外泌体源LncRNA ZEB2-AS1调控PRRX1促进胃癌循环肿瘤细胞EMT的机制研究

Circulating tumor cell (CTC) from primary cancers hold great potential that initiates distant metastasis. Although CTC are extraordinarily rare cancer cells, our established techniques effective for their detection and identification ensure us to analyze valuable biological information carried by them and to explore cellular signal pathways related to the blood-borne metastatic mechanism. Our previous studies have shown that CTC exhibited characteristics of epithelial-mesenchymal transition (EMT), and the number of CTC was positively associated with advanced stage of hepatocellular carcinoma. EMT has been considered as a critical process for tumor cells to gain invasive phenotype. Therefore, studies focusing on CTC’ research would achieve a more comprehensive spectrum of metastatic mechanism by combining with EMT-mediated metastasis. LncRNAs have been demonstrated to act as master regulators of some key players that are involved in many important biological pathways of EMT, exosomes may act as the intercellular communication by transmitting intracellular cargoes to confer LncRNAs to active EMT pathway. Our preliminary findings suggested that the expression level of peripheral exsomal LncRNA ZEB2-AS1 was correlated with the number of CTCs with EMT characteristic, also we found that LncRNA ZEB2-AS1 acted as a novel player in modulating tumor cell EMT through interacting with PRRX1. Based on our preliminary results, this project aims to systematically determine the essential in vitro and in vivo roles for LncRNA ZEB2-AS1/PRRX1 mediated EMT in gastric CTC formation and invasion, finally identifying the underlying mechanisms responsible for these observations. These novel results obtained from this work would reveal mechanistic insights to highlight the importance of lncRNA ZEB2-AS1/PRRX1 mediated EMT in gastric cancer CTC invasion, and provide promising intervention targets for lowering the incidence of metastasis among patients with gastric cancer.

循环肿瘤细胞（CTC）是一类存在于外周血中的肿瘤细胞，与肿瘤的远处转移密切相关；CTC存在上皮间质化（EMT）特点，EMT是肿瘤侵袭转移一个重要过程，结合CTC研究EMT机制，可以对恶性肿瘤侵袭转移获得更为全面的认识。在EMT调控过程中，外泌体可通过传递LncRNA在EMT表型调控中起到关键性作用，我们前期发现胃癌外周血外泌体源LncRNA ZEB2-AS1与CTC的EMT状态相关，上调LncRNA ZEB2-AS1可以诱导胃癌细胞的EMT，而且可能通过ceRNA功能调控PRRX1实现。关于LncRNA ZEB2-AS1在EMT中的作用和机制、外泌体源LncRNA ZEB2-AS1/PRRX1通路促进CTC EMT均未见报道。我们拟根据初步发现，以机制研究为出发点，动物模型、临床样本为落脚点，系统阐明外泌体介导LncRNA ZEB2-AS1/PRRX1通路促进CTC EMT的作用机理。

转移是导致胃癌患者主要死亡原因，上皮间质化（EMT）与胃癌侵袭转移密切相关。lncRNA在其中起关键作用，本项目通过临床样本、稳定转染细胞系和动物模型，系统研究ZEB2-AS1在胃癌EMT中的作用。临床样本结果显示，在胃癌组织中，ZEB2-AS1表达水平显著高于癌旁正常组织，且在转移灶的表达量显著高于原位癌，同时，胃癌患者血浆外泌体中ZEB2-AS1显著高于正常人，外泌体ZEB2-AS1高表达与CTC/CTCM+相关；与癌组织中PRRX1表达呈正相关，提示ZEB2-AS1和PRRX1可能影响CTC形成及胃癌侵袭转移。稳转细胞实验结果表明，分别在MKN45中敲低/MGC803中过表达ZEB2-AS1后，胃癌细胞的迁移和侵袭能力显著减弱/增强。同时，收集过表达ZEB2-AS1的MGC803外泌体，其中ZEB2-AS1表达明显升高，外泌体处理后，空白MGC803细胞侵袭，迁移能力明显增强，检测EMT标志物发现E-cadherin明显降低，Vimentin明显升高，证实发生EMT的肿瘤细胞可以通过外泌体传递ZEB2-AS1至相邻肿瘤细胞，形成EMT的级联放大反应。生信分析发现，ZEB2-AS1与miR-204-5p/PRRX1存在共同结合位点，miR-204-5p可靶向结合PRRX1，转录后水平调控PRRX1的表达。在过表达ZEB2-AS1细胞系中同时过表达miR-204-5p，胃癌细胞的迁移和侵袭能力受到明显的抑制，且抑制了胃癌细胞的EMT。这提示我们，ZEB2-AS1可通过miR-204-5p/PRRX1通路，调控EMT。同时敲低PRRX1后，ZEB2-AS1表达降低，双荧光素酶报告，CHIP实验证实PRRX1可作用于ZEB2-AS1的promoter区域，促进ZEB2-AS1表达。动物模型结果表明，过表达ZEB2-AS1可上调PRRX1的表达，诱导胃癌细胞EMT，增强胃癌细胞成瘤能力，促进CTC形成，增加肝转移几率，证实ZEB2-AS1在裸鼠体内也能影响胃癌细胞的侵袭转移。本研究充分表明，外泌体来源LncRNA ZEB2-AS1可靶向PRRX1形成正反馈回路调控EMT信号通路，级联放大肿瘤细胞的EMT进程，介导CTC形成，影响胃癌的侵袭转移。本研究成果揭示了胃癌侵袭转移机制之一，为胃癌的精准诊疗提供理论基础和治疗策略。