水通道蛋白9在结直肠癌干细胞分化过程中的作用及机制研究

The efficacy of chemotherapy in metastatic colorectal cancer has no breakthrough. One of the reasons is colorectal cancer stem cells (CRC-CSCs) can escape therapy which results in tumor relapse and metastasis. It is a potential way to improve the chemosensitivity through inducing CRC-CSCs differentiation. However, the key factors that initiate differentiation are not well known. WNT signaling pathway controls CRC-CSCs self-renewing and differentiation, GSK-3β is the negative modulator of WNT signaling pathway. Our preliminary results showed patients with high aquaporin 9(AQP9) expression had better response to 5-Fluorouracil, and CRC cells with AQP9 transfection had increased p-GSK-3β expression, decreased WNT activity and decreased stem cell markers. Given both AQP9 and GSK-3β induce gluconeogenesis, and both of them regulated by PI3K/AKT pathway, it is assumed that positive correlation exists between the two factors. So we hypothesize that AQP9 activates GSK-3β, which results in WNT inhibition, and initiating CRC-CSCs differentiation. After treating CRC-CSCs with AQP9 transfection, AQP9 siRNA and phloretin inhibition, the change of cell cloning ability, in VIVO oncogenicity， stemness and differentiation markers, GSK-3β and WNT activity is detected, in order to illustrate the key importance of AQP9 in inducing CRC-CSCs differentiation. In addition, by mRNA and miRNA array, integrative network analysis is used to dig out the molecular network interaction. The sensitivity of 5-FU and AQP9 expression are measured after treating CRC-CSCs with PI3K/AKT inhibitors in vitro and in vivo. The whole project aims to providing the potential targets and treatment strategy for targeting CRC-CSCs and enhancing chemosensivity.

晚期结直肠癌化疗至今无突破性进展，与结直肠癌干细胞（CRC-CSCs）对化疗不敏感有关。诱导CRC-CSCs分化是潜在提高疗效的方法，但其启动分化的机制尚未明确。本课题组前期研究发现，水通道蛋白9(AQP9)在化疗敏感患者中高表达，CRC-CSCs转染AQP9后WNT信号通路活性降低。基于AQP9和GSK-3β均刺激糖异生并受PI3K/AKT负调控，两者存在正相关，我们假设AQP9激活GSK-3β,后者抑制WNT信号通路活性，细胞启动分化。本项目拟通过基因转染、基因沉默AQP9等方法处理CRC-CSCs，检测细胞成瘤能力、干性/分化标志物变化，系统阐述AQP9在诱导CRC-CSCs分化过程中的关键作用，再通过生物信息学方法挖掘其分子机制；通过体内体外实验，阐述PI3K/AKT抑制剂诱导AQP9表达，促进细胞分化、增强5-FU疗效的作用。为靶向CRC-CSCs提供潜在的新靶点和治疗策略。

晚期结直肠癌化疗至今无突破性进展，与结直肠癌细胞对化疗不敏感有重要关系。寻找化疗耐药相关靶点并进行阻滞是潜在提高化疗疗效的方法。前期研究发现AQP9与结直肠癌细胞耐药有潜在关系，但具体机制尚未明确。本项目通过基因转染、基因沉默AQP9等方法处理CRC细胞，并检测细胞对化疗敏感性的变化。系统研究AQP9在诱导肠癌细胞对化疗敏感的过程中的关键作用，再通过生物信息学方法挖掘其分子机制；通过体内体外实验，阐述AQP9表达与5-FU疗效的作用。为靶向CRC细胞提供潜在的新靶点和治疗策略。研究结果证实AQP9可作为CRC辅助化疗的独立预测标志物。 具有高AQP9表达的患者具有比具有低AQP9表达的患者更高的无病生存率（DFS）。 AQP9的上调与体外和体内对5-氟尿嘧啶（5-FU）的增强的化学敏感性相关。 AQP9的过表达导致CRC细胞中5-FU的细胞内水平增加，因此在5-FU处理后导致更高百分比的细胞凋亡。 此外，AQP9与CRC中的RAS活化和其他下游信号分子正相关。 AQP9过表达导致p21上调并诱导S期停滞。 总之，AQP9通过激活RAS信号传导同时诱导S期阻滞并促进药物摄取，增强了对CRC细胞中5-FU的细胞毒性反应。我们的研究结果表明，AQP9可能是CRC中基于5-FU的化疗有益的新型预测因子。 鉴定AQP9诱导的肿瘤对5-FU的敏感性突出了AQP9在调节CRC中化学敏感性中的作用。