HIF-1α/BRCA1介导的双链DNA损伤修复对乏氧环境下乳腺癌干细胞放射敏感性的调控机制研究

Hypoxia is a key factor affecting curative effect of tumor radiotherapy, but the mechanism is not clear. Our previous studies demonstrated, in hypoxia condition, the percentage of breast cancer stem cells increased, the radiotherapy sensitivity decreased, and the expression of HIF-1α hypoxia inducible transcription factor increased. It was reported that the high efficiency of DNA damage repair is one of the important mechanisms of radiation resistance in the glioma tumor stem cell, thus in the hypoxia condition, the mechanism of radiosensitivity regulation of breast cancer stem cell maybe relate to the changes of DNA damage repair. Thus, we detected the expression of proteins taking part in DNA damage repair, under hypoxia, after ion radiation, and found that the expression difference of BRCA1 is significant between stem cell and non-stem cell. BRCA1 is a key protein involved in double-strand DNA damage repair, and there are mutual regulation between HIF-1 α and BRCA1, thus in this project, we plan to study, under hypoxia condition, after ion radiation, interfering expression or activity of HIF-1α, BRCA1, the changes of general biological characteristics, radiosensitivity and DNA damage repair response, HR and NHEJ repair pathways and functions in breast cancer stem cells and the corresponding xenografts in nude mice. This study is beneficial for elucidating the mechanism of hypoxic in regulating radiosensitivity of breast cancer cell, exploiting strategies targeting breast cancer stem cells, sensitizing radiotherapy of breast cancer.

乏氧是影响肿瘤放疗疗效的重要不利因素，而调控机制不清。我们前期研究证实，乏氧情况下，乳腺癌干细胞比例增加，放疗敏感性下降，乏氧诱导转录因子HIF-1α表达增加。据报道脑胶质瘤干细胞高效率DNA修复是其放疗抵抗的重要机制之一，那么乏氧情况下，乳腺癌干细胞放疗敏感性是否与DNA损伤修复反应改变有关？我们检测了乏氧时放射处理后干细胞中DNA损伤相关蛋白的表达，发现BRCA1表达差异显著。BRCA1是双链DNA损伤修复的关键蛋白，与HIF-1α之间存在相互调控，因此本项目拟以HIF-1α/BRCA1为切入点，考察乏氧情况下，干预HIF-1α、BRCA1表达的乳腺癌干细胞及其对应的裸鼠移植瘤，在放射处理后生物学特性、放射敏感性和DNA损伤修复反应（应答、HR和NHEJ修复途径和功能）的变化。本研究为阐明乏氧调控乳腺癌细胞放疗敏感性的相关机制、寻找针对干细胞增敏乳腺癌放疗的干预措施奠定理论和实验基础。

乏氧是影响肿瘤放疗疗效的重要不利因素，而调控机制不清。我们前期研究证实，乏氧情况下，乳腺癌干细胞比例增加，放疗敏感性下降，乏氧诱导转录因子HIF-1α表达增加。据报道脑胶质瘤干细胞高效率DNA修复是其放疗抵抗的重要机制之一，那么乏氧情况下，乳腺癌干细胞放疗敏感性是否与DNA损伤修复反应改变有关？本项目以HIF-1α为切入点，首先构建沉默HIF-1α表达的慢病毒质粒，转染悬浮培养的乳腺癌干样MCF-7S细胞，考察沉默HIF-1α表达的乳腺癌干细胞，在放射线处理下，细胞凋亡细胞比例、细胞微核率和克隆形成率等的差异；考察DNA损伤修复功能（彗星实验和γ-H2AX斑点实验）的变化；及其沉默HIF-1α表达后MCF-7S细胞的DNA损伤应答(ATM、ChK1) ，双链DNA损伤修复的HR和NHEJ通路中的关键蛋白（BRCA1, RAD51, XRCC4,DNA-PKCs等）的表达差异；发现沉默HIF-1α表达后细胞对放疗的敏感性增加，DNA损伤应答蛋白ATM和HR通路的蛋白RAD51表达变化显著，而NHEJ通路蛋白DNA-PK变化不明显；本项目同步构建裸鼠移植瘤模型，在体情况下，证实沉默HIF-1α表达的裸鼠移植瘤，成瘤时间延长和成瘤率降低，对放疗的敏感性增加，与其调控DNA损伤反应和修复蛋白的表达有关。本研究为阐明乏氧调控乳腺癌细胞放疗敏感性的相关机制、寻找针对干细胞增敏乳腺癌放疗的干预措施奠定理论和实验基础。