沉默表皮生长因子受体的表达对肺腺癌A549细胞自噬的影响

目的：研究降低表皮生长因子受体（EGFR）的表达对肺腺癌A549细胞自噬活性的影响。方法：A549细胞中加入EGF处理，分为4组，即未处理组和10、25、50 ng/mL EGF处理组，用Western blot方法检测EGFR的蛋白表达；采用转染siRNA降低肺腺癌细胞A549细胞EGFR的表达，将A549细胞分为4组，即未处理组、EGFR siRNA-1组、EGFR siRNA-2组和EGFR siRNA-3组，分别采用Real-time RT-PCR和Western blot方法检测EGFR mRNA和蛋白表达水平来评价3个siRNA的沉默效果；将A549细胞分为转染EGFR siRNA组、转染空载体组、转染试剂组（只加入等量转染试剂）及未处理组，且4组均加入等量的50 ng/mL EGF，用Western blot检测LC3Ⅱ/LC3Ⅰ蛋白表达，观察沉默EGFR表达对自噬活性变化；A549细胞分为转染EGFR siRNA组和转染空载体组，用透射电子显微镜观察自噬小体。结果：转染siRNA后A549细胞EGF蛋白的表达降低，LC3Ⅰ向LC3Ⅱ的转化水平升高（P < 0.05），细胞内出现较多自噬小体，自噬活性明显增加。结论：降低EGFR的表达可以提高肺腺癌A549细胞自噬活性。

Silencing EGFR expression influenced autophagy activity in A549 cells

OBJECTIVE:The role of EGFR in autophagy activity in lung adenocarcinoma A549 cells was investigated using small interference RNA to silence EGFR expression. METHODS:A549 cells were divided into four groups:untreated,EGFR siRNA-1,EGFR siRNA-2 and EGFR siRNA-3 groups. Real-time RT-PCR and Western blot were used to detect degradation of EGFR after transfection with EGFR siRNA. In addition,A549 cells were treated with different EGF concentrations:0,10,25 and 50 ng/mL. Protein expression of EGFR was detected using Western blot. Furthermore,A549 cells were divided into four groups:EGFR siRNA,negative control siRNA,transfection reagent (only add equivalent transfection reagent) and untreated groups. All four groups also received 50 ng/mL EGF. Western blot was used to detect expression of LC3Ⅱ/LC3Ⅰ which was observed as the effect on silencing EGFR expression on autophagy activity. A549 cells were divided into EGFR siRNA transfection and negative control siRNA groups,and autophagosomes were observed by transmission electron microscope. RESULTS:After treatment of A549 cells with the three different EGFR siRNAs,the mRNA and protein expression of EGFR were significant reduced. Western blot indicates that increased LC3-Ⅱ/LC3-Ⅰ ratios were induced by down-regulation of EGFR. Transmission electron microscopy shows that EGFR siRNA treatment,compared with control siRNA treatment,induced marked formation of autophagsomes. CONCLUSION:Reducing the expression of EGFR increased autophagy activities of lung adenocarcinoma A549 cells.