三阴乳腺癌预后极差，辅助放射治疗能否改善预后与不同辐射剂量诱发肿瘤细胞自噬效应有关，但机制不明。我们前期工作发现，高剂量辐射可诱导三阴乳腺癌细胞AMPK上调，PI3K/Akt抑制，自噬相关基因表达增强；抑制三阴乳腺癌复发、转移。我们推测："不同辐射剂量、时间可能是通过AMPK和PI3K-Akt通路影响mTOR信号诱发三阴乳腺癌细胞自噬或自噬性死亡"。阐明该问题，可为放射治疗三阴乳腺癌提供剂量和时间学理论依据。课题拟采用人三阴乳腺癌细胞系、建立荷瘤动物模型等方法，进一步研究明确：1.不同剂量辐射三阴乳腺癌细胞对mTOR信号通路分子表达及自噬相关基因影响。 2.选择性干预Akt、AMPK信号对辐射后三阴乳腺癌细胞的生存、增殖和自噬的影响及荷瘤动物放疗抵抗或增敏效应。该项目预期阐明不同辐射剂量对mTOR信号激活与三阴乳腺癌细胞辐射反应的分子机制，为三阴乳腺癌放射治疗模式提供理论支持和依据。

Triple negative breast cancer presents a poor prognosis,whether adjuvant radiation therapy can improve the prognosis depends on different dose of radiation induce autophagy effect of tumor cells, however its accurate mechanism remains unclear. Our previous work has demonstrated that high doses of radiation could increase AMPK activity, repress PI3K/Akt and enhance autophagy-related gene expression, meanwhile inhibition of recurrence and metastasis in triple negative breast cancer. We hypothesize: "Different radiation doses and time maybe regulate mTOR signaling through AMPK and PI3K-Akt pathway induced autophagy or autophagic death on triple negative breast cancer cell". To clarify the issue, which can provide theoretical support for radiotherapy dose and time in triple negative breast cancer, is urgent problem at present. The proposed triple negative breast cancer cell lines, tumor-bearing animal models, RNA interference experiments will be established, the research further promotes to demonstrate: 1) Different doses of radiation influence mTOR signaling molecule expression and autophagy-related genes in triple negative breast cancer cells. 2) Selectively interfere Akt, AMPK signaling to influence triple negative breast cancer cell survival, proliferation, colony and autophagy. Blocking or activating AMPK or PI3K/Akt signal pathway influence radioresistance of tumor-bearing animal with clinical dose radation. The project is expected to clarify the biological response of different-dose or -time radiation for activation of mTOR signaling on triple negative breast cancer, its molecular mechanisms will provide radiant theoretical support and scientific basis for triple negative breast cancer.

三阴乳腺癌预后极差，辅助放射治疗能否改善预后，尚存争议。我们采用三阴乳腺癌细胞和荷瘤动物等方法观察不同辐射剂量诱发肿瘤细胞自噬与辐射效应的关系。RT-PCR、Western blot、免疫荧光检测AMPK、PI3K、Akt及自噬相关基因；流式细胞术检测活性氧（ROS）表达和细胞周期；膜片钳检测Ca2+和Cl-电流；免疫组化检测血管形成、巨噬细胞激化及自噬、代谢相关基因改变。结果：⑴高剂量辐射可诱导三阴乳腺癌细胞ROS和AMPK上调，PI3K/Akt抑制，自噬相关基因表达增强；可激活VSOR Cl-通道，诱发Ca2+超载，从而引起氧化应激损伤。⑵自噬特异性抑制剂3MA可抑制自噬，降低细胞自噬性死亡发生率，mTOR抑制剂RAPA增加自噬性死亡发生率。⑶6MV X 线0Gy、1Gy、3Gy、5Gy照射三阴乳腺癌MDA-MB-231细胞，随辐射剂量增加，ROS表达和自噬逐渐增强，而LAMP2表达逐渐降低，细胞自噬性死亡增加。同时降低细胞DSB修复能力，细胞侵袭性逐渐降低。ELISA法更动态显示了LC3II与LAMP2改变与辐射剂量的关系，并发现褪黑素可能影响溶酶体数量和LAMP2表达。⑷不同剂量辐射能促使MDA-MB-231细胞不同LncRNA表达， 其中高剂量辐射可抑制HOTAIR表达，解除mir34a启动子甲基化状态，抑制MDA-MB-231侵袭性；⑸荷留动物实验显示，高辐射能明显促进裸鼠三阴乳腺癌肿瘤组织自噬增加，肿瘤相关巨噬细胞向M1极化，血管生成减少，重塑肿瘤代谢途径，抑制糖酵解，抑制瘤体增长等。结论: 放射治疗三阴乳腺癌主要通过上调AMPK，抑制PI3K和Akt磷酸化诱发三阴乳腺癌细胞自噬性死亡，同时改变肿瘤代谢微环境，抑制肿瘤生长。