miR-494/ZEB2通路调控的循环肿瘤细胞EMT在结直肠癌肝转移中的作用

Circulating tumor cells (CTCs) from primary cancers hold great potential that initiates distant metastasis. Although CTCs 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 CTCs exhibited characteristics of epithelial-mesenchymal transition (EMT), and the number of EMT-CTCs was positively associated with advanced stage of colorectal cancer. EMT has been considered as a critical process for tumor cells to gain invasive phenotype. Therefore, studies focusing on CTCs’ research would achieve a more comprehensive spectrum of metastatic mechanism by combining with EMT-mediated metastasis. MicroRNAs (miRNAs) have been demonstrated to act as master regulators of some key players that are involved in many important biological pathways of EMT. Our preliminary findings suggested that miR-494 act as a novel player in modulating tumor cell EMT through regulating ZEB2 expression. Based on our preliminary results, this project aims to systematically determine the essential in vitro and in vivo roles for miR-494/ZEB2 signaling-mediated tumor cell EMT in colorectal CTCs formation and invasion, finally indentifying the underlying mechanisms responsible for these observations. These novel results obtained from this work would reveal mechanistic insights to highlight the importance of miR494/ZEB2 signaling-mediated EMT in colorectal CTCs’ liver metastatic potential, and provide promising intervention targets for lowering the incidence of metastasis among patients with colorectal cancer.

循环肿瘤细胞(CTCs)是一类存在于外周血中稀有的肿瘤细胞，与肿瘤的远处转移密切相关，针对此类细胞的研究可以阐明转移的重要机制。我们前期针对CTCs的研究发现，CTCs存在上皮间质化（EMT）特点，EMT是肿瘤侵袭转移一个重要过程，肿瘤细胞通过EMT获得侵袭能力，结合CTCs研究EMT的机制，可以对恶性肿瘤侵袭转移获得更为全面的认识；microRNA在EMT调控过程中起关键性作用，我们前期研究发现miR-494下调可以诱导肿瘤细胞的EMT，且可能通过特异性调节ZEB2实现。关于miR-494在EMT中的作用和机制、miR-494/ZEB2调控的EMT对CTCs侵袭转移的影响均未见报道。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miR-494/ZEB2调控EMT的作用机制,以及miR-494/ZEB2通路对CTCs肝转移潜能的影响。

转移是导致结直肠癌治疗失败的主要原因，上皮间质化（EMT）与恶性肿瘤侵袭转移密切相关。miRNA在EMT调控过程中起关键作用，我们前期研究发现miR-494下调可诱导结肠癌细胞EMT，且可能通过特异性调控ZEB2实现。为进一步明确其调控作用和机制，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miR-494在结直肠癌EMT中的作用、miR-494/ZEB2调控结直肠癌EMT对侵袭转移的影响。临床样本结果显示，在结直肠癌组织侵袭前缘中，miR-494表达水平显著低于肿瘤中心，ZEB2的表达量明显高于肿瘤中心，二者表达水平呈显著负相关；miR-494低表达和ZEB2高表达与CTC/CTM+相关；CTCM检出率与miR-494表达量呈负相关，与ZEB2表达量呈正相关，这提示miR-494和ZEB2可能影响CTC形成及结直肠癌侵袭转移。稳定转染细胞系结果表明，在HCT116中miR-494被抑制后，结肠癌细胞的增殖、迁移和侵袭能力显著增强；在SW480中miR-494过表达后，结肠癌细胞的增殖、迁移和侵袭能力显著降低。生信分析结果表明，miR-494与ZEB2存在潜在结合位点。进一步研究发现，miR-494可靶向结合ZEB2，影响ZEB2 mRNA的稳定性，调控ZEB2的表达。沉默ZEB2后结肠癌细胞的增殖、迁移和侵袭能力受到明显的抑制，消除了miR-494被抑制对结肠癌细胞增殖、迁移和侵袭的促进作用，抑制结肠癌细胞的EMT。这提示我们，miR-494在体外可靶向ZEB2，调控EMT信号通路，影响结肠癌细胞的侵袭转移。动物模型结果表明，miR-494被抑制后可上调ZEB2的表达，诱导结肠癌细胞EMT，进而增强结肠癌细胞成瘤能力，促进CTC形成，增加肝转移和肺转移几率，证实miR-494在裸鼠体内环境中也能影响结肠癌细胞的侵袭转移。本研究充分表明，miR-494可靶向ZEB2调控EMT信号通路，介导CTC形成，影响结直肠癌的侵袭转移。本研究成果揭示了结直肠癌侵袭转移机制之一，为结直肠癌的精准治疗提供理论基础和治疗策略。