姜黄素通过Hedgehog/Gli信号通路调控肿瘤干细胞干性及EMT抑制乳腺癌肺转移分子机制研究

Breast cancer stem cells (BCSCs) are the origin of breast cancer recurrence and metastasis. Studies showed that proliferation and stemness maintenance of BCSCs are closely correlated to the Hedgehog (Hh) signaling pathway. Activation of downstream nuclear transcription factor of Hh pathway Gli can cause EMT in breast cancer cells. Our previous study found that curcumin reduced BCSCs suspension mammospheres formation and differentiation ability and the stem cell surface marker CD44 +CD24- / low cell subpopulation, effectively inhibiting its stemness features and epithelial-mesenchymal transition (EMT). Therefore, we hypothesize that the genes regulate EMT exist great difference between BCSCs and the adherent parental cells and curcumin may prevent breast cancer metastasis by regulating the Hh/Gli signaling pathway and inhibiting the progression of BCSCs and EMT. In this project, EMT gene expression PCR chips were used to comprehensively detect the EMT characteristics of BCSCs for the first time. Then in vitro, curcumin modulates the function of BCSCs and EMT through Hh signaling pathway-related molecules and use lentiviral vector to construct BCSCs-luc2 cell line with stable expressed luciferase to prepare nude mice model with breast cancer lung metastasis. Small animal living imaging technology will be used to observe the effect of curcumin on tumor growth and metastasis of BCSCs-luc2 tumor-bearing nude mice and to explore its molecular mechanism. It is helpful for us to find targets of curcumin on breast cancer metastasis and to lay the experimental foundation for the research and application of curcumin in the field of breast cancer metastasis.

乳腺癌干细胞（BCSCs）是乳腺癌复发转移的根源，研究表明BCSCs干性与Hedgehog（Hh）通路密切相关，该通路核转录因子Gli激活可引起乳腺癌细胞上皮间质转化（EMT）。我们前期研究发现姜黄素能降低BCSCs悬浮球形成能力及CD44+CD24-细胞亚群，抑制干细胞基因，调节 EMT标志物表达。由此我们推测BCSCs与贴壁细胞存在EMT相关高度差异表达关键基因；姜黄素可能通过Hh/Gli通路靶向BCSCs调控EMT进程阻止癌转移。本项目首次应用EMT基因PCR芯片分析BCSCs的EMT特征，继而体外研究姜黄素是否通过Hh通路分子靶向调节BCSCs干性及EMT；并创新性应用慢病毒载体构建稳定表达荧光素酶BCSCs-luc2，制备乳腺癌肺转移裸鼠模型，小动物活体成像追踪观察姜黄素对BCSCs-luc2荷瘤裸鼠肿瘤生长及转移干预作用并研究其分子机制，为姜黄素在乳腺癌转移领域的应用奠定基础。

项目背景：乳腺癌发病率逐年递增，其复发转移仍然是目前临床治疗的难题。乳腺癌干细胞（BCSCs）具有较强的致瘤性、分化潜能和自我更新能力，可能是导致癌转移的关键因素。Hedgehog (Hh)信号通路与乳腺癌细胞上皮间质转化密切相关，其中Gli1是重要的转录因子，在Hh通路激活过程中起重要作用；姜黄素来源于传统活血化瘀中药姜黄根茎，具有抑制血管生成，抗肿瘤，抗氧化等多种药理活性。.主要研究内容：本项目首先研究姜黄素对高转移乳腺癌细胞MDA-MB-231和MDA-MB-468增殖、侵袭和迁移能力的影响，进而采用无血清悬浮法培养MDA-MB-231 干细胞球，经过流式分析悬浮球细胞表面标志物及通过小鼠皮下移植瘤实验鉴定其干性及致瘤性，采用EMT基因PCR芯片全面检测悬浮球细胞与贴壁细胞高度差异表达EMT相关基因；运用慢病毒转染构建Gli1过表达稳转MDA-MB-231贴壁细胞和干细胞株，观察Gli1抑制剂GANT61及姜黄素对乳腺癌干细胞Gli1核表达、定位及对EMT进程影响和姜黄素干预效果。采用MDA-MB-231-luc悬浮球稳转细胞株建立小鼠乳腺癌干细胞肺转移模型，活体成像动态监测肺转移及姜黄素靶向Gli1干预效果。.结果、数据及意义：研究结果表明姜黄素可抑制MDA-MB-231和MDA-MB-468细胞增殖、侵袭和迁移能力；MDA-MB-231悬浮球细胞干性较强，其与贴壁细胞具有高度差异表达EMT基因；Gli1过表达MDA-MB-231细胞较阴性对照细胞克隆增殖能力及侵袭迁移能力增强并可被姜黄素抑制；Gli1过表达乳腺癌干细胞EMT基因N-cadherin，Vimentin；Hh通路基因SMO、Gli2及干性基因SOX2、OCT4蛋白表达增加，经过姜黄素处理后，上述蛋白表达均有下调。此外，激光共聚焦显微镜观察发现Gli1主要表达在细胞核中，姜黄素可抑制MDA-MB-231细胞中Gli1表达及干细胞中因Gli1过表达诱导的升高。进一步在体实验研究表明，姜黄素及GANT61处理后均能抑制小鼠乳腺癌干细胞肺部转移灶形成，且姜黄素降低肺转移灶Nanog等干性基因表达。研究结果表明Hh/Gli1通路与BCSCs干性及EMT密切相关；姜黄素能够靶向Gli1抑制乳腺癌干性及EMT进程，为探索应用姜黄素协同防治乳腺癌肺转移提供实验基础。