生物钟基因Clock经外泌体途径促进结直肠癌细胞肝转移的作用与机制研究

Liver metastasis of colorectal cancer (CRC) has a high incidence but poor prognosis, of which the mechanism remains to be revealed. In previous research, we found that the expression of circadian gene Clock was abnormally high in both CRC tissues and liver metastases. Furthermore, up-regulating Clock gene expression promoted migration and invasion of CRC, while Clock gene silencing may suppress metastatic of CRC cells. The underlying molecular mechanisms remain to be revealed. Recent studies have demonstrated that exosomes play a vital role in guiding the directional transfer of tumor cells. Combined with our further investigation, it is found that the up-regulated expression level of Clock correlated with that of EMT-induced protein and integrin α6β1 and α6β4 on CRC cells’ exosomes, suggesting that Clock may play the role in CRC progression by regulating exosomes. In summary, we propose that on the one hand, over expression of Clock promotes the EMT, migration and invasion of CRC cells by promoting exosomes’ secretion. On the other hand, over expression of integrin α6β1 and α6β4 on cancer cells and exosomes caused by Clock will promote intake of exosomes in liver tissues. In this study, we aim to verify the above assumption by a series of molecular biology experiments on both cells and animal level, together with analysis in clinical samples to clarify the relationship between Clock, exosomes and liver metastasis of CRC. This study will also provide a new direction and target for the treatment of CRC.

结直肠癌肝转移发生率高且预后差，机制复杂。前期研究中我们发现生物钟基因Clock在结直肠癌和肝转移灶中异常高表达，且上调/沉默Clock表达可诱导/抑制癌细胞转移，但Clock促进结直肠癌肝转移的分子机制尚未阐明。近来认为外泌体是引导肿瘤细胞定向转移的重要介质，结合我们前期研究发现Clock高表达与外泌体携带的EMT诱导蛋白及整合素α6β1、α6β4分泌上调相关，提示Clock基因可能通过调节外泌体来发挥作用。综上，我们提出：Clock基因过表达一方面通过促进结直肠癌细胞分泌外泌体，诱导癌细胞EMT转化，增强其迁移侵袭能力；另一方面上调癌细胞和外泌体上整合素α6β1、α6β4的表达水平，促使肝组织增加对外泌体的摄取。本项目拟通过临床样本分析及细胞、动物水平的分子生物学实验验证以上设想，阐明Clock、外泌体与结直肠癌肝转移之间的关系，为结直肠癌肝转移治疗提供新方向和靶点。

本课题设旨在通过研究生物钟基因通过外泌体途径促进结直肠癌转移的作用机制，阐明Clock基因-外泌体-结直肠癌转移之间的关系。.首先，我们通过在线数据库分析以及细胞水平的分子生物学实验，验证了生物钟基因Bmal1及其分子伴侣Clock参与促进结直肠癌的转移过程，并且Bmal1的作用更为明显。然后，通过构建Bmal1敲低或过表达的SW480、SW620、HCT116CRC稳转株，并纯化出外泌体，与结直肠癌细胞、人脐静脉内皮进行共孵育，检测细胞迁移情况，阐明了Bmal1通过促进外泌体的分泌，从而促进结直肠癌细胞和人脐静脉内皮细胞的迁移。再通过敲低不同结直肠癌细胞中外泌体分泌的关键酶基因Rab27a，提纯外泌体，与结直肠癌细胞和内皮细胞共孵育，阐明Rab27a调节外泌体的分泌从而调节促细胞迁移的作用。最后通过荧光素酶报告基因验证了Bmal1和Clock通过结合在Rab27a启动子区，激活Rab27a转录过程，揭示了Bmal1基因通过调控Rab27a转录，从而调节外泌体分泌，影响结直肠癌转移的分子机制。.本实验为干预结直肠癌的转移的基础和临床研究提供新思路，为结直肠癌转移的风险评估提供新的分子标记物，并为临床治疗提供新靶点。后续计划进行外泌体的RNA测序，继续深入探索Bmal1调节外泌体，进而影响结直肠癌的转移和耐药的分子机制，并进行动物模型的验证，同时发掘基于该机制基础上的时辰治疗的潜力。