NEDD4促进非小细胞肺癌继发厄洛替尼耐药

NEDD4 promotes acquired erlotinib resistance in non-small cell lung cancer

目的探讨NEDD4(neural precursor cell expressed developmentally downregulated protein 4)在非小细胞肺癌(non-small cell lung cancer,NSCLC)继发厄洛替尼耐药中的作用。方法以HCC827细胞及耐厄洛替尼的HCC827(HCC827/ER)细胞为工具,CCK-8法检测HCC827/ER细胞的耐药指数;实时荧光定量PCR(quantitative real-time,q PCR)及Western blot检测厄洛替尼处理48 h后,NEDD4 mRNA和蛋白在两种细胞中的表达及PI3K/AKT信号通路活化情况。NEDD4小干扰RNA(si NEDD4)转染HCC827/ER细胞,比较处理前后HCC827/ER细胞的厄洛替尼IC50变化以及PI3K/AKT信号通路活化情况。裸鼠成瘤实验在活体水平上进一步验证NEDD4在NSCLC继发厄洛替尼耐药中的作用。结果HCC827/ER细胞的耐药指数为(118.23±23.77);HCC827/ER细胞NEDD4 mRNA和蛋白以及PI3K/AKT信号通路活化水平均高于HCC827细胞;HCC827/ER细胞成功转染si NEDD4后,转染组的PI3K/AKT信号通路活化水平降低,且厄洛替尼IC50值明显低于对照组(P<0.05)。裸鼠成瘤实验中转染组肿瘤对厄洛替尼的敏感性明显增加,与阴性对照组比较,药物处理组肿瘤生长受到明显的抑制。结论NEDD4通过激活PI3K/AKT信号通路促进NSCLC继发厄洛替尼耐药。

Objective To investigate the role of neural precursor cell expressed developmentally downregulated protein 4 （NEDD4） to erlotinib acquired resistance in nonsmall cell lung cancer （NSCLC）. Methods HCC827 and HCC827/ER cells （erlotinib resistant cells induced from HCC827 cells） were sub-cultured to evaluate erlotinib resistance index using CCK-8 kit. After treated with erlotinib for 48 h, the mRNA and protein levels of NEDD4 in these cells were detected by quantitative real-time PCR （qPCR） and Western blotting respectively. The activity of PI3K/Akt signaling pathway was measured at the same time. After knocking down NEDD4 in HCC827/ER with si-NEDD4, changes of erlotinib IC50 and PI3K/Akt signaling pathway activity were detected again. In vivo experiments were taken to further validate the role of NEDD4 in NSCLC acquired erlotinib resistance in nude mice. Results The resistance index of HCC827/ER to erlotinib was 118.23±23.77. Compared with the HCC827 cells, the HCC827/ER cells had higher mRNA and protein levels of NEDD4, and stronger activation of PI3K/Akt signaling pathway. NEDD4 knockdown by siNEDD4 transfection resulted in lowered activation of PI3K/Akt signaling pathway and reduced erlotinib IC50 than the control cells （P〈0.05）. In vivo experiments indicated that the nude mice bearing siNEDD4-transfected HCC827/ER cells had obviously enhanced sensitivity to erlotinib, and remarkably inhibited tumor growth when compared with the mice bearing the untrasfected cells. Conclusion NEDD4 promotes erlotinib acquired resistance in NSCLC through activation of PI3K/Akt signaling pathway.