胃癌循环肿瘤细胞EMT过程中PI3K-AKT-miRNA-200s信号通路异常表达的分子调控机制研究

Gastric cancer ranks second in cancer mortality, mainly due to metastasis and treatment resistance. Circulating tumor cells (CTCs) and epithelial-mesenchymal transition (EMT) both plays an important role in blood metastasis and treatment resistance. We have successfully isolated CTCs from gastric cancer patients and cultured them in vitro . we found that gastric CTCs occurred EMT. In the process of EMT, PI3K-AKT-miRNA-200s signaling pathway was abnormal expression: expression of miRNA-200s was defect, whereas, inhibition of p-AKT, raised the expression of miRNA-200s. However, how AKT regulate the expression of miRNA-200s and upstream molecules that activated PI3K/AKT are to be further elucidated. Based on the previous research of gastric CTCs and EMT, the study will use the technology of isolation and culture of gastric CTCs, to elucidate the molecular regulation of abnormal expression of PI3K/AKT signaling pathway, to elucidate the molecular mechanisms of abnormal expression of miRNA-200s from the perspective of epigenetics, as well as to clarify the regulatory mechanisms of PI3K/AKT signaling pathway on the abnormal expression of miRNA-200s from the transcriptional level point of view, in the EMT process in gastric CTCs. It will provide a theoretical basis for understanding of gastric cancer metastasis and treatment resistance, and will provide new ideas to targeted therapy for gastric cancer.

胃癌死亡率居恶性肿瘤第二位，主要原因是转移和治疗抵抗，而循环肿瘤细胞（CTCs）和上皮间质转化（EMT）在血行转移和治疗抵抗中扮演重要角色。我们已成功分离并体外培养胃癌CTCs，并发现胃癌CTCs发生了EMT，在该过程中，PI3K-AKT-miRNA-200s信号异常表达：miRNA-200s表达缺失，而抑制p-AKT，上调了miRNA-200s的表达。然而，AKT调控miRNA-200s的分子机制及激活PI3K/AKT的上游分子有待阐明。本项目在前期胃癌CTCs及EMT研究的基础上，利用前期CTCs的分离培养技术，阐明胃癌CTCs EMT过程中PI3K/AKT信号异常表达的分子调控，从表观遗传学角度阐明miRNA-200s异常表达的分子机制，从转录水平调控角度阐明PI3K/AKT信号对miRNA-200s异常表达的调控机制，为理解胃癌转移及治疗抵抗提供理论基础，为胃癌靶向治疗提供新思路。

胃癌死亡率居恶性肿瘤第二位，主要原因是转移和治疗抵抗，而循环肿瘤细胞（CTCs）和上皮间质转化（EMT）在血行转移和治疗抵抗中扮演重要角色。我们已成功分离并体外培养胃 癌CTCs，并发现胃癌CTCs发生了EMT，在该过程中，PI3K-AKT-miRNA-200s信号异常表达：miRN A-200s表达缺失，而抑制p-AKT，上调了miRNA-200s的表达。小G蛋白RhoA参与了肿瘤的发生发展多个过程。然而，AKT调控miRNA-200s的分子机制及激活PI3K/AKT的上游分子有待阐明。本项目在前期胃癌CTCs及EMT研究的基础上，利用多种小分子抑制剂及小干扰RNA技术，western blot及免疫荧光验证胃癌CTCs EMT过程中PI3K/AKT/miR200s信号异常表达的分子调控，我们发现在胃癌CTCs中加入RhoA抑制剂 Rhosin，RhoA下游分子ROCK1抑制剂Y27632，PI3K及 AKT抑制剂及干扰RhoA后细胞的的EMT现象得到逆转，同时细胞的侵袭及迁移能力下降，这表明RhoA及其下游信号分子同PI3K/AKT/miR200s一起参与了EMT过程。转染miR200b/c后RhoA及其下游信号分子ROCK1，DIAP1下调，E-Cadherin上调；抑制RhoA后, E-Cadherin表达上调，vimentin表达轻度上调，N-cadherin 表达变化不著，AKT及p-AKT表达下降；抑制AKT后RhoA表达量下调。上述研究表明RhoA及其下游信号分子同PI3K/AKT/ miR200s一起参与胃癌CTCs EMT过程的分子调控，结合我们既往的研究，miR200s调控RhoA, 同时受AKT的调控，而RhoA与PI3K/AKT之间也存在相互调控，因此我们推测胃癌CTCs EMT的分子调控机制中可能存在RhoA, miR200s, PI3K/AKT的分子调控环路。