NOP14对胰腺癌侵袭转移的调控作用及其相关分子机制和预后价值研究

发布时间：2017-12-26 16:13

  本文关键词：NOP14对胰腺癌侵袭转移的调控作用及其相关分子机制和预后价值研究　出处：《北京协和医学院》2017年博士论文　论文类型：学位论文

  更多相关文章： 胰腺癌 核仁蛋白14 微小RNA p53突变 肿瘤转移

【摘要】：肿瘤转移是导致胰腺癌患者预后差的关键因素。p53突变体的富集可以促进肿瘤的侵袭和转移,然而,目前有关胰腺癌中突变型p53蛋白富集及相关信号通路持续活化的分子机制仍不清楚。本课题组前期研究发现核仁蛋白14(NOP14)在胰腺癌转移过程中发挥重要功能,至今有关NOP14表达异常与恶性肿瘤发生及进展的关系鲜有报道。本研究主要分为三个部分:功能研究篇我们首先利用免疫组织化学方法检测了配对的胰腺导管腺癌原发灶、转移灶及癌旁组织中NOP14的表达,进而在体外水平利用划痕实验、Transwell侵袭和迁移实验研究NOP14对细胞运动能力的影响,并进一步在体内水平通过建立胰腺癌小鼠皮下和原位移植瘤模型及尾静脉注射模型系统探究NOP14在胰腺癌侵袭转移中的功能。机制探索篇我们首先利用RNA-seq分析联合qPCR验证筛选和鉴定NOP14功能发挥的下游靶点,并进一步利用RNA稳定性实验、荧光素酶报告基因实验及染色质免疫共沉淀实验等深入阐释NOP14、p53突变体、miR-17-5p和P21等相关信号分子之间的调控作用,最终揭示NOP14介导胰腺癌转移的分子机制。预后分析篇我们利用组织芯片探讨了胰腺癌中NOP14表达与临床病理参数之间的相关性及其预后价值。功能研究发现NOP14在胰腺导管腺癌及其转移灶中的表达水平明显升高,上调NOP14表达促进了胰腺癌细胞侵袭和迁移,反之抑制其表达则导致细胞的侵袭和迁移明显下降,体内实验结果亦表明NOP14是一种介导胰腺癌侵袭和转移的正调控因子。机制探索方面,我们证实miR-17-5p/P21信号通路是介导NOP14功能发挥的下游分子,NOP14可以通过与p53突变体mRNA结合增强其稳定性实现对下游miR-17-5p/P21信号通路的调控。预后分析表明NOP14高表达与胰腺癌局部侵袭和淋巴结转移密切相关,是胰腺癌患者预后不良的危险因素。本研究阐释了 NOP14在p53突变驱动的胰腺癌转移中的功能和分子机制,加深了我们对胰腺癌细胞中突变型p53蛋白富集及其相关信号通路持续活化机制的理解。靶向抑制NOP14的功能可能通过一种p53突变体依赖的方式减少胰腺癌转移,表明NOp14可以作为阻止p53突变驱动的胰腺癌转移的潜在靶点。
[Abstract]:Tumor metastasis is a key factor in the poor prognosis of patients with pancreatic cancer. The enrichment of p53 mutants can promote tumor invasion and metastasis. However, the molecular mechanism of mutant p53 protein enrichment and related signaling pathways in pancreatic cancer is still unclear. In our previous study, nucleolus protein 14 (NOP14) played an important role in the metastasis of pancreatic cancer. Until now, the relationship between NOP14 expression abnormality and the occurrence and progression of malignant tumor has rarely been reported. This research is mainly divided into three parts: the function research we use immunohistochemical method to detect the expression of pancreatic ductal adenocarcinoma matched primary tumors, NOP14 metastasis and tumor adjacent tissues, and in vitro invasion and migration level using scratch test and Transwell experimental research on the influence of NOP14 on cell motility further, through the establishment of subcutaneous and orthotopic pancreatic cancer xenografts in mice model and intravenous injection of NOP14 model system on the invasion and metastasis of pancreatic cancer in function in vivo. We explore the mechanism of downstream targets using RNA-seq analysis combined with qPCR verify the screening and identification of NOP14 function, and using the RNA stability test, luciferase reporter assay and chromatin immunoprecipitation experiments further explain the regulation effect between NOP14 and p53 mutants, miR-17-5p and P21 and other related signal molecules, suggesting that NOP14 is the final the molecular mechanism of metastasis of pancreatic cancer. We used tissue microarray to investigate the correlation between NOP14 expression and clinicopathological parameters in pancreatic cancer and its prognostic value. Functional studies showed that the expression level of NOP14 in pancreatic ductal adenocarcinoma and metastasis increased, the up-regulated expression of NOP14 migration and invasion of pancreatic cancer cells promoted, whereas inhibition of its expression in the invasion and migration of cells decreased significantly in vivo experimental results also show that NOP14 is a positive regulatory factor mediated invasion and metastasis of pancreatic cancer the. In terms of mechanism exploration, we confirmed that miR-17-5p/P21 signaling pathway is a downstream molecule that mediates NOP14 function. NOP14 can enhance its stability by combining with p53 mutant mRNA to control downstream miR-17-5p/P21 signaling pathway. The prognosis analysis shows that the high expression of NOP14 is closely related to the local invasion and lymph node metastasis of pancreatic cancer, which is a risk factor for poor prognosis of pancreatic cancer. This study explains the function and molecular mechanism of NOP14 in the metastasis of pancreatic cancer driven by p53 mutation, and deepens our understanding of the mutagenesis of p53 protein and the activation mechanism of related signaling pathways in pancreatic cancer cells. Targeted inhibition of NOP14 may reduce metastasis of pancreatic cancer through a p53 mutant dependent manner, indicating that NOp14 can be used as a potential target to prevent p53 mutation driven pancreatic cancer metastasis.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.9
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本文编号：1337931