小ras同源蛋白Ⅰ基因在胰腺癌中的抑癌作用及其对表皮生长因子-Ras-Raf-促分裂原活化蛋白激酶/细胞外信号调节激酶信号转导通路的影响

Inhibitory Effect of A Ras Homologue Member Ⅰ on Pancreatic Cancer and Its Modulation on Epithelium Growth Factor Receptor-RasRaf-Mitogen-Activated Protein Kinase/Extracellular Regulated Protein Kinase/1/2 Pathway

目的探讨抑癌蛋白小ras同源蛋白I(ARHI)转染对胰腺癌细胞Panc-1细胞增殖的影响,评估其对表皮生长因子(EGF)-Ras通路相关信号蛋白表皮生长因子受体(EGFR)、细胞外信号调节激酶(ERK)1/2、pan-Ras的作用。方法采用重组质粒pIRES2-EGFP-ARHI瞬时转染Panc-1细胞,观察EGF刺激下ARHI基因对细胞增殖的影响。酶联免疫吸附试验(ELISA)检测ARHI基因对细胞膜和细胞质中EGFR表达情况的影响,Western blot法检测不同EGF浓度刺激下ARHI蛋白的表达对Panc-1细胞内EGF-EGFR-Ras-Raf-MAPK/ERK1/2通路蛋白pan-Ras和P-ERK的影响,并与未转染Panc-1细胞进行比较。结果转染pIRES2-EGFP-ARHI后,Panc-1细胞增殖显著受抑。转染组和未转染组Panc-1细胞中EGFR的表达不存在明显差异,但受EGF作用的影响。转染组pan-Ras表达显著低于未转染组(P<0.05)。以50 ng/ml EGF刺激Panc-1瞬时转染细胞,结果显示在24 h内P-ERK1/2有短暂激活,长期(72 h内)则呈下降趋势。结论 ARHI和EGF可能共同作用于EGF-EGFR-Ras-Raf-MAPK/ERK通路,影响EGFR、pan-Ras和P-ERK表达。

Objective To investigate the regulation of epithelium growth factor receptor （EGFR）, pan-Ras, and extracellular regulated protein kinase （ERK） with both a ras homologue member I （ARHI） sup- pression and epithelium growth factor （EGF） stimulation. Methods After identification and implication, the constructed plasmid pIRES2-EGFP-ARHI was transfected into Panc-1. The untransfected cell was also explored as controls. The growth curve was drawn to indicate the proliferation effect of ARHI. EGFR-ELISA wasperformed to investigate the expression of EGFR. Western blot analysis was used to investigate the expression of protein MAPK/ERK1/2, pan-Ras in Panc-1. Results The proliferation rate of Panc-1 was inhibited by ARHI compared with both empty plasmid and untransfected cell. The amount of EGFR was parallel in both transfected and untrasfected cell but affected by EGF stimulation. The amount of pan-Ras was decreased after ARHI trans- fection. The optimum concentration of EGF effect on P-ERK was 50 ng/ml. Conclusion Both ARHI and EGF play roles in the EGF-EGFR-Ras-Raf-MAPK/ERK1/2 pathway.