丹参人参组分配伍调控PTEN相关信号通路抑制肺癌EMT和转移的机制研究

The epithelial-mesenchymal transition (EMT) is closely related to tumor metastasis. Our preliminary study found that the effective component formula of Salvia Miltiorrhiza and Panax Ginseng can specially inhibits the proliferation of tumor cells , EMT, and metastasis in lung cancer cells. The molecular studies indicate the regulation of PTEN by PI3K/AKT and MEK//ERK signaling pathways could be the mechanism. We made a hypothesis: the effective component formula of Salvia Miltiorrhiza and Panax Ginseng could regulate the EMT and metastasis through targeting PTEN in lung cancer. According to this hypothesis, firstly we will study the effect of effective component formula of Salvia Miltiorrhiza and Panax Ginseng to the biological behaviors and molecular expression of EMT and metastasis in lung cancer, and detect the PTEN-related signaling pathways that regulate EMT, meanwhile, test and verify with the corresponding kinase inhibitors. For drug-targeted signaling pathways, we will study the regulatory relationship among drug-targeted pathways and analyze the interaction among molecules by Co-IP, CRISPR/Cas 9 and biofilm polarization techniques. These researches provide mechanistic insights into the effective component formula of Salvia Miltiorrhiza and Panax Ginseng regulates the EMT and metastasis through targeting PTEN in lung cancer. We may discover novel signaling loops. The project has the source innovation, which can provide ideas and methods for the innovation medicine research and anti-lung cancer therapy.

上皮-间质转化(EMT)与肿瘤转移密切相关。丹参人参组分配伍是我们前期研究获得的有效组分复方，能抑制肺癌细胞EMT和转移，分子水平研究提示：通过调控PTEN活性影响PI3K/AKT和MEK/ERK信号通路是其可能的药效机制。据此提出假说：“丹参人参组分配伍通过PTEN相关信号通路调控肺癌细胞EMT抑制肿瘤转移”。围绕此假说，项目拟首先研究丹参人参组分配伍影响肺癌EMT和转移的细胞生物学行为和分子表达；再检测药物调控EMT的PTEN相关信号通路，并以相应的激酶抑制剂加以验证；进而针对药物靶向的信号通路，应用Co-IP、CRISPR/Cas9、生物膜偏振技术，研究药物共靶向通路间的调控关系，分析分子间的相互作用，从而阐明丹参人参组分配伍通过PTEN相关信号通路抑制肺癌EMT和转移的分子调控机制，并有可能发现新的信号环路途径。项目具源头创新性，可为相关治法方药创新研究提供思路与方法。

丹参人参组分配伍（OCF）是我们前期研究优选获得的抗肿瘤有效组分配伍复方。转移是恶性肿瘤治疗失败和患者死亡的主要原因，上皮-间质转化(EMT)与肿瘤转移密切相关。本项目通过体内外实验研究OCF抑制肺癌EMT和转移的作用及分子调控机制。体外实验结果表明OCF可以显著抑制肺癌细胞的活性及侵袭转移能力；Signal Transduction PathwayFinder PCR基因芯片及qRT-PCR结果显示OCF可参与调控肺癌细胞wnt/β-catenin通路并逆转肺癌细胞EMT进程；生物膜干涉技术及蛋白印迹实验证实OCF与AKT蛋白具有直接的相互作用，抑制AKT磷酸化和下游GSK-3β、β-catenin蛋白活性；进一步AKT磷酸化抑制剂MK2206、激动剂SC-79与OCF联合作用实验结果显示OCF与MK2206具有协同抑制肺癌细胞侵袭转移、逆转肺癌EMT进程，抑制AKT/GSK-3β/β-catenin通路的作用，而SC-79能够逆转OCF对肺癌细胞转移、EMT及AKT/GSK-3β/β-catenin通路的抑制作用；通过Balb/c裸鼠尾静脉注射绿色荧光标记的H1299细胞构建体内肺癌转移模型，小动物活体成像（IVIS）实验证实OCF可有效抑制体内肺癌细胞生长及转移性肿瘤结节的形成；肺转移结节的免疫荧光检测结果显示，OCF可抑制N-cadherin、vimentin蛋白表达，上调E-cadherin蛋白表达，抑制EMT；免疫组化结果提示，OCF可下调肺转移肿瘤组织中p-AKT，p-GSK-3β、β-catenin蛋白的表达，抑制AKT/GSK-3β/β-catenin信号通路。综上，本研究证实了丹参人参组分复方抑制肺癌EMT和转移的作用，明确其通过与AKT蛋白的直接结合，抑制AKT/GSK-3β/β-catenin信号通路，发挥抑制肺癌EMT发生和转移的分子机制。