毛蕊异黄酮通过干预ERβ/miR-17/PTEN信号通路抗结直肠癌的作用研究

Colorectal cancer (CRC) is one of the most common malignancies and a leading cause of cancer deaths worldwide. Recently, numerous studies demonstrate a protective role for estrogen and estrogen receptor beta (ER β) in the initiation and progression of CRC. Calycosin is a main active component of the herb Radix Astragali, and is considered as a phytoestrogen. Because of the structural similarity with estrogen, it can produce estrogenic effects. It is reported that calycosin had inhibitory effects on the proliferation and invasion of breast cancer cell, and the inhibitory effects might be related to the level of ER β and the inactivation of PI3K/Akt signaling pathway. However, the roles and potential mechanisms of calycosin in CRC are still unknown. Our previous study first found that calycosin could induce cell apoptosis, inhibit cell proliferation and invasion in CRC, and downregulated the level of miR-17 and upregulated the level of ER β and PTEN protein. Furthermore, we found that overexpression of ER β in CRC cell could supress the expression of miR-17. Based on the above information, we hypothesize that ERβ/miR-17/PTEN pathwey, followed by regulation of PI3K/Akt signaling, may be responsible for the anti-tumor effect induced by calycosin in CRC. In the present study, by MTT assay, flow cytometry, Western blot, real-time PCR, siRNA, Xenograft tumor growth,and immunohistochemistry, we will elucidate the relationship among calycosin, ERβ, miR-17, and PTEN/PI3K/Akt pathway in CRC. And identify that calycosin inhibite growth and metastasis of CRC via interrupting the ERβ/miR-17/PTEN pathway. The results will not only help to further ascertain calycosin-mediated signaling pathway, but also provide experimental foundation for future development of calycosin for treatment of CRC.

结直肠癌（CRC）是常见的消化系统恶性肿瘤，近年来研究发现雌激素及雌激素受体β（ERβ）具有预防和抑制CRC发生发展的作用。毛蕊异黄酮是中药黄芪的主要活性成分，属于异黄酮类植物雌激素，能与ER结合发挥雌激素样效应。有研究显示毛蕊异黄酮可通过干预ERβ及PI3K/Akt信号通路，抑制乳腺癌等肿瘤细胞生长，但其在CRC的作用还不清楚。课题组前期研究首次发现毛蕊异黄酮能明显诱导人CRC细胞凋亡，抑制细胞增殖及侵袭力，且可降低miR-17表达，升高ERβ及PTEN水平。据此，我们推测毛蕊异黄酮可通过干预ERβ/miR-17/PTEN通路及下游PI3K/Akt信号通路分子，抑制CRC的生长转移。本项目将结合体内外模型，首次以miR-17为切入点，揭示ERβ、miR-17及PTEN/PI3K/Akt通路的信号级联，探讨毛蕊异黄酮抗CRC作用及信号转导通路，从而为CRC的防治药物研究提供新思路。

结直肠癌（colorectal cancer，CRC）是常见的消化系统恶性肿瘤，近年来研究发现雌激素及雌激素受体β（ERβ）具有预防和抑制CRC发生发展的作用。毛蕊异黄酮是中药黄芪的主要活性成分，属于异黄酮类植物雌激素。有研究显示毛蕊异黄酮可通过作用于ERβ抑制激素依赖性肿瘤生长，但其在CRC的作用尚不清楚。本课题通过体内外实验观察毛蕊异黄酮对人CRC细胞增殖、周期、凋亡、迁移力及小鼠移植瘤生长的影响。同时利用RNA干扰技术，沉默CRC细胞中ERβ和miR-17表达，分析转染前后细胞生物学行为及相关基因表达的改变。研究发现毛蕊异黄酮能明显抑制人CRC细胞及小鼠移植瘤生长，诱导细胞凋亡，阻滞细胞周期及抑制细胞迁移。同时毛瑞异黄酮能剂量依耐性的提高ERβ、PTEN表达；降低miR-17及P-AKT、Bcl-2、cyclinD1、MMP-9 蛋白表达。而干扰ERβ的表达后能明显拮抗毛蕊异黄酮对CRC细胞的抑制作用及对PTEN/PI3K/Akt 通路蛋白的调控作用。本课题研究结果提示毛蕊异黄酮可能是通过上调ERβ，降低 miR-17 表达，抑制PTEN/PI3K/Akt 通路活化，从而发挥抗CRC作用。项目研究结果将为毛蕊异黄酮的开发和应用提供理论依据。