Gli抑制剂GANT61抑制食管腺癌细胞发生上皮-间质转化

Gli inhibitor GANT61 inhibits epithelial-mesenchymal transition in esophageal adenocarcinoma

目的:研究Gli抑制剂GANT61对人食管腺癌细胞OE19和OE33发生上皮-间质转化(epithelial-mesenchymal transition,EMT)的影响,并探讨其可能的抗肿瘤机制。方法:用GANT61(10μmol/L)和重组sonic hedgehog(Shh)蛋白(0.5mg/mL)(阳性对照组)处理OE19和OE33细胞24 h后,分别采用实时荧光定量PCR法和蛋白质印迹法检测OEl9和OE33细胞中Gli1、Gli2、波形蛋白(vimentin)、N-钙黏连蛋白(N-cadherin,N-cad)和E-钙黏连蛋白(E-cadherin,E-cad)mRNA和蛋白表达的变化,划痕愈合实验观察OE19和OE33细胞迁移能力的变化。结果:实时荧光定量PCR和蛋白质印迹法检测结果显示,GANT61处理OE19和OE33细胞24 h后,可以明显下调细胞中Gli1、Gli2、vimentin和N-cad mRNA和蛋白的表达水平,而上调E-cad mRNA和蛋白的表达水平(P值均<0.01)重组Shh蛋白可以上调Gli1、Gli2、vimentin和N-cad mRNA及蛋白的表达水平,下调E-cad mRNA和蛋白表达(P值均<0.01)。划痕愈合实验结果显示,GANT61可以显著降低OE19和OE33细胞的迁移能力,而重组Shh蛋白则促进肿瘤细胞的迁移能力(P值均<0.01)。结论:GANT61可以通过抑制Hedgehog信号通路中Gli1和Gli2的表达来影响食管腺癌发生EMT,从而导致肿瘤细胞迁移能力的降低。预测Gli可成为有效抑制食管腺癌细胞转移的新的分子靶点。

Objective： To investigate the effect of Gli inhibitor GANT61 on epithelial-mesenchymal transition （EMT） of the human esophageal adenocarcinoma OE19 and OE33 cells, and to explore its possible mechanism. Methods： OE1 9 and OE33 cells were treated with GANT61 （1 0 μmol/L） and recombinant sonic hedgehog （Shh） protein （0.5 mg/mL） （as positive control） for 24 h. The mRNA and protein expression levels of Glil, Gli2, N-cadherin （N-cad）, E-cadherin （E-cad） and vimentin were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The migration of OE19 and OE33 cells was measured by wound-healing assay. Results： The results of real-time fluorescent quantitative PCR and Western blotting showed that treatment with GANT61 for 24 h, the mRNA and protein expression levels of Gill, Gli2, vimentin and N-cad in OE19 and OE33 cells were significantly decreased, but the expression levels of E-cad mRNA and protein in OE19 and OE33 cells were significantly increased （all P 〈 0.01）. After stimulation of Hedgehog pathway with recombinant Shh protein, the mRNA and protein expression levels of Glil, Gli2, vimentin and N-cad in OE19 and OE33 cells were significantly increased, but the mRNA and protein expression levels of E-cad in OE19 and OE33 cells were significantly decreased （all P 〈 0.01）. The result of wound-healing assay showed that GANT61 could inhibit the migration abilities of OE19 and OE33 cells, but recombinant Shh could increase the migration abilities of OE19 and OE33 cells （all P 〈 0.01）. Conclusion： GANT61 can effectively inhibit EMT of esophageal adenocarcinoma by down- regulating the expressions of Glil and Gli2, which lead to reduce migration abilities of esophageal adenocarcinoma cells. Gli may be a new and potential target for treatment of esophageal adenocarcinoma.