EGFR过表达降低人子宫内膜癌细胞孕激素敏感性的研究

Over-expression EGFR reduced sensitivity of human endometrial carcinoma cells to progesterone

目的:检测表皮生长因子受体(EGFR)基因在子宫内膜癌孕激素敏感细胞株Ishikawa及孕激素不敏感细胞株KLE的表达,探讨EGFR基因过表达对人子宫内膜癌细胞孕激素敏感性的影响。方法:实时定量PCR法和蛋白印迹法检测Ishikawa和KLE细胞中EGFR及PR-BmRNA和蛋白的表达。将EGFR全长cDNA真核表达质粒在脂质体介导下转染至Ishikawa细胞,同时以转染空载体和未转染的Ishikawa细胞为对照,分别应用实时定量PCR检测各组细胞EGFR、PR-BmRNA表达的变化,应用蛋白印迹法检测各组细胞EGFR、PR-B蛋白表达的变化;CCK-8法观察转染EGFR基因后Ishikawa细胞对孕激素敏感性的变化。结果:(1)Ishikawa细胞中,EGFRmRNA和蛋白的表达明显低于KLE细胞(P<0.001),而PR-BmRNA和蛋白的表达则显著高于KLE细胞(P<0.001);(2)稳定转染EGFR基因后,Ishikawa细胞中EGFRmRNA和蛋白的表达水平明显高于转染空载体和未转染的Ishikawa细胞(P<0.001),而PR-BmRNA和蛋白的表达水平则显著降低;(3)10-8、10-7、10-6、10-5mol/L的MPA对未转染和转染空载体的Ishikawa细胞的抑制作用显著(P<0.05),但对稳定过表达EGFR的Ishikawa细胞无明显抑制作用(P>0.05)。结论:转染EGFR基因能有效提高Ishikawa细胞内EGFR基因的表达,但可下调PR-B基因的表达使Ishikawa细胞对MPA不敏感。

Objective：To examine expressions of EGFR gene in endometrial carcinoma progesterone-sensitive cell line Ishikawa cells and progesterone-resistant cell line KLE cells,and evaluate the effect of over-expression EGFR gene on progesterone sensitivity of Ishikawa cells.Methods：The expressions of EGFR,PR-B mRNA and protein in Ishikawa and KLE cells were detected by real-time PCR and Western blot.Recombinant eukaryotic expression plasmid containing human hole length cDNA of EGFR gene was transfected into Ishikawa cells by lipofectamine 2000.The expressions of EGFR,PR-B mRNA were monitored by real-time PCR and the expressions of EGFR,PR-B protein were analyzed by Western blot in EGFR transfected and untransfected Ishikawa cells.Proliferation and sensitivity of cells to progesterone were measured by Cell Counting Kit-8（CCK-8）.Results：（1）The expressions of EGFR mRNA and protein in Ishikawa cells were significantly lower than those in KLE cells（P0.001）.However the expressions of PR-B mRNA and protein were converse（P0.001）.（2）After stably transfected with EGFR gene,the expressions of EGFR mRNA and protein in Ishikawa cells were higher than those in Ishikawa cells tranfected with empty vector and untransfected Ishikawa cells（P0.001）.Interestingly,the expressions of PR-B mRNA and protein in overexpression EGFR Ishikawa cells were significantly decreased（P0.001）.（3）MPA treatment reduced the growth of parental Ishikawa cells and Ishikawa cells tranfected with empty vector in a dose-dependent manner：when MPA added at 10-7,10-6,10-5mol/L,the inhibitory effects were significantly respectively（P0.05）.But,the inhibitory effects in over-expression EGFR Ishikawa cells were not obvious（P0.05）.Conclusion：Transfection of EGFR can increase the expression of EGFR in Ishikawa cells,and decrease the sensitivity of MPA of human endometrial carcinoma cell line Ishikawa by reducing the expression of PR-B.