of plasma source or glioma cell line, and its expression is along with glioma WHO disease. The expression of miR-221 in high grade (III and IV) glioma was significantly higher than that of low grade (II grade) glioma, and the difference was statistically significant positive correlation between the expression of.2 miR-221 and the pathological grade of glioma WHO, indicating that the exocrine miR-221 may be used in the clinical detection of glioma. Second part U87MG cells The effect of source miR-221 on biological activity of SHG-44 cells: To study the effects of U87MG cell derived exocrine miR-221 on proliferation, invasion and chemotherapy resistance of SHG-44 cells. Methods: 1 culture U87MG and SHG-44 cell lines, and the extraction of U87MG cell line exocrine.2 to SHG-44 cell lines for anti-miR-221 (miR-221) Expression of miR-221 in low cell and transfection of anti-miR-NC (Ctrl ASO) as control. In the experimental group SHG-44 cell culture medium also added exosecreting.3 to carry out CCK-8 experimental study to reduce the proliferation ability of glioma cells after miR-221 expression and.4 performed by flow cytometry to reduce the apoptosis ability of miR-221 expression to glioma cells and temozolomide Drug resistance changes.5 conducted Transwell experiment and scratch test to reduce the invasion ability of glioma cells after miR-221 expression. Results: 1 qRT-PCR results showed that after transfection of anti-miR-221, the expression of miR-221 in SHG-44 cells decreased significantly (P0.01).2 CCK-8 experimental results, anti-miR-NC+Exo group was the highest proliferation activity. Anti-miR-2 The proliferation activity of the 21 groups was the lowest, that is, after the expression of low miR-221, the proliferation ability of SHG-44 in glioma cells decreased significantly (P0.01).3 flow cytometry results showed that the apoptosis ability of SHG-44 cells was not significantly changed after the knockout of low miR-221 expression, but the sensitivity to temozolomide increased significantly (P0.01).4 Transwell experiment and scratch test results. The invasion ability of SHG-44 cells decreased significantly (P0.01) after the knockout of miR-221. Conclusion: the proliferation and invasion ability of SHG-44 cells decreased significantly and the apoptosis ability was not changed, but the sensitivity to temozolomide increased, but.MiR-221 could affect the proliferation of glioma cells, invasion and chemotherapy of temozolomide. Resistance. The regulation of the downstream target gene DNM3 by part third miR-221 and the effect of RELA on the transcriptional ability of miR-221: to find the downstream target of miR-221 and the transcriptional regulator of miR-221, and to understand the mechanism of the effect of miR-221 on the biological effects of glioma. Method: 1 to predict the downstream effect of miR-221 with Targetscan database. Target, and collect relevant literature screening meaningful results.2 construction of miR-221 overexpression lentivirus plasmid, miR-221 overexpression of stable SHG-44 cell line, stable cell line screening,.3 double luciferase reporter gene detection is used to verify the relationship between miR-221 and DNM3,.4 application qRT-PCR and Western blot (Western blot) method for detecting M The expression of iR-221 and DNM3 in SHG-44 cells and the correlation of expression.5 through CCK-8 experiments, flow cytometry, Transwell migration and invasion experiments, the effect of up regulation of DNM3 on the proliferation, invasion and apoptosis of glioma SHG-44 cells,.6 through bioinformatics methods, and prediction of miR-221 promoter region The transcriptional factor binding site; using luciferase reporter assay to verify the binding of the transcription factor to the miR-221 promoter in SHG-44 cells. By up or down this transcription factor, the real-time quantitative PCR method was used to detect the changes in the expression of miR-221. Results: 1 according to the prediction results of Targetscan and the related literature, It was found that there were two target sites on DNM3 with miR-221,.2 successfully constructed the miR-221 overexpressed lentivirus and glioma cell lines, and the expression level of miR-221 increased significantly (P0.01), and the results of the 45.5 times.3 double Luciferase Report of the control group showed that miR-221 could significantly inhibit the activity of luciferase (P0.0) compared with miR-NC (P0.0). 1), and the expression of DNM3 in glioma tissues was detected after the mutation target site (P0.01).4 qRT-PCR: the expression of DNM3 expression decreased with the histopathological level of glioma and decreased.Spearman rank correlation analysis, indicating that the expression of DNM3 in glioma cells was negatively correlated with the expression of miR-221 (Spearman r=-0.908, P0.01).5. After the carrier, the expression of DNM3 was low in the cells with high expression of miR-221. The CCK-8 experiment suggested that the proliferation of the cells decreased significantly after the increase of the expression of DNM3. Flow cytometry detected that DNM3 promoted the early apoptosis of glioma cells (P0.01).Transwell experiment and scratch test. The results showed that the transfection of DNM3 was inhibited. The migration and invasion ability of glioma cells.6 predicted the presence of RELA binding sites in the promoter region of the miR-221 encoding gene by bioinformatics method, and the double luciferase reporter gene detection confirmed that RELA could be combined with the promoter region of the miR-221 gene. The real-time quantitative PCR results showed that after transfection of RELA, miR-221 could be induced by the transfection of RELA. Expression. Conclusion: the targeting site of miR-221 in glioma is found in DNM3 mRNA. Transcription factor RELA regulates the transcription of miR-221 gene. Fourth part miR-221 promotes the growth of SHG-44 human glioma cell line in vivo. The objective of the study is to confirm the effect of miR-221 on the biological activity of glioma in vivo. Method: 1 stable transfection of miR-221 SHG- 44 glioma cell lines were screened for stable cell lines. The control group was transfected with miR-NC.2 using reverse transcriptional polymerase chain reaction (qRT-PCR) to detect the expression of miR-221 in SHG-44 cells. The growth of.4 tumor was observed in the subcutaneous human glioma model of.3 nude mice. The length of a tumor was measured every 4 days with a vernier caliper. A) and wide diameter (b). Tumor volume was calculated. Results: 1 the miR-221 overexpressing SHG-44 glioma cell line was successfully constructed, and the stable cells were detected by qRT-PCR.

【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R739.41

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