Gli抑制剂GANT61对肺癌细胞上皮-间质转化作用的研究

摘要

目的：研究GANT61对人肺癌细胞 H1703和 A549的上皮‐间质转化（EMT）的影响，并初步探讨其作用机制。方法以二甲基亚砜（DMSO）作为对照（DMSO组），用GANT61处理H1703和A549细胞24 h后，采用实时荧光定量PCR法检测Gli‐1、Gli‐2、E‐cadherin和Vimentin基因变化；蛋白质印迹法（Western blotting）观察GANT61作用于 H1703和A549细胞后对E‐cadherin和Vimentin蛋白表达的影响，划痕愈合实验观察GANT61作用于H1703和A549细胞后对肿瘤细胞侵袭能力的影响。结果与DMSO组比较，实时荧光定量PCR结果显示GANT61可下调H1703和A549细胞Gli‐1、Gli‐2及Vimentin mRNA的表达，升高E‐cadherin mRNA的表达（P＜0．01）；Western blotting显示，GANT61可以下调H1703和A549细胞Vimentin蛋白表达，升高E‐cadherin蛋白表达（P＜0．01）；划痕愈合实验显示，GANT61处理组 H1703和A549细胞的侵袭能力明显下降（P＜0．01）。结论肺癌的EMT与 Hedgehog信号转导通路中Gli‐1和Gli‐2的异常激活有关，GANT61通过下调Gli‐1和Gli‐2的表达影响肺癌细胞的EM T能力，Gli可能成为抑制肺癌细胞转移的新的分子靶点。%Objective To research the effect of GANT61 on epithelial‐mesenchymal transition (EMT) in human lung cancer H1703 and A549 cells lines ,and to preliminarily investigate its action mechanism .Methods DMSO was used as the control(DMSO group) .After treating H1703 and A549 cells with GANT61 for 24 h ,the gene changes of Gli‐1 ,Gli‐2 ,E‐cadherin and Vimentin were detected by using the real time fluorescence quantitative PCR method .The influence of GANT61 on the expression of E‐cad‐herin and Vimentin protein after acting on H1703 and A549 cells was observed by using the Western blotting assay .The scratch healing test was performed to evaluate the effect of GANT 61 on the tumor cell invasion ability after acting on H1703 and A549 cells .Results The real time fluorescence quantitative PCR showed that ,compared with the DMSO group ,GANT61 down‐regulated the mRNA expression of Gli‐1 ,Gli‐2 and Vimentin mRNA of H1703 and A549 cell lines and elevated the expression of E‐cadherin protein(P<0 .01);the Western blotting showed that GANT61 down‐regulated the expression of Vimentin of H1703 and A549 cell lines ,and elevated the expression of E‐cadherin(P<0 .01);the scratch healing test revealed the invasion ability of H 1703 and A549 cells in the GANT61 treatment group was significantly decreased (P<0 .01) .Conclusion EMT in lung cancer is related with aber‐rant activations of Gli1 and Gli2 in Hedgehog signal transduction pathway ,GANT61 could influence the EMT ability in lung cancer cells by down‐regulating the expression of Gli‐1 and Gli‐2 .Gli could become a new molecular target for inhibiting the lung cancer cell metastasis.