解毒三根汤调控Hippo信号通路与 Akt/GSK-3β信号通路串话抑制结肠癌侵袭转移的机制研究

K-ras gene downstream of EGFR is considered as the key to start the canceration and the overexpression of YAP gene in Hippo-YAP signal pathway may promote tumor proliferation as well as the process of EMT, while the transcriptional coactivator TAZ upstream of YAP is regulated by Akt / GSK-3β signal pathway. In the previous studies, we found that Jiedu Sangen Decoction (JSD, consisting of Polygonum cuspidatum, Geum japonicum thumnb, Radix actinidiae) plays an inhibitory effect in the migration, invasion of SW480 cells and reverses SW480 cells’ EMT by EGFR / Hippo / YAP axis. After Lats1 silenced, However, JSD still has the ability to inhabiting invasion and metastasis. It supposes that JSD intervenes TAZ by Akt / GSK-3β signal pathway thereby inhibiting EMT.. Using EMT vitro model of colon cancer intervened by JSD to detect the ability of cell invasion, metastasis and the expression levels of phosphorylated of Hippo singal pathway, Akt / GSK-3β signal pathway, metastasis-associated protein, etc. To construct colon cancer with liver metastasis model in nude mice, analyze the effect to the potential of colon cancer metastasis by the expression of each gene in Hippo singal pathway and Akt / GSK-3β signal pathway and the action of JSD, verify JSD’s inhibition of invasion and metastasis of colon cancer by regulating crosstalk between Hippo singal pathway and Akt / GSK-3β signal pathway.

EGFR下游的K-ras基因被认为是启动癌变的关键，Hippo-YAP信号通路中YAP基因的过表达可促使肿瘤增殖及EMT过程，而YAP上游的转录共激活子TAZ受Akt/GSK-3β信号通路的调控。前期研究中，我们发现解毒三根汤（藤梨根、虎杖根和水杨梅根）通过EGFR/Hippo/YAP轴发挥抑制SW480细胞的迁移、侵袭以及逆转SW480细胞EMT的作用，但是Lats1沉默后，解毒三根汤仍然具有抗侵袭转移能力。所以我们猜测解毒三根汤可能通过Akt/GSK-3β信号通路作用干预TAZ进而抑制EMT的发生。本项目选用解毒三根汤应用体外结肠癌EMT模型，检测细胞侵袭转移能力及Hippo通路与Akt/GSK-3β通路蛋白、转移相关蛋白等磷酸化及表达水平，构建裸鼠结肠癌肝转移模型，分析Hippo信号通路与Akt/GSK-3β信号通路各基因的表达情况对结肠癌转移潜力的影响及解毒三根汤的作用。

解毒三根汤（JSD）是一种具有抗结肠癌侵袭转移作用的中药经验方，本研究旨在探讨JSD抑制结肠癌侵袭转移的机制。在体外，我们用EGF诱导结肠癌细胞，成功构建了EMT模型，再给予JSD干预，行划痕、transwell和western blot方法观察细胞侵袭、转移能力及上皮间质转化相关蛋白和hippo、AKT1/GSK-3β信号通路相关蛋白的表达变化。进一步，我们成功地在SW480和SW620细胞系中构建了AKT1基因敲减（si-AKT1）或过表达（oe-AKT1）的细胞稳定株。然给予JSD干预。采用划痕、transwell和western blot方法观察细胞侵袭、转移能力及上皮间质转化相关蛋白和AKT1/GSK-3β信号通路相关蛋白的表达变化。体内接种SW480细胞建立结肠癌肝转移模型小鼠。在JSD干预后，进行活体荧光显像和体重测定，观察JSD对结肠癌肝转移的抑制作用。采用HE染色、免疫组化和western blot方法观察组织学特征及蛋白表达情况。研究结果发现JSD治疗或AKT1基因敲减可明显抑制结肠癌的侵袭转移和EMT，AKT1过表达可增强其侵袭转移能力。当AKT1被敲减时，JSD诱导的抑制作用明显减弱，而当AKT1过度表达时，抑制作用明显增强。此外，JSD可导致结肠癌细胞E-cadherin表达增加，N-cadherin、Vimentin、p-AKT1、AKT1、p-GSK-3β、YAP、TAZ、Snail、Slug和Twist表达减少。由此可以得出，JSD可能同时通过hippo、AKT/GSK-3β信号途径多靶点逆转EMT，抑制结肠癌的侵袭和转移。