NOP14介导的miRNA加工在胰腺癌转移中的作用机制研究

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a high mortality rate. As a silent killer, the molecular mechanisms underlying PDAC recurrence and metastasis remain unclear. Our previous data demonstrated that NOP14 depicted a significant over-expression and several point mutations in the metastatic PDAC tissues by using exosome capturing sequencing. A preliminary investigation in PDAC cell lines and mouse model indicated that knock-down of NOP14 led to an inhibitory cell growth and invasion, which suggest that NOP14 is a potential therapeutic target for PDAC. However, the molecular mechanism was not well-established. Base on the finding of RNA sequencing employed in PDAC cells with NOP14 over-expression or inhibition, we observed that several miRNAs were regulated by NOP14, which suggest that NOP14 led to enhanced cell invasion , at least, partly through miRNA-mediated signaling. In this project, we aim to clarify the molecular basis of NOP14-mediated miRNA expression and processing in the metastatic cascade of PDAC. Firstly, we want investigate the NOP14 protein complex by using an improved two-hybrid system and evaluate the subcellular localization of NOP14 and its partners. Meanwhile, we seek to indicate the biological function of NOP14 in nucleus and cytoplasm. Secondly, the regulatory roles of NOP14 in miRNA biogenesis will be investigated and the correlation between NOP14-induced miRNA processing and PADC invasion will be studied in cell lines and animal models. Thirdly, we will focus on the functional mutation of NOP14 and elucidate the biological significance of specific mutation in PADC tumorigenesis and development through demonstrating their impact on the cell phenotypic alterations in vitro and in vivo. This study will identify potential targets for PDAC metastasis suppression and lay a foundation for personalized therapy.

早期转移是导致胰腺癌预后差、死亡率高的主要原因之一，至今相关分子机制仍待明确。申请者课题组通过外显子捕获测序技术发现，NOP14在胰腺癌转移组织中表达上调且存在多个位点突变，初步功能实验表明NOP14介导了胰腺癌细胞体内外侵袭与转移，但作用机制尚不清楚。前期研究结果显示，NOP14可以通过调控多个肿瘤相关miRNA的表达，影响胰腺癌细胞的转移。本项目拟利用酵母双杂交系统结合蛋白质相互作用的研究方法，明确NOP14蛋白质作用复合物在细胞内的定位及其功能，深入研究NOP14调控miRNA转录及加工的作用机制，揭示NOP14促进胰腺癌转移的分子基础；同时通过探讨NOP14突变对其功能的影响，从病因学角度阐明胰腺癌转移相关突变的生物学意义，并在细胞及动物水平考察关键性突变对NOP14-miRNA信号通路及胰腺癌表型的影响，最终发现有效抑制胰腺癌细胞转移的新靶标，为胰腺癌个体化治疗研究奠定基础。

早期转移是导致胰腺癌预后差、死亡率高的主要原因之一，但是相关分子机制仍不明确。负责人课题组通过外显子捕获测序技术发现，NOP14在胰腺癌转移组织中表达上调且存在多个位点突变，初步功能研究表明，NOP14介导了胰腺癌细胞体侵袭与转移，但分子机制尚不清楚。前期研究结果显示，NOP14可以通过调控多个肿瘤相关miRNA的表达，影响胰腺癌细胞的转移。本项目利用RNA干扰、慢病毒系统介导的NOP14过表达或抑制等策略在PANC-1、MIA PaCa-2等胰腺癌细胞中进行表型分析，细胞划痕、transwell、3D-invasion等实验结果显示，过表达NOP14可以促进胰腺癌细胞的迁移和侵袭，抑制NOP14则明显降低了胰腺癌的运动能力。后续体内模型则在皮下移植瘤、原位移植瘤、尾静脉注射的体内转移实验体系中加以验证，结果显示，NOP14表达抑制明显降低瘤细胞的原位浸润、远端转移等。既往研究显示NOP14是一个核仁蛋白，其功能主要集中于核糖体合成加工等，本课题利用高通量RNA测序技术对转录组、小RNA等进行测序分析，结果发现NOP14可以影响多个与肿瘤发生有关的关键信号分子，其中包括p53。进一步研究发现NOP14对p53调控发生在RNA水平，即NOP14可以结合TP53 mRNA发挥稳定mRNA的作用，从而增加p53蛋白的输出。在对miRNA的分析的显示，miR-17-92家族受到NOP14调控，但是NOP14与该miRNA的前体RNA分子并不存在直接相互作用。已有报道显示，miR-17-92家族受p53调控，推测NOP14对该家族的调节可能是通过p53实现。后续拯救实验则证实NOP14通过增强突变型TP53 mRNA稳定性，在影响某些信号分子的同时也可以通过miR-17负调控p21活性促进胰腺癌的进展。在揭示NOP14-TP53-miR-17-p21调控轴的分子机制与功能后，本课题利用含82例胰腺癌样本的组织芯片中对NOP14的表达与胰腺癌临床相关性进行深入分析，结果显示，NOP14在胰腺癌组织中表达明显上调（P=0.033），NOP14高表达样本呈现明显原位浸润（P=0.035）和远端淋巴结转移（P=0.034）。此外，NOP14高表达患者的生存期明显低于NOP14低表达组，提示NOP14在胰腺癌中发挥癌基因的作用，可作为潜在的治疗靶点，尤其针对携带p53突变的患者。