长链非编码RNA-LINC01116在脑胶质瘤中的表达及其肿瘤生物学功能与机制的研究

发布时间：2018-04-02 05:35

本文选题：胶质瘤　切入点：基因芯片　出处：《第二军医大学》2017年博士论文

【摘要】：脑胶质瘤是中枢神经系统最常见的恶性肿瘤,约占全部颅内肿瘤的40%-65%,是颅内肿瘤患者的主要死因之一。其中低级别胶质瘤患者的预后往往较好,10年的总体生存率可以达到47%左右,而绝大多数高级别胶质瘤患者预后较差,其中多形性胶质母细胞瘤(glioblastoma multiforme,GBM)的恶性程度最高,其5年的存活率甚至小于3%,明确诊断后的平均生存时间只有12月左右。尽管医学界对胶质瘤患者的治疗已经做出了巨大的努力,但是对胶质瘤患者的预后改善仍然较差。造成胶质瘤治疗效果欠佳的原因不仅包括肿瘤本身增长迅速,血管密集,血运丰富,对放化疗的的耐受性,也包括肿瘤细胞向周围正常脑组织侵润的特性。胶质瘤的发生发展是一个非常复杂的生物学过程,其中涉及到了非常多的基因和细胞生物学行为的改变,为提高胶质瘤的治疗效果,新的治疗手段如基因靶向治疗开始兴起并应用于胶质瘤的治疗,然而目前临床上应用的药物,如替莫唑胺、贝伐单抗等,在患者中的有效率仍然很低。因此,只有全面深入地了解胶质瘤发生发展的分子机制,才能为临床对胶质瘤的诊治提供效率更高的分子靶向药物和更为精准的辅助诊断标志物。随着基因测序、基因芯片和基因组学等研究技术的发展,越来越多的长链非编码RNA(long non-coding RNA,lncRNA)被发现参与到了中枢神经系统的病理生理过程中,在中枢神经系统的发展及疾病的发生过程中起到了重要的作用。长链非编码RNA的异常表达可能会导致一系列中枢系统疾病的发生,其对神经元细胞的分化影响巨大。并且长链非编码RNA在脑胶质瘤病理生理过程中的重要作用也越来越被广泛接受,其不仅可通过调控多种通路及与不同分子的相互作用来调控胶质瘤的发生发展,且在胶质瘤的转移和复发、分型和预后,及维持胶质瘤细胞干性上都发挥了重要的作用。因此本研究通过lncRNAs芯片,表达谱芯片等高通量筛选技术,筛选出在胶质瘤发生发展过程中可能发挥重要作用的长链非编码RNA分子LINC01116,并结合生物信息学分析及一系列经典分子生物学技术等,在胶质瘤临床样本、体外胶质瘤细胞系、体内裸鼠荷瘤模型中对LINC01116分子在胶质瘤临床样本中的表达情况,以及其在胶质瘤发生发展过程中的所起到的具体的生物学功能及相关的分子机制进行全面而深入的研究。通过本课题的研究,我们期待明确LINC01116在胶质瘤发生发展过程中的功能与机制,并明确其在胶质瘤临床诊断和评估预后上的作用,为胶质瘤的诊断和预后提供新的标志物,并为其作为潜在药物作用靶点提供有力的实验依据。第一部分正常脑组织和脑胶质瘤肿瘤组织中长链非编码rna与mrna的芯片检测和分析研究目的:观察在正常脑组织和胶质瘤肿瘤组织中差异表达的长链非编码rna分子和信使rna(messengerrna,mrna)分子的表达变化情况,挑选关键的长链非编码rna分子作为进行深入研究的对象。研究方法:收集2014年12月至2015年5月间在上海长征医院神经外科收治并手术切除的5例胶质母细胞瘤临床组织样本和5例正常脑组织样本,使用arraystar人类lncrna芯片v3.0芯片系统地检测长链非编码rna和信使rna的表达,筛选差异倍数≥2的长链非编码rna和信使rna,并应用配对t检验计算长链非编码rna和信使rna在两组间差异的显著性,p≤0.05时差异具有统计学意义,最终取差异倍数≥2,且p≤0.05的长链非编码rna和信使rna,并随机挑选12个分子采用实时定量pcr(quantitativerealtimepolymerasechainreaction,qrt-pcr)在原5对正常脑组织和胶质母细胞瘤样本验证芯片数据的可靠性。利用生物信息学靶定与胶质瘤发生发展密切相关的长链非编码rna分子。结果:共有4502条lncrna和5492条mrna在两组样品间表达差异明显。其中2601条lncrna和2693条mrna表达上调,1901条lncrna和2799条mrna表达下调。qrt-pcr的结果提示挑选出的12个分子的表达与lncrna和mrna芯片结果的表达趋势一致。其中长链非编码linc01116在肿瘤组织中显著上调表达,提示它可能对胶质瘤的发生发展具有重要作用。结论:(1)通过基因芯片检测发现,对比正常脑组织,在脑胶质瘤组织中存在大量差异表达的长链非编码rna和信使rna,这些差异表达的分子可能与胶质瘤的发生发展和迁移侵袭过程密切相关,为后续进一步筛选与胶质瘤发生发展相关的关键长链非编码rna提供了基础。(2)qrt-pcr验证了基因芯片的结果,证明lncrna和mrna芯片结果是可靠的。(3)生物信息学分析靶定了可能在胶质瘤发生发展过程中发挥关键作用候选长链非编码rna分子linc01116。第二部分长链非编码rna-linc01116在人脑胶质瘤临床样本中的表达情况及临床意义研究目的:扩大临床样本验证linc01116分子在不同级别胶质瘤中的表达情况,分析其与胶质瘤患者的临床病理指标及生存预后是否存在关联,并为其是否能作为胶质瘤诊断和治疗的靶点及评估胶质瘤患者预后的指标提供有力的实验依据。研究方法:收集2004年1月至2015年12月间上海长征医院收治并手术的89例不同级别的脑胶质瘤患者的临床样本与临床资料及13例正常脑组织样本,通过qrt-pcr检测linc01116分子在个样本中的表达。采用独立样本t检验、单因素分析、多因素分析、卡方检验、相关性分析及生存分析等统计学方法分析其与脑胶质瘤患者临床病理指标及生存预后的相关性。结果:对比正常脑组织,qrt-pcr结果提示linc01116在脑胶质瘤样本中呈显著上调表达,其在高级别胶质瘤中的表达明显高于低级别(p0.001)。通过将linc01116在本组胶质瘤临床样本中的表达与本组脑胶质瘤患者的生存与预后进行统计分析我们发现:linc01116的表达与本组胶质瘤患者的完全无进展生存期(progressionfreesurvival,pfs)及总体生存期(overallsurvival,os)均存在显著的统计学关系(p=0.043,p=0.008),linc01116表达量低的患者,其复发和总体生存情况均较好。结论:长链非编码rna分子linc01116在脑胶质瘤组织中呈显著高表达,且其与脑胶质瘤患者的临床病理级别及生存预后情况密切相关,具备成为脑胶质瘤辅助性诊断分子标志物和评估脑胶质瘤患者生存预后指标的潜能。第三部分长链非编码rna-linc01116在脑胶质瘤发生发展过程中的生物学功能与分子机制研究研究目的:明确长链非编码rna分子linc01116在胶质瘤发生发展过程中的生物学功能;初步探索linc01116分子发挥生物学功能的分子机制及其下游可能的调控靶点,以期为胶质瘤的分子诊断和临床治疗提供新的药物作用靶点和思路,并提供相应的理论和有力的实验依据。研究方法:通过在胶质瘤细胞系u251、u87mg、a172、u118mg中利用慢病毒载体构建稳定表达的干扰或过表达linc01116分子的胶质瘤细胞系,并在体外细胞实验中利用流式细胞技术检测细胞周期、cck8实验检测细胞的增殖能力、transwell小室实验检测细胞的迁移侵袭能力,及小管形成实验检测linc01116分子对huvec细胞小管形成能力的影响,在u87mg细胞系中构建干扰linc01116分子的稳转细胞系进行裸鼠皮下荷瘤检测linc01116分子在体内实验中对肿瘤形成的影响。通过在干扰linc01116分子的u251稳转细胞系中构建表达谱基因芯片,结合qrt-pcr、westernbolt、双荧光素酶报告系统等实验技术靶定linc01116分子的下游靶基因及相关信号通路。结果:通过实验我们发现干扰linc01116的表达后,在体外细胞水平提示:胶质瘤细胞生长周期阻滞在g1期,肿瘤细胞生长减慢,增殖能力下降,同时肿瘤细胞的迁移和侵袭能力显著下降。在体内裸鼠皮下荷瘤提示:干扰linc01116分子的表达后,裸鼠皮下成瘤能力显著下降,对生成的肿瘤进行血管内皮生长因子(vascular endothelial growth fact A,VEGFA)、CD31等分子的免疫组化发现,VEGFA、CD31的表达显著下降,肿瘤内部血管生成能力显著下降。通过分析表达谱芯片结果并结合生物信息学分析,我们发现VEGFA为LINC01116分子的下游靶基因,且LINC01116分子与VEGFA分子的3’UTR区均存在miR-31-5p的结合位点,双荧光素酶报告实验提示LINC01116与VEGFA分子的3’UTR区均可吸附miR-31-5p分子。Western bolt提示干扰LINC01116后VEGFA的蛋白表达下降。结论:长链非编码RNA-LINC01116可以通过调控胶质瘤细胞的生长周期进而调控胶质瘤细胞的增殖能力,且其在维持胶质瘤细胞的迁移和侵袭能力方面发挥着重要作用;LINC01116可通过竞争性吸附miR-31-5p在转录后水平调节VEGFA分子的表达,从而影响胶质瘤血管的生成,进而影响肿瘤的生成。
[Abstract]:Glioma is the most common malignant tumors of the central nervous system, accounting for 40%-65% of all intracranial tumors, is one of the main causes of death in patients with intracranial tumors. The prognosis of patients with low grade gliomas often better overall 10 year survival rate can reach about 47%, and most of the high level of poor prognosis in patients with glioma. The glioblastoma multiforme (glioblastoma multiforme, GBM) of the highest degree of malignancy, the survival rate at 5 years or even less than 3%, clear the average survival time after diagnosis only around December. Although the treatment medicine for glioma patients has made great efforts, but to improve the prognosis of glioma patients is still poor. The cause of poor therapeutic effect on glioma includes not only the rapid growth of the tumor, vascular density, abundant blood supply, tolerance to chemotherapy, including tumor cells to surrounding it The characteristics of ordinary brain tissue invasion. The development of glioma is a complex biological process, which involves the genetic and cell biological behavior is very much changed, in order to improve the treatment of glioma, new therapeutic methods such as gene targeting therapy began to rise and application in the treatment of glioma. However, the current clinical application of drugs, such as temozolomide, bevacizumab, in patients with efficiency is still low. Therefore, only a thorough understanding of the molecular mechanism of the occurrence and development of glioma, for clinical diagnosis and treatment of glioma molecular target to provide higher efficiency and more auxiliary drug the precise diagnosis markers. With the development of gene sequencing, gene chip technology and genomics, more and more non long chain encoding RNA (long non-coding RNA, lncRNA) were found in the central nervous system disease Physical and physiological process, in the process of development and diseases of the central nervous system plays an important role. The abnormal expression of long chain non encoding of RNA may lead to a series of central nervous system diseases and its influence on the differentiation of neurons is huge. And long chain non important role in the pathophysiological process of RNA encoding glioma is more and more widely accepted, it can not only regulate the development of glioma by regulating various pathways and interactions with different molecules, and the metastasis and recurrence of glioma, typing and prognosis of glioma cells, and maintain the dry have played an important role. Therefore this study through the lncRNAs chip, such as microarray high-throughput screening technology, selection of long chain may play an important role in the development of glioma in the non encoding RNA molecule LINC01116, and Bioinformatics Analysis and a series of classic molecular biology technology, in clinical samples of glioma, glioma cell lines in vitro, tumor bearing nude mice model of LINC01116 molecule expression in glioma clinical samples, and its occurrence in the development of gliomas by up to specific biological functions and related molecules the mechanism of comprehensive and in-depth study. Through this study, we look forward to developing LINC01116 function and mechanism in the development of glioma, and clearly in the glioma clinical diagnosis and prognosis assessment on the role, to provide new markers for diagnosis and prognosis of glioma, and to provide experimental evidence the potential drug targets for the long chain non RNA encoding and mRNA chip detection and analysis to the first part of normal brain tissue and glioma tumor tissues were observed in the normal brain: The long chain of differentially expressed RNA molecules and non encoding messenger RNA tissues and glioma tumor tissues (messengerrna, mRNA) expression of long chain molecules, select key non encoding RNA molecules as the object of in-depth study. Research methods: from December 2014 to May 2015 in 5 cases of glioblastoma clinical tissue samples the Department of Neurosurgery of Shanghai Changzheng Hospital were resected and 5 cases of normal brain tissue samples, using arraystar human lncrna chip V3.0 system to detect expression of long chain non encoding RNA and RNA Messenger, more than 2 fold difference screening of long chain non encoding RNA and RNA Messenger, and the application of significant paired t test calculation of long chain non encoding RNA and the messenger RNA in the difference between the two groups, statistically significant difference when p is less than or equal to 0.05, then more than 2 fold difference, and the long chain P is less than or equal to 0.05 of non encoding RNA and RNA Messenger, and random selection of 12 A molecule by quantitative real-time PCR (quantitativerealtimepolymerasechainreaction, qRT-PCR) on the reliability of the original 5 of normal brain tissues and glioblastoma samples verify the microarray data. Long chain using bioinformatics and targeting glioma is closely related to the development of non encoding RNA molecules. Results: a total of 4502 lncrna and 5492 mRNA expression. In two groups of samples. Among which 2601 lncrna and 2693 mRNA expression, consistent expression trend of 12 molecules down regulated the expression of.Qrt-pcr results of the 1901 lncrna and 2799 mRNA were selected and the expression of lncrna and mRNA chip. The results of long chain non encoding linc01116 expression was significantly up-regulated in tumor tissues. Suggesting that it may play an important role in the development of glioma. Conclusion: (1) by gene chip detection showed that compared with normal brain tissue in glioma tissues are large The amount of long chain expression differences of RNA and non encoding messenger RNA molecules may differentially expressed with glioma is closely related to the occurrence and development of migration and invasion, non encoding RNA provides the basis for further screening and glioma occurrence and development of long chain related key. (2) qRT-PCR to verify the microarray results. Lncrna and mRNA chip results are reliable. (3) the bioinformatics analysis of the target in the development of glioma may play a key role in the process of long chain non candidate RNA molecules encoding the second part linc01116. of long chain non objective to study the expression of rna-linc01116 encoding in clinical samples of human glioma and its clinical significance in expanding the expression: the clinical sample validation of linc01116 molecules in different grade gliomas, and to analyze the clinicopathological parameters and prognosis of glioma patients is related, and it is It can be used as indicators of prognosis of patients with glioma target for diagnosis and treatment of glioma and evaluation provide powerful experimental basis. Methods: clinical samples and clinical data and 13 cases of normal brain tissue samples collected from January 2004 to December 2015 in Shanghai Changzheng Hospital were analyzed and surgery in 89 cases with different levels of brain glioma patients, through the detection of expression linc01116 molecule qRT-PCR in the samples. The independent sample t test, single factor analysis, multi factor analysis, chi square test, correlation analysis and survival analysis were used to analyze its correlation with glioma patients with clinicopathological parameters and prognosis. Results: compared with normal brain tissue, the results of qRT-PCR showed that linc01116 was significantly increase in glioma samples in the expression, the expression in high grade gliomas was significantly higher than that of low level (p0.001). The linc01116 in this group of glue The survival and prognosis of the expression of stromal tumor in clinical samples and the group of brain glioma patients for statistical analysis we found that the expression of linc01116 and the group of glioma patients was progression free survival (progressionfreesurvival, PFS) and overall survival (overallsurvival, OS) were statistically significant relationship (p=0.043, p=0.008) the expression of linc01116, patients with low volume, recurrence and overall survival were better. Conclusion: long chain non encoding RNA molecule expression of linc01116 was significantly higher in glioma tissues, and the patients with glioma clinical pathological grade and prognosis are closely related, and have become the evaluation of brain glioma the survival prognostic indicators of potential markers of brain glioma assisted molecular diagnosis. The third part of long chain non encoding rna-linc01116 biological function and molecular mechanism in the development of brain glioma Objective: To study the biological function of long chain non explicit encoding RNA molecule linc01116 in the process of the occurrence and development of glioma; preliminary exploration of linc01116 molecules play a biological function of molecular mechanism and its possible downstream targets, in order to provide drug targets and new ideas for clinical treatment and molecular diagnosis of glioma, and to provide the corresponding theory and powerful experimental basis. Research methods: through U251, in U87MG glioma cell line, A172, interference vector construction and stable expression or overexpression of linc01116 glioma cell line by lentiviral u118mg molecules, and the use of flow cytometry to detect the cell cycle in vitro, CCK8 assay cell proliferation and migration of Transwell cell invasion assay, and tube formation experimental detection of linc01116 molecules forming ability of HUVEC tubules. Sound construction effect of interfering linc01116 molecules of stabletransfection cell lines for detection of linc01116 molecules in nude mice subcutaneous tumor formation in vivo of tumor in U87MG cell lines. The interference in the linc01116 molecule of U251 stable cell lines to construct the gene chip, combined with qRT-PCR, westernbolt and downstream target gene target experiment technology luciferase reporter system such as linc01116 molecules and signaling pathways. Results: through the experiment we found that interfering the expression of linc01116, suggesting that in vitro cellular level: glioma cell cycle arrest in G1 phase, the growth of tumor cell proliferation decreased at the same time, slow down, migration and invasion of tumor cells in vivo suggest that decreased significantly. Subcutaneous tumor: interfering expression of linc01116 molecules after subcutaneous tumor formation ability decreased significantly, the generation of tumor vascular endothelial growth factor (vascular endothelial growth fact sub A, VEGFA), group of immune CD31 molecules found that VEGFA, CD31 expression significantly decreased tumor angiogenesis capacity decreased significantly. The microarray and bioinformatics analysis by analyzing the expression, we found VEGFA LINC01116 downstream target gene, miR-31-5p binding sites there are 3 UTR region and the LINC01116 and VEGFA molecules, dual luciferase reporter experiments showed that LINC01116 molecules of VEGFA and VEGFA protein expression was decreased 3 UTR region can be adsorbed miR-31-5p.Western bolt interference LINC01116. Conclusion: after a long chain of non encoding RNA-LINC01116 can regulate cell proliferation through growth cycle glioma cells, and in the maintenance of glioma cell migration and invasion ability plays an important role in LINC01116 by competitive; The adsorption of miR-31-5p at the post transcriptional level regulates the expression of VEGFA molecules, which affects the angiogenesis of glioma, and then affects the formation of the tumor.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R739.41

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