类黄酮化合物是植物性食物中的一类低分子有机活性成分，其抑癌作用广受关注。本课题组前期研究从类黄酮化合物中筛选出抗肿瘤活性强的3,6-二羟黄酮（3,6-DHF），通过体内外研究发现其能下调miR-21、上调miR-34a的表达抑制乳腺癌发生，但相关机制不清。基于研究进展和预实验结果我们提出：3,6-DHF通过表遗传调控miR-21和miR-34a的表达，进而促进自噬发挥抑癌效应。本课题利用体内外乳腺癌发生模型，采用MeDIP-seq、CHIP-on-chip、MSP、CHIP-qPCR、RNAi等技术，观察乳腺癌发生中miR-21和miR-34a DNA甲基化、组蛋白修饰的动态变化及3,6-DHF干预的影响，研究3,6-DHF调控其表达的机制，进而观察3,6-DHF通过调控两者表达促进自噬的作用，并研究相关作用机制，特别是对PI3K/Akt/mTOR通路的影响，企在进一步揭示类黄酮抑癌机理。

Breast cancer risk is closely related to dietary factors. Flavonoids, a class of organic active compounds with low molecular weight, occurring ubiquitously in fruits and vegetables, have been identified as potential cancer-preventive components. We have previously selected a promising anti-cancer agent 3,6-dihydroxyflavone (3,6-DHF) in pharmacodynamic experiments from a lot of dietary flavonoid compounds. Our study showed 3,6-DHF is a potent chemopreventive agent, and miR-34a and miR-21 play important roles in the anticancer effect of it, yet the mechanisms are unclear. Recent studies have shown that the expressions of miR-21 and miR-34a were regulated by epigenetics, and they may play important role in autophagy. Effects of flavonoids on epigenetics and autophagy have become hot issues in cancer prevention. Using the breast carcinogenesis experiment models in vivo and in vitro, the study investigates effects of 3,6-DHF on epigenetics, and explore the mechanism of 3,6-DHF regulating miR-21 and miR-34a expression, by methods of MeDIP-seq, MSP, CHIP-on-chip, CHIP-qPCR, RNAi, et al. Furthermore, the study observes influences of 3,6-DHF on autophagy and PI3K/Akt/mTOR pathway by regulating the expression of miR-21 and miR-34a, and investigates the related mechanisms and the role of it in chemoprevention. The study is aimed to further reveal the molecular mechanisms of breast cancer-preventive effects of dietary flavonoids.

乳腺癌是严重危害生命健康的常见恶性肿瘤，其发病率在我国呈快速上升趋势，防治压力日益增大。乳腺癌的发生发展与膳食因素关系密切，探讨通过膳食途径有效预防乳腺癌的发生具有重要的理论和现实意义。类黄酮化合物是植物食物中广泛存在的一类抑癌成分，本课题组前期从膳食黄酮化合物中筛选出抑癌活性强的3,6-二羟黄酮（3,6-DHF），本课题旨在深入探讨3,6-DHF抑制乳腺癌发生的效应和分子机制。本项目研究结果表明，3,6-DHF能够有效抑制致癌剂（MNU、NNK和B[a]P）诱导的大鼠乳腺肿瘤发生和体外乳腺上皮细胞的癌转化；在乳腺肿瘤发生过程中，miR-21的表达水平显著升高，而miR-34a的水平显著降低，3,6-DHF可通过表遗传途径调控两者的表达：3,6-DHF通过降低miR-34a基因启动子区DNA甲基化水平提高其表达，同时通过影响miR-21基因启动子区组蛋白乙酰化修饰（H3K9-14ac、H3K27ac和H3K27me3等）降低miR-21的表达。进一步研究发现，乳腺癌发生过程中，细胞内DNA甲基化-去甲基化平衡发生显著变化，DNA甲基化酶DNMT1的表达和活性升高，去甲基化酶TET1的表达和活性下降，3,6-DHF干预可显著抑制DNMT1的表达和活性，提高TET1的表达，调节细胞内DNA甲基化-去甲基化的平衡，进而调控miR-34a等基因的DNA甲基化和表达水平；DNA甲基化酶活性检测和分子对接模拟分析显示，3,6-DHF有可能直接嵌入DNMT1的疏水活性结构域，抑制其活性。另外，我们的研究发现，PI3K/Akt/mTOR信号通路在乳腺癌发生中发挥关键作用，致癌剂可诱导此通路持续激活，促进细胞快速生长、增殖和癌特性转化，3,6-DHF干预可在体内外有效抑制致癌剂诱导的PI3K/Akt/mTOR活化，同时抑制乳腺癌细胞PI3K/Akt/mTOR通路，其作用机制可能涉及通过miR-34a和miR-21调节PTEN、Notch-1、Hes-1等关键上游蛋白的表达，进而抑制此信号通路。本项目研究结果表明，3,6-DHF可通过影响DNA甲基化和组蛋白修饰，促进miR-34a表达，抑制miR-21表达，并进一步调节Notch-1 和PTEN等关键蛋白的表达，抑制PI3K/Akt/mTOR信号通路，对乳腺癌的发生和发展发挥显著抑制效应。