PGC-1β协同FOXA2经由LKB1/AMPK/mTOR通路调控乳腺肿瘤干细胞凋亡的分子机制研究

The immortal characteristics of tumor cells are the result of cell apoptosis inhibition, and this apoptosis inhibition is regulated by multiple genes and cell signaling pathways. Many scientists have been trying to investigate the mechanisms of the signaling pathway that regulates cell apoptosis, hoping to find molecules that could be used to antagonize apoptosis resistance, with the hope to induce tumor cell apoptosis as a better way for tumor therapy.In the previous experiment, our research group found the interaction between PGC-1β and FOXA2 for the first time, which through the mTOR/AKT signaling pathway in breast cancer stem cell proliferation. To test this hypothesis, we used the human breast cancer cells and breast cancer in nude mice animal model through the following experimental validation. 1) We used the GST pull-down, Co-IP, confocal laser, PGC-1βdeletion mutant and subcellular localization to detect the interaction. 2) We used Western blot, flow cytometry, nude mice tumor formation to investigate the PGC-1β and FOXA2 co-expression on the growth of breast cancer cell. 3) Through the LKB1/AMPK/mTOR signal pathway analysis, we detected the proliferation of breast cancer cells between PGC-1βand FOXA2, which lay the basement for targeted clinical treatment of breast cancer.

肿瘤无限生长特性是其细胞凋亡受抑制的结果，研究肿瘤细胞凋亡的分子调控机制，通过诱导肿瘤细胞凋亡来治疗肿瘤，是临床治疗的目标之一。课题组前期实验发现PGC-1β与FOXA2存在相互作用关系，推测其通过LKB1/AMPK/mTOR信号通路调控乳腺癌干细胞的增殖。为了验证该推测，采用人乳腺癌细胞系和裸鼠乳腺荷瘤模型为对象，分别进行：①GST pull-down、Co-IP、激光共聚焦、缺失突变体检测，验证PGC-1β与FOXA2的作用关系及其亚细胞定位；② Western blot、流式细胞术、裸鼠成瘤等，探讨PGC-1β与FOXA2共表达对乳腺癌细胞凋亡的影响；③与LKB1/AMPK/mTOR信号通路相关的基因和蛋白分析，明确PGC-1β与FOXA2在促进乳腺肿瘤细胞凋亡的分子机制。研究结果将为阐明PGC-1β协同FOXA2调控乳腺肿瘤干细胞凋亡的分子机制，及对乳腺癌靶向临床治疗研究提供基础。

背景：从全球癌症发病率统计数据来看，乳腺癌是女性中最常见的恶性肿瘤之一。 目前研究表明过氧化物酶体γ共激活因子1β（PGC-1β）与乳腺癌的发生发展密切相关。然而，PGC-1β在乳腺癌中的发病机制尚未阐明，有待进一步证实。.方法：利用qRT-PCR和Western Blotting法分别检测乳腺癌组织和细胞系中PGC-1β和FOXA2 mRNA和蛋白的表达情况。同时采用免疫化学和免疫荧光染色方法检测PGC-1β和FOXA2的表达和定位。其次，稳定转染PGC-1β干扰和FOXA2过表达载体，通过CCK8，克隆形成，transwell和划痕实验方法评估PGC-1β和FOXA2对乳腺肿瘤细胞增殖和迁移的影响；裸鼠异种移植瘤模型评估其对体内肿瘤生长的影响。另外，通过Western Blotting方法检测参与调控乳腺癌发展的PI3K-AKT-mTOR信号通路中相关蛋白的表达。.结果：研究结果表明，PGC-1β在乳腺癌组织和细胞系中表达上调，FOXA2表达下调，且二者相互作用形成复合物。同时，我们发现PGC-1β干扰和FOXA2过度表达联合作用显著抑制了体外细胞的增殖、迁移以及体内肿瘤的生长。我们进一步证实PGC-1β和FOXA2与PI3K-AKT-mTOR信号通路密切相关，其通过激活该信号通路发挥生物学功能。.结论：PGC-1β低表达与FOXA2过表达联合作用通过调节PI3K-AKT-mTOR信号通路抑制乳腺癌的发生发展。

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