MiR200c-PDE7B-cAMP在乳腺癌转移EMT中的作用机制及清热解毒药对干预的研究

Metastasis is the main cause of the high mortality rate of breast cancer. Previous works show that miR200c-PDE7B-cAMP pathway plays the key regulatory role during epithelial-mesenchymal transition (EMT) process in breast cancer metastasis with the aid of pharmacology and genomics tools..Couplet medicines that constituted of oldenlandia diffusa (Willd.)Roxb and Scutellaria barbata D.Don with clearing away heat and toxic property had demonstrated that it is crucial for clinical use in breast cancer treatment. .We found that the anti-inflammatory active faction from this couplet medicines by in vitro screening, also can effectively inhibit metastasis of breast cancer cells, and PDE7B and NLRP3 inflammasome signaling pathways were be involved. Based on the above results, the use of cell co-culture system to mimic microenvironment, bone metastasis in mice model and lung metastasis in nude mice model were applied to study the active fraction, component compatibility and PDE7B inhibitor on breast cancer metastasis in vivo and in vitro, to establish the role of cAMP and its downstream signaling pathways PKA / VASP on breast cancer metastasis , to clarify the impact of the interaction with NLRP3 inflammasome and to characterize the profile of active fraction meanwhile. .Project results will be beneficial to elaborate the material basis and inflammatory related mechanisms of couplet medicines.

乳腺癌转移是造成乳腺癌患者死亡率居高不下的主要原因。前期工作中经多种乳腺癌细胞株间比较、临床样本验证，同时利用药理学和基因学手段，发现miR200c-PDE7B-cAMP在乳腺癌转移的上皮细胞间质化(EMT)进程中起关键调控作用。白花蛇舌草和半枝莲配伍是乳腺癌治疗临床使用的关键药对，体外筛选发现等比配伍的抗炎活性组分能有效抑制乳腺癌细胞转移，且干预PDE7B和NLRP3炎症小体信号通路。本项目基于以上结果，利用细胞共培养模拟微环境、乳腺癌骨转移小鼠模型和乳腺癌肺转移裸鼠模型考察药对活性组分、成分配伍和PDE7B抑制剂对乳腺癌体内体外转移的影响，确立cAMP对乳腺癌转移的治疗作用并阐明其下游信号通路PKA/VASP及与炎症小体NLRP3的交互作用对EMT的影响，同时对组分进行化学表征。项目结果将有利于阐述有效方剂清热解毒药对控制乳腺癌转移的物质基础和干预肿瘤相关性炎性发挥疗效的作用机制。

三阴性乳腺癌(TNBC)恶性程度高，预后差，迄今治疗手段有限。miRNA-200家族中的miR-200c通过ZEB1/2调控上皮细胞间质化EMT进程，经多种乳腺癌细胞株间比较和临床样本验证，同时利用药理学和基因学手段，我们发现miR-200c不依赖ZEB1/2机制调控TNBC增殖生长的新机制。TNBC细胞中miR-200c的缺失，导致了其靶基因磷酸二酯酶PDE7B的表达提高，PDE7B作为环磷酸腺苷cAMP的特异性水解酶，使细胞内cAMP含量降低，从而使cAMP抑制细胞增殖和诱导凋亡的作用减弱。该研究结果揭示PDE7B为干预TNBC增殖生长的潜在靶点，为开发中药源PDE7B抑制剂或miR-200c诱导剂提供理论基础。此外，miR-200c还靶向色氨酸2，3-加二氧酶TDO2，失活STAT3通路，提高MDA-MB-231胞内抗癌代谢产物水平，促使代谢表型从依赖糖酵解为主向有氧呼吸型转化，从而影响MDA-MB-231细胞的恶性生物学行为。白花蛇舌草和半枝莲是临床使用多年抗炎抗肿瘤的核心药对，我们发现等比提取白花蛇舌草半枝莲水提物的乙酸乙酯组分(EA11)具有抗炎、抑制TNBC增殖和生长、抑制乳腺癌骨转移细胞迁移、调控巨噬细胞表型极化、抑制破骨细胞分化的活性，进一步研究发现EA11通过提高miR-200c，抑制其靶基因PDE7B，提高胞内cAMP含量，失活MAPK和AKT信号通路而发挥抑制TNBC细胞4T1的体外增殖及体内生长的作用；通过miR-155调控巨噬细胞M1/M2及破骨分化进程；通过miR-181c抑制乳腺癌骨转移细胞的体外迁移，并结合高分辨质谱和超高效液相鉴定含对羟基苯乙酮、野黄芩苷、木犀草素和芹菜素 4 种成分。该研究结果为该清热解毒药对的进一步开发包括标准提取制剂制备及质控和扩大临床适应症提供实验依据。