靶向抑制表皮生长因子受体对胰腺癌细胞上皮-间质转化的影响及机制

Suppression of epidermal growth factor receptor inhibits epithelial-mesenchymal transition in hu- man pancreatic cancer cells

目的观察靶向抑制表皮生长因子受体（EGFR）对人胰腺癌细胞（PANC-1）上皮-间质转化（EMT）过程的影响，探讨其机制。方法设计并合成针对EGFR基因特异的RNA干扰靶点，运用慢病毒介导的RNA干扰技术沉默人胰腺癌PANC-1细胞EGFR基因表达。应用划痕试验及基质胶侵袭试验检测EGFR干扰后细胞运动、侵袭能力的变化；应用实时定量聚合酶链反应（Real-timePCR）及Westernblot法检测转染后EMT相关标志物E-钙粘蛋白（E-Cadherin）、N-钙粘蛋白（N—Cadherin）、波形蛋白（Vimentin）、纤维粘连蛋白（Fibronectin）等的表达变化，并检测EMT相关转录因子的表达变化；应用细胞免疫荧光共聚焦显微镜检测上述蛋白的定位及细胞形态的变化。结果应用慢病毒介导的RNA干扰技术可特异性抑制人胰腺癌PANC-1细胞EGFR基因的表达。抑制EGFR后PANC-1细胞的运动及侵袭能力显著降低；上皮表型标志物E-Cadherin表达明显上调，间质表型N-Cadherin、Vimentin、Fibronectin等的表达显著下调；与对照组比较，EGFR干扰组细胞由纺锤形变成立方形，伪足消失；EGFR干扰组中EMT相关转录因子Snail及Slug表达显著下调。结论应用慢病毒介导的RNA干扰技术特异性沉默EGFR基因的表达，可显著抑制PANC-1细胞的EMT过程，其机制可能与下调转录因子Snail及Slug表达有关。

Objective To determine the role of epidermal growth factor receptor （EGFR） in mediating epithelial-mesenchymal transition （EMT） in pancreatic cancer cells （PANC-1）. Methods PANC-1 cells were transfected with small interfering RNA of EGFR using 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 assay were used to analyze cell migration and invasion. Real-time polymerase chain reaction （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, Twistl, and Sipl in PANC-1, NC-PANC-1, and si-PANC-1 cells. hnmunofluorescent staining with the above antibodies and confocal microscopy were applied to observe their cellular location and morphologic changes. Results After RNA interference of EGFR, the migration and invasion ability of si-PANC-1 ceils were decreased significantly. The expression of epithelial phenotype marker E-Cadherin was increased and that of mesenchymal phenotype markers N-Cadherin, Vimenfin, and Fibronectin was decreased, indicating reversion of EMT. The expression of transcription factors Snail and Slug in si-PANC-1 cells was decreased significantly. Conclusion Suppression of EGFR expression can significantly inhibit EMT of pancreatic cancer PANC-1 ceils probably through the down-regulation of the expression of transcription factors Snail and Slug.