术藤合剂通过HIF-1α/Smad7/Snail通路调控EMT抑制结直肠癌侵袭转移的机制研究

Invasion and metastasis of colorectal cancer is the main cause of death. Epithelial-mesenchymal transition (EMT) plays a key role in this respect.Our previous studies suggested that HIF-1a could induce EMT to promote invasion and metastasis of colorectal cancer under hypoxia, but how its downstream molecules regulated EMT was still unclear. Some studies had shown that HIF-1a could induce Smad complex to combine with target gene and inhibited Snail gene expression in cells in order to promote EMT. However, whether the same mechanism existed in colorectal cancer had not been reported. Our another study found that Zhuteng Mixture could inhibit the invasion and metastasis of colorectal cancer by regulating EMT through HIF-1a, but whether the compound could also regulate the expression of Smad7 and Snail needed further studied. Therefore, we proposed the hypothesis that Zhuteng Mixture regulated EMT through HIF-1a/Smad7/Snail pathway to inhibit metastatic recurrence of colorectal cancer. This project intended to: 1) verify the role of HIF-1a/Smad7/Snail pathway in regulating EMT in the invasion and metastasis of colorectal cancer; 2) focus the molecular mediated Zhuteng Mixture to inhibit EMT and metastatic recurrence of colorectal cancer.

结直肠癌侵袭转移是其相关死亡的主要原因。上皮-间质转化（EMT）在其中扮演着关键角色。我们前期研究提示缺氧条件下HIF-1α能诱导EMT促进结直肠癌侵袭转移，但其下游分子如何调控EMT尚不清楚。有研究表明HIF-1α能诱导Smad复合体与靶基因结合，引起细胞内Snail表达被抑制，从而促进EMT。然而该机制是否同样存在于结直肠癌，尚未见研究报道。我们另一项研究发现术藤合剂能通过HIF-1α调控EMT抑制结直肠癌侵袭转移。但是该复方是否能调控其下游Smad7、Snail的表达仍需进一步验证。因此，我们提出了“术藤合剂通过HIF-1α/Smad7/Snail通路调控EMT抑制结直肠癌侵袭转移“的假说。本项目拟研究：1）验证HIF-1α/Smad7/Snail通路调控EMT影响结直肠癌侵袭转移的作用；2）重点研究该通路上是哪些分子介导术藤合剂发挥抑制结直肠癌EMT和侵袭转移的作用。

目的：我们拟通过本项目验证HIF-1α/Smad7/Snail通路调控EMT影响结直肠癌侵袭转移的作用；2）重点研究该通路上是哪些分子介导术藤合剂发挥抑制结直肠癌EMT和侵袭转移的作用。方法：通过免疫组化芯片筛选人肠癌和正常肠组织配对标本的差异性表达基因；结合慢病毒转染、Transwell、划痕实验、实时定量PCR、Western blot等检测HIF-1α/Smad7/Snail通路各分子对EMT及肠癌细胞（HCT116、HCT8）的影响；CCK-8筛选术藤合剂所需浓度，并结合上述方法及裸鼠肠转移模型观察该方对通路各分子表达的影响及对肿瘤的抑制作用。结果：1）在正常组织中HIF-1α下调，Smad7下调，Snail表达未检出。HIF-1α、Smad7表达与肿瘤侵袭性呈正相关。2）Smad7敲减后Vimentin表达降低，E-cadherin、Sanil和Claudin-4表达水平升高，HCT116细胞迁移能力降低；沉默Snail后，Vimentin表达升高，E-cadherin、Claudin-4表达水平降低，HCT116细胞迁移能力提高。3）HCT8的IC50值是90.68mg/ml，HCT116的IC50值是115.4mg/ml。术藤合剂干预后HIF-1α表达下降，Smad7表达下降，Snail表达上升，上皮标记物Claudin-4、E-cadherin的表达上升，间质标记物Vimentin的表达下降。结论：术藤合剂能通过介导HIF-1α表达，调控Smad7/Snail途径抑制EMT从而降低结直肠癌侵袭转移。