Department of General Surgery, Peking University First Hospital, 8th Xishiku Street, Xicheng District, Beijing 100034, People's Republic of China.
Dig Dis Sci. 2012 May;57(5):1181-9. doi: 10.1007/s10620-012-2036-4. Epub 2012 Jan 21.
Aberrant expression of epidermal growth factor receptor (EGFR) has been detected in pancreatic cancer; however, the mechanisms of EGFR in inducing pancreatic cancer development have not been adequately elucidated. The objective of this study was to determine the role of EGFR in mediating epithelial-mesenchymal transition (EMT) in pancreatic cancer cells.
Pancreatic cancer cell line PANC-1 was transfected with small interfering RNA of EGFR by use of a lentiviral expression vector to establish an EGFR-knockdown cell line (si-PANC-1). PANC-1 cells transfected with lentiviral vector expressing negative control sequence were used as negative control (NC-PANC-1). Scratch assay and transwell study were used to analyze cell migration and invasion. Real-time PCR and Western blotting were used to detect the expression of EMT markers E-cadherin, N-cadherin, vimentin, and fibronectin and transcription factors snail, slug, twist1, and sip1 in PANC-1, NC-PANC-1, and si-PANC-1 cells. Immunofluorescent staining with these antibodies and confocal microscopy were used to observe their cellular location and morphologic changes.
After RNA interference of EGFR, the migration and invasion ability of si-PANC-1 cells decreased significantly. The expression of epithelial phenotype marker E-cadherin increased and the expression of mesenchymal phenotype markers N-cadherin, vimentin, and fibronectin decreased, indicating reversion of EMT. We also observed intracellular translocation of E-cadherin. Expression of transcription factors snail and slug in si-PANC-1 cells decreased significantly.
Suppression of EGFR expression can significantly inhibit EMT of pancreatic cancer PANC-1 cells. The mechanism may be related with the down-regulation of the expression of transcription factors snail and slug.
表皮生长因子受体(EGFR)在胰腺癌中表达异常;然而,EGFR 诱导胰腺癌发展的机制尚未充分阐明。本研究旨在确定 EGFR 在介导胰腺癌细胞上皮-间充质转化(EMT)中的作用。
通过慢病毒表达载体转染胰腺癌细胞系 PANC-1 的 EGFR 小干扰 RNA,建立 EGFR 敲低细胞系(si-PANC-1)。转染表达阴性对照序列慢病毒载体的 PANC-1 细胞作为阴性对照(NC-PANC-1)。划痕实验和 Transwell 研究用于分析细胞迁移和侵袭。实时 PCR 和 Western blot 用于检测 EMT 标志物 E-钙黏蛋白、N-钙黏蛋白、波形蛋白和纤连蛋白以及转录因子 snail、slug、twist1 和 sip1 在 PANC-1、NC-PANC-1 和 si-PANC-1 细胞中的表达。用这些抗体进行免疫荧光染色和共聚焦显微镜观察,观察它们的细胞内位置和形态变化。
EGFR 的 RNA 干扰后,si-PANC-1 细胞的迁移和侵袭能力显著下降。上皮表型标志物 E-钙黏蛋白的表达增加,间充质表型标志物 N-钙黏蛋白、波形蛋白和纤连蛋白的表达减少,表明 EMT 逆转。我们还观察到 E-钙黏蛋白的细胞内转位。si-PANC-1 细胞中转录因子 snail 和 slug 的表达显著下降。
抑制 EGFR 表达可显著抑制胰腺癌细胞 PANC-1 的 EMT。其机制可能与转录因子 snail 和 slug 的表达下调有关。