白术经表观遗传调控Nrf2/Beclin1通路及其乳腺癌化学预防作用的机理研究

In recent years, the incidence of breast cancer has increased year by year, and the population has become progressively younger. The prevention and treatment of breast cancer is particularly urgent. As a hotspot in cancer prevention research, chemoprevention is considered as one of the important means for early intervention of tumorigenesis. Our previous studies showed that Atractylodes macrocephala Koidz (AMK) could prevent mammary tumorigenesis by inducing Nrf2 expression. To further elucidate the underlying mechanisms, we put forward the hypothesis that AMK could activate Nrf2/Beclin1 pathway via epigenetic regulation, thus preventing mammary tumorigenesis based on the previous research. In this proposal, 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 regulation of Beclin1. The bisulfite genomic sequencing (BGS), methylated DNA Immunoprecipitation-qPCR (MeDIP-qPCR) and ChIP to explore the epigenetic regulation of Nrf2 by AMK in human mammary epithelial cells. Furthermore, the effects of AMK on mammary tumorigenesis would be validated in vitro and in vivo. The results from current project would not only provide the scientific evidence for AMK effect on mammary tumorigenesis, but also broaden the area of future application of traditional Chinese medicine (TCM) on chemoprevention.

近几年乳腺癌发病率逐年升高且发病人群日益年轻化，针对乳腺癌的防治工作显得尤为迫切。作为肿瘤防治研究领域的热点，化学预防被认为是早期干预肿瘤发生的重要手段之一。本课题组前期研究显示白术可预防乳腺癌发生，并初步证实该作用与诱导Nrf2表达有关。为进一步揭示其内在机理，本课题组在已有研究基础上围绕白术是否通过表观遗传诱导Nrf2表达，进而激活Nrf2/Beclin1通路，对乳腺癌的化学预防作用开展研究。研究工作采用定点突变结合报告基因系统、凝胶迁移实验与染色质免疫共沉淀技术，揭示Nrf2调控Beclin1的机理；应用亚硫酸氢盐测序、DNA甲基化免疫共沉淀与染色质免疫共沉淀技术，探究白术对Nrf2的表观遗传调控机制；并结合乳腺癌癌前病变细胞与动物模型，阐明白术乳腺癌化学预防作用的内在机理。研究工作将为白术的临床应用提供科学依据，也为中药的肿瘤化学预防作用的机理研究开阔思路。

化学预防是乳腺癌防治的重要手段之一，从我国传统中药中发现新的化学预防药物对于预防乳腺癌的发生至关重要。文献报道，炎症与氧化应激能诱导肿瘤的发生发展，作为体内重要的内源性抗炎抗氧化因子Nrf2与细胞自噬可通过抑制炎症与氧化应激，进而抑制乳腺癌的发生。本项目通过构建乳腺癌癌前病变细胞与动物模型，发现白术内酯III可延长大鼠乳腺癌发生的潜伏期、降低乳腺癌的多发率和肿瘤体积，并改善乳腺癌对大鼠体重的影响，进一步研究阐释了白术内酯III可激活正常乳腺上皮细胞Nrf2/ARE通路，诱导细胞自噬发生，并抑制大鼠乳腺癌组织炎症与氧化应激水平；本项目还阐明了白术内酯I乳腺癌化学预防作用与TLR4介导的NF-κB通路有关，作为TLR4拮抗剂，白术内酯I可通过TLR4介导的NF-κB通路抑制大鼠乳腺癌的发生，抑制乳腺癌细胞的增殖、迁移与侵袭，并诱导其凋亡。此外，我们还通过生物信息学手段，进行了乳腺癌ceRNA网络的构建及潜在表观遗传机制的探索。这些研究为乳腺癌化学预防和治疗靶点的发现开拓了思路，也为白术内酯I与白术内酯III的临床应用提供了理论依据。