LncRNA-SNHG16/YAP1正反馈回路调控CTC EMT促进结直肠癌肝转移的机制研究

Circulating tumor cells (CTCs) from primary cancers hold great potential that initiates distant metastasis. Although CTCs are extraordinarily rare cancer cells, our established techniques could effectively enrich CTCs for further analysis of blood-borne metastatic mechanism. Previous studies by us showed 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’ EMT would achieve a more comprehensive spectrum of metastatic mechanism. LncRNA(Long non-coding RNA) 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 SNHG16 act as a novel player in modulating tumor cell EMT through regulating YAP1 expression, and YAP1 may play its role of transcriptional coactivator in the regulation of SNHG16. YAP1 and SNHG16 may form a positive feedback loop to promote EMT-mediated colorectal cancer invasion and metastasis. Based on our preliminary results, this project aims to systematically determine the essential in vitro and in vivo roles for SNHG16/YAP1 positive feedback loop-mediated tumor cell EMT in colorectal CTCs 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 SNHG16/YAP1 positive feedback loop-mediated EMT in colorectal CTCs’ metastatic potential, and further illuminate the role and molecular mechanism of CTCs EMT in liver metastasis of 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肿瘤转移的重要机制。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；LncRNA在EMT调控过程中起关键性作用，LncRNA-SNHG16上调可以诱导肿瘤细胞的EMT，且可能通过发挥ceRNA功能特异性调节YAP1；同时YAP1表达改变可直接影响SNHG16的表达水平。我们依据前期工作基础，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SNHG16/YAP1正反馈回路调控EMT的作用机制，以及SNHG16/YAP1正反馈回路对CTCs转移潜能的影响，进一步阐明CTCs EMT在结直肠癌肝转移中的作用和机制。

中文摘要.目的：转移是癌症相关死亡的主要原因。越来越多的研究表明，循环肿瘤细胞（circulating tumor cell，CTC）是癌症转移的重要先导和前驱。结直肠癌患者血液中的CTC可通过EMT克服转移过程的瓶颈，实现肿瘤的远处转移。然而，EMT的潜在机制还有待进一步研究。.方法：分别利用ISH和qRT-PCR分别检测组织切片和提取RNA的表达水平。我们利用免疫组化和western blot分别检测组织切片和提取蛋白的表达水平。基因敲除和过表达的方法被用来研究各种基因改变对结直肠癌进展的影响。同时，我们利用划痕、transwell实验，克隆形成实验，CCK8实验研究基因表达改变对肿瘤迁徙、侵袭、克隆形成及增殖等生物学行为的影响。然后，RNA免疫共沉淀、染色质免疫共沉淀、蛋白质免疫共沉淀、GST pull-down、生物素标记的RNA pull-down以及双荧光素酶实验被用来研究分子之间的相互作用。CTC主要通过CTCBIOPSY®捕获。最后，裸鼠异种移植瘤实验被用来评估SNHG16对体内肿瘤进展的影响。.结果：与结直肠正常组织和正常细胞系相比，SNHG16在结直肠癌组织和癌细胞系中表达均明显升高。临床统计分析显示，SNHG16表达上调与结直肠癌患者TNM分期、远处转移明显正相关。而且，SNHG16高表达组患者预后不良的风险明显增大。体内外进一步研究表明，SNHG16能通过YAP1促进肿瘤的克隆形成、增殖、迁徙、侵袭、EMT及间质性CTC的产生。机制研究进一步证实，SNHG16能发挥miRNA海绵作用沉默miR-195-5p，从而保护YAP1免遭miR-195-5p介导的降解。更重要的是，高表达的YAP1能与TEAD1形成YAP1/TEAD1复合物结合在SNHG16的启动子的两个位点上，从而促进SNHG16的转录。在促进转录的过程中，YAP1与TEAD1互相依赖缺一不可。最后，我们发现SNHG16能逆转YAP1引起的促癌作用。.结论：在此，我们在结直肠癌中发现了SNHG16与YAP1/TEAD1之间一个迄今尚未研究的正反馈回路。通过这个正反馈回路，SNHG16促进了结直肠癌CTC的产生及EMT过程，从而持续促进结直肠癌的肝转移，对患者造成不可逆损伤。这些发现为结肠癌肝转移的治疗提供了新的视野和潜在的治疗靶点.关键词：SNHG16；YAP1；miR-195-5p