猕猴桃根抗肿瘤有效组分调节miR-34a/Axl/NF-κB轴抑制胃癌侵袭性生长的作用研究

How to stop the invasion and metastasis of tumor has been a hotspot and difficulty of the gastric cancer prevention and control research. We have confirmed that the main component of actinidia chinensis actinidia polysaccharides and total threeterpenoids can block the gastric cancer cell invasion and metastasis through the .inhibition of EMT . We have also determine the optimal ratio of Radix Actinidiae total three terpenoids and total polysaccharide, and consider the compatibility of actinidia chinensis as antitumor effective components，We have also found that the antitumor active group of actinidia chinensis can inhibit the metastasis of gastric cancer cells by upregulating miR-34a. The miR-34a target gene Axl in human gastric cancer cells often expressed abnormally and it can induce EMT through NF- κ B pathway, Thus we speculate that the antitumor active group of actinidia chinensis can block EMT on invasion and metastasis of gastric cancer cells by miR-34a/Axl/NF- κ B axis. The project will study the role of antitumor active group of actinidia chinensis in the regulation of miR-34a and its downstream Axl /NF- κ B pathway. We will confirm miR-34a/Axl/NF- κ B axis is necessary for the blocking of EMT in human gastric cancer cell lines. Finally, We will carry out the animal experiments to prove the antitumor active group of actinidia chinensis may inhibit the invasion and metastasis of gastric cancer through the miR-34a/Axl/NF- κ B axis. The new project will be completed for the mechanism of anti-tumor medicine and enriched the Chinese medicine tumor treatment content.

阻止肿瘤侵袭转移一直是当前胃癌防控研究的热点和难点，项目组前期研究基础中已证实猕猴桃根的主要成分猕猴桃多糖和总三萜类化合物均可以通过抑制EMT阻断胃癌细胞转移侵袭，确定了总三萜与总多糖抗肿瘤的最佳配比，为猕猴桃根抗肿瘤有效组分；并发现猕猴桃根抗肿瘤有效组分具有上调miR-34a表达抑制胃癌细胞转移的作用，miR-34a靶基因Axl在胃癌细胞中异常表达，Axl通过NF-κB通路诱导EMT，推测有效组分可以通过miR-34a/Axl/NF-κB轴阻断EMT抑制胃癌的侵袭转移。项目将系统研究猕猴桃根抗肿瘤有效组分对表达miR-34a及其下游Axl/NF-κB通路的调控作用，明确miR-34a/Axl/NF-κB轴是其阻断胃癌细胞EMT所必需的；通过动物模型验证其通过miR-34a/Axl/NF-κB轴抑制胃癌细胞侵袭性生长，为中医抗肿瘤机理提供新的实验资料，丰富了中医肿瘤治疗学的内容。

阻止肿瘤侵袭转移一直是当前胃癌防控研究的热点和难点，项目组开展了大量对猕猴桃根的研究工作，为充分挖掘猕猴桃根有效组分的抗肿瘤活性，本研究主要由四部分组成：①确定猕猴桃根抗肿瘤有效组分（熊果酸、齐墩果酸、粗多糖）浓度最佳配比，发现有效组分可降低BGC-823、MKN-7、MGC803和SGC-7901细胞的迁移，能显著诱导细胞凋亡（34.77%）；基因芯片技术分析有效组分干预前后的胃癌细胞BGC823 miRNA差异表达谱，筛选出24种差异表达miRNA，其中miR-630上调最为显著；RT-PCR重复验证，miR-630表达也是增加的，与芯片结果相符；②细胞药物毒性实验中齐墩果酸对HGC-27细胞抑制作用，熊果酸对BGC823和HGC-27细胞有抑制作用；MTT法测定齐墩果酸、熊果酸在一定浓度下可抑制肿瘤生长；RT-PCR法测定熊果酸、齐墩果酸作用于BGC823和HGC-27细胞后miR-630表达降低；流式结果显示：熊果酸能显著诱导细胞凋亡，熊果酸联合齐墩果酸能显著诱导细胞凋亡，且诱导效果优于单药熊果酸，三药联合能显著诱导细胞凋亡，且诱导效果略微优于单药熊果酸，我们推测是熊果酸起到诱导细胞凋亡的主要作用；③Transwell检测熊果酸对BGC823、AGS、MGC803细胞有明显的抑制迁移作用；WB检测熊果酸对细胞中EMT相关蛋白蛋白表达水平，熊果酸能激活BGC823细胞中Axl、IKKα/β的表达，抑制MGC803细胞中p65、Axl的活化，我们推测熊果酸能抑制Axl/NF-κB通路活性，从而阻断胃癌EMT及转移；④在原位移植瘤的研究中，HE染色发现各组脏器中有不同程度的炎症反应，提示炎症和胃癌转移之间可能存在相互联系；免疫组织化学实验和Western blot实验，以齐墩果酸与熊果酸联合应用，N-cadherin与Snail下调表达最为显著，表明猕猴桃根抗肿瘤有效组分的抑瘤机制可能与下调EMT标志蛋白N-cadherin、和转录因子Snail表达有关。. 探讨中医药治疗胃癌的作用机理，并在此基础筛选和研制治疗胃癌的高效低毒中药新制剂，已经成为公认的可能提高胃癌综合防治水平的新的突破口。本项目的实施可为中医药防治EMT介导的胃癌侵袭转移提供新思路和新方法，为中药治疗胃癌提供有效的临床依据。