R-spondin1/Lgr5信号通路对胃癌细胞侵袭和转移影响及其分子调控机制研究

The survival rate of gastric cancer patients remains very low because of invasion and metastasis. Invasion and metastasis are known to be the major biological characteristics of gastric cancer, and responsible for poor prognosis. However, the detailed molecular mechanisms of this process have not been fully elucidated. The R-spondin1/Lgr5 signaling pathway system was identified and confirmed recently. This system could activate and enhance the classical Wnt/β-catenin signaling, and was associated with the tumorigenesis and progression. However, To the best of our knowledge, there has been no published study reporting the relationship between R-spondin1/Lgr5 signaling pathway and these invasion/metastasis-related factors in gastric cancer. The detail regulatory mechanisms are not clear. Our previous studies indicated that R-spondin1 was cointernalized with Lgr5 by confocal immunofluorescence analysis. Both R-spondin1 and Lgr5 expression were correlated with depth of invasion, lymph node metastasis, distance of metastasis, and β-catenin expression levels. Inhibition of Lgr5 could decreased the migration of gastric cancer cell significantly. Therefore, we hypothesize that R-spondin1/Lgr5 signaling pathway could affect invasion and metastasis of gastric cancer through Wnt/β-catenin signaling. In this study, we will firstly investigate the correlation between R-spondin1/Lgr5 and the biological characteristics of invasion and metastasis. Then different metastatic potential gastric cancer cell lines and nude mice model were used to perform the in vitro/in vivo experiments. After the targeting R-spondin1 and Lgr5 siRNA adenovirus plasmids were constructed, RNAi echnology was used to block the R-spondin1/Lgr5 signaling pathway, and the inhibition effect of invasion and metastasis of gastric cancer was evaluated. The subject could provide a better understanding the mechanisms of invasion and metastasis, and will assist in elucidating novel therapeutic strategies against gastric cancer.

侵袭转移特性是胃癌预后极差的首要原因，但具体调控机制尚不明确。R-spondin1/Lgr5是近两年被证实的配体受体信号通路系统，他们可以激活经典的Wnt/β-catenin信号通路，与肿瘤的发生发展密切相关。然而，该通路与胃癌侵袭转移之间的关系及其调控机制，迄今未有报道。前期研究显示：R-spondin1、Lgr5之间存在空间共定位，该通路与胃癌浸润深度、淋巴结转移、远处转移及β-catenin表达相关，沉默胃癌细胞中Lgr5可抑制胃癌侵袭能力。由此推测：R-spondin1/Lgr5通路可能通过经典的Wnt/β-catenin信号通路传导，影响侵袭转移。本课题拟借助RNAi、裸鼠成瘤等实验技术方法，探究R-spondin1/Lgr5信号通路在胃癌侵袭转移中的作用及其机制。本研究开展，有利于进一步明确胃癌侵袭转移相关分子机制，为下一步研发抗胃癌侵袭转移的生物靶向治疗方案奠定理论基础。

近年来的研究显示，R-spondin1/Lgr5配体受体信号通路系统可以激活经典的Wnt/β-catenin信号通路，并且与肿瘤的发生发展密切相关。基于前期研究，本课题提出了R-spondin1/Lgr5可能影响胃癌的侵袭转移的设想。因此，本课题通过免疫组化等检测R-spondin1和Lgr5的表达和共定位，以及其与临床病理特征的关系；我们进而使用小干扰RNA敲低Lgr5，加入R-spondin1后通过胃癌细胞悬浮成球，Transwell和人脐静脉内皮细胞（HUVECs）管腔形成能力检测，随后构建了裸鼠移植瘤模型，验证了敲低Lgr5后对细胞体内外成瘤能力的影响。结果显示R-spondin1与Lgr5有共定位，Lgr5同血管生成标记物显著相关；并且敲低Lgr5后胃癌细胞的悬浮成球能力、侵袭转移和血管生成能力均显著下降，加入R-spondin1后能够一定程度上逆转该下调现象。机制探索发现Lgr5表达与Wnt信号通路的变化具有较高的一致性；敲低Lgr5可以下调Wnt信号通路的关键靶点β-cateinin。体内实验证实敲低Lgr5可以降低裸鼠体内成瘤能力。以上结果均提示R-spondin1/Lgr5通过影响Wnt/β-catenin信号通路作为促进胃癌细胞侵袭和转移的重要上游调节分子靶点，具有一定的潜在靶点研发价值。