ADAMTS5抑制Ets-1介导的血管生成调控胃癌侵袭转移的机制研究

ADAMTS5 is involved in the invasion and metastasis of gastric cancer, however, the underlying mechanism remains unknown. Our previous studies suggest that ADAMTS5 may inhibit invasion and metastasis by suppression of the Ets-1-induced angiogenesis via inactivating PI3K/AKT pathway of gastric cancer. Based on this hypothesis, IHC and qRT-PCR were used to detect the expression and analyze the correlation of ADAMTS5, Ets-1 and CD31 in gastric cancer tissues and gastric cancer cells. Stable cells with over-expression/knock-down of ADAMTS5 or Ets-1 were established and subsequently used to explore the effects of ADAMTS5 on invasive ability of gastric cancer cells, PI3K/AKT and Ets-1 expression and angiogenesis by biological methods. Methods of RNA interference, pathway activator were employed to assess the roles of PI3K/AKT pathway and Ets-1 on the inhibition of angiogenesis and invasion and metastasis by ADAMTS5 of gastric cancer; Methods of luciferase reporter gene and immunoprecipitation were adopted to evaluate the mechanism of ADAMTS5 regulate PI3K/AKT pathway and Ets-1. Finally, animal models were conducted to verify that ADAMTS5 down-regulates Ets-1 to reduce MVD and inhibits invasion and metastasis of gastric cancer. In vitro and in vivo experiments demonstrated the molecular mechanism of ADAMTS5 inhibits PI3K/AKT/Ets-1 pathway to reduce angiogenesis and suppress the invasion and metastasis of gastric cancer. The clinical implication of this study is to provide some evidence to elucidate the molecular mechanism of invasion and metastasis of gastric cancer, and to seek a potentially novel target predicting metastasis in gastric cancer.

我们发现ADAMTS5与胃癌侵袭转移相关，但机制不详。前期研究提示ADAMTS5可能通过PI3K/AKT通路抑制Ets-1诱导的血管生成调节胃癌侵袭转移。基于此假说，IHC和qRT-PCR检测ADAMTS5、Ets-1和CD31在胃癌中的表达及相关性；构建ADAMTS5、Ets-1高/低表达细胞，分子生物技术检测胃癌细胞侵袭能力、PI3K/AKT和Ets-1的表达及血管生成；RNA干扰、通路激活剂等方法研究ADAMTS5抑制Ets-1介导的血管生成调节胃癌侵袭转移；荧光素酶报告基因等法探讨ADAMTS5通过PI3K/AKT通路调控Ets-1的详细机制。动物模型验证ADAMTS5下调Ets-1减少MVD抑制胃癌侵袭转移。体内外实验阐明ADAMTS5调节PI3K/AKT/Ets-1通路减少血管生成抑制胃癌侵袭转移的分子机制。本研究对补充胃癌侵袭转移的分子机制，探寻新的转移标志物有一定的意义。

我们在前期的研究中发现ADAMTS5在胃癌组织与细胞中低表达，且与胃癌侵袭转移相关，ADAMTS5低表达预示胃癌不良预后，但详细的机制不详。前期研究提示ADAMTS5可能通过PI3K/AKT通路抑制Ets-1诱导的血管生成调节胃癌侵袭转移。基于此假说，我们设计一系列实验，利用IHC和qRT-PCR检测ADAMTS5、Ets-1和CD31在胃癌中的表达及相关性；构建ADAMTS5高/低表达细胞，分子生物技术检测胃癌细胞侵袭能力、PI3K/AKT和Ets-1的表达及血管生成；RNA干扰、通路激活剂等方法研究ADAMTS5抑制Ets-1介导的血管生成调节胃癌侵袭转移；荧光素酶报告基因等法探讨ADAMTS5调控Ets-1的详细机制。动物模型验证ADAMTS5调节血管生成、抑制胃癌侵袭转移。研究结果表明，ADAMTS5在胃癌中低表达，胃癌组织中ADAMTS5蛋白与CD31蛋白、Ets-1蛋白表达呈负相关，提示ADAMTS5抑制胃癌组织中血管生成，且可能是通过调节Ets-1蛋白发挥作用。体外实验结果表明，ADAMTS5过表达后可抑制胃癌细胞迁移侵袭，并可抑制共培养人脐静脉内皮细胞的小管形成，证明其可抑制血管生成。在体实验表明，ADAMTS5可抑制小鼠体内肿瘤生长，并且促进肿瘤内部血管正常化。进一步探讨其机制，ADAMTS5通过抑制PI3K/AKT/Ets-1通路而发挥上述作用。mRNA芯片以及双荧光素酶实验结果表明，ADAMTS5通过作用于转录因子RasGRP1启动子区域，调控RasGRP1转录水平，从而下调CHI3L1表达，而调节PI3K/AKT/Ets-1通路。本研究初步阐明了ADAMTS5调节胃癌侵袭转移、血管生成的分子机制，对进一步深入研究胃癌发生发展的分子机制，以及研究开发新的胃癌治疗药物有一定的意义。