汉黄芩素经表观遗传调控Nrf2/HIF-1α通路及逆转乳腺癌放疗抵抗的作用机制研究

放疗是临床治疗乳腺癌的重要手段，但肿瘤细胞常常出现放疗抵抗，引起肿瘤的复发及转移。本课题组的前期研究结果初步提示，汉黄芩素具有潜在的逆转乳腺癌放疗抵抗的作用。为揭示该现象产生的机理，本项目在已有的工作基础上围绕汉黄芩素是否通过表观遗传影响Nrf2的表达，继而抑制Nrf2/HIF-1α信号通路，对乳腺癌的放疗抵抗发挥逆转作用开展研究。研究工作采用定点突变结合报告基因系统、凝胶迁移实验、染色质免疫共沉淀等方法，揭示Nrf2调控HIF-1α的机理；应用甲基化特异性PCR、硫化测序PCR和DNA甲基化免疫共沉淀技术探索汉黄芩素对乳腺癌细胞中Nrf2的表观遗传调控机制；结合细胞克隆存活实验和裸鼠移植瘤模型，宏观呈现汉黄芩素对乳腺癌放疗抵抗的逆转作用。研究工作将揭示汉黄芩素逆转乳腺癌放疗抵抗的分子机制，为汉黄芩素的临床应用提供科学依据，也为中药单体在肿瘤放疗中发挥增效作用的分子机制研究开阔思路。

Radiotherapy is an important means in the treatment of breast cancer. However, the tumor cells often appear to be resistant to radiation, causing tumor recurrence and metastasis. Our previous studies showed that wogonin could reverse radioresistance of breast cancer cells. The inhibition of Nrf2/HIF-1α via epigenetic regulation by wogonin, thus reversing radioresistance could be a potential mechanism. In this proposal, the following experiments will be carried out to identify the exact mechanisms. The site-directed mutagenesis combined with luciferase reporter gene system, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP) would be utilized to verify the mechanisms underlying Nrf2 regulating HIF-1α. Methylation specific PCR (MSP), bisulfite sequencing PCR (BSP) and methylation DNA immunoprecipitation (MeDIP) to explore the epigenetic regulation of Nrf2 by wogonin in breast cancer cells. Furthermore, the cell clonogenic survival assay and nude mice xenograft model would be performed to validate the effects of wogonin on breast cancer radioresistance. The results from current project would not only provide the scientific evidence for wogonin effect on reversing radioresistance, but also broaden the area of future application of traditional Chinese medicine (TCM) monomer composition - radiotherapy combination.