TGF-β1诱导胃癌细胞上皮间质转化与CD133表达研究

TGF- β1 induces epithelial- mesenchymal transition in gastric cancer cells and its mechanism

目的研究转化生长因子-β1(TGF-β1)促进胃癌侵袭能力,诱导胃癌MKN-45细胞发生上皮间质转化(EMT)的机制及其与PI3K/Akt信号通路调控肿瘤起始细胞标志物CD133表达的关系。方法设空白对照,应用TGF-β1处理MKN-45细胞(TGF-β1处理组),观察其对细胞形态学的影响;Transwell检测细胞侵袭能力的改变;RT-PCR及Western blot检测细胞EMT相关因子Snail、E-cadherin、N-cadherin、p-Akt及CD133的表达水平;PI3K特异性抑制剂LY294002预处理后再应用TGF-β1处理细胞(TGF-β1+LY294002处理组),检测p-Akt与CD133表达水平的变化;免疫磁珠分选CD133+与CD133-亚群细胞,检测CD133+组与CD133-组细胞EMT相关因子的表达差异。结果TGF-β1处理72h后,TGF-β1处理组细胞由上皮形态转化为间质形态。TGF-β1处理组Snail、N-cadherin mRNA、蛋白表达水平均高于对照组(均P<0.05);而E-cadherin mRNA、蛋白表达水平均低于对照组(均P<0.05);Transwell检测发现TGF-β1处理组穿膜细胞数高于对照组(P<0.05);TGF-β1处理组p-Akt蛋白与CD133mRNA、蛋白表达水平均高于对照组(均P<0.05);TGF-β1+LY294002处理组p-Akt蛋白与CD133 mRNA、蛋白表达水平均低于TGF-β1处理组(均P<0.05)。CD133+组Snail、N-cadherin mRNA、蛋白表达水平均高于CD133-组(均P<0.05),而E-cadherin mRNA、蛋白表达水平均低于CD133-组(均P<0.05)。结论TGF-β1诱导胃癌细胞发生EMT,并通过PI3K/Akt信号通路调控CD133表达从而增强胃癌MKN-45细胞的侵袭能力。

Objective To investigate the effect of TGF-β1 on the cancer cells and its mechanism. Methods Human gastric cancer epithefial-mesenchymal transition （EMT） in gastric MKN-45 cells were treated with TGF-β 1. The micromorphological changes were observed and the invasive potency of MKN-45 cells were examined with Transwell method; the mRNA and protein expressions of Snail, E-cadherin, N-cadherin, p-Akt and CD133 were detected by RT-PCR and Western blot, respectively. The MKN-45 cells were pretreated with PI3K special inhibitor LY294002, then treated with TGF-β1, and the expressions of p-Akt and CD133 were detected. CD133＋ and CD133 cells were sorted by MACS and the expression of EMT-related proteins were measured and compared between two subsets of MKN-45 cells. Results The micromorphology of culture cells was changed to mesenchymal profiles 72h after TGF-β1 treatment; and the mRNA and protein expression levels of Snail and N-cadherin were higher than those in control group （P〈0.05）, the mRNA and protein expression levels of E-cadherin were significantly lower than those in control group （P〈0.05）. The invasion ability of TGF-β 1 treated group was higher than that of control group （P〈0.05）. The relative protein expression levels of p-Akt and CD133 in TGF-β1 treated group were higher than those in control group（P〈0.05）. In LY294002 pretreated cells, the p-Akt and CD133were down-regulated（P〈 0.05）. The relative mRNA and protein expression levels of Snail, N-cadherin in CD133＋ group were higher than those in CD133 group （P〈0.05）, the relative mRNA and protein expression levels of E-cadherin in CD133＋ group were lower than those in CD133- group（P〈0.05）. Conclusion TGF-β1 can induce EMT in MKN-45 cells, enhance the invasion ability of MKN-45 cells and up-regulate the expression of CD133 via Pi3K/Akt pathway.