本文關(guān)鍵詞：NOP14對胰腺癌侵襲轉(zhuǎn)移的調(diào)控作用及其相關(guān)分子機制和預(yù)后價值研究　出處：《北京協(xié)和醫(yī)學(xué)院》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 胰腺癌 核仁蛋白14 微小RNA p53突變 腫瘤轉(zhuǎn)移

【摘要】：腫瘤轉(zhuǎn)移是導(dǎo)致胰腺癌患者預(yù)后差的關(guān)鍵因素。p53突變體的富集可以促進腫瘤的侵襲和轉(zhuǎn)移,然而,目前有關(guān)胰腺癌中突變型p53蛋白富集及相關(guān)信號通路持續(xù)活化的分子機制仍不清楚。本課題組前期研究發(fā)現(xiàn)核仁蛋白14(NOP14)在胰腺癌轉(zhuǎn)移過程中發(fā)揮重要功能,至今有關(guān)NOP14表達異常與惡性腫瘤發(fā)生及進展的關(guān)系鮮有報道。本研究主要分為三個部分:功能研究篇我們首先利用免疫組織化學(xué)方法檢測了配對的胰腺導(dǎo)管腺癌原發(fā)灶、轉(zhuǎn)移灶及癌旁組織中NOP14的表達,進而在體外水平利用劃痕實驗、Transwell侵襲和遷移實驗研究NOP14對細胞運動能力的影響,并進一步在體內(nèi)水平通過建立胰腺癌小鼠皮下和原位移植瘤模型及尾靜脈注射模型系統(tǒng)探究NOP14在胰腺癌侵襲轉(zhuǎn)移中的功能。機制探索篇我們首先利用RNA-seq分析聯(lián)合qPCR驗證篩選和鑒定NOP14功能發(fā)揮的下游靶點,并進一步利用RNA穩(wěn)定性實驗、熒光素酶報告基因?qū)嶒灱叭旧|(zhì)免疫共沉淀實驗等深入闡釋NOP14、p53突變體、miR-17-5p和P21等相關(guān)信號分子之間的調(diào)控作用,最終揭示NOP14介導(dǎo)胰腺癌轉(zhuǎn)移的分子機制。預(yù)后分析篇我們利用組織芯片探討了胰腺癌中NOP14表達與臨床病理參數(shù)之間的相關(guān)性及其預(yù)后價值。功能研究發(fā)現(xiàn)NOP14在胰腺導(dǎo)管腺癌及其轉(zhuǎn)移灶中的表達水平明顯升高,上調(diào)NOP14表達促進了胰腺癌細胞侵襲和遷移,反之抑制其表達則導(dǎo)致細胞的侵襲和遷移明顯下降,體內(nèi)實驗結(jié)果亦表明NOP14是一種介導(dǎo)胰腺癌侵襲和轉(zhuǎn)移的正調(diào)控因子。機制探索方面,我們證實miR-17-5p/P21信號通路是介導(dǎo)NOP14功能發(fā)揮的下游分子,NOP14可以通過與p53突變體mRNA結(jié)合增強其穩(wěn)定性實現(xiàn)對下游miR-17-5p/P21信號通路的調(diào)控。預(yù)后分析表明NOP14高表達與胰腺癌局部侵襲和淋巴結(jié)轉(zhuǎn)移密切相關(guān),是胰腺癌患者預(yù)后不良的危險因素。本研究闡釋了 NOP14在p53突變驅(qū)動的胰腺癌轉(zhuǎn)移中的功能和分子機制,加深了我們對胰腺癌細胞中突變型p53蛋白富集及其相關(guān)信號通路持續(xù)活化機制的理解。靶向抑制NOP14的功能可能通過一種p53突變體依賴的方式減少胰腺癌轉(zhuǎn)移,表明NOp14可以作為阻止p53突變驅(qū)動的胰腺癌轉(zhuǎn)移的潛在靶點。
[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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R735.9
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本文編號：1337931