核仁小RNA-SNORA74B通过PTB/ZEB1信号轴诱导胆囊癌细胞EMT的作用及机制研究

Epithelial-mesenchymal transition (EMT) promoting tumor invasion and metastasis is a negative predictor of gallbladder carcinoma (GBC), so deconstructing the mechanisms of EMT is of great significance to improve prognosis of GBC patients. Small nucleolar RNA (snoRNA) is a class of non-coding RNAs which have been demonstrated to play important regulatory roles during tumor development and progression. In our previous study, we found SNORA74B is overexpressed in GBC tissues and cell lines, and it may promote cell migration and invasion through inducing EMT. However, the underlying mechanisms have not been revealed yet. Our further study suggests that SNORA74B mediates the binding of polypyrimidine tract binding protein (PTB) to ZEB1 mRNA, enhancing the mRNA stability and promoting the translation of ZEB1 mRNA. Eventually, the upregulation of ZEB1 protein promotes EMT, migration and invasion of GBC cells. In the present study, we will study the roles of SNORA74B, PTB and ZEB1 in EMT of GBC cells at the molecular, cellular, animal, clinical levels, and also evaluate the clinical significance of SNORA74B as a diagnostic and prognostic biomarker in GBC. The present study investigates the effects and mechanisms of SNORA74B on migration and invasion of GBC cells at post-transcriptional level, which may provide potential targets for GBC therapy from a new perspective.

上皮间质转化（EMT）是促进胆囊癌侵袭转移以及影响预后的重要因素，阐明其发生机制并进行干预对改善患者预后意义重大。核仁小RNA（snoRNA）是一类非编码RNA，现已证实其在肿瘤发生发展过程中发挥重要调控作用。我们前期研究发现SNORA74B在胆囊癌组织和细胞中表达异常增高，并可诱导EMT促进胆囊癌细胞迁移和侵袭，但机制不清。我们进一步研究发现SNORA74B可能促进多聚嘧啶串结合蛋白PTB与EMT诱导因子ZEB1 mRNA结合，增强mRNA稳定性，促进蛋白翻译，导致ZEB1蛋白水平升高最终诱导EMT。本项目拟从分子、细胞、组织和动物等层面探讨SNORA74B、PTB和ZEB1基因在胆囊癌细胞EMT中的作用和机制，评估SNORA74B作为胆囊癌肿瘤标志物的临床应用价值。本项目从snoRNA这个新视点探讨SNORA74B转录后水平调控胆囊癌侵袭转移的分子机制，将为胆囊癌靶向治疗提供新思路。

本项目围绕核仁小RNA（snoRNA）与胆囊癌发生发展之间的关系开展一系列的研究，研究结果说明snoRNA-SNORA74B可以促进胆囊癌细胞的某些恶性生物学行为尤其是侵袭转移和增殖，并初步阐明其中的分子机制。研究内容主要包括以下三个：1）通过CCK-8、Transwell、动物实验等方法检测SNORA74B对胆囊癌细胞迁移侵袭和增殖的调控作用；2）通过病毒转染、western blotting、qRT-PCR等方法检测SNORA74B促进胆囊癌细胞侵袭转移的分子机制；3）通过免疫组化、qRT-PCR等方法检测研究SNORA74B在胆囊癌患者组织中的表达水平，并分析其表达水平与胆囊癌患者临床病理特征之间的相关性。通过以上研究，我们获得以下研究结果：1、SNORA74B在胆囊癌细胞中表达水平显著增高，并且沉默其表达还可以显著抑制细胞增殖、迁移和侵袭；提高SNORA74B表达可以促进胆囊癌细胞NOZ迁移和侵袭，而沉默其表达则抑制胆囊癌细胞迁移和侵袭。2、SNORA74B基因可以显著抑制E-cadherin蛋白表达，上调Vimentin蛋白表达；PTB基因可以促进Zeb1蛋白表达，而沉默其表达则抑制Zeb1蛋白表达；SNORA74B基因可以促进ZEB1蛋白表达，沉默其表达则抑制Zeb1蛋白表达，上调SNORA74B表达且同时沉默PTB基因表达却无法促进ZEB1蛋白表达；SNORA74B基因可以促进PTB蛋白表达，而沉默其表达则抑制PTB蛋白表达。3、SNORA74B在胆囊癌患者组织中的表达水平增高，其表达水平越高，患者总体生存期和无进展生存期越短；且其表达水平与胆囊癌局部浸润、淋巴结转移、远处转移、TNM分期以及CA19-9呈显著正相关。