恶性脑胶质瘤TRAIL敏感性相关微小RNA的功能性筛选及靶向协同治疗策略的建立

发布时间：2018-04-09 10:33

  本文选题：microRNA-7　切入点：XIAP　出处：《第四军医大学》2017年博士论文

【摘要】：第一部分 恶性胶质瘤细胞TRAIL敏感性相关的microRNA的筛选、功能鉴定及机制研究【背景】恶性胶质母细胞瘤(GBM)是最具侵袭性的脑肿瘤之一,中位生存时间不超过16个月。尽管对于它的治疗已取得很大的进展,但它仍然是最具挑战性的疾病之一。TRAIL(TNF相关的凋亡诱导配体)能够特异性地杀伤肿瘤而不影响正常细胞,因此是很有潜力的抗癌药物。但是已有研究表明,许多肿瘤对其耐受,且在临床试验中,TRAIL因其半衰期短以及体内的不稳定致使其实际应用受到了很大限制。然而,最近的研究表明,使用间充质干细胞(MSCs)表达TRAIL可将TRAIL特异性地运送到肿瘤局部,这可在一定程度上补偿这一缺点。MicroRNA,一类在进化上高度保守的非编码小RNA,可以通过与靶基因的3'非翻译区(UTRs)结合发挥转录后调控作用,进而抑制mRNA翻译或降解mRNA。越来越多的证据表明,miRNA在肿瘤细胞的恶性表型的调控中发挥着关键作用,而且已有研究证实,细胞可以外泌体的形式分泌miRNA,进而下调受体细胞中靶基因的表达。因此,这提示我们,联合使用MSCs、microRNA、TRAIL进行肿瘤靶向治疗的策略或许会有很好的治疗前景。【目的】找出可以增敏TRAIL的microRNA,并使用MSCs作为运载工具开展靶向治疗。【方法】为了找到在胶质瘤中可增敏TRAIL的关键microRNA,我们首先对对TRAIL表现不同敏感程度的胶质瘤细胞系进行mi RNA表达谱的分析,从中找到与TRAIL诱导的凋亡呈现显著相关性的miRNAs。之后在不同的细胞中过表达或干涉该microRNA,检测TRAIL诱导细胞凋亡的情况,进而确认增敏TRAIL的关键microRNA。然后通过生物信息学预测、双荧光素酶报告基因实验、实时定量PCR和蛋白印迹实验,找到该miRNA的下游靶基因。同时,通过功能缺失和恢复实验,进一步确定这样一条调控轴线。然后对MSCs进行改造,使其携带TRAIL和该microRNA,与胶质瘤细胞系U87进行体外共培养实验,验证基因修饰后的MSCs是否可以显著杀伤肿瘤细胞。同时,利用外泌体的抑制剂来进行分析,这种影响是否通过外泌体介导的分子传递来完成的。之后分别通过transwell实验和免疫组化实验在体外和体内实验中分析,携带TRAIL和microRNA的MSCs是否具有较好的肿瘤趋向性。最后建立裸鼠皮下荷瘤模型,使用TRAIL和microRNA联合过表达的MSCs进行尾静脉治疗实验,确认治疗效果。【结果】我们发现,miR-7在胶质瘤细胞系中的表达情况与TRAIL诱导的细胞凋亡存在显著的正相关关系。之后,功能缺失和恢复实验证实,miR-7确实是胶质瘤细胞系中增敏TRAIL关键分子。随后,在机制研究中,我们发现,XIAP是其介导该作用的关键的下游靶基因,进一步的研究发现,miR-7-XIAP这种调控关系存在于多种肿瘤中。而且,体内体外实验证实,携带TRAIL和microRNA-7的间充质干细胞具备很好的肿瘤趋向性。同时,通过外泌体抑制实验证实,MSCs可以以外泌体的形式分泌microRNA-7进而下调受体细胞中的XIAP,从而增强受体细胞的凋亡。最后,更重要的是,在裸鼠移植瘤的治疗模型中,TRAIL和microRNA-7联合过表达的间充质干细胞表现出很好的促凋亡作用,从而抑制肿瘤的生长,达到较好的治疗效果。【结论】在肿瘤细胞中,miR-7可以通过下调XIAP显著增强TRAIL诱导的细胞凋亡。携带TRAIL和miR-7的MSCs可以特异性地趋向于肿瘤局部,抑制肿瘤的生长。第二部分 microRNA-7在肝癌细胞中的抑癌新机制研究【背景】Micro RNA(miRNA)分子是一类在多细胞生物中广泛转录的、普遍存在的非编码小RNA分子。成熟的mi RNA分子可通过序列特异性的碱基互补配对结合到m RNA的3’非编码区,形成RNA沉默复合物,进而导致靶m RNA的翻译抑制或直接将其降解。更重要的是,一个mi RNA可以同时调节上百种m RNA,这也使得其能够参与错综复杂的分子调控网络,进而影响细胞的生命活动。越来越多的研究表明,mi RNA的正常表达是生命活动正常运转所必需的,而它的失调可能会导致包括肿瘤在内的多种疾病的发生。近来研究发现,在肝癌的发生发展过程中伴随着一些抑癌mi RNA的下调,而在这些mi RNA中,mi R-7在肝癌组织中显著低表达,且mi R-7的过表达能够抑制肝癌的增殖和转移。也有研究证实,mi R-7可以通过靶向PI3K/AKT通路抑制肝癌的生长和转移。但是,这些都还不能充分揭示mi R-7在肝癌中发挥的关键抑癌作用。【目的】阐明microRNA-7在HCC细胞中的新的抑癌机制。【方法】首先,在肝癌细胞中瞬时转染miR-7,48小时后使用流式细胞术检测细胞周期。之后,通过生物信息学预测工具分析预测mi R-7的可能下游靶基因,随后通过双荧光素酶报告基因实验初步确认mi R-7的下游靶基因。然后通过实时定量PCR和Western blot检测mi R-7过表达后的靶基因的m RNA和蛋白表达水平的变化,同时通过功能缺失和恢复实验进一步确认靶基因与细胞周期的关系。最后在肿瘤临床样品和肿瘤细胞系中分析mi R-7与靶基因的RNA表达水平的相关性。【结果】首先,我们发现,在肝癌细胞中过表达miR-7可以显著导致细胞周期G1期阻滞。之后通过生物信息学预测、报告基因实验、实时定量PCR和蛋白印迹实验证实,细胞周期蛋白CCNE1是mi R-7发挥该抑制作用的关键下游靶基因。进一步的研究证实,干涉CCNE1与过表达mi R-7导致相似的细胞周期变化,同时CCNE1的恢复过表达可以消除mi R-7引起的细胞周期G1期阻滞。最后,临床组织样品和细胞系的结果显示,mi R-7与CCNE1的表达水平存在着显著的负相关关系。【结论】miR-7可以直接靶向下调细胞周期蛋白CCNE1的表达,进而导致细胞周期G1期阻滞,从而抑制肝癌细胞增殖。
[Abstract]:The first part of the screening of malignant glioma microRNA cells related to TRAIL sensitivity, function identification and mechanism study [background] malignant glioblastoma (GBM) is one of the most aggressive brain cancer, the median survival time of less than 16 months. Although the treatment has made great progress, but it is still one of the most challenging disease.TRAIL (TNF related apoptosis inducing ligand) can specifically kill tumor cells without affecting normal, so it is a potential anticancer drug. But studies have shown that the tolerance of many tumors, and in clinical trials, TRAIL because of its short half-life and in vivo the instability resulting in its practical application is limited. However, recent studies suggest that the use of mesenchymal stem cells (MSCs) expression of TRAIL TRAIL can be specifically delivered to the tumor, which can be compensated to a certain extent. The disadvantages of.MicroRNA, a class of evolutionarily conserved non small RNA encoding, with target gene 3'untranslated region (UTRs) combines post transcriptional regulation, thereby inhibiting translation or degradation of mRNA. mRNA growing evidence that miRNA plays a key role in the regulation of tumor cell malignant phenotype in, and studies have confirmed that cells can form the exosome secretion of miRNA, and down regulate the expression of receptor cells in target genes. Therefore, this indicates that the combined use of MSCs, microRNA, TRAIL of tumor targeted therapy strategies may have good prospects for treatment. [Objective] to find out can increase sensitive TRAIL microRNA, and uses MSCs as a vehicle to carry out targeted therapy. [method] in order to find the key of microRNA in glioma can be sensitized by TRAIL, we first on TRAIL performance of different sensitivity of glioma fine Cell lines mi RNA expression profiles, from apoptosis induced by TRAIL and found a significant correlation between the expression of miRNAs. or the microRNA interference in different cells, detect the apoptosis induced by TRAIL, and then confirm the key microRNA. sensitized TRAIL and then through bioinformatics prediction, dual luciferase reporter gene the experiment, real time quantitative PCR and Western blot to find the downstream target gene of miRNA. At the same time, the loss of function and recovery experiments, further confirmed such a regulation for the transformation of the MSCs axis. Then, the carrying TRAIL and the microRNA, and in vitro U87 glioma cell line co culture experiments, verify whether gene the modified MSCs can effectively kill tumor cells. At the same time, with the analysis of the molecular inhibitors of exosomes, whether this effect by exosome mediated transfer to complete . after respectively by Transwell test and immunohistochemical experiments in vitro and in vivo experiments, whether carrying TRAIL and microRNA MSCs have good tumor tropism. Finally, establish the model of tumor bearing nude mice, using TRAIL and microRNA combined with MSCs over expression of caudal vein treatment experiment, confirm the therapeutic effect. [results] we found that there was a significant positive correlation between miR-7 apoptosis in glioma cell lines and the expression induced by TRAIL. After the loss of function and recovery experiments confirmed that miR-7 is indeed sensitized TRAIL key molecules in glioma cell lines. Then, in the mechanism study, we found that XIAP is a downstream target the key genes mediating this effect, further research found that the miR-7-XIAP of this regulation relationship exists in a variety of tumors. Moreover, confirmed in vivo and in vitro experiments, carrying TRAIL and microRNA-7 between Mesenchymal stem cells have good tumor tropism. At the same time, through the exosome inhibition experiments confirmed that MSCs can form outside the urinary secretion of microRNA-7 and down-regulation of receptor XIAP in the cells, thereby enhancing apoptosis of receptor cells. Finally, more importantly, in the treatment model in nude mice, and TRAIL microRNA-7 co expression of mesenchymal stem cells showed apoptosis promoting effect is very good, to inhibit the growth of tumor, achieve better therapeutic effects. [Conclusion] in tumor cells, miR-7 can downregulate XIAP enhanced TRAIL induced apoptosis with TRAIL and miR-7. MSCs can specifically tend to the local tumor, inhibit the growth of tumor. The second part of microRNA-7 in HCC tumor suppressor mechanism study [background] Micro RNA (miRNA) is a class of molecules in multicellular organisms are ubiquitous transcription. Non small RNA molecules. Mi encoding mature RNA molecules by sequence specific base pairing with m RNA 3 'non encoding region, the formation of RNA silencing complex, leading to the target m RNA translational repression or directly degrade. More importantly, a mi RNA can be adjusted at the same time hundreds of M RNA, which was able to participate in the molecular regulation of network and perplexing, cellular activity. More and more studies show that the normal expression of MI RNA is necessary for the normal operation of life activity, and its imbalance may lead to various diseases including tumors. Recent research found that and in the occurrence and development of hepatocellular carcinoma with tumor suppressor mi downregulation of RNA, and in these mi RNA, MI R-7 in hepatocellular carcinoma was significantly lower expression of MI and overexpression of R-7 can inhibit the proliferation and metastasis of hepatocellular carcinoma. There is also evidence Actually, MI R-7 can inhibit the growth and metastasis of liver cancer through the PI3K/AKT pathway to the target. However, these are not fully reveal the key mi R-7 play in hepatocellular carcinoma and inhibition of cancer. [Objective] to elucidate microRNA-7 in HCC cells of the new tumor suppressor mechanism. [method] firstly, transient transfection in hepatocellular carcinoma cells miR-7,48 hours after the cell cycle was detected by flow cytometry. Then through bioinformatics prediction possible downstream target gene prediction analysis tool R-7 MI, then the preliminary confirmation of downstream target gene mi R-7 by dual luciferase assay. Then the expression of M RNA and protein expression of target genes and quantitative real-time PCR Western blot detection of MI after R-7, at the same time through the loss of function and recovery experiments to further confirm the relationship between target gene and cell cycle. Finally, in clinical samples and tumor cell lines in Mi analysis of R-7 and target gene expression level of RNA gene. [results] first, we found that overexpression of miR-7 could obviously induce G1 cell cycle arrest in HepG2 cells. Through bioinformatics prediction, reporter gene assay, confirmed by quantitative real-time PCR and Western blotting experiments, cyclin CCNE1 is a key downstream the target gene mi R-7 play the role of inhibition. Further studies showed that CCNE1 interference and overexpression of MI R-7 leads to cell cycle changes similar to that at the same time the recovery of CCNE1 overexpression can eliminate the G1 phase of the cell cycle blocking Mi induced by R-7. Finally, the clinical tissue samples and cell lines showed that the expression level of Mi R-7 with CCNE1 there is a significant negative correlation. [Conclusion] miR-7 can directly target the expression of cyclin CCNE1, which leads to G1 cell cycle arrest, thereby inhibiting liver cancer cell Cell proliferation.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R739.41
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本文编号：1726065